Synthetic phosphorylation signaling networks

Abstract

Presented herein are synthetic protein phosphorylation signaling networks.

Description

SYNTHETIC PHOSPHORYLATION SIGNALING NETWORKSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional application 63 / 663,492, filed on June 24, 2024, the contents of which are incorporated by reference in their entirety.SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The present application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated herein by reference in its entirety. Said computer readable file, was created on June 23, 2025 is named 130492-847322 Sequence Listing. xml and is 96 kilobytes in size.GOVERNMENT INTERESTS

[0003] This invention was made with government support under Grant No. N00014-21-1- 4006, awarded by the Department of Defense, Office of Naval Research, and Grant Nos. R01EB029483 and R01EB032272, awarded by the National Institutes of Health. The United States government has certain rights in the invention.FIELD OF THE INVENTION

[0004] This invention is directed to polypeptides comprising a synthetic domain or fragment thereof. In some aspects, the polypeptide comprises a synthetic kinase domain (synKin) or fragment thereof. In some aspects, the polypeptide comprises a synthetic phosphorylatable domain (synSub) or fragment thereof. In some aspects, the polypeptide comprises a synthetic phosphatase domain (synPhos) or fragment thereof.BACKGROUND

[0005] Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Cells universally use protein phosphorylation signaling networks to adapt to chemical and physical cues from their external environment. In metazoan cells, these networks consist of multilayered pathways that rapidly and reversibly convert signals detected by cell surface receptors into diverse responses such as cell movement, secretion, metabolism, and gene expression.SUMMARY OF THE INVENTION

[0006] In some aspects, provided herein is a polypeptide comprising: a synthetic kinase domain or fragment thereof; and a dimerization domain.

[0007] In some aspects, the polypeptide further comprises a protein tag. In some aspects, the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag. In some aspects, the protein tag is a FLAG tag. In some aspects, the FLAG tag comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0008] In some aspects, the synthetic kinase domain comprises a kinase involved in activation of T Cell Receptor or B Cell Receptor signaling or an Src family member. In some aspects, the synthetic kinase domain is selected from the group consisting of TCR, ZAP70, Syk, Lyn, Lek, and ABL. In some aspects, the synthetic kinase domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:2-16. In some aspects, the synthetic kinase domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:2-16.

[0009] In some aspects, the polypeptide further comprises a linker. In some aspects, the linker comprises a GS linker. In some aspects, the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0010] In some aspects, the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R. In some aspects, the dimerization domain comprises a Leucine Zipper E. In some aspects, the Leucine Zipper E comprises an amino acid sequence set forth in SEQ ID NO: 18.

[0011] In some aspects, the polypeptide comprises a Flag tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0012] Also provided herein, in some aspects, is a polypeptide comprising: a synthetic phosphorylatable domain or fragment thereof; and a dimerization domain.

[0013] In some aspects, the polypeptide further comprises a protein tag. In some aspects, the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag. In some aspects, the polypeptide comprises a MYC tag and a GST tag.

[0014] In some aspects, the synthetic phosphorylatable domain or fragment thereof comprises an immune tyrosine activation motif (ITAM) or an immune tyrosine inhibition motif (ITIM). In some aspects, wherein the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:21-32. In some aspects, the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:21-32.

[0015] In some aspects, the polypeptide further comprises a linker. In some aspects, the linker comprises a GS linker. In some aspects, the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0016] In some aspects, wherein the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R. In some aspects, the dimerization domain comprises a Leucine Zipper R. In some aspects, the Leucine Zipper R comprises an amino acid sequence set forth in SEQ ID NO:33.

[0017] In some aspects, the polypeptide comprises a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a GST tag comprising the amino acid sequence set forth in SEQ ID NO:20, a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0018] Also provided herein, in some aspects, is a polypeptide comprising: a synthetic phosphatase domain or fragment thereof; and a dimerization domain.

[0019] In some aspects, the polypeptide further comprises a protein tag. In some aspects, the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag,a Protein A / G tag, and a Halo tag. In some aspects, the protein tag is an HA tag. In some aspects, the HA tag comprises an amino acid sequence set forth in SEQ ID NO:34.

[0020] In some aspects, the synthetic phosphatase domain or fragment thereof comprises a tyrosine phosphatase. In some aspects, the tyrosine phosphatase is PTPN1, PTPN4, or PTPN6. In some aspects, the synthetic phosphatase domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:35-43. In some aspects, the synthetic phosphatase domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:35-43.

[0021] In some aspects, the polypeptide further comprises a linker. In some aspects, the linker comprises a GS linker. In some aspects, the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0022] In some aspects, the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R. In some aspects, the dimerization domain comprises a Leucine Zipper E. In some aspects, the Leucine Zipper E comprises an amino acid sequence set forth in SEQ ID NO: 18.

[0023] In some aspects, the polypeptide comprises a protein tag comprising the amino acid sequence set forth in SEQ ID NO:34, a synthetic phosphatase domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0024] Also provided herein, in some aspects, is a polypeptide comprising a synthetic cellular receptor, wherein the polypeptide comprises an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain.

[0025] In some aspects, synthetic cellular receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:53.

[0026] In some aspects, the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a FRB(K2095P) domain comprising the amino acid sequence set forth in SEQ ID NO:46, a transmembrane domain comprising the amino acidsequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0027] In some aspects, the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:49, an FKBP domain comprising the amino acid sequence set forth in SEQ ID NO:50, transmembrane domains comprising the amino acid sequences set forth in SEQ ID NO:47, SEQ ID NO:51, and SEQ ID NO:52, and a Leucine Zipper E comprising the amino acid sequences set forth in SEQ ID NO: 18.

[0028] In some aspects, the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-ot scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0029] In some aspects, the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and a Leucine Zipper E domain comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0030] Also provided herein, in some aspects, is a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:2-16, or an amino acid sequence at least 90% identical thereto, fused to a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-32, or an amino acid sequence at least 90% identical thereto.

[0031] In some aspects, the polypeptide further comprises an amino acid sequence according set forth in any one of SEQ ID NOs:35-43 or an amino acid sequence at least 90% identical thereto.

[0032] Also provided herein, in some aspects, is a fusion protein comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:54 or 76-86.

[0033] Also provided herein, in some aspects, is a polypeptide comprising: a nuclear localization signal (NLS); one or more DNA binding polypeptides; a nuclear export signal (NES); and a synthetic phosphorylatable domain or fragment thereof.

[0034] In some aspects, the NLS comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:57.

[0035] In some aspects, the one or more DNA binding polypeptides comprise one or more zinc finger domains. In some aspects, the one or more DNA binding polypeptides comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:59.

[0036] In some aspects, the NES comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 62.

[0037] In some aspects, the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:63.

[0038] In some aspects, the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:21-32.

[0039] In some aspects, the polypeptide further comprises one or more linkers.

[0040] Also provided herein, in some aspects, is a polypeptide comprising: one or more nuclear localization signals; a recruitment motif; and a transcriptional activator.

[0041] In some aspects, the one or more nuclear localization signals comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NO:66 or SEQ ID NO:75.

[0042] In some aspects, the recruitment motif comprises an engineered SH2 domain. In some aspects, the recruitment motif comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs:67-72.

[0043] In some aspects, the transcriptional activator comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 74.

[0044] In some aspects, the polypeptide further comprises one or more linkers.

[0045] Also provided herein, in some aspects, is a vector comprising the nucleic acid disclosed herein.

[0046] Also provided herein, in some aspects, is a cell comprising the vector disclosed herein.

[0047] Also provided herein, in some aspects, is a fusion protein comprising a polypeptide with kinase activity fused to a polypeptide capable of being phosphorylated. In some aspects, the fusion protein further comprises a polypeptide with phosphatase activity.

[0048] Also provided herein, in some aspects, is a synthetic phosphorylation circuit comprising a polypeptide described herein. In some aspects, the synthetic phosphorylation circuit can convert an extracellular signal into an intracellular response.

[0049] In some aspects, synthetic phosphorylation circuit comprises a TNFa-sensing / IL-10 response circuit.

[0050] In some aspects, the synthetic phosphorylation circuit responds to TNF-a by a proportional secretion of IL- 10.

[0051] In some aspects, the conversion of an extracellular signal into an intracellular response comprises fast timescale transcription.

[0052] Also provided herein, in some aspects, is a cell comprising the synthetic phosphorylation circuit disclosed herein.

[0053] Also provided herein, in some aspects, is a method of treating a disease, disorder or medical condition, the method comprising administering to the subject a cell disclosed herein. In some aspects, the disease, disorder or medical condition comprises inflammatory diseases, autoimmune diseases, and / or metabolic diseases.BRIEF DESCRIPTION OF THE FIGURES

[0054] FIG. 1A provides data showing building and tuning synthetic phosphorylation cycles in human cells. Panel A provides a schematic showing phosphorylation cycles, where kinase and phosphatase activities mutually regulate (red arrows) phosphorylation equilibrium (black arrows) of a substrate. These cycles are fundamental units that make up phospho-signalingnetworks. Panel B provides a data showing engineering synthetic kinase (synKin) and substrate (synSub) pairs. Plasmids (grey rectangles) encoding synKin and synSub proteins are transfected into HEK293T cells and measured for expression and phosphorylation via immunofluorescence flow cytometry (green dotted lines) after 36 h. Leucine zippers (LZs) mediate interactions (cyan dashed line) between synKin and synSub proteins (left). Expression and phosphorylation data for synKin / synSub allele combinations are shown on the right as hexagonal-hit-and-heat (HHH), through the whole study the expression space was uniformly binned into grids, the hexagon size indicates the cell counts in each bin, the largest hexagon size represents highest cell density, while the smallest hexagon size represents lowest density, with hexagon sizes in between representing loglO-normalized cell counts proportionally (FIG. 9). Values associated with HHH plots are mean phosphorylation (AU) ± standard error of the mean (SEM) (n=3). Panel C provides data showing tuning synSub phosphorylation. Phosphorylation was measured for synKin-synSub compositions featuring parts that tune LZ binding affinity, synKin expression level, and synKin activity. The top panel shows behavior of the default synKin-synSub from FIG. IB for comparison. Numbers associated with HHH plots, mean phosphorylation (AU) ± SEM (n=3). Panel D provides data showing complete synthetic phosphorylation cycle. LZ-recruited synPhos dephosphorylates synSub (left). Values adjacent to HHH plots of synPhos variants indicate mean phosphorylation (AU) ± SEM (n=3) (right). Panel E provides data showing modeling synthetic phosphorylation cycle equilibrium. LZ interactions Kws, synKin-synSub affinity [W, “writer”; S, “substrate”]; KES, synPhos-synSub affinity [E, “eraser”; , “substrate”) and component activity (kws, normalized phosphorylation rate; k ES, normalized k dephosphorylation rate) normalized dephosphorylation rate) of the cycle (left). Phosphorylation cycle data from FIG. IB to FIG. ID were used for fitting. Model- predicted phosphorylation distributions (red) are plotted against experimentally measured distributions (grey). Kolmogorov-Smirnov divergence DKS) values comparing model to experiment are shown for each plot, (norm., normalized). Panel F provides data showing model-predicted phosphorylation cycle behavior space. The beeswarm plot shows phosphorylation values for predicted (grey dots) and training (red dots) part compositions (216 total). HHH plots for compositions from across design space (indicated with black dotted lines) comparing predicted (red borders) and measured phosphorylation (black borders).Compositions of highlighted circuits are identical to synPhos composition in ID (strong synKin and synPhos LZ, strong synKin and synPhos activity, strong synKin and synPhos expression) except for the following: bottom left, low synKin expression, dead synPhos; bottom middle,medium synKin expression, weak synPhos LZ; top right, low synPhos; bottom right, weak synPhos activity, medium synKin LZ.

[0055] FIG. 2A provides data showing building and tuning phosphorylation cycle networks. Panel A provides data showing two-step phosphorylation cascade circuit. To create a synthetic phosphorylation network connection, an upstream cycle (red) can be coupled to a downstream cycle (blue) using a phosphorylation-dependent interaction (left). The upstream synKin phosphorylates a “phospho-couple” (PC) protein, which functions as synSub for the upstream cycle and synKin for the downstream one (right). The PC contains a rigid linker domain (cyan outline) that prevents cis substrate motif phosphorylation while supporting SH2-mediated recruitment of a downstream substrate (cyan dashed line; upper right). Panel B provides data showing two-step circuit validation. HHH plots for PC (left column) and synSub (right column) phosphorylation are shown for various circuit compositions. All circuit compositions contain synPhos. Brackets indicate the number of IT AM motif repeats. Values in each plot are mean phosphorylation (AU) ± SEM (n=3). Panel C provides data showing model-predicted circuit behavior space for two-step circuit. The modeling framework was used to fit steady-state phosphorylation of two-step push-pulls from FIG. 2B (See FIG. 26A). Scatter plot shows predicted mean phosphorylation for compositions with (y-axis) and without (x-axis) upstream synKin (red dots, training set compositions from FIG. 26B; grey dots, model -predicted compositions [3,456 total]). Region of >10x fold-change (+ / - synKin) is shown in cyan. Four high gain circuits (indicated with black dotted lines) were constructed and tested for synSub phosphorylation. HHH plots for model -predicted (red border) and experimental measurements (black border) are shown. Compositions of highlighted circuits are identical with the multivalent composition in FIG. 2B, except for the following: #1, medium PC kinase activity, high synPhos activity; #2, low PC expression; #3, high synKin expression, low PC expression; #4, high PC expression, high synPhos expression. Values at the top of each plot indicate mean phosphorylation (AU) ± SEM (n=3) (right). DKS values are shown at the bottom of each experimental plot. Predicted (red) and experimentally measured (black) fold change values are shown to the right of the plots.

[0056] FIG. 3A provides data showing constructing receptor-coupled sensor circuits. Panel A provides a schematic showing sensor circuit design. Reversible ligand binding to extracellular receptors triggers phosphorylation cycle activation (left). The circuit is encoded as 4 genes on 3 plasmids; it includes two receptor chains (FRB*-TMkinase domain and FKBP-TM-LZ), asynSub with 3 motif repeats, and a synPhos. Dimerization of extracellular FRB* and FKBP domains induced by ligand (AP21967) triggers colocalization of receptor-appended kinase with synSub (cyan box), leading to phosphorylation (right). Panel B provides data showing testing receptor-induced phosphorylation. HHH plots depict flow-cytometry data from sensor circuit compositions analyzed 12 h after treatment with 200 nM ligand (+ lig) or a carrier-only control (-lig). Values in each plot indicate mean phosphorylation (AU) ± SEM (n=3). Phosphorylation fold-change values are next to each set of plots. Dose response plot (bottom right) shows synSub phosphorylation as a function of ligand concentration. Individual triplicate measurements (circles) with mean ± SEM (error bars) are shown. Data were fitted (magenta line) to equation 150 (rin, Hill coefficient; ECso, input at 50% activation). Panel C provides data showing measuring pathway activation using an LLP condensate co-localization reporter. The reporter consists of EGFP-tagged synSub and an mCherry-tagged SH2 domain tagged with an intrinsically disordered protein (IDP); phosphorylation leads synSub-EGFP recruitment to condensates and EGFP / mCherry colocalization (left). For activation experiments, cells were cultured for 24 h and then time-lapse images were taken every 10 mins following ligand addition over an 80 min time course to track pathway activation (left time course plot). For deactivation experiments 200 nM ligand was added for 90 mins followed by addition of the inhibitor imatinib mesylate (10 pM), and then time-lapse images were taken every 5 mins following inhibitor addition over a 60 min time course (right time course plot). Data are plotted as single cell trajectories (thin pink lines) for activation (n=15 cells) and deactivation (n=10 cells), with mean values (thick pink line) ± SEM (shaded pink bands). For selected time points (0, 30, and 70 min for activation and 0 and 30 min for deactivation), images of EGFP and mCherry (false-colored green and red, right middle) are shown for representative single cells, with the cell boundaries (dotted white outlines) as determined by custom segmentation software. Histograms (right top) show max-normalized EGFP and mCherry intensities along the straight white dashed lines drawn in images, and intensity is plotted for each channel in the same plot, with the black dashed line representing max-normalized cytoplasmic EGFP intensity; shaded regions, outside the cytoplasm. Scale bars, 5 pm.

[0057] 4 provides data showing phosphorylation circuit-mediated closed-loop sense-and- response function. Panel A provides data showing the use of phospho-signaling to connect extracellular sensing to transcriptional output. The phosphosensor circuit (FIG. 3A) was coupled to a two-step amplifier circuit (FIG. 2 A), which was in turn coupled to aphosphorylation-dependent transcriptional reporter module (FIG. 23), yielding a membrane-to- nucleus signaling pathway. The PC is tagged with nuclear localization (NLS) and nuclear export signals (NES) to enable shuttling between the nucleus and cytoplasm. HHH plots for PC phosphorylation (left column) and EGFP expression (right column) are shown for cells + / - ligand for compositions with and without NLS / NES tags. Values in the plots indicate mean fluorescence (AU) ± SEM (n=3). Fold-change values are shown to the right of each set of plots. Panel B shows the cytokine sensor circuit. Receptor chains in FIG. 3A are appended to (singlechain variable fragments) scFvs that bind to human tumor necrosis factor alpha (TNF-a). TNF- a binding induced synSub phosphorylation. HHH plots depict flow-cytometry data analyzed 12 h after treatment with 20 ng / mL TNF-a (+lig) or a carrier-only control (-lig). Values in each plot indicate mean phosphorylation (AU) ± SEM (n=3). Panel C provides data showing engineering a phospho-signaling pathway for closed-loop therapeutic control. HEK293T cells expressing a circuit that can sense TNF-a (green arrow) and respond by secreting IL-10 (blue arrow) (top left) are placed in transwell coculture with activated T cells (bottom left) for 60 h, with media collected every 12 h to measure cytokine levels. T cell proliferation was assessed by EdU assay at 60 h. TNF-a and IL-10 time courses are shown for empty HEK293T cells (no circuit), constitutive IL- 10 expression driven by a non-receptor synKin driving phosphorylation of 2-step cascade (open loop), and the sense-and-respond circuit (closed loop). Each circle represents a different PBMC donor (black line, mean values; shaded regions, ± SEM) (middle). Measurements of CD4+ and CD8+ proliferation, with circles representing data from 3 PBMC donors (error bars, indicating mean values ± SEM, n=3 measurements; upper dashed line, proliferation [maximum EdU signal] of activated T cells alone with no HEK293T cell; lower dashed line, proliferation of activated T cells inhibited with 500 ng / ml IL- 10 [minimum EdU signal]).

[0058] FIG. 5 provides a schematic showing the cloning workflow. Multi-level hierarchical cloning workflow for constructing the plasmids described in Appendix A. For level 1 assemblies, PCR-amplification of DNA fragments or plasmids containing “sub-parts” (e.g., protein domains or promoter fragments) were used as inputs for assembly into ccdB-containing, AmpR entry vectors using Bsal (pink) to generate plasmids containing a promoter, open reading frame (ORF), or terminator. For Level 2 assemblies, EUs were constructed by assembling promoter, ORF, and terminator part plasmids into a KanR destination vector containing a dual ccdB-mCherry2 expression cassette using Esp3I (blue). In the final level 3assemblies, EUs are combined into a destination vector containing an AmpR marker. This occurs via Bbsl-mediated assembly (purple) to replace a Crt operon marker, resulting in multi- EU arrays where genes are ordered based on Level 2 entry vector overhangs. Lower-case Greek and upper-case Roman letters represent different overhangs for type IIS assembly. Arrows on the vectors show the orientations of type IIS cutting.

[0059] FIG. 6 provides data showing native pathways as sources of synthetic signalling circuit parts. Panel A provides kinetic constants for various pS / T and pY kinases involved in signal transduction as measured in vitro against peptide substrates. Panel B provides a schematic showing TCR pathway activation. During TCR signalling, the Y residues of TCR IT AMs are phosphorylated after Y kinases (Lek, ZAP70) are physically recruited to the complex. The Y phosphatases are also recruited to the complex to dephosphorylate the pYs. The pYs can then serve as docking sites for specific tandem SH2 domains, mediating downstream signal transduction, which includes phosphorylation of the scaffold protein SLP76. Panel C provides a schematic showing the domain parts. The component properties of the domains comprising native signalling pathway proteins allow them to be appropriated and modified to create parts for constructing synthetic phosphorylation cycles.

[0060] FIG. 7 provides native tyrosine kinase sequence alignments. Amino acid sequences and 2° structure elements for all kinases tested in this study are shown. ZAP70 and Syk are aligned with each other. Src family kinases (Lyn and Lek) are also aligned. ABL is shown separately. Red boxes indicate predicted domain boundaries for SH2, SH3, and kinase domains (Uniprot predictions). 2° structure features (a-helix and P-strand) are also indicated. Purple lollipops indicate boundaries for tested truncation variants. Highlighted Ys indicate known phosphorylation sites.

[0061] FIG. 8 provides synthetic kinase and substrate designs. Amino acid sequences of the synKin and synSub pair featured in FIG. IB and available part variants are shown. The synKin contains a 3x FLAG epitope tag on the N terminus, an engineered pY kinase domain, a flexible lOx GS repeat linker, and an acidic leucine zipper (LZ-E) on the C terminus. A synKin derived from the kinase domain of ZAP70 (AAs 326-619) is shown (FIG. 10 A). The synSub features (from the N- to C-terminus) a 3x MYC epitope tag, GST, a Y-containing substrate motif derived from CD3Z (AAs 131-164), and a cognate basic zipper (LZ-R). GS linkers are inserted between GST and the substrate motif, as well as the substrate motif and the LZ-R. Brackets indicate part sequences that are varied to tune component function, including LZ affinity (3total LZ-E variants) and substrate identity (SLP76-derived motif [AAs 108-154] and / or multiple motif repeats). Mutated kinase domain residues that tune catalytic activity are shown in purple, including the residue that was altered to make the kinase dead allele (K369R). Residues that tune LZ affinity are orange. Y residues that are phosphorylated by synKin are shown as dark red. Dots indicate residues (Y142 for CD3Z and Y128 for SLP76) that are recognized in their phospho form by a-pY antibodies.

[0062] FIG. 9 shows a multi-color flow cytometry data processing workflow. Panel A provides data showing gating and plotting strategy. Events (l-2xl05) are size gated by forward (FSC-A) and side scatter area (SSC-A). Single cells were then gated by forward scatter height (FSC-H) versus area (FSC-A) to yield ~8.4xl04events. Cells were gated for BFP+ events to identify transfected cells. Adjacent box plots show event counts distribution for all experimental replicates reported in the study (n = 99). Transfection-gated cells are plotted as a two-dimensional scatter plot of component expression (e g., synKin vs. synSub) and then replotted as an HH plot, where event count is indicated by hexagon size. Average counts per hexagon across all HHH plots in the study are shown in the box plots. HHH plots are created by overlaying a colormap indicating mean phosphorylation level for events in each hexagon. Data shown are representative expression and phosphorylation levels for cells expressing a synKin and synSub (FIG. IB, upper right panel). Panel B provides data showing measuring spillover between fluorescence channels. Plasmids expressing individual components harboring epitopes for each antibody. Data from the channel corresponding to the emission of each fluorophore is colored and other histograms are grey (dotted lines, mean intensity from blank cells in each channel). Top, unmixing of a 6-color experiment. Bottom, unmixing of a 4-color experiment. Panel C provides data showing comparison of transfection reagents. Experimental groups from FIG. IB were transfected using PEI and jetPRIME and data plotted as HHH plots (number in each HHH plot, phosphorylation geometric mean).

[0063] FIG. 10A provides data showing screening kinase domain truncation variants for synKin optimization. Panel A provides a schematic showing synKin activity screen design. Truncation variants are fused to a cognate or non-binding (nb) LZ and co-expressed with synSub in HEK293T cells. Flow cytometry is used to measure variant expression levels (a- FLAG, APC) and phosphorylation signal (a-pY, PE). a-pY antibody signal from non-specific kinase activity is assessed by co-expression with a non-phosphorylatable (np) synSub. Recruitment-independent phosphorylation is assessed by co-expressing synSub with kinasevariants fused to a non-binding LZ. Recruitment-dependent activity is tested by expressing synSub with active kinase variants fused to cognate LZ. Panel B provides data showing background signal for phosphorylation level and component expression measured for empty cells and cells expressing synSub only. Panel C provides data showing phosphorylation signal and expression level for each truncated variant are measured for non-specific, unrecruited, and recruited kinase configurations. Numbers indicate the geometric mean of the measured histograms. Dotted lines indicate background measurements for cells transfected with BFP only. Purple box indicates the truncation allele chosen for further synKin engineering. Panel D provides data showing Screening synKin point mutants for optimized expression and activity profiles. Potentially phosphorylatable Y residues in selected ZAP70 and ABL truncation variants are mutated to F or E, fused to a cognate LZ, and co-expressed with synSub containing a single repeat of a CD3Z-derived IT AM motif (AAs 131-164). Phosphorylation level and kinase expression were measured for all the mutants. Numbers indicate histogram geometric means. Dotted lines indicate background measured for empty cells. Purple boxes indicate the mutant alleles chosen as synKins for use throughout the study.

[0064] FIG. 11 provides data showing testing the specificity of synthetic signalling components. Panel A provides data showing testing the two synKin designs identified in FIG. 10B (ZAP70326-619 FF and ABL228-540 FF) against synSubs harboring motifs derived from CD3Z and SLP76 (see FIG. 8), number in each HHH plot indicates phosphorylation geometric mean. Panel B provides Western blot analysis of synKin and synSub components (see FIG. IB), along with controls. Lysates were generated for indicated samples and probed for general a-pY, a-pY synSub, a-Flag, and a-Myc. In the pY blot, magenta bar plots display normalized intensity values relative to the loading control for the area highlighted by magenta boxes, with the dashed line showing the value from empty cells. In the pY synSub blot, pink bar plots display normalized intensity values for the area highlighted by pink boxes, while magenta bar plots show values for the area highlighted by magenta boxes subtracted by those highlighted by pink boxes to represent background signaling from the pY synSub antibody. The values above the bar plots indicate the fold change in synSub phosphorylation between the compared samples. Panel C provides data showing cells expressing synSub were treated with various Y kinase inhibitors and NaaVO4. Histograms of phosphorylation level is shown for each condition, with values showing mean phosphorylation (AU) ± SEM (n=2). Panel D provides data showing growth burden of synthetic signaling components on HEK293T cells.Cell populations transfected with constructs encoding expression of the indicated proteins were measured for viability and cell density every 12 h for the experimental groups in FIG. IB, with error bars representing mean ± SEM (n=3).

[0065] FIG. 12 provides native Y phosphatase sequences. Amino acid sequences with 2° structures for all the phosphatases tested in the study. PTPN1, PTPN3, and PTPN6 were chosen to represent the three PTP subfamilies. Red boxes indicate the boundaries for PTP domains and regulatory domains such as PDZ and SH2 (Uniprot predictions). 2° structure features (a-helix and P-strand) are also indicated. Purple lollipops indicate boundaries for tested truncation variants.

[0066] 13 provides data showing synthetic phosphatase designs and truncation variant screening. Panel A provides amino acid sequences of parts comprising synPhos. synPhos contains a 3x HA epitope tag, truncated phosphatase domain, flexible l Ox GS linker, and an LZ-E. A synPhos derived from PTPN1 is shown (FIG. 12). The purple residue indicates the position of the mutations used for the phosphatase dead allele (D181A, R221M). Panel B provides data showing phosphatase truncations are fused with a cognate LZ and co-expressed with the synKin and synSub shown in FIG. IB. The phosphorylation levels of synSub and expression levels of phosphatase domain truncation variants are measured by flow cytometry. Numbers next to the histogram indicate the geometric mean of the measured distribution. Dotted lines indicate the mean intensity from blank cells for phosphorylation and phosphatase expression. Purple box highlights the chosen variant for synPhos used in circuit construction throughout the study.

[0067] FIG. 14 provides design of constructs for fluorescence signal conversion. Constructs used for converting antibody-conjugated fluorophore signal to EGFP units are shown. The FLAG-to-EGFP construct replaces the synKin LZ from FIG. 8 with EGFP. The HA-to-EGFP construct replaces the synPhos LZ from FIG. 13 with EGFP. A single construct supports pCD3Z-to-EGFP and MYC-to-EGFP conversion; it has a 3x MYC epitope, ZAP70 kinase domain (from FIG. 8), lx CD3Z substrate motif, EGFP, and a cognate LZ-R. The V5-to-EGFP construct contains a 3x V5 epitope tag, GST, lx SLP76 substrate motif, EGFP, and LZ-R. 20x GS linkers separate the substrate motif from the synKin and EGFP. The pSLP76-to-V5 construct has 3x V5 epitope tag, ZAP70 kinase domain (from FIG. 8), lx SLP76 peptide, a cognate LZ-R, a 20x GS linker are inserted between kinase domain and SLP76, and a lOx GS linker between SLP76 and LZ. Constructs were expressed in HEK293T cells and used toconvert ab-conjugated fluorescence into EGFP signal. The left column shows the distribution of fluorescence signals from each of the constructs (represented by colored histograms) alongside empty cells (depicted in grey-shaded histograms). Middle column, the 2D scatter plot of the fluorescence vs. EGFP expression from each construct, with dashed lines denoting the powerlaw fit from ab-conjugated fluorescence to EGFP. Right column, the distribution of the EGFP signal from the corresponding construct (light green histograms) and empty cells (depicted in dark green histograms).

[0068] FIG. 15 provides a computational workflow for model fitting and phosphorylation prediction. On the left (from top to bottom), antibody-conjugated fluorescence values obtained from flow cytometry experiments are converted into stoichiometrically equivalent EGFP units (see FIG. 14) and then background fluorescence values are subtracted, yielding model-operable concentration unit (MOCU) distributions that are then used for fitting. On the right (from bottom to top), parameters iteratively generated from the model fitting via MLE are used to generate predicted phosphorylation levels in EGFP units, which then have background signal added back to them. Finally, predicted EGFP signals are transformed back into phosphoantibody fluorescence for comparison with experimental measurements.

[0069] FIG. 16A provides data showing fit parameters for one-layer synthetic phosphorylation cycle. Panel A provides a schematic showing parameterization for background- normalized synKin / synPhos activity rates (k) and binding affinity for the LZs (K). All catalytic rates are normalized by the background dephosphorylation rate (kbg). Panel B provides data showing a summary of thermodynamic model parameters. Each parameter is plotted with two-sided error bars. Solid line error bars indicate parameters with upper and lower limits, and the dashed line indicates parameters with only an upper or lower limit. For affinity and rate constants, fitted, lower limit, and upper limit values are shown in three columns. Affinity values are expressed as arbitrary units and as Kd values calculated based on in vitro measured data value for the medium affinity LZ value. Other In vitro measured Kd values are shown in the last column.

[0070] FIG. 17 provides data showing the modeling of phosphorylation cycle behavior space. Panel A provides a schematic defining the part-allowed composition space. Part combinations that include kinase domains with different activities (active site mutations), synKin expression level (different Kozak sequences), and LZ-E affinities resulted in a final set of 216 compositions. Panel B provides data showing predicted phosphorylation levels for all compositions in phosphorylation cycle design space. Beeswarm plot corresponds to thegeometric mean of phosphorylation for each circuit composition. Red dots indicate experimentally measured compositions that the model was trained on (n=12, data from FIG. IB to FIG. ID) while grey dots indicated model-predicted behavior for the rest of design space (n=204). Below the behavior space is a part frequency analysis of part parameter values for three different scatter regions, corresponding to low, medium, and high levels of predicted phosphorylation.

[0071] FIG. 18 provides data showing sensitivity analysis of phosphorylation cycle design space. Panel A provides data showing behavior of circuit compositions in FIG. ID in Goldbeter-Koshland-style sensitivity plots, with [synKin] / [syn-Phos] ratio as x axis and fraction synSub phosphorylation as the y axis. Grey dots, noise model-inferred measurements acquired from FIG. ID for single cells; magenta lines, predicted dose response curve based on the sampled expression level space for [synKin] / [synPhos]. nH, effective hill coefficient; EC so, [synKin] / [synPhos] values at half maximal activation (unitless). (Panel B) Left, scatter plot of EC50 and nn for all part-allowed circuit configurations from FIG. IF, color scale shows the maximum synSub phosphorylation level. Circuits from Panel A are highlighted as a, b, and c. One circuit from high rm region and one from the high ECso region are also highlighted. Predicted single-cell fraction phosphorylation levels and predicted curves are shown in Goldbeter-Koshland plots, with detailed part usage displayed below. Panel C provides sensitivity analysis for expanded parameter space, nn heatmaps are shown while varying synKin (top) or synPhos (bottom) activity vs. LZ affinity, shown for three distinct synSub concentations. Kws and KES are plotted with AU, kWS and kES are unitless, as shown in FIG. 16B. Purple boxes indicate current parts allowed space from FIG. 16B.

[0072] FIG. 19 shows using a transcriptional reporter to measure phospho-dependent tSH2 binding. Panel A shows the reporter design. A phospho-dependent transcriptional reporter system leverages the interaction between a pY motif and a tSH2 domain to activate transcription. It consists of two parts: 1) a transactivator-tSH2 fusion, which contains an SV40 NLS, VPR activation domain, and a tSH2 domain; 2) a synTF-pY motif fusion, which contains of a 3x MYC epitope, SV40 NLS, 3x ZF array, GSF, a 3x substrate motif, and LZ-R. Panel B provides data showing the use of the reporter to test phospho-dependent tSH2 recruitment. Phosphorylation by synKin can result in binding between VPR-tSH2 and synTF-pY ITAM-LZ- R activates the reporter gene expression. Binding specificity was assessed by screening tandem tSH2 domains from ZAP70, Syk, SHP1, SHP2, and p85a against IT AM motifs from the TCR,BCR and Fc receptor, as well as tandem pY motifs from PDGFR, and SHPS-1. Amino acid sequences for each motif are shown with phosphorylated Ys highlighted in red. Geometric means are shown beside histograms. tSH2 / motif pairs chosen for later use are indicated with purple boxes. Panel C provides design and validation of LZ-recruited transcriptional activation system. The LZ-recruited transcriptional activation module consists of two parts: an activator comprising a VPR-LZ-E fusion with an N-terminal NLS; 2) a synTF made up of a 3x MYC epitope tag, SV40 NLS, 3x ZF array, and LZ-E. Panel D provides data showing binding of VPR-LZ-E to synTF-LZ-R activates reporter gene expression. EUs encoding activator, synTF, and mCherry reporter were assembled into one plasmid (grey box) using the workflow shown in FIG. 5. Flow cytometry data for the mCherry reporter expression on the right for the 3-EU combination, empty cells as well as reporter only, synTF only, and negative controls without recruited activator (non-cognate LZ). Numbers indicate geometric means for mCherry expression. The dotted line indicates background mCherry fluorescence.

[0073] FIG. 20 provides the design of a phospho-dependent binder for a SLP76-derived pY motif. Panel A provides the design of a transactivator-tSH2 fusion for binding to a 3x SLP76108-154 motif, which contains an SV40 NLS, VPR, and a 2x repeat of a synthetic Vavl- ITK SH2 fusion. Panel B provides the assessment of pY-SLP76-motif-dependent tSH2 recruitment using the reporter system. The same reporter system used in FIG. 19 was employed to evaluate the interaction between synthetic SH2 domains and 3x SLP76108-154. Following phosphorylation by synKin, the binding capabilities of various synthetic SH2 domains with 3x SLP76108-154 were examined by measuring the mCherry reporter expression. Geometric means for phosphorylation are shown beside the histograms. The optimized synthetic tSH2 chosen for later use in circuit construction is indicated with the purple box.

[0074] FIG. 21 provides a design and optimization of signaling proteins to interconnect phosphorylation cycles. Panel A shows the middle substrate features a 3x MYC tag, synKin, 3x CD3Z substrate motif, and LZ-R. A rigid linker domain is inserted between synKin and CD3Z to avoid cis-phosphorylation and GS linkers are inserted between the ITAM and the LZ. The SH2-substrate protein features a 3x V5 epitope tag, GST, 3x SLP76-dervied substrate motif, and 2x ZAP70 SH2. Panel B shows optimizing the PC rigid linker to limit cis-phosphorylation. Interconnected two-step circuit is shown. The placement of a rigid linker protein between the kinase domain and substrate motif within the PC is designed to limit intramolecular phosphorylation. Proteins are expressed on three plasmids (grey boxes). Panel C provides datashowing the testing of the effects of rigid linkers on cis phosphorylation. Phosphorylation levels were measured for configurations where the PC contained different rigid linkers or a flexible linker, with and without the upstream synKin. Dotted lines indicate mean background phosphorylation levels. The purple box indicates the rigid linker selected for further engineering.

[0075] FIG. 22A provides data showing fitting paramaters for the two-step phosphorylation circuit. Panel A provides a schematic showing parametrization for the activity for both synKin and synPhos, binding affinity for the LZs between synKin / PC and synPhos / PC, and the binding affinity between phosphorylated CD3Z and ZAP70 SH22-254. Similar to FIG. 14, all activity terms are normalized by the background dephosphorylation rate for fitting. Panel B provides data showing the goodness of fit for the two-step phosphorylation circuit. The comparison between experimental and fitted phosphorylation levels for both substrates is shown for all the circuit configurations in FIG. 2B. K-S was calculated for each set of comparisons between experimental and fitting data and DKS is shown on the top-right of each group of histograms. Panel C provides a summary of thermodynamic model parameters. The two-sided error bars and the parameters are calculated and plotted the same way as FIG. 16B.

[0076] FIG. 23 provides data showing the design basis of behavior space for coupled phosphorylation cycles. Panel A provides a schematic showing the defining of configuration space. Part variants were modeled for all part-allowed configurations of the two-step push-pull circuit, resulting in a final set of 3,456 compositions. Panel B provides data showing predicted behavior space. The behavior space is divided into 6 regions based on phosphorylation of the second synSub. Panel C provides data showing parameter frequency analysis for the 6 regions of circuit behavior space. The number of compositions in each region is labeled in the icon for each region (left).

[0077] FIG. 24 provides receptor design optimization for the phosphor-sensor circuit. Panel A shows the kinase domain and LZ from a synKin is appended to two receptor chains that are fused with membrane-localized FKBP and FRB* (K2095P, bold grey) domains. The FRB* chain has an extracellular FLAG epitope tag and C-terminal kinase domain. The FKBP features an extracellular V5 epitope tag and a LZ-E at the C terminus. Both chains feature a CD28 TM flanked by lOx GS linkers and a signal peptide from IgGX. Panel B provides a design for the phospho-sensor circuit. The histograms to the right show background phosphorylation for empty cells and cells transfected with synSub only. Panel C provides data showing flexible GSlinkers before and after the TM for both receptor chains were combinatorially tested to identify linker lengths that yield high fold-change of ligand-inducible phosphorylation. Three linker lengths were tested for each receptor chain. Phosphorylation levels were measured for each combination with or without the addition of ligand. Geometric means are indicated to the right of the histograms, and fold change was calculated by dividing ligand-induced phosphorylation by uninduced. Black dotted lines indicate the geometric mean of phosphorylation for blank cells. The purple box indicates linker combinations with the highest ligand-inducible fold change in phosphorylation. Panel C provides data showing the testing of mutations in the TM for both receptor chains to identify TM combinations that yield high fold change ligandinducible phosphorylation. Sequence details for the TMs are shown in Panel A. TM variants for the FRB*-synKin chain were tested against variants in the FKBP-LZ-E. Phosphorylation levels and fold change values were calculated as before. Purple box indicates the TM combinations with the highest fold change.

[0078] FIG. 25 provides data showing a condensate co-localization reporter design. Panel A provides data showing the reporter uses a synSub design in which GST is replaced with an EGFP (see FIG. 8). Condensates were formed using and an mCherry-PopZ tag fused to a 2x ZAP70 tSH22-259 domain. Expression of the PopZ-mCherry fusion in HEK293T cells results in spontaneous formation of cytoplasmic condensates (top right). Panel B provides a workflow for time-lapse microscopy and data processing. Left: transfected HEK293T cells are loaded into an multi-well chamber slide and imaged using time-lapse microscopy. Left-middle: single-cell segmentation is performed for both EGFP and mCherry channels to identify single-cell masks and trajectories. Right: To assess EGFP / mCherry co-localization, masks (white dotted line) were used to quantitate subcellular fluorescence for both colors. The resulting pixel intensity distribution was fit to a normal distribution (blue histogram) and the threshold for the condensate (dotted line) is placed at the 99th quantile (TEGFP', Tmcherry). EGFP pixel intensities below TEGFP (cytoplasmic EGFP white mask) are summed to calculate cytoplasmic intensity. mCherry pixel intensities above Tmcherry are considered part of the condensate and used as a mask (condensate mCherry mask) to quantitate overlappling EGFP pixel intensity for pixels > TF.GFP (condensate EGFP mask). Representative images from the beginning (t=0 min) and the end (t=80 min) of the time-lapse experiment are shown in FIG. 3C. Panel C provides data showing EGFP / mCherry co-localization measured for deficient circuits and compared with the full circuit, error bars indicated mean values ± SEM. Number of replicates are labelled on topof each group. Bottom, EGFP and mCherry merged images are shown for representative single cells, with the cell boundaries that were determined by custom segmentation software represented as dotted white outlines. Panel D provides data showing phosphorylation level measured in the condensate reporter circuit with or without the addition of ligand. HHH maps are used to plot the component expression and phosphorylation. The geometric mean for phosphorylation is noted in the bottom right of the maps and the fold change is labeled on the right.

[0079] FIG. 26A provides data showing obtaining kinetic parameters for the phospho-sensor circuit. (Panel A) The off kinetics of the phospho-sensor circuit were measured with synPhos absent. The red dashed line the mean deactivation data of the complete circuit shown in FIG. 3C. For the circuit lacking synPhos, trajectories for 10 cells are shown (light pink lines), with mean values (thick pink line) ± SEM (shaded pink band). Green dashed line indicates the addition of ABL inhibitor imatinib mesylate (10 pM). Panel B provides data showing the kinetic model of activation and deactivation dynamics of the phospho-sensor. ODEs are used to account for receptor activation and phospho-dependent condensate co-localization. Bottom left, fitting experimental data (circles) from FIG. 3C and Panel A to the model (pink line) and TI, 2 values were calculated from the fitted model parameters. Bottom right, rate constants were obtained from fitting for activation and deactivation data. Background-normalized rate constants are compared with values obtained from fitting in FIG. 22C. Panel C provides data showing activation kinetics for native pathways. Localization reporter data from native signaling pathways are plotted using data obtained from the cited references to compare with the dynamics of the ligand-inducible phosphorylation circuit. All activation curves are normalized to the maximum output signal. T1 / 2 values are estimated based on data from the indicated references.

[0080] FIG. 27 provides data showing the design of sense-and-respond signalling circuit components. Panel A shows the same design strategy from FIG. 21 was used to connect the phosphorylation of the second substrate to the transcriptional activation (TA) domain. The TA- SELZ fusion features SV40 NLS, VPR, and a double repeat of the synthetic Vavl-ITK SH2, while the synTF-ITAM consists of a 3x MYC epitope tag, SV40 NLS, 10-1 zinc finger array, GST, a 3x SLP76 peptide and LZ-R. (Panel B) To enhance signaling from the cell membrane to the nucleus, subcellular localization tags were added to the PC. Dashed lines show thegeometric mean of empty cell phosphorylation. The purple box indicates the configuration that was used for sense-and-respond circuit engineering in FIG. 4A.

[0081] FIG. 28 provides data showing validating design choices for the sense-and-respond circuit. (Panel A)To test whether the design principle provided by the model prediction from FIG. 2C can be used to guide sense-and-respond circuit design, all high-gain circuit compositions from FIG. 2C (#1-4) along with lower-gain compositions (#5-8) were implemented as part of the sense-and-respond circuit. EGFP expression before and after ligand addition is plotted as HHH plots. The model-predicted fold change for the circuits (red values) and the experimentally-measured EGFP fold-change (green values) are shown to the right of the plots. The composition selected for sense-and-respond circuit engineering (FIG. 4A) is plotted on the top left (#2) while non-optimal compositions (#1, 3-8) are plotted below, with specific changes in part composition shown to the left of each set of plots. Panel B shows dose response for composition #2. For each concentration, each circle representing mean EGFP value for one of the three replicates, with error bars representing mean value ± SEM. EC50 and nu were extracted by fitting to equation 150.

[0082] FIG. 29A provides data showing the expand ligand sensing capabilities with a TNF-a sensor module. Panel A provides the FRB* / FKBP domains from the rapalog-sensing receptor pair were swapped for an scFv specific for TNF-a. Panel B shows dose response for circuit depicted in FIG. 4B. For each concentration, circles mean phosphorylation level for each three replicates, with error bars representing mean values ± SEM. EC50 and nu were extracted by fitting to equation 150.

[0083] FIG. 30 shows data processing for PBMC phenotyping. Panel A shows the workflow for processing PBMC flow cytometry data. Similar to HEK293T data processing (FIG. 9), PBMCs are gated by forward (FSC-A) and side scatter (SSC-A) area for cell population, and single cells are gated by forward scatter height (FSC-H) and area (FSC-A). Live cells are gated using the lower peak of Ghost Dye™ Violet 450 area. T cells are gated using CD3+, then CD4+ area is plotted against CD8+ area to analyze T cell subsets. The top row shows an example of the PBMC sample treated with CD3 / CD28 Dynabeads™, while the bottom row shows the profile for untreated PBMCs. Values indicate mean ± SEM for bead-activated samples. Panel B provides data showing quantitating T cell proliferation by EdU assay. At the conclusion of the co-culture time course experiments (60 h) shown in FIG. 4B, cells were assessed for proliferation. Gated CD4+ and CD8+ subsets are replotted as Andy Fluor™ 488histograms. The red gate is used to calculate the percentage of the population that is proliferative. Values indicate the mean ± SEM for n=3 donors. Panel C provides data showing endpoint cytokine secretion profiles. At the conclusion of the co-culture time course, concentrations for IL-10, TNF-a, and IFN-y were measured by ELISA for different circuits. Error bars show mean ± SEM for n=3.

[0084] FIG. 31 provides data showing fitting cytokine dynamics in the cytokine control circuit co-culture. A quantitative model describing cytokine secretion dynamics for the transwell culture experiments depicted in FIG. 4B is provided. ODEs are used to account for changes in receptor activation and cytokine concentration. Left, circuit schematic. Middle, corresponding ODEs. Right, fitting the cytokine secretion data (circles) from FIG. 4B to the dynamic model (green or blue lines). Bottom, rate constants were obtained from a global fit of the time course data, and constrained by TNF-a dose-response curve from FIG. 29B.

[0085] FIG. 32 provides data predicting cytokine secretion for circuits with slower dynamic profiles. Panel A provides data showing secretion of TNF-a (green) and IL-10 (blue) for coculture systems in which sense-and-respond circuits have reduced activation or deactivation rates (k*). Model -predicted time courses for TNF-a (dark green) and IL- 10 (dark blue) are shown for 10-fold lower rates. Panel B provides data showing comparison to simulated systems featuring versions of the circuit depicted in FIG. 4B that are activated by transcription or proteolysis. Values used in the simulations for proteolysis and transcription are taken from the literature as described herein.

[0086] FIG. 33 provides data showing predicted immunogenicity of cytokine control circuit components compared to other commonly used synthetic signaling parts. T Cell Class I pMHC Immunogenicity tool from the Immune Epitope Database (IEDB) was used to evaluate the immunogenicity scores of all 9-mer peptide sequences within the six engineered proteins in the cytokine control circuit, along with three commonly-used synthetic receptors, two proteases and three FDA-approved fully humanized monoclonal antibodies (mAbs). Subdomains are labelled above the score heatmaps. Protein sizes are indicated by the residue number label on the bottom. A higher immunogenicity score (hotter color) for a peptide indicates that its composition more closely resembles that of immunogenic peptides, and thus indicates a higher probability of eliciting an immune response. A summary of immunogenicity scores of all 9-mer peptide sequences within all synthetic components from this research and other parts is shown in the tables on the left, with the total number of 9-mer peptides, the percentage of 9-mer scores>0, and the percentage of 9-mer scores >0.328 calculated. TVMVp, tobacco vein mottling virus protease. TEVp, tobacco etch virus protease.

[0087] FIG. 34 provides data showing signaling circuit behavior in therapeutically-relevant cells. Panel A provides data showing behavior of receptor-coupled sensor circuit in human APRE-19 cells and human UC-MSCs. HHH plots depict flow-cytometry data of the complete sensor circuit from FIG. 3B in different cell types with 200 nM ligand (+ lig) or a carrier-only control (- lig). Values in each plot indicate mean phosphorylation (AU) ± SEM (n=3). Phosphorylation fold-change values are next to each set of plots. Panel B provides data showing behavior of the sense-and-response signaling circuit in ARPE-19 . PC expression, PC phosphorylation signal and EGFP level were measured. Numbers indicate the geometric mean values (AU) ± SEM (n=3).

[0088] FIG. 35 provides time-lapse fluorescence microscopy images showing activation of two representative single cells harboring the phospho-sensor circuit and condensate reporter. The time-lapse (80 min total, each frame is lOmin) was initiated at t=- 10 min and ligand AP21967 was added to the culture at t=0 min to a final concentration of 200 nM. Panel A provides an image at t=- 10 min. Panel B provides an image at t=0 min. Panel C provides an image at t= 10 min. Panel D provides an image at t=20 min. Panel E provides an image at t=30 min. Panel F provides an image at t-=40 min. Panel G provides an image at t=50 min. Panel H provides an image at t=60 min. Panel I provides an image at t=70 min. Images are presented in an EGFP- and mCherry-merged format, in false colors (green and red, respectively). A timestamp is displayed on the top left of each frame throughout the time-lapse. Scale bar, 5 pm.

[0089] FIG. 36 provides time-lapse fluorescence microscopy showing deactivation of two representative single cells harboring the phospho-sensor circuit and condensate reporter. The time-lapse (60 min total, each frame is 5 min) was initiated at t=-5 min and ABL inhibitor imatinib mesylate was added to the culture at t=0 min a final concentration of 10 pM. Panel A provides an image at t=-5 min. Panel B provides an image at t=0 min. Panel C provides an image at t=5 min. Panel D provides an image at t=10 min. Panel E provides an image at t=l 5 min. Panel F provides an image at t-=20 min. Panel G provides an image at t=25 min. Panel H provides an image at t=30 min. Panel I provides an image at t=35 min. Panel J provides an image at t=-40 min. Panel K provides an image at t=45 min. Panel L provides an image at t=50 min. Panel M provides an image at t=55 min. Images are presented in a GFPandmCherry-merged format, in false colors (green and red, respectively). A timestamp is displayed on the top left of each frame throughout the time-lapse. Scale bar, 5 pm.

[0090] FIG. 37A shows a schematic diagram depicting a direct sense and respond circuit comprising an equilibrium translocon (SEQ ID NO: 54).

[0091] FIG. 37B shows a bar graph depicting eGFP fluorescence following rapalog induced circuit activation in human hUC-MSC cells.

[0092] FIG. 37C shows bar graphs depicting eGFP fluorescence for a control cell lacking a synthetic kinase with and without expression of the equilibrium translocon (top), a cell expressing a constitutively active synthetic kinase (middle), and a cell expressing a rapalog inducible receptor (bottom).DETAILED DESCRIPTION OF THE INVENTION

[0093] Aspects of the invention are drawn to a polypeptide as described herein.

[0094] Detailed descriptions of one or more aspects are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0095] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0096] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0097] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0098] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0099] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0100] Aspects of the invention are drawn to polypeptides comprising a synthetic domain or fragment thereof. In some aspects, the polypeptide can comprise a synthetic kinase domain (synKin) or fragment thereof. In some aspects, the polypeptide can comprise a synthetic phosphorylatable domain (synSub) or fragment thereof. In some aspects, the polypeptide can comprise a synthetic phosphatase domain (synPhos) or fragment thereof.

[0101] "Polypeptide" as used herein can encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" can refer to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, can refer to "polypeptide" herein, and the term "polypeptide" can be used instead of, or interchangeably with any of these terms.

[0102] "Polypeptide" can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarilytranslated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0103] As to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant". In some aspects, the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0104] Aspects of the invention are drawn to the engineering of synthetic phosphorylation networks in which “push-pull” motifs — reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities — are assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells (See FIG. 1A).

[0105] Without wishing to be bound by theory, the composability of the design scheme described herein enables model-guided tuning of circuit function and the ability to make diverse network connections. As described herein, synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression.

[0106] Phosphorylation plays critical roles in the regulation of many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways. Phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell. While phosphorylation has been observed in bacterial proteins, it is considerably more pervasive in eukaryotic cells. It is estimated that one-third of the proteins in the human proteome are substrates for phosphorylation at some point. Indeed, phosphoproteomics has been established as a branch of proteomics that focuses solely on the identification and characterization of phosphorylated proteins.

[0107] While phosphorylation is a prevalent post-translational modification (PTM) for regulating protein function, it only occurs at the side chains of three amino acids, serine, threonine and tyrosine, in eukaryotic cells. These amino acids have a nucleophilic (-OH) group that attacks the terminal phosphate group (y-PO32-) on the universal phosphoryl donor adenosine triphosphate (ATP), resulting in the transfer of the phosphate group to the aminoacid side chain. This transfer is facilitated by magnesium (Mg2+), which chelates the y- and P- phosphate groups to lower the threshold for phosphoryl transfer to the nucleophilic (-OH) group. This reaction is unidirectional because of the large amount of free energy that is released when the phosphate-phosphate bond in ATP is broken to form adenosine diphosphate (ADP).

[0108] For a large subset of proteins, phosphorylation is tightly associated with protein activity and is a key point of protein function regulation. Phosphorylation regulates protein function and cell signaling by causing conformational changes in the phosphorylated protein. These changes can affect the protein in two ways. First, conformational changes regulate the catalytic activity of the protein. Thus, a protein can be either activated or inactivated by phosphorylation. Second, phosphorylated proteins recruit neighboring proteins that have structurally conserved domains that recognize and bind to phosphomotifs. These domains show specificity for distinct amino acids. For example, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains show specificity for phosphotyrosine (pY), although distinctions in these two structures give each domain specificity for distinct phosphotyrosine motifs.Phosphoserine (pS) recognition domains include MH2 and the WW domain, while phosphothreonine (pT) is recognized by forkhead-associated (FHA) domains. The ability of phosphoproteins to recruit other proteins is critical for signal transduction, in which downstream effector proteins are recruited to phosphorylated signaling proteins.

[0109] Protein phosphorylation is a reversible PTM that is mediated by kinases and phosphatases, which phosphorylate and dephosphorylate substrates, respectively. These two families of enzymes facilitate the dynamic nature of phosphorylated proteins in a cell.

[0110] The intensity and duration of phosphorylation-dependent signaling can be regulated by three mechanisms: 1) removal of the activating ligand; 2) kinase or substrate proteolysis; and 3) phosphatase-dependent dephosphorylation.[OHl] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0112] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patentdocument or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.A. Kinases

[0113] Aspects of the invention are drawn to a polypeptide comprising a synthetic kinase domain (synKin) or fragment thereof

[0114] Kinases are enzymes that facilitate phosphate group transfer to substrates. Protein kinases are a large family of proteins which play a critical role in the regulation of a wide variety of cellular processes and functions. ATP is the co-substrate for almost all protein kinases, although guanosine triphosphate is used by a small number of kinases. ATP is the ideal structure for the transfer of a-, - or y-phosphate groups for nucleotidyl-, pyrophosphoryl- or phosphoryltransfer, respectively. While the substrate specificity of kinases varies, the ATP- binding site is generally conserved.

[0115] Protein kinases can be broken down into two classes, the protein tyrosine kinases and the serine-threonine kinases.

[0116] In some aspects, the synthetic kinase domain comprises a tyrosine kinase.

[0117] Protein tyrosine kinases (PTKs) are enzymes which phosphorylate tyrosine residues in peptides and proteins and which play a key role in the regulation of cell signaling including cell proliferation and cell differentiation.

[0118] Protein kinase substrate specificity is based not only on the target amino acid but also on consensus sequences that flank it. These consensus sequences allow some kinases to phosphorylate single proteins and others to phosphorylate multiple substrates (>300).Additionally, kinases can phosphorylate single or multiple amino acids on an individual protein if the kinase-specific consensus sequences are available.

[0119] In some aspects, the polypeptide can comprise a synthetic, or engineered, kinase (See FIG. 8).

[0120] In some aspects, the synthetic kinase can comprise a kinase involved in activation of T Cell Receptor (TCR) and B Cell Receptor signaling (e.g., ZAP70 and Syk) and / or an Src family member (e.g., Lyn, Lek, ABL) (See Table 1 and FIG. 10A).

[0121] T Cell Receptor (TCR) activation promotes a number of signaling cascades that ultimately determine cell fate through regulating cytokine production, cell survival, proliferation, and differentiation. An early event in TCR activation is phosphorylation ofimmunoreceptor tyrosine-based activation motifs (IT AMs) on the cytosolic side of the TCR / CD3 complex by lymphocyte protein tyrosine kinase (Lek). The CD45 receptor tyrosine phosphatase modulates the phosphorylation and activation of Lek and other Src family tyrosine kinases. Zeta-chain associated protein kinase (ZAP70) is recruited to the TCR / CD3 complex where it becomes activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins. Phosphorylation of SLP-76 by ZAP-70 promotes recruitment of Vav (a guanine nucleotide exchange factor), the adaptor proteins NCK and GADS, and an inducible T cell kinase (Itk). Phosphorylation of phospholipase C yl (PLCyl) by Itk results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates PKC9 and the MAPK / Erk pathways, both promoting transcription factor NF-KB activation. IP3 triggers the release of Ca2+ from the ER, which promotes entry of extracellular Ca2+ into cells through calcium release-activated Ca2+ (CRAC) channels. Calcium-bound calmodulin (Ca2+ / CaM) activates the phosphatase calcineurin, which promotes IL-2 gene transcription through the transcription factor NF AT. Feedback regulation at several points within these pathways allows for different outcomes, depending on the cell type and environment. The incorporation of signals from additional cell surface receptors (such as CD28 or LFA-1) further regulates cellular response.

[0122] The B cell antigen receptor (BCR) is composed of membrane immunoglobulin (mlg) molecules and associated Iga / IgP (CD79a / CD79b) heterodimers (a / p). The mlg subunits bind antigen, resulting in receptor aggregation, while the a / p subunits transduce signals to the cell interior. BCR aggregation rapidly activates the Src family kinases Lyn, Blk, and Fyn as well as the Syk and Btk tyrosine kinases. This initiates the formation of a ‘signalosome’ composed of the BCR, the aforementioned tyrosine kinases, adaptor proteins such as CD 19 and BLNK, and signaling enzymes such as PLCy2, PI3K, and Vav. Signals emanating from the signalosome activate multiple signaling cascades that involve kinases, GTPases, and transcription factors. This results in changes in cell metabolism, gene expression, and cytoskeletal organization. The complexity of BCR signaling permits many distinct outcomes, including survival, tolerance (anergy) or apoptosis, proliferation, and differentiation into antibody-producing cells or memory B cells. The outcome of the response is determined by the maturation state of the cell, the nature of the antigen, the magnitude and duration of BCR signaling, and signals from other receptors such as CD40, the IL-21 receptor, and BAFF-R. Many other transmembrane proteins,some of which are receptors, modulate specific elements of BCR signaling. A few of these, including CD45, CD19, CD22, PIR-B, and FcyRIIBl (CD32), are indicated here in yellow. The magnitude and duration of BCR signaling are limited by negative feedback loops including those involving the Lyn / CD22 / SHP-1 pathway, the Cbp / Csk pathway, SHIP, Cbl, Dok-1, Dok- 3, FcyRIIBl, PIR-B, and internalization of the BCR. In vivo, B cells are often activated by antigen-presenting cells that capture antigens and display them on their cell surface. Activation of B cells by such membrane-associated antigens requires BCR-induced cytoskeletal reorganization. Please refer to the diagrams for the PI3K / Akt signaling pathway, the NF-KB signaling pathway, and the regulation of actin dynamics for more details about these pathways.

[0123] In some aspects, provided herein are polypeptides comprising: a protein tag, a synthetic kinase domain or fragment thereof, a linker, and a dimerization domain.

[0124] In some aspects, the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag. In other aspects, the protein tag is a FLAG tag. In some aspects, the FLAG tag comprises the amino acid sequence set forth in SEQ ID NO:1.

[0125] In some aspects, the linker comprises a GS linker. In some aspects, the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0126] In some aspects, the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R. In some aspects, the dimerization domain comprises a Leucine Zipper E. In some aspects, the Leucine Zipper E domain comprises an amino acid sequence set forth in SEQ ID NO: 18.

[0127] In some aspects, the polypeptide described herein can contain a synthetic kinase domain or fragment thereof. Table 1 provides the amino acid sequences for the components of exemplary synKin proteins. The synKin can contain a Flag tag on the N terminus, an engineered pY kinase domain, a flexible GS linker, and an acidic leucine zipper (Leucine Zipper E) on the C terminus. FIG. 8, for example, refers to an exemplary embodiment of the synKin protein.Table 1. Synthetic Kinase (synKin) Protein Structure. Amino acid sequences for the components of the synKin protein structure are provided. The components of the synKinprotein structure can include a Flag tag, a synthetic kinase domain or fragment thereof, a GS linker, and a Leucine Zipper E.

[0128] In some aspects, the synKin protein comprises a synthetic kinase domain or fragment thereof comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:2-16. In other aspects, the synKin protein comprises a synthetic kinase domain or fragment thereof comprising an amino acid sequence set forth in any one of SEQ ID NOs:2-16 comprising one or more amino acid substitutions. In other aspects, the synKin protein comprises a synthetic kinase domain or fragment thereof comprising an amino acid sequence set forth in any one of SEQ ID NOs:2-16.

[0129] In some aspects, the synKin protein structure comprises a Flag tag comprising the amino acid sequence set forth in SEQ ID NO:1, a synthetic kinase domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0130] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0131] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:2, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0132] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:2, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0133] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:3, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0134] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth inSEQ ID N0:3, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0135] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:4, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0136] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:4, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0137] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:5, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0138] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:5, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0139] In some aspects, the synKin protein structure comprises a a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:6, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0140] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:6, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0141] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 7, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0142] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:7, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0143] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1 , a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:8, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0144] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:8, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0145] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:9, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0146] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:9, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0147] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereofcomprising the amino acid sequence set forth in SEQ ID NO: 10, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0148] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 10, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0149] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 11, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0150] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 11, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0151] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 12, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0152] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 12, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0153] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 13, a GS Linker comprising theamino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0154] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 13, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0155] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 14, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0156] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 14, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0157] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 15, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0158] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 15, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0159] In some aspects, the synKin protein structure comprises a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0160] In some aspects, the synKin protein structure comprises, from N-terminus to C- terminus, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.B. Substrates

[0161] Aspects of the invention are further drawn to a polypeptide comprising a synthetic phosphorylatable domain or fragment thereof. For example, the synthetic phosphorylatable domain or fragment thereof can comprise a synthetic substrate (synSub) as described herein.

[0162] In some aspects, the polypeptide can comprise an immune tyrosine activation motif (IT AM) and / or an immune tyrosine inhibition motif (ITIM) (See Table 2 and FIG. 19).

[0163] ITAMs and ITIMs are phosphorylation motifs found in a large number of receptors or adaptor proteins. An IT AM is a highly conserved region in the cytoplasmic domain of signaling chains and receptors and is a critical mediator of intracellular signals. Phosphorylated ITAMs serve as docking sites for tandem SH2 domains of Syk family kinases, whereas phosphorylated ITIMs recruit tyrosine phosphatases. Signaling through IT AM-bearing receptors usually results in cell activation, while engagement of ITIM-bearing receptors is usually inhibitory, although exceptions have been described.

[0164] In some aspects, the polypeptide described herein can contain a synthetic substrate domain or fragment thereof. Table 2 provides the amino acid sequences for the components of the synSub protein. The synSub can contain (from the N- to C-terminus) a 3x MYC epitope tag, a GST tag, a synthetic phosphorylatable domain or fragment thereof, and a cognate basic zipper (Leucine Zipper R). FIG. 8, for example, refers to an embodiment of the synSub protein.Table 2. Synthetic Substrate (synSub) Protein Structure. Amino acid sequences for the components of the synSub protein structure are provided. The components of the synSub protein structure can include a MYC tag, a GST tag, a synthetic phosphorylatable domain or fragment thereof, a GS linker, and a Leucine Zipper R.

[0165] In some aspects, the synSub protein comprises a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:21-32. In other aspects, the synSub protein comprises a truncated substrate domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-32 comprising one or more amino acid substitutions. In other aspects, the synSub protein comprises a truncated substrate domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-32.

[0166] In some aspects, the synSub protein comprises a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO 31, andSEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0167] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0168] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:21, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0169] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:21, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0170] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:22, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0171] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:22, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0172] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereofcomprising the amino acid sequence set forth in SEQ ID NO:23, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0173] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:23, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0174] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:24, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0175] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:24, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0176] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:25, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0177] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:25, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0178] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:26, a GS linker comprising theamino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0179] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:26, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0180] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:27, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0181] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:27, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0182] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:28, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0183] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:28, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0184] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:29, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0185] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:29, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0186] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:30, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0187] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:30, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0188] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:31, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0189] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:31, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0190] In some aspects, the synSub protein comprises, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

[0191] In some aspects, the synSub protein comprises, from N-terminus to C-terminus, a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a syntheticphosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.C. Phosphatases

[0192] Aspects of the invention are further drawn to a polypeptide comprising a synthetic phosphatase domain (synPhos) or fragment thereof.

[0193] A protein phosphatase is a phosphatase enzyme that removes a phosphate group from the phosphorylated amino acid residue of its substrate protein. The human proteome is estimated to contain approximately 150 protein phosphatases, which show specificity for pS / pT and pY residues. While dephosphorylation is the end goal of these two groups of phosphatases, they do it through separate mechanisms. Serine / threonine phosphatases mediate the direct hydrolysis of the phosphorus atom of the phosphate group using a bimetallic (Fe / Zn) center, while tyrosine phosphatases form a covalent thiophosphoryl intermediate that facilitates removal of the tyrosine residue.

[0194] In some aspects, the phosphatase can comprise a tyrosine phosphatase.

[0195] In some aspects, the phosphatase can comprise PTPN1, PTPN4, and / or PTPN6 (See Table 3 and FIG. 13).

[0196] In some aspects, the polypeptide described herein can contain a synthetic phosphatase domain or fragment thereof. Table 3 provides the amino acid sequences for the components of the synPhos protein. The synPhos can contain a 3x HA epitope tag, a truncated phosphatase domain, a GS linker, and an LZ-E. FIG. 13, for example, refers to an embodiment of the synPhos protein.Table 3. Synthetic Phosphatase (synPhos) Protein Structure. Amino acid sequences for the components of the synPhos protein structure are provided. The components of the synPhos protein structure can include a 3x HA tag, a truncated phosphatase domain, a GS linker, and a leucine zipper E.

[0197] In some aspects, the synPhos protein comprises a truncated phosphatase domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:35-43. In other aspects, the synPhos protein comprises a truncated phosphatase domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:35-43 comprising one or more amino acid substitutions. In other aspects, the synPhos protein comprises a truncated phosphatase domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:35-43.

[0198] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0199] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0200] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:35, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0201] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:35, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0202] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:36, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0203] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:36, a GSlinker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0204] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:37, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0205] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:37, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0206] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:38, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0207] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:38, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0208] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:39, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0209] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:39, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0210] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:40, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0211] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:40, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0212] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:41, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0213] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:41, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0214] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:42, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0215] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:42, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0216] In some aspects, the synPhos protein comprises a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising theamino acid sequence set forth in SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0217] In some aspects, the synPhos protein comprises, from N-terminus to C-terminus, a 3x HA Tag comprising the amino acid sequence set forth in SEQ ID NO:34, a truncated phosphatase domain comprising the amino acid sequence set forth in SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.D. Receptors

[0218] Aspects of the invention are further drawn to a polypeptide comprising a synthetic cellular receptor. In some aspects, the polypeptide can comprise an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain.

[0219] In preferred In some aspects, the antigen recognition domain can comprise an antibody or fragment thereof.

[0220] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides.

[0221] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, can refer to a portion of an antibody such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized bythe intact antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “antibody fragment” can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab'and F(ab’)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti -idiotypic (anti-Id) antibodies.

[0222] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide- encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883).

[0223] In some aspects, the regions can be connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. In some aspects, the linker is a GS linker. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0224] In some aspects, the antibody or fragment thereof can be specific for TNFa.

[0225] Tumor necrosis factor alpha (TNFa) is a pro-inflammatory cytokine mediating the regulation of diverse inflammatory, infectious and immune-related processes and diseases, TNFa being considered the most important mediator responsible for inflammatory pathology.

[0226] TNFa is a 17 kD molecular weight protein, initially synthesized as a transmembrane protein arranged in stable trimers, then cleaved by metalloprotease-TNFa converting enzyme (TACE) to form the homotrimeric soluble TNF (sTNF) which engages to its cognate receptors (TNFRI, p55 and TNFRII, p75), expressed ubiquitously. The ubiquitous TNF receptors provide the basis for the wide variety of TNF-alpha mediated cellular responses.

[0227] TNFa induces a wide variety of cellular responses, many of which result in deleterious consequences, such as cachexia (loss of fat and whole body protein depletion, leading to anorexia, common in cancer and AIDS patients) and septic shock. Elevated secretion of TNF- alpha has been implicated in a variety of human diseases including diabetes, allograft rejection,sepsis, inflammatory bowel diseases, osteoporosis, in many autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, psoriatic arthritis, hypersensitivity, immune complex diseases, and even in malaria, cancer and lung fibrosis.

[0228] In some aspects, a circuit that senses tumor necrosis factor a (TNF-a) — a cytokine secreted by T cells that drives adverse inflammatory response — and responds by secreting interleukin 10 (IL-10), a cytokine that inhibits T cell activation, proliferation, and TNF-a production, is engineered. Without wishing to be bound by theory, this circuit can establish an anti-inflammatory control loop that suppresses T cell activation while maintaining low concentrations of both cytokines. (See FIG. 4A). In some aspects, single chain antibody fragments (scFvs) that recognize TNF-a can be appended to the receptors (FIG. 4B, FIG. 29 A), and replaced the EGFP reporter with IL-10.

[0229] In some aspects, the receptor can be amenable to any extracellular binding domain (e.g., and scFv or other binder) and can be linked to many varied intracellular responses.

[0230] In some aspects, the polypeptide described herein can contain a synthetic cellular receptor. Table 4 provides the amino acid sequences for the components of the synthetic signaling protein structure described herein. FIG. 24, for example, refers to an embodiment of the synthetic signaling protein design comprising a synthetic cellular receptor.Table 4. Synthetic Signaling Protein Structure: Receptors. Amino acid sequences for the components of the synthetic signaling protein structure are provided.

[0231] In some aspects, the synthetic signaling protein comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO:45, a FRB<K2095P) domain comprising the amino acid sequence set forth in SEQ ID NO:46, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228 -540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0232] In some aspects, the synthetic signaling protein comprises, from N-terminus to C- terminus, a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO:45, a FRB K2095P) domain comprising the amino acid sequence set forth in SEQ ID NO:46, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0233] In some aspects, the synthetic signaling protein comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a V-5 tag comprising the amino acid sequence set forth in SEQ ID NO:49, an FKBP domain comprising the amino acid sequence set forth in SEQ ID NO:50, transmembrane domains comprising the amino acid sequences set forth in SEQ ID NO:47, SEQ ID NO:51, and SEQ ID NO:52, and a Leucine Zipper E comprising the amino acid sequences set forth in SEQ ID NO: 18.

[0234] In some aspects, the synthetic signaling protein comprises, from N-terminus to C- terminus, a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a V- 5 tag comprising the amino acid sequence set forth in SEQ ID NO:49, an FKBP domain comprising the amino acid sequence set forth in SEQ ID NO:50, transmembrane domains comprising the amino acid sequences set forth in SEQ ID NO:47, SEQ ID NO:51, and SEQ ID NO:52, and a Leucine Zipper E comprising the amino acid sequences set forth in SEQ ID NO: 18.

[0235] In some aspects, the synthetic signaling protein comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acidsequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0236] In some aspects, the synthetic signaling protein comprises, from N-terminus to C- terminus, a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

[0237] In some aspects, the synthetic signaling protein comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO 45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and a Leucine Zipper E domain comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0238] In some aspects, the synthetic signaling protein comprises, from N-terminus to C- terminus, a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a FLAG tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and a Leucine Zipper E domain comprising the amino acid sequence set forth in SEQ ID NO: 18.E. Regulation of Transcription

[0239] Aspects of the invention are further drawn to a synthetic phosphorylation circuit capable of phosphorylation signaling that results in reporter transcriptional activation in a cell. In some aspects, the synthetic phosphorylation circuit is capable of a two-step phosphorylation signaling circuit that results in reporter transcriptional activation. In other aspects, the synthetic phosphorylation circuit is capable of a direct phosphorylation signaling circuit that results in reporter transcriptional activation.

[0240] In some aspects the synthetic phosphorylation circuit comprises a polypeptide comprising a synthetic transcription factor and a polypeptide comprising a synthetic reader.

[0241] In some aspects, the synthetic transcription factor comprises one or more nuclear localization signal (NLS), one or more DNA binding polypeptides, one or more nuclear exportsignal (NES), one or more synthetic phosphorylatable domain, one or more dimerization domain, and one or more linker sequences.

[0242] In some aspects, the synthetic transcription factor comprises a protein tag, an NLS, a DNA binding domain, an NES, a synthetic phosphorylatable domain or fragment thereof, a dimerization domain, and one or more linker sequences. In some aspects, the DNA binding domain is a zinc finger domain. In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs:21-32. In some aspects the dimerization domain is a Leucine Zipper domain. In some aspects, the dimerization domain is a Leucine Zipper R domain.

[0243] In some aspects, the synthetic transcription factor comprises, from N terminus to C terminus, a linker amino acid sequence, a protein tag, a linker amino acid sequence, an NLS, a linker amino acid sequence, a zinc finger domain comprising the amino acid sequence set forth in SEQ ID NO: 59, a linker amino acid sequence, a GST domain comprising the amino acid sequence set forth in SEQ ID NO:20, a linker amino acid sequence, an NES, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:63, a linker amino acid sequence, and a dimerization domain. In some aspects the dimerization domain is a Leucine Zipper domain. In some aspects, the dimerization domain is a Leucine Zipper R domain.

[0244] In some aspects, the synthetic transcription factor comprises, from N terminus to C terminus, a linker amino acid sequence, a protein tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a linker amino acid sequence, an NLS comprising the amino acid sequence set forth in SEQ ID NO:57, a linker amino acid sequence, a zinc finger domain comprising the amino acid sequence set forth in SEQ ID NO:59, a linker amino acid sequence, a GST domain comprising the amino acid sequence set forth in SEQ ID NO:20, a linker amino acid sequence, an NES comprising the amino acid sequence set forth in SEQ ID NO:62, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:63, a linker amino acid sequence, and a dimerization domain comprising the amino acid sequence set forth in SEQ ID NO:33 or SEQ ID NO:65. In some aspects the dimerization domain is a Leucine Zipper domain. In some aspects, the dimerization domain is a Leucine Zipper R domain.

[0245] In some aspects, the synthetic transcription factor comprises a linker comprising the amino acid sequence set forth in SEQ ID NO:55, a protein tag comprising the amino acidsequence set forth in SEQ ID NO: 19, a linker comprising the amino acid sequence set forth in SEQ ID NO:55, an NLS comprising the amino acid sequence set forth in SEQ ID NO:57, a linker comprising the amino acid sequence set forth in SEQ ID NO:58, a zinc finger domain comprising the amino acid sequence set forth in SEQ ID NO:59, a linker comprising the amino acid sequence set forth in SEQ ID NO:60, a GST domain comprising the amino acid sequence set forth in SEQ ID NO:20, a linker comprising the amino acid sequence set forth in SEQ ID NO:61, an NES comprising the amino acid sequence set forth in SEQ ID NO:62, a synthetic phosphorylatable domain or fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:63, a linker comprising the amino acid sequence set forth in SEQ ID NO:64, and a dimerization domain comprising the amino acid sequence set forth in SEQ ID NO:33 or SEQ ID NO:65. In some aspects the dimerization domain is a Leucine Zipper domain. In some aspects, the dimerization domain is a Leucine Zipper R domain.

[0246] In some aspects, the synthetic transcription factor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:54. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:54.

[0247] In some aspects, the synthetic reader comprises one or more NLSs, a recruitment motif, and a transcriptional activator. In some aspects, the recruitment motif comprises an engineered SH2 domain. In other aspects, the recruitment motif comprises a synthetic kinase domain or fragment thereof comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:2-16. In other aspects, the recruitment motif comprises a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth as SEQ ID NOs:21-32.

[0248] In some aspects, the synthetic reader comprises, from N-terminus to C-terminus, an NLS, a recruitment motif, a linker amino acid sequence, a transcriptional activator, and an NLS.

[0249] In some aspects, the synthetic reader comprises, from N-terminus to C-terminus, an NLS, a recruitment motif comprising an amino acid sequence set forth in any one of SEQ ID NOs: 67-72, a linker amino acid sequence, a transcriptional activator, and an NLS.

[0250] In some aspects, the synthetic reader comprises, from N-terminus to C-terminus, an NLS, a recruitment motif comprising an amino acid sequence set forth in any one of SEQ ID NOs:67-72, a linker amino acid sequence, a transcriptional activator comprising an amino acid sequence set forth in SEQ ID NO:74, and an NLS.

[0251] In some aspects, the synthetic reader comprises, from N-terminus to C-terminus, an NLS comprising the amino acid sequence set forth in SEQ ID NO:66, a recruitment motif comprising an amino acid sequence set forth in any one of SEQ ID NOs:67-72, a linker amino acid sequence, a transcriptional activator comprising an amino acid sequence set forth in SEQ ID NO:74, and an NLS comprising the amino acid sequence set forth in SEQ ID NO:75. In some aspects, the linker amino acid sequence comprises an amino acid sequence set forth in SEQ ID NO: 73.

[0252] In some aspects, the transcriptional activation of the synthetic phosphorylation circuit results in the expression of a therapeutic protein.F. Modularity of the System

[0253] The development of a collection of protein domain parts — kinases, phosphatases, substrates, transcription factors and obligate and phosphorylation-dependent interaction domains — to support synthetic cycle construction, tuning, and interconnection into networks is described herein. This set of parts can be used to compose synthetic kinases, phosphatase, and substrates as described herein, which respectively are referred to as synKin, synPhos, and synSub. Each of these components can comprise a catalytic activity or substrate domain module that is involved in signal transmission, as well as an interaction module that directs its specificity.

[0254] The engineering of artificial phosphorylation networks in which reversible enzymatic phosphorylation cycles can be assembled from such modular protein domain parts and wired together to create synthetic phosphorylation circuits in human cells is described herein (See FIG. 1A). The amino acid sequences of the modular protein domain parts described herein are provided in Tables 1-4.

[0255] This design scheme is modular, thereby allowing highly immunogenic parts to be swapped out for less reactive parts that retain similar biophysical properties (e.g., new proteinprotein interaction domains that bind with equivalent, tunable affinity). For example, the design scheme can allow for a synthetic phosphorylation circuit comprising a polypeptide as describedherein that can convert an extracellular signal into an intracellular response. For example, the receptor can be amenable to any extracellular binding domain (e.g., and scFv or other binder) and that can be linked to many varied intracellular responses.

[0256] Without wishing to be bound by theory, the highly modular and scalable design of engineered proteins comprising the modular protein domain parts described herein enables flexible inputs and outputs such as: 1) sense and report; and 2) sense and response. In some aspects, sense and report can refer to a condensate-based reporter circuit to assess the time scale of synthetic phosphorylation activation. In some aspects, sense and response can refer to a dynamic cytokine converter circuit that can sense TNFa and produce IL- 10, shown to modulate T cell proliferation (See FIG. 4).

[0257] Aspects of the invention are further drawn to a synthetic phosphorylation circuit comprising one or more of a polypeptide described herein. In some aspects, the synthetic phosphorylation circuit can convert an extracellular input into an intracellular or secreted output. For example, the extracellular input can comprise a synthetic molecule, such as rapalog, or a native biomolecule, such as TNFa. For example, the intracellular or secreted output can comprise a reporter or a therapeutic molecule, such as IL-10. This conversion can occur via fast timescale transcription, and can be used as a cell therapy for immune modulation, production of cancer therapeutics in situ, and / or metabolic disorders. Non-limiting examples of immune modulation comprise graft vs. host disease, traumatic brain injury, and autoimmune disease. In some aspects, the synthetic phosphorylation circuit comprises the TNFa-sensing / IL-10 response circuit. In some aspects, the synthetic phosphorylation circuit can respond to TNF-a by a proportional secretion of IL-10.

[0258] In some aspects, the synthetic phosphorylation circuit can convert an extracellular signal into an intracellular response.

[0259] In some aspects, the extracellular signal comprises a polypeptide, a hormone, a growth factor, a neurotransmitter, or a cytokine. In some aspects, the hormone is selected from the group consisting of growth hormone, insulin, glucagon, thyroxine (T4), triiodothyronine (T3), cortisol, estrogen, testosterone, follicle-stimulating hormone (FSH), or luteinizing hormone (LH). In some aspects, the neurotransmitter comprises acetylcholine, dopamine, epinephrine, norepinephrine, serotonin, gamma-aminobutyric acid (GABA), glutamate, glycine, or histamine. In some aspects, the cytokine comprises an interleukin such as IL-1, IL-2, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-17, IL-18, IL-22, IL-35, IL-37, or IL-38. In other aspects,the cytokine comprises a tumor necrosis factor (TNF) such as TNFa, TNFp, or IFNa. In other aspects, the cytokine comprises an interferon (IFN) such as IFNp, or IFNy. In other aspects, the cytokine comprises a cology stimulating factor such as G-CSF, FM-CSF, or M-CSF. In some aspects, the cytokine comprises Transforming Growth Factor-beta (TGF-P). In some aspects, the cytokine comprises a chemokine. In some aspects, the chemokine comprises CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL11 (eotaxin), CCL19, CCL21, CXCL1, CXCL8 (IL-8), CXCL9, CXCL10 (IP- 10), CXCL12 (SDF-1), or CX3CL1.

[0260] In some aspects, the intracellular response comprises the induction of gene transcription. In other aspects, the intracellular response comprises the activation of a kinase cascade or the mobilization of intracellular calcium. In some aspects, the intracellular response comprises the transcription of a cytokine such as IL-10 or IL-35.G. Fusion Proteins

[0261] Aspects of the invention are further drawn to a fusion protein. In some aspects, the fusion protein can comprise one or a combination of the polypeptides described herein. In some aspects, a fusion protein can comprise a polypeptide comprising a synthetic kinase domain or fragment thereof as described herein (See Table 1), a polypeptide comprising a synthetic phosphorylatable domain or fragment thereof as described herein (See Table 2), a polypeptide comprising a synthetic phosphatase domain or fragment thereof (See Table 3), a polypeptide comprising a synthetic cellular receptor as described herein (See Table 4), or any combination thereof.

[0262] The term "fusion protein" can refer to a protein that comprises at least one first protein that is genetically linked to at least one second protein. Fusion proteins are made through the binding of two or more genes that originally encode separate proteins. Thus, fusion proteins can include multimers of the same or different proteins that are represented as a single linear polypeptide. In some aspects, the fusion protein described herein can comprise one or more tags. In some aspects, the fusion protein described herein can comprise one or more labeling molecules. In some aspects, the fusion protein described herein can comprise one or more linkers. In some aspects, the fusion protein described herein can comprise a leucine zipper.

[0263] The terms “tag” or “epitope tag” can refer to short peptide sequences that are introduced into a protein of interest, and can allow the purification and detection of the targetproteins through their recognition by well-characterized and validated corresponding tag antibodies. In some aspects, the tag can comprise a Flag-tag or a 3x HA tag.

[0264] The term “linker” can refer to a short amino acid sequence created in nature to separate multiple domains in a single protein. In some aspects, the linker can comprise a GS linker. The term “GS linker” can refer to a linker consisting of repeats of glycine and serine residues.

[0265] The term “leucine zipper” or “LZ” can refer a structural motif typically comprised of five successive heptads of amino acids with a signature leucine at every seventh position Leucine zippers play a central role in the dimerization of bZIP family of transcription factors and their subsequent binding to the DNA promoter regions of target genes.

[0266] In some aspects, the fusion protein can comprise a polypeptide with kinase activity (See Table 1) fused to a polypeptide capable of being phosphorylated (See Table 2). The polypeptide with kinase activity can correspond to ZAP70, Syk, Lyn, Lek, ABL kinase, or a fragment or variant thereof. In some aspects, the polypeptide with kinase activity can comprise ZAP70FL(1-619), ZAP70(255-619), Z AP70(309-619), ZAP70(326-619), Or ZAP70 (333-619), LckFL(l-509), Lck(225-509), SykFL(l-635), Syk(260-635), Syk(342-635), Syk(359-635), Syk(366-635), LynFL(l-512) or Lyn(227- 512) (See FIG. 10A).

[0267] In some aspects, the polypeptide with kinase activity can comprise a variant. The variant can comprise ZAP70YY, ZAP70EE, ZAP70Y493E, ZAP70Y492E, ZAP70FF, ZAP70Y493F, ZAP70Y492F, ABLYY, ABLEE, ABLY393E, ABLY226E, ABLFF, ABLY393F, or ABLY226F (See FIG. 10A). The polypeptide capable of being phosphorylated can comprise CD3Z, SLP76, or a fragment or variant thereof (See FIG. 19).

[0268] In some aspects, the fusion protein can further comprise a polypeptide with phosphatase activity (See Table 3). The polypeptide with phosphatase activity can comprise PTPN1, PTPN3, PTPN6, or a fragment or variant thereof. In some aspects, the polypeptide with phosphatase activity can comprise PTPNl(i-268), PTPNl(i-3i9), PTPN3(583-9i3), PTPN3(606- 913), PTPN3(628-9i3), PTPN6(2i4-595), PTPN6(24i-595), PTPN6(2i4-527), or PTPN6(2i4-527) (See 13).

[0269] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:76. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:76, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:76.

[0270] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:77. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:77, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:77.

[0271] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:78. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:78, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:78.

[0272] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:79. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:79, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:79.

[0273] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:80. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 80, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:80.

[0274] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:54. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 54, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:54.

[0275] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:81. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:81, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:81.

[0276] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth inSEQ ID NO:82. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 82, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:82.

[0277] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:83. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:83, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:83.

[0278] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:84. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:84, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:84.

[0279] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:85. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 85, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:85.

[0280] In some aspects, the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:86. In other aspects, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 86, comprising one or more amino acid substitutions. In some aspects, the synthetic transcription factor comprises an amino acid sequence set forth in SEQ ID NO:86.H. Methods of Treatment

[0281] In some aspects, presented herein is a method of treating a disease, disorder or medical condition, the method comprising administering to the subject a cell comprising one or more polypeptides described herein. In some aspects, the cell comprises a synthetic phosphorylation circuit described herein.

[0282] In some aspects, the cell comprising the synthetic phosphorylation circuit comprises a stem cell. In other aspects, the cell comprising the synthetic phosphorylation circuit comprises a primary cell. In some aspects, the cell comprising the synthetic phosphorylation circuit isselected from the group consisting of mesenchymal stem cells (MSCs), retinal pigment epithelial (RPE) cells, and induced pluripotent stem cells (iPSCs). In some aspects, the cell comprises an iPSC. In some aspects, the cell comprises a cell derived from an iPCS. In other aspects, the cell comprises a hematopoietic stem cell, or a derivative thereof. In other aspects, the cell comprises a Peripheral Blood mononuclear cell (PBMC) or a derivatives thereof, such as for example, T cells, B cells, natural killer cells, monocytes, and dendritic cells.

[0283] In some aspects, the cell comprises a cell line. In some aspects, the cell line is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney 293 (HEK293) cells, Human Fibrosarcoma (HT-1080) cells, K562 cells, Jurkat cells, PER.C6 cells, and HuH-7 cells.

[0284] In some aspects, the disease, disorder or medical condition comprises an inflammatory disease, an autoimmune disease, and / or a metabolic disease.

[0285] In some aspects, the inflammatory disease comprises fatty liver disease, endometriosis, asthma, inflammatory bowel disease (including ulcerative colitis and Crohn’s disease), and rheumatoid arthritis. In some aspects, the autoimmune disease comprises type 1 diabetes, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), Graves’ disease, Sjogren’s syndrome, myasthenia gravis, celiac disease, and systemic lupus erythematosus.

[0286] In some aspects, the metabolic disease comprises type 2 diabetes, Gaucher’s disease, hemochromatosis, phenylketonuria (PKU), and mitochondrial disorders.

[0287] In some aspects, the disease comprises a neurodegenerative disease. In some aspects, the neurodegenerative disease comprises Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic Lateral Sclerosis (ALS), Huntington’s Disease, Multiple Sclerosis (MS), Lewy Body Dementia, Frontotemporal Dementia, or Spinal Muscular Atrophy (SMA).

[0288] In some aspects, the disease comprises a traumatic brain injury (TBI), a spinal cord injury (SCI), inflammatory bowel disease (IBD), acute inflammatory disorders (such as appendicitis and acute pancreatitis), allergies, infectious diseases (such as influenza, hepatitis, and tuberculosis), and trauma-induced conditions.

[0289] In other aspect, the disease comprises cancer. In some aspects, the synthetic phosphorylation circuit is designed to sense and response to a tumor microenvironment and express or deliver a cytotoxic agent. In other aspects, synthetic phosphorylation circuit is designed to convert a cold tumor to a hot tumor by recruiting immune cells.Other Aspects

[0290] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0291] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EQUIVALENTS

[0292] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.EXAMPLES

[0293] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific aspects disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1: Engineering synthetic phosphorylation signaling networks in human cells

[0294] Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Described herein are artificial phosphorylation networks in which “push-pull” motifs — reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities — were assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells. As shown herein, this design scheme enables model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression. These capabilities were leveraged to engineer cellbased cytokine controllers that dynamically sense and suppress activated T cells. Thus, disclosed herein is a generalizable approach for designing and building phosphorylation signaling circuits that enable user-defined sense-and-respond functions for diverse biosensing and therapeutic applications.

[0295] In some aspects, the disclosure herein could be classified into any of the following categories or a combination of them:

[0296] New process:• Kinase truncation and screening assay to identify candidates with optimal recruitmentdependent activity• Conversion of antibody fluorescence to GFP units for use in modeling• Quantitative model for predictive tuning of circuit behavior• Transcriptional reporter assay for identifying tSH2 / pY motif pairs that showed synKin phosphorylation-dependent interactions.

[0297] Composition of matter:• Suite of engineered proteins that combine to enable a synthetic phosphorylation circuito Recruitment dependent synthetic kinase (synKin) and phosphatase (synPhos) to create a synthetic push-pull for changing phosphorylation equilibrium o Two-layer push-pull circuit architecture with rigid linker o Synthetic receptor push-pull scaffold with rapid and reversible signal transduction• Highly modular and scalable design of these engineered proteins enables flexible inputs and outputs such as: o Sense and report: A condensate-based reporter circuit to assess the time scale of synthetic phosphorylation activation o Sense and respond: A dynamic cytokine converter circuit that can sense TNFa and produce IL- 10, shown to modulate T cell proliferation

[0298] Device: (or an extension of composition of matter if these do not meet the definition of device)• An engineered cell with the “sense and report” circuit used as a cell-based “device” to sense extracellular ligands and report through a fluorescence output• An engineered cell with the “sense and respond” circuit used as a cell-based “device” to sense biological signals and respond by transcribing a therapeutic output (e.g., IL- 10)

[0299] The disclosure presented herein represents a pioneering framework for engineering synthetic phosphorylation signaling circuits that achieve the dynamic and complex signaling capabilities found in nature. This approach resulted in a robust design for multi-layered networks with rapid dynamics and customizable input / output configurations. The disclosure presented herein therefore has profound implications for cell-based therapeutics, enabling autonomous, rapid response to specific physiological signals or disease markers.

[0300] First, this approach established a synthetic “push-pull” cycle that relies on recruitment dependent kinase and phosphatase activities that dynamically changed the phosphorylation equilibrium of a synthetic substrate (FIG. 1A). This required sourcing protein domains from multiple native proteins to create a synthetic kinase (synKin), a synthetic substrate (synSub), and a synthetic phosphatase (synPhos). These proteins were truncated and mutated to establish recruitment dependent interactions that created a complete push-pull (FIG. ID). This required screening kinase substrate pairs (FIG. IB, FIG. 6 to FIG. 8, FIG. 10A to FIG. 12), tuning the phosphorylation through the leucine zipper affinity, synKin activity, and synKin expression (FIG. 1C), and introducing a synPhos (FIG. 15 to FIG. 16A).

[0301] Next, two push-pull circuits were connected through the use of a synthetic “phosphocouple” protein that uses a rigid linker to limit cis-phosphorylation (FIG. 2A, and FIG. 21). This enabled a phosphorylation event to be propagated to a second substrate. Importantly, this enabled the creation of a synthetic receptor that could trigger the push-pull circuit upon extracellular ligand binding (FIG. 3A to FIG. 3B, and FIG. 24). This “sense-and-report” system was propagated into the cell using a system that forms detectable condensates in the cell cytoplasm (FIG. 3C, FIG. 25).

[0302] Finally, a “sense-and-respond” circuit was developed by connecting a transcriptional amplifier to the circuit to convert the binding of extracellular ligand to a transcriptional output. In this circuit, the extracellular ligand binding leads to phosphorylation, which is propagated through the cytoplasm using the phospho-couple protein, triggering the recruitment of a synthetic transcription factor to a synthetic promoter, resulting in the expression of a gene of interest (FIG. 4A, FIG. 19 to FIG. 20, FIG. 27 to FIG. 28). Because this design was inherently modular, both the surface receptor for the extracellular ligand and the transcriptional output could be changed to therapeutically relevant biomolecules (TNFa and IL 10 respectively) (FIG. 4B, FIG. 29A).

[0303] This approach also established quantitative computational models for the single layer and two-layer push-pulls in order to predict circuit behavior and inform which configurations should be tested for optimal circuit performance (FIG. IE to FIG. IF, FIG. 2C, FIG. 16A to FIG. 17, FIG. 22A to FIG. 23). Critical to that model was the creation of a “model-operable concentration unit” (MOCU) that converts antibody-conjugated fluorescence to stoichiometrically equivalent GFP units (FIG. 14 to FIG. 15). Additionally, a model was created to characterize the activation and deactivation of the phospho-circuit and found that it performed similarly to other native phosphorylation pathways (FIG. 26A). The full signaling pathway was modeled (FIG. 28), including the secretion dynamics of the TNFa to IL10 pathway (FIG. 31 to FIG. 32).

[0304] Basic science: This approach can be used to study native pathways to better understand biological processes such as T cell biology and develop future therapies.

[0305] Biotechnology: The “sense-and-report” concept can be used as a biotechnology sensor either in vitro cell culture or in an implanted device.

[0306] lell therapy: The “sense-and-respond” concept can be used as a therapeutic directly either by using the TNFot to IL10 cytokine controller directly, or by replacing the inputs and outputs with other indication specific molecules.

[0307] Without wishing to be bound by theory, this approach solves the problems of timescale and orthogonality.

[0308] Timescale: this circuit operates on the order of seconds to minutes instead of minutes to hours as compared to other “state of the art” synthetic biology circuits. The circuits presented herein are reversible, unlike transcriptional or protease-based circuits. This dramatically improves the on / off kinetics of the circuit performance, which improves the therapeutic output and makes it more responsive and dynamic to the input (FIG. 32B).

[0309] Orthogonality: The circuits presented herein are entirely recruitment dependent and do not rely on other endogenous factors, insulating the circuit from the surrounding cellular environment. Thus, the circuits presented herein are sheltered from the cellular microenvironment, a direct contrast to other circuits that utilize endogenous pathways.

[0310] This approach outperforms other competing circuits in its fast timescale, reversibility, and orthogonality. After modeling protease- and transcriptional-based circuits, only the circuits described herein were able to dynamically control TNFa without overproducing IL-10.EXAMPLE 2: Engineering synthetic phosphorylation signaling networks in human cells

[0311] Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Presented herein are artificial phosphorylation networks in which reversible enzymatic phosphorylation cycles were assembled from modular protein domain parts and wired together to create synthetic phosphorylation circuits in human cells. This design scheme enabled model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, and downstream connections can regulate gene expression. Cell-based cytokine controllers were engineered to dynamically sense and suppress activated T cells. This approach allowed for the design of signaling circuits that enabled user-defined sense-and-respond functions for diverse biosensing and therapeutic applications.

[0312] Cells universally use protein phosphorylation signaling networks to adapt to chemical and physical cues from their external environment. In metazoan cells, these networks consist ofmultilayered pathways that rapidly and reversibly convert signals detected by cell surface receptors into diverse responses such as cell movement, secretion, metabolism, and gene expression. The ability to design artificial phospho-signaling circuits that exhibit native-like signaling behavior, yet can be programmed with custom-defined input / output connectivity, could be used to create powerful biotechnology, including human cell-based therapeutics that autonomously sense and respond to specific physiological signals or disease markers on a fast timescale.

[0313] Despite this potential, engineering of phospho-signaling circuits has lagged behind that of genetic circuits, for which advances in both microorganismal and mammalian settings have been enabled by design frameworks that leverage the intrinsic modularity of promoters and coding regions, as well as carefully benchmarked sets of genetic parts — features that have facilitated scaling of circuit complexity and fine tuning of circuit behavior with predictive quantitative models. Progress to date in engineering phospho-signaling circuits has included development of two-component phosphorylation pathways as programmable sense and- respond modules in bacteria and mammalian cells, and complex fast timescale phosphorylation circuitry in yeast. In human cells, rewiring of native phosphosignaling networks has been used to create compact therapeutic programs and sense-and respond circuits that connect surface receptors to transcriptional outputs. The polypeptides described herein facilitated the de novo design of multi-layered synthetic phosphorylation circuitry in human cells with programmable input-output connectivity and signal processing.

[0314] Changes in cycle equilibrium occur rapidly in response to input signals and are quickly reversed upon input removal, enabling cells to adapt to environmental changes on timescales of seconds to minutes. These features motivated the design of tunable, interconnectable phosphorylation cycles as elementary units for constructing synthetic signaling circuits. To accomplish this, the intrinsic structural modularity of signaling proteins was utilized. Such signaling proteins are typically composed of discrete domains that either carry out catalytic function (e g., kinase or phosphatase domains) or specify interactions with other signaling components (e.g., PDZ or SH3 domains). Orthogonal interaction domains were used to mutually direct kinase and phosphatase domains to act on protein substrate targets, thereby establishing synthetic cycles of phosphorylation and dephosphorylation that operated separately from native signaling networks.

[0315] To validate this design strategy, a protein domain part set was designed and included engineered catalytic domains derived from components of immune phosphotyrosine (pY) signaling pathways (FIG. 5 to FIG. 6). These proteins naturally utilize recruitment-dependent mechanisms of signaling activation and are weakly expressed in most non-immune cell types. As an initial test, various pY kinase domains (FIG. 7) were fused to leucine zippers (LZs) — small, highly specific heterodimerizing protein interaction domains with tunable interaction affinities — to create synthetically targeted kinases (synKins) (FIG. 8). Synthetic substrate proteins (synSubs) for the synKins were constructed by fusing cognate LZs with ITAMs (immunoreceptor tyrosine-based activation motifs), conserved pY motifs involved in immune signaling pathway activation (FIG. IB and FIG. 8). Plasmid constructs encoding pairs of epitope-tagged synKins and synSubs (FIG. 8) were transfected into human embryonic kidney cells (HEK293T), and multi-color flow cytometry was used to simultaneously measure component expression (staining of epitopes) and synSub phosphorylation (staining of phosphorylated ITAMs) in single cells (FIG. 9). To optimize synKin function, numerous kinase domain boundaries and point mutations were tested (FIG. 10A). Variants were identified that showed strong expression and demonstrated phosphorylation activity toward synSub that was highly dependent on LZ-mediated kinase domain recruitment, as evidenced by non-binding, catalytically-inactive, and unrecruited (non-cognate LZ) controls all showing little or no phosphorylation (FIG. IB, right and 11 A to 11C). Minimal crosstalk with host cell machiner was observed for these components: expression of synKin showed limited non-specific Y phosphorylation, while synSub phosphorylation in the absence of synKin expression was negligible (1 IB and C). Further, expression of the engineered proteins showed no significant limiting effect on cell viability or growth rate (FIG. 1 ID).

[0316] The activity of synKin toward synSub was tuned by altering the molecular properties of the domain parts. We constructed several sets of synKin variants: LZ sequence variants were introduced to tune binding affinity to synSub, the catalytic turnover rate was adjusted with pY kinase domain active site mutations (FIG. 8), and expression levels were tuned by introducing Kozak sequence variants to synKin expression constructs, resulting in differential rates of protein translation. When these engineered proteins were tested with each synKin part set, modulation of synSub phosphorylation was observed across a 10-to-20 fold range (FIG. 1C). To create complete cycles of reversible synSub phosphorylation, synthetically targeted pY phosphatases (synPhos) were engineered. A similar approach as was used for the synKins,identifying domain variants derived from pY phosphatases involved in immune signaling (FIG. 12 to FIG. 13) and fusing them to the same LZ species as the synKin. When co-expressed with a synKin / synSub pair, synPhos dephosphorylated synSub in a recruitment- and phosphatase activity-dependent manner (FIG. ID). Taken together, these results validated the design strategy for constructing synthetic phosphorylation cycles and demonstrated that a simple part set consisting of catalytic and interactions domain variants could be used to rationally control intracellular phosphorylation equilibrium.

[0317] A model was developed to quantitatively describe the relationship between phosphorylation cycle equilibrium and part biophysical properties. To achieve this, single-cell fluorescence values were converted into stoichiometric equivalents for all phosphorylation cycles depicted in FIG. IB to FIG. ID by normalizing different color fluorophores to an EGFP reference (FIG. 14 to FIG. 15). These transformed data were then fit to a non-equilibrium thermodynamic model (FIG. IE and FIG. 16A) to obtain part-specific parameters for LZ variant interaction affinities (Kws, KES) and catalytic turnover rates of synKin and synPhos variants (k"WS, k" ES) (FIG. 16A). The parameterized model was then used to predict phosphorylation states for all part combinations within our design space (n=216 total compositions) (FIG. IF and FIG. 17). To validate these predictions, compositions were constructed and measured from across the predicted behavior distribution; all showed excellent overall agreement with the model (FIG. IF), demonstrating that the functional modularity inherent in this design scheme enabled the prediction of regulated phosphorylation behavior based on individual parts properties. Finally, the model was used to analyze the part set for compositions that could undergo sharp, ultrasensitive transition in phosphorylation upon changes in component activity, expression levels, and LZ affinity (FIG. 18). From this analysis, the majority of the compositions in our part design space contain part compositions were determined to fall in a linear regime, though tuning to higher sensitivity was possible using higher synKin LZ affinity, lower synPhos LZ affinity, and higher synSub expression.

[0318] Native signaling networks convert protein phosphorylation into molecular outputs through various mechanisms, including allosteric regulation of protein activity, changes in protein localization and stability, and formation of new protein-protein interactions. In the latter case, phospho-specific binding domains recognize phosphorylated substrate motifs, forming interactions that facilitate downstream signaling. To form circuit connections between phosphorylation cycles engineered SH2 domains were used. These domains bind to pY-containing motifs and are conserved among metazoans (FIG. 2A). A transcriptional reporter (FIGs. 19A, B) was used to not only validate synKin activity-dependent SH2-pY motif interactions, but also to identify part sets with orthogonal interaction specificities (FIG. 19D): tandem SH2 (tSH2) domains and an engineered multivalent SH2 respectively bound synKin- phosphorylated IT AMs (FIG. 19C) and a dual pY motif derived from the immune signaling protein SLP76 (SH2-domain containing leukocyte protein of 76 kDa) (FIG. 20A), with no observable crosstalk (FIG. 20B).

[0319] To test whether these phospho-dependent interactions could be used to link two phosphorylation cycles together, a “phospho-couple” (PC) protein was engineered that integrated the functions of an upstream synSub and downstream synKin by fusing a kinase domain to three substrate motifs and placing a rigid linker domain between them to limit cisphosphorylation (FIG. 2A, right). This design facilitated sequential activation of phosphorylation cycles: when a 4-protein system (upstream synKin, PC, synPhos, and downstream SH2-synSub) was expressed in HEK293T cells, phosphorylation of PC by the upstream synKin led to recruitment and phosphorylation of a downstream, tSH2-fused synSub (FIG. 2B). Sequential phosphorylation was dependent upon upstream synKin recruitment, PC activity, and SH2-mediated recruitment, as well as 3x substrate motif valency (FIG. 2B, right).

[0320] One important systems-level property of native phosphorylation cascades is their ability to stoichiometrically amplify weak input signals into macroscopic cellular outputs. To determine whether our two-step circuit architecture could be tuned to maximize amplification of an upstream input, the quantitative model was expanded to fit data from FIG. 2B, obtaining part-specific parameters (FIG. 22A) that allowed behavior predictions across two-step circuit combinatorial design space (n=3,456 compositions) (FIG. 2C, left). A region of behavior space was identified with compositions predicted to show a >10x fold-change in downstream synSub phosphorylation upon addition of an upstream synKin (n=261 compositions). Circuits from this high-gain region were enriched for features that are consistent with stoichiometric amplification, including a low PC:SH2-synSub ratio and strong synPhos activity (FIG. 23). To validate these model predictions, several amplifier circuit compositions from this region were selected to experimentally measure, demonstrating the general agreement of their behavior with model predictions (FIG. 2C, right). These results indicated that the part set and predictive modeling framework could be extended to guide the design of multi-phosphorylation cycle networks with programmed signal-processing properties.

[0321] Having developed approaches for building, interconnecting, and predictively tuning synthetic phosphorylation cycles, surface receptors were engineered that could couple extracellular ligand binding to changes in phosphorylation cycle equilibrium (FIG. 3A, left). A pair of synthetic receptor scaffolds (FIG. 24) were constructed consisting of flexible intracellular and extracellular linker sequences and transmembrane helices. Kinase and LZ domains were appended to the cytoplasmic termini of the scaffolds, and FKBP (FK506 binding protein) and FRB* (FKBP-rapamycin-binding domain of mTOR kinase, K2095P) — domains that heterodimerize upon binding to the rapamycin analog AP21967 — to their extracellular termini. This architecture enabled ligand-induced synSub phosphorylation as a result of receptor dimerization-enforced proximity between the synKin and LZ (FIG. 3A, right). After transfecting a 4-protein system consisting of the receptor pair, synSub, and synPhos proteins, a ~20-fold change in phosphorylation upon ligand addition was observed. Circuit induction was dependent on both LZ-mediated synSub recruitment and synKin activity. Additionally, elimination of synPhos activity resulted in a lower fold-change response (6.9x), demonstrating the importance of phosphorylation cycle reversibility for optimizing circuit performance (FIG. 3B). Finally, the dose response profile for the circuit was highly linear (HH= 1.1)

[0322] To assess the timescale of activation for the receptor-mediated phospho-sensor circuit, a reporter system was engineered to track the accumulation of phosphorylated synSub in real time by fluorescence microscopy (FIG. 3C). The reporter was created by fusing an SH2 domain and mCherry fluorescent protein to a tag derived from Polar Organizing Protein Z, an intrinsically disordered protein that can sequester fused client proteins into cytoplasmically- localized liquid-liquid phase condensates (FIG. 25A). This enabled monitoring circuit activation by quantifying the co-localization of enhanced green fluorescent protein (EGFP)- fused synSub to the condensates as a proxy for phosphorylation (FIGs. 25B, C and FIG. 35). Results showed eGFP / mCherry co-localization within 10 mins following ligand addition and steady state was reached after ~1 hour, whereas addition of a synKin inhibitor to the fully active pathway led to rapid synPhos-dependent de-localization (FIG. 3C and FIG. 36). In a circuit in which synPhos was absent, de-localization occurred >10x slower, demonstrating that synPhos was critical for rapid circuit reversibility (FIG. 26A). Fitting these data to a dynamic model (FIG. 26B) yielded circuit activation and deactivation half-times of 28.8 and 22.8 min, respectively — rapid dynamics similar to those measured for JAK / STAT (Janus kinase / signaltransducer and activator of transcription) and TGF-P (transforming growth factor beta) cytokine signaling pathway (26C).

[0323] Next, a combination of the phospho-sensor and two-step amplifier modules were tested to create a sense-and-respond circuit that would convert an extracellular input signal into expression of a transgene (FIG. 4A, left and FIG. 27A). To promote membrane-to-nucleus signal propagation, an amplifier composition (#2 from FIG. 2C) was selected that showed both high gain and high downstream phosphorylation, and appended nuclear localization sequence (NLS) and nuclear exporting sequence (NES) to the PC to promote shuttling between the cytoplasm and nucleus. Regulation of transcriptional activation was implemented by fusing the second substrate to a synthetic zinc finger transcription factor (synTF) to facilitate phosphodependent recruitment of an SH2-fused transcriptional activation domain (TAD), resulting in initiation of EGFP reporter expression. This 6-protein, 7-gene circuit was tested in HEK293T cells and observed PC phosphorylation (19x fold-change) and EGFP expression (16x foldchange) in response to ligand addition (FIG. 4A, right) was noted. Non-NLS / NES-tagged (FIG. 27B) and lower-gain circuit designs (28 A) showed <10x fold-change, underscoring the importance of shuttling and amplification as circuit design features. The circuit also demonstrated a linear dose response profile (HH=0.97) (28B).

[0324] As a demonstration of the translational potential of our framework, a circuit was engineered to sense tumor necrosis factor a (TNF-a) — a cytokine secreted by T cells that drives adverse inflammatory response — and respond by secreting interleukin 10 (IL- 10), a cytokine that inhibits T cell activation, proliferation, and TNF-a production, but has toxic side effects that limit its clinical utility. This circuit was therefore designed to established an antiinflammatory control loop that suppressed T cell activation while maintaining low concentrations of both cytokines. To achieve this, the sense-and-respond circuit in FIG. 4A was adapted by appending single chain antibody fragments (scFvs) that recognized TNF-a to the receptors (FIG. 4B, FIG. 29A), and replaced the EGFP reporter with IL-10. HEK293T cells containing this circuit were introduced into a transwell co-culture with CD3 and CD28- activated human PBMCs, and cytokine production and T cell proliferation were assessed across a 60-hour time course (FIG. 4C, left). Co-cultures containing cells with no circuit showed rapid accumulation of TNF-a and robust T cell proliferation, whereas cells containing a circuit driving constitutive IL- 10 expression (open-loop composition) inhibited TNF-a secretion and T cell proliferation (4C, right and FIG. 30). Cells containing the sense-and-respond circuit(closed-loop composition) also suppressed T cell proliferation but reached low steady-state concentrations of both TNF-a and IL- 10 after ~12 hours. As indicated by modeling the dynamics of this system (FIG. 31), this rapid setpoint convergence is likely dependent on the fast activation and deactivation rates of our phospho-signaling circuit, and may not be achievable with circuits that use molecular mechanisms that operate on slower timescales (FIG. 31 and FIG. 32).

[0325] The sense and respond circuit was further developed by designing a ligand-inducible, phosphorylation-based transcriptional activator in MSCs. A protein was engineered, termed the ‘equilibrium translocon’ (FIG. 37A, SEQ ID NO:54), from the available parts described herein, such as the phospho-couple protein described in Appendix A (Supplemental Figure 23B describing a protein comprising an NLS, NES, CD3z, and leucine zipper) fused to parts from the phospho-activated synTF described in Appendix A (Supplemental Figure 23A describing a protein comprising a myc, zinc finger, and GST). This new protein bypassed the two-step phosphorylation and resulted in direct transcriptional activation by “reporting” the state of phosphorylation equilibrium (FIG. 37B, and FIG. 37C).

[0326] Synthetic phospho-signaling circuits were then engineered using a simple design logic in which phosphorylation cycles were used as building blocks, and circuit connectivity and information flow were defined through programmed protein-protein interactions. This design framework enabled predictive tuning of circuit behavior and the use of nonequilibrium thermodynamic modeling to guide circuit design. While the part set used herein to demonstrate the practicability of the design scheme consisted largely of domains and motifs repurposed from native human immune signaling, our framework should facilitate incorporation of domains drawn from other sources or generated by computational design. Catalytic domains could be engineered to enhance circuit performance through activity tuning, or by introducing allosteric regulation. Because the functional specificities of the components were determined by recruitment, scaling to greater circuit complexity could be enabled by simply expanding the number of orthogonal interaction domains in the part set. This could facilitate construction of circuit topologies that carry out advanced signal processing functions, such as Boolean logic enabled through multi-site phosphorylation, feedback connections that tune circuit dynamics or introduce ultra sensitivity, or multi-input-output circuits that can perceive and compute internal or external states.

[0327] Finally, because the circuits described herein are post-translational and signal rapidly and reversibly, they could potentially support a broad array of cell-based diagnostic and therapeutic applications that require sensing of minute-scale physiological or pathological events. The plug-and-play configurability of such circuits could enable their coupling to diverse receptor inputs capable of sensing small molecules, bioactive factors, or disease markers. Because of their temporal responsiveness, the circuits may complement or offer advantages over other highly programmable circuit design schemes that signal by slower- turnover molecular mechanisms (e.g., transcription or proteolysis). Additionally, since the circuits described herein operate in parallel to native signaling pathways, they offer opportunities for programming signal-processing functions that are not possible for signaling circuits that harness native components to propagate signal. Furthermore, because they can be configured with human-derived protein domains and are relatively compact, circuits constructed using our design framework are likely to have low immunogenicity (FIG. 33) and could potentially be delivered to clinically-relevant cell types (FIG. 34) to enable therapeutic sense-and-respond function.EXAMPLE 3

[0328] See Appendix A.EXAMPLE 4

[0329] See Appendix B.EXAMPLE 5

[0330] See Appendix C.EXEMPLARY EMBODIMENTS1 . A polypeptide comprising a synthetic kinase domain or fragment thereof.2. The polypeptide of embodiment 1, wherein the polypeptide comprises an amino acid sequence according to Table 1 or an amino acid sequence at least 90% identical thereto.3. A polypeptide comprising a synthetic phosphorylatable domain or fragment thereof.4. The polypeptide of embodiment 3, wherein the polypeptide comprises an amino acid sequence according to Table 2 or an amino acid sequence at least 90% identical thereto.5. A polypeptide comprising a synthetic phosphatase domain or fragment thereof.6. The polypeptide of embodiment 5, wherein the polypeptide comprises an amino acid sequence according to Table 3 or an amino acid sequence at least 90% identical thereto.7. A polypeptide comprising a synthetic cellular receptor, wherein the polypeptide comprises an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain.8. The polypeptide of embodiment 7, wherein the polypeptide comprises an amino acid sequence according to Table 4 or an amino acid sequence at least 90% identical thereto.9. A polypeptide comprising an amino acid sequence according to Table 1, or an amino acid sequence at least 90% identical thereto, fused to a polypeptide comprising an amino acid sequence according to Table 2, or an amino acid sequence at least 90% identical thereto.10. The polypeptide of embodiment 9, wherein the polypeptide further comprises an amino acid sequence according to Table 3 or an amino acid sequence at least 90% identical thereto.11. A fusion protein comprising the polypeptide according to embodiment 1, the polypeptide according to embodiment 3, the polypeptide according to embodiment 5, or any combination thereof.12. A nucleic acid encoding the polypeptide according to embodiment 1, the polypeptide according to embodiment 3, the polypeptide according to embodiment 5, the polypeptide according to embodiment 7 or the fusion protein according to embodiment 11.13. A vector comprising the nucleic acid of embodiment 12.14. A cell comprising the vector of embodiment 13.15. A fusion protein comprising a polypeptide with kinase activity fused to a polypeptide capable of being phosphorylated.16. The fusion protein of embodiment 15, wherein the fusion protein further comprises a polypeptide with phosphatase activity.17. A synthetic phosphorylation circuit comprising a polypeptide of any one of the above embodiments.18. The synthetic phosphorylation circuit of embodiment 17, wherein the synthetic phosphorylation circuit can convert an extracellular signal into an intracellular response.19. The synthetic phosphorylation circuit of embodiment 17, wherein the synthetic phosphorylation circuit comprises the TNFa-sensing / IL-10 response circuit.20. The synthetic phosphorylation circuit of embodiment 17, wherein the synthetic phosphorylation circuit responds to TNF-a by a proportional secretion of IL-10.21. The synthetic phosphorylation circuit of embodiment 18, wherein the conversion of an extracellular signal into an intracellular response comprises fast timescale transcription.22. A cell comprising the synthetic phosphorylation circuit of embodiment 17.23. A method of treating a disease, disorder or medical condition, the method comprising administering to the subject the cell of embodiment 22.24. The method of embodiment 23, wherein the disease, disorder or medical condition comprises inflammatory diseases, autoimmune diseases, and / or metabolic diseases.SEQUENCES

Claims

CLAIMS1. A polypeptide comprising: a synthetic kinase domain or fragment thereof; and a dimerization domain.

2. The polypeptide of claim 1, wherein the polypeptide further comprises a protein tag.

3. The polypeptide of claim 2, wherein the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag.

4. The polypeptide of claim 2 or 3, wherein the protein tag is a FLAG tag.

5. The polypeptide of claim 4, wherein the FLAG tag comprises the amino acid sequence set forth in SEQ ID NO: 1.

6. The polypeptide of any one of claims 1 to 5, wherein the synthetic kinase domain comprises a kinase involved in activation of T Cell Receptor or B Cell Receptor signaling or an Src family member.

7. The polypeptide of any one of claims 1 to 6, wherein the synthetic kinase domain is selected from the group consisting of TCR, ZAP70, Syk, Lyn, Lek, and ABL.

8. The polypeptide of any one of claims 1 to 7, wherein the synthetic kinase domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth asSEQ ID NOs:2-16.

9. The polypeptide of any one of claims 1 to 8, wherein the synthetic kinase domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:2-16.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide further comprises a linker.

11. The polypeptide of claim 10, wherein the linker comprises a GS linker.

12. The polypeptide of claim 11, wherein the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

13. The polypeptide of any one of claims 1 to 12, wherein the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R.

14. The polypeptide of any one of claims 1 to 13, wherein the dimerization domain comprises a Leucine Zipper E.

15. The polypeptide of claim 14, wherein the Leucine Zipper E comprises an amino acid sequence set forth in SEQ ID NO: 18.

16. The polypeptide of any one of claims 1 to 15, wherein the polypeptide comprises a Flag tag comprising the amino acid sequence set forth in SEQ ID NO: 1, a synthetic kinase domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, a GS Linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

17. A polypeptide comprising: a synthetic phosphorylatable domain or fragment thereof; and a dimerization domain.

18. The polypeptide of claim 17, wherein the polypeptide further comprises a protein tag.

19. The polypeptide of claim 18, wherein the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag.

20. The polypeptide of claim 18 or 19, wherein the polypeptide comprises a MYC tag and a GST tag.

21. The polypeptide of any one of claims 17 to 20, wherein the synthetic phosphorylatable domain or fragment thereof comprises an immune tyrosine activation motif (IT AM) or an immune tyrosine inhibition motif (ITIM).

22. The polypeptide of any one of claims 17 to 21, wherein the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth asSEQ ID NOs:21-32.

23. The polypeptide of any one of claims 17 to 22, wherein the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:21-32.

24. The polypeptide of any one of claims 17 to 23, wherein the polypeptide further comprises a linker.

25. The polypeptide of claim 24, wherein the linker comprises a GS linker.

26. The polypeptide of claim 25, wherein the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

27. The polypeptide of any one of claims 17 to 26, wherein the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R.

28. The polypeptide of any one of claims 17 to 27, wherein the dimerization domain comprises a Leucine Zipper R.

29. The polypeptide of claim 28, wherein the Leucine Zipper R comprises an amino acid sequence set forth in SEQ ID NO:33.

30. The polypeptide of any one of claims 17 to 29, wherein the polypeptide comprises a MYC tag comprising the amino acid sequence set forth in SEQ ID NO: 19, a GST tag comprising the amino acid sequence set forth in SEQ ID NO:20, a synthetic phosphorylatable domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID N0:31, and SEQ ID NO:32, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper R comprising the amino acid sequence set forth in SEQ ID NO:33.

31. A polypeptide comprising: a synthetic phosphatase domain or fragment thereof; and a dimerization domain.

32. The polypeptide of claim 31, wherein the polypeptide further comprises a protein tag.

33. The polypeptide of claim 32, wherein the protein tag is selected from the group consisting of an HA tag, a MYC tag, a FLAG tag, a His tag, a GST tag, an MBP tag, a Strep-tag, a TAP tag, an HSV tag, a KT3 epitope tag, a LacZ tag, a Protein A / G tag, and a Halo tag.

34. The polypeptide of claim 32 or 33, wherein the protein tag is an HA tag.

35. The polypeptide of claim 34, wherein the HA tag comprises an amino acid sequence set forth in SEQ ID NO:34.

36. The polypeptide of any one of claims 31 to 35, wherein the synthetic phosphatase domain or fragment thereof comprises a tyrosine phosphatase.

37. The polypeptide of claim 36, wherein the tyrosine phosphatase is PTPN1, PTPN4, or PTPN6.

38. The polypeptide of any one of claims 31 to 37, wherein the synthetic phosphatase domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth asSEQ ID NOs:35-43.

39. The polypeptide of any one of claims 31 to 38, wherein the synthetic phosphatase domain or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs:35- 43.

40. The polypeptide of any one of claims 31 to 39, wherein the polypeptide further comprises a linker.

41. The polypeptide of claim 40, wherein the linker comprises a GS linker.

42. The polypeptide of claim 41, wherein the GS linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

43. The polypeptide of any one of claims 31 to 42, wherein the dimerization domain comprises a Leucine Zipper E or Leucine Zipper R.

44. The polypeptide of any one of claims 31 to 43, wherein the dimerization domain comprises a Leucine Zipper E.

45. The polypeptide of claim 44, wherein the Leucine Zipper E comprises an amino acid sequence set forth in SEQ ID NO: 18.

46. The polypeptide of any one of claims 31 to 45, wherein the polypeptide comprises a protein tag comprising the amino acid sequence set forth in SEQ ID NO:34, a synthetic phosphatase domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, a GS linker comprising the amino acid sequence set forth in SEQ ID NO: 17, and a Leucine Zipper E comprising the amino acid sequence set forth in SEQ ID NO: 18.

47. A polypeptide comprising a synthetic cellular receptor, wherein the polypeptide comprises an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain.

48. The polypeptide of claim 47, wherein synthetic cellular receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:53.

49. The polypeptide of claim 47 or 48, wherein the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a FRB(K2095P) domain comprising the amino acid sequence set forth in SEQ ID NO:46, a transmembrane domaincomprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

50. The polypeptide of claim 47 or 48, wherein the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:49, an FKBP domain comprising the amino acid sequence set forth in SEQ ID NO:50, transmembrane domains comprising the amino acid sequences set forth in SEQ ID NO:47, SEQ ID NO:51, and SEQ ID NO:52, and a Leucine Zipper E comprising the amino acid sequences set forth in SEQ ID NO: 18.

51. The polypeptide of claim 47 or 48, wherein the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and an ABL228-540FF domain comprising the amino acid sequence set forth in SEQ ID NO:48.

52. The polypeptide of claim 47 or 48, wherein the synthetic cellular receptor comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:44, a protein tag comprising the amino acid sequence set forth in SEQ ID NO:45, a TNF-a scFv domain comprising the amino acid sequence set forth in SEQ ID NO:53, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:47, and a Leucine Zipper E domain comprising the amino acid sequence set forth in SEQ ID NO: 18.

53. A polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:2- 16, or an amino acid sequence at least 90% identical thereto, fused to a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-32, or an amino acid sequence at least 90% identical thereto.

54. The polypeptide of claim 53, wherein the polypeptide further comprises an amino acid sequence according set forth in any one of SEQ ID NOs:35-43 or an amino acid sequence at least 90% identical thereto.

55. A fusion protein comprising the polypeptide according to claim 1, the polypeptide according to claim 17, the polypeptide according to claim 31, or any combination thereof.

56. The fusion protein of claim 55, wherein the fusion protein comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:54 or 76-86.

57. A polypeptide comprising: a nuclear localization signal (NLS); one or more DNA binding polypeptides; a nuclear export signal (NES); and a synthetic phosphorylatable domain or fragment thereof.

58. The polypeptide of claim 57, wherein the NLS comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 57.

59. The polypeptide of claim 57 or 58, wherein the one or more DNA binding polypeptides comprise one or more zinc finger domains.

60. The polypeptide of any one of claims 57 to 59, wherein the one or more DNA binding polypeptides comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 59.

61. The polypeptide of any one of claims 57 to 60, wherein the NES comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:62.

62. The polypeptide of any one of claims 57 to 61, wherein the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 63.

63. The polypeptide of any one of claims 57 to 61, wherein the synthetic phosphorylatable domain or fragment thereof comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth inSEQ ID NOs:21-32.

64. The polypeptide of any one of claims 57 to 63, wherein the polypeptide further comprises one or more linkers.

65. A polypeptide comprising: one or more nuclear localization signals; a recruitment motif; and a transcriptional activator.

66. The polypeptide of claim 65, wherein the one or more nuclear localization signals comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NO:66 or SEQ ID NO:75.

67. The polypeptide of claim 65 or 66, wherein the recruitment motif comprises an engineered SH2 domain.

68. The polypeptide of any one of claims 65 to 67, wherein the recruitment motif comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs:67-72.

69. The polypeptide of any one of claims 65 to 68, wherein the transcriptional activator comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:74.

70. The polypeptide of any one of claims 65 to 69, wherein the polypeptide further comprises one or more linkers.

71. A nucleic acid encoding the polypeptide according to claim 1, the polypeptide according to claim 17, the polypeptide according to claim 31, the polypeptide according to claim 47 or the fusion protein according to claim 55.

72. A vector comprising the nucleic acid of claim 71.

73. A cell comprising the vector of claim 72.

74. A fusion protein comprising a polypeptide with kinase activity fused to a polypeptide capable of being phosphorylated.

75. The fusion protein of claim 74, wherein the fusion protein further comprises a polypeptide with phosphatase activity.

76. A synthetic phosphorylation circuit comprising a polypeptide of any one of claims 1 to 54.

77. The synthetic phosphorylation circuit of claim 76, wherein the synthetic phosphorylation circuit can convert an extracellular signal into an intracellular response.

78. The synthetic phosphorylation circuit of claim 76, wherein the synthetic phosphorylation circuit comprises a TNFa-sensing / IL-10 response circuit.

79. The synthetic phosphorylation circuit of claim 76, wherein the synthetic phosphorylation circuit responds to TNF-a by a proportional secretion of IL-10.

80. The synthetic phosphorylation circuit of claim 77, wherein the conversion of an extracellular signal into an intracellular response comprises fast timescale transcription.

81. A cell comprising the synthetic phosphorylation circuit of claim 76.

82. A method of treating a disease, disorder or medical condition, the method comprising administering to the subject the cell of claim 81.

83. The method of claim 82, wherein the disease, disorder or medical condition comprises inflammatory diseases, autoimmune diseases, neurodegenerative diseases, traumatic brain injuries, and / or metabolic diseases.

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