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Integrin-linked kinase and its uses

a technology of integrin-linked kinase and kinase, which is applied in the field of integrin-linked kinase, can solve the problems that to date no cellular protein kinase has been identified that has been demonstrated to bind

Inactive Publication Date: 2007-08-30
SUNNYBROOK HEALTH SCI CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no cellular protein kinase has been identified to date that has been demonstrated to bind to an integrin molecule under physiological conditions.

Method used

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  • Integrin-linked kinase and its uses
  • Integrin-linked kinase and its uses
  • Integrin-linked kinase and its uses

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of ILK cDNA

[0107] A partial cDNA, BIT-9, was isolated in a two-hybrid screen using a bait plasmid expressing the cytoplasmic domain of the β1 integrin subunit The BIT-9 insert was used to isolate clones from a human placental cDNA library. A 1.8 kb clone, Plac5, was found to contain a high degree of similarity to cDNAs encoding protein kinases (FIG. 1 a-c), and recognized a widely expressed transcript of 1.8 kb in Northern blots (FIG. 1 d). Deduced amino acid residues 186-451 from Plac5 comprise a domain which is highly homologous with the catalytic domains of a large number of protein tyrosine and serine / threonine kinases (FIG. 1b). Residues 33-164 comprise four repeats of a motif originally identified in erythrocyte ankyrin (FIG. 1 c), likely defining a domain involved in mediating additional protein-protein interactions. Affinity-purified anti-ILK antibodies (see methods described in Example 3) were used in Western blot analyses of mammalian cell extracts, and detected...

example 2

[0110] For analysis of kinase activity in, vitro, a bacterially-expressed fusion protein, GST-ILK132, was SDS-PAGE band puified, and incubated with [γ-32P]ATP in the presence or absence of the exogenous substrate myelin basic protein (FIG. 2). GST-ILK132 autophosphorylated and labeled MBP efficiently in these assays (FIG. 2a). Anti-GST-ILK132 (antibody 91-3) immunoprecipitates of PC3 cell lysates were incubated with [γ-32P]ATP, similar to experiments performed with purified recombinant GST-ILK132. ILK immune complexes labeled a protein of apparent Mr of 59 kDa (FIG. 2b) corresponding to p59ILK, as well as cellular proteins of apparent Mr 32 kDa and 70 kDa, which may be endogenous ILK substrates (FIG. 2b). Cellular phosphoproteins (serine / threonine) of approximately 32 kDa and 70 kDa, were also seen in β1 integrin-specific immune complex kinase assays.

[0111] In ILK immune complex kinase assays a synthetic peptide representing the β3 cytoplasmic domain was phosphorylated, while a sim...

example 3

Association of ILK and β Integrin in Mammalian Cells

[0114] Immunofluorescence experiments indicated that ILK and β integrin co-localize in focal plaques. In order to test further for this association in intact mammalian cells, we performed co-immunoprecipitation assays in lysates of PC3 cells, in which integrin expression has been well-characterized. PC3 cell lysates were immunoprecipitated with specific anti-integrin antibodies, and immune complexes analyzed by Western blotting with the anti-ILK antibody, 92-2. The specificities of the anti-ILK antibodies were tested by immunoprecipitation and Western blotting (FIGS. 3a, b). We detected p59ILK in immune complexes obtained with anti-fibronectin receptor (FNR, α5 / α3 β integrin), and anti-vitronectin receptor (VNR, αvβ3 / β5 integrin) antibodies, but not in those obtained with non-immune serum (FIG. 3c). Three anti-β1 monoclonal antibodies also co-precipitated p59ILK from PC3 lysates, confirming the β integrin specificity of p59ILK int...

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Abstract

Methods for isolating ILK genes are provided. The ILK nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as identification of cell type based on expression, and the like.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a a continuation of U.S. patent application Ser. No. 09 / 967,854, filed Sep. 28, 2001, which is a Divisional of U.S. patent application Ser. No. 09 / 390,425, filed Sep. 3, 1999, which is a Continuation of U.S. patent application Ser. No. 09 / 035,706, filed Mar. 5, 1998, now issued as U.S. Pat. No. 6,001,622 on Dec. 14, 1999, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 955,841, filed Oct. 21, 1997, now issued as U.S. Pat. No. 6,013,782 on Jan. 11, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 752,345, filed Nov. 19, 1996, now abandoned which claims priority to provisional patent application No. 60 / 009,074, filed Dec. 21, 1995.INTRODUCTION [0002] 1. Background [0003] Proteins of the extracellular matrix (ECM) act to influence fundamental cell and tissue behaviors. ECM regulates cell structure, growth, survival, differentiation, motility and, at the organismal level...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K2/00A61K38/00C12N9/12C12Q1/48
CPCA61K38/00C12N9/12G01N2333/9121C12Q1/48G01N2333/70546C12N9/1205
Inventor DEDHAR, SHOUKATHANNIGAN, GREG
Owner SUNNYBROOK HEALTH SCI CENT
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