Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading.
The C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.
Bergman
M
M
Department of Pathology, University of Helsinki, Finland.
Joukov
V
V
Virtanen
I
I
Alitalo
K
K
D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007700:Kinetics; D051419:Paxillin; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D019030:Receptors, Vitronectin; D017384:Sequence Deletion; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D019061:src-Family Kinases
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D016023:Integrins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C000631346:CSK protein, human; D013752:Tetracycline