Adhesion-induced tyrosine phosphorylation of the p130 src substrate.
Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of which have now been identified as the focal adhesion proteins pp125FAK, paxillin and tensin. In addition, we have previously noted the adhesion-induced tyrosine phosphorylation of a fourth protein, with an apparent molecular mass of 130. As in the case of FAK, paxillin and tensin, a 130 kDa protein is also found to be highly tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed cells. This protein forms a stable complex with pp60src and is directly phosphorylated by activated forms of c-src. Using a monoclonal antibody (mAb 4F4) specific for the src-associated p130 we show that p130 is also phosphorylated in response to cell adhesion. Immunoprecipitation of p130 followed by an anti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fibroblasts (REF52) to fibronectin (FN) led to a significant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cell lysates before and after immunoprecipitation confirmed the absence of tyrosine phosphorylated p130 from lysates immunoprecipitated with mAb 4F4. Immunofluorescence staining of REF52s revealed that p130 is found in focal adhesions as well as along stress fibers in a pattern reminiscent of that exhibited by alpha-actinin. In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130. Finally, unlike pp125FAK, p130 does not appear to be itself a kinase as evidence by immune-complex kinase assays carried out in the presence or absence of exogenous substrates.
Petch
L A
LA
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599, USA.
Bockholt
S M
SM
Bouton
A
A
Parsons
J T
JT
Burridge
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D001358:Avian Sarcoma Viruses; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004622:Embryo, Mammalian; D005347:Fibroblasts; D005353:Fibronectins; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016391:Genes, src; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D005353:Fibronectins; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); C496052:Ptk2 protein, rat