Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.
Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix.
Sabe
H
H
Laboratory of Molecular Oncology, Rockefeller University, New York, NY 10021.
Hata
A
A
Okada
M
M
Nakagawa
H
H
Hanafusa
H
H
D000818:Animals; D001483:Base Sequence; D000081247:CSK Tyrosine-Protein Kinase; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D019061:src-Family Kinases
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D051419:Paxillin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases