title: The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.
abstract: CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.
authors.affiliation: Graduate Program in Neurosciences, University of California, San Diego, La Jolla 92093, USA.
authors.forename: K
authors.initials: K
authors.lastname: Neet
authors.forename: T
authors.initials: T
authors.lastname: Hunter
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D000081247:CSK Tyrosine-Protein Kinase; D002451:Cell Compartmentation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis* / Q000235:genetics; D010449:Peptide Mapping; D010748:Phosphopeptides; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017385:Sequence Homology; D014170:Transformation, Genetic; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases
chemical_list: D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; C074561:GAP-associated protein p62; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases