Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases.
When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.
Risk Assessment, Detection and Intervention Program, The H. Lee Moffitt Cancer Center and Research Institute, MRC 3 West, 12902 Magnolia Drive, Tampa, FL 33612, USA. monteian@moffitt.usf.edu
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Monteiro
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D047468:Immunoprecipitation; D051575:Proto-Oncogene Proteins c-yes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; D002118:Calcium