Tyrosine phosphorylation of beta2-chimaerin by Src-family kinase negatively regulates its Rac-specific GAP activity.
beta2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. beta2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of beta2-chimaerin remains largely unknown. Here we report that beta2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of beta2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type beta2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates beta2-chimaerin Rac-GAP activity.
Kai
Masahiro
M
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556, Japan.
Yasuda
Satoshi
S
Imai
Shin-Ichi
S
Kanoh
Hideo
H
Sakane
Fumio
F
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion / Q000235:genetics; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism*
D009363:Neoplasm Proteins; C079844:beta-chimaerin; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D010649:Phenylalanine; D004815:Epidermal Growth Factor; D019061:src-Family Kinases; D020744:rac GTP-Binding Proteins