EVIDENCE OF A NOVEL REDOX-LINKED ACTIVATION MECHANISM FOR THE SRC KINASE WHICH IS INDEPENDENT OF TYROSINE 527-MEDIATED REGULATION

The kinase activity of p60(c-src) has been shown to be basically regul ated through phosphorylation and dephosphorylation of Y527. We found t hat catalytic activity of the immunoprecipitated c-Src kinase from NIH 3T3 cells was elevated several folds by exposure to 0.5-50 mu M of sul fhydryl-reactive Hg2+. V-max of the kinase was increased whereas K-m w as decreased. N-acetylcysteine neutralized this Hg2+ effect, suggestin g a critical role of the Hg2+-mediated sulfhydryl modification of the kinase in the mechanism. Addition of protein tyrosine phosphatase inhi bitor Na3VO4 into the reaction mixture did not inhibit the Hg2+-mediat ed activation. Further study revealed that Hg2+ was capable of activat ing the v-Src kinase lacking Y527 and the c-Src kinase from mutant cel ls defective of the Y527-phosphorylating Csk kinase. Cyanogen bromide cleavage maps of radiolabeled Src proteins showed that Hg2+ selectivel y promoted the autophosphorylation at Y416 and that the previously in vivo radiolabeled phosphorous on Y527 was not deleted during the promo tion of Y416 autophosphorylation by Hg2+, Phosphoamino acid analysis d emonstrated selective promotion of phosphorylation at tyrosine but not at serine/threonine. Not like bivalent Hg2+, monovalent p-chloromercu ribenzenesulfonic acid was incapable of activating c-Src kinase. These results suggest a novel Y416 phosphorylation-linked activation pathwa y for Src kinases which is initially triggered independent of Y527-med iated or serine/threonine phosphorylation-linked regulation, possibly through sulfhydryl-based protein structural modification for functiona l alteration.