Evidence of a novel redox-linked activation mechanism for the Src kinase which is independent of tyrosine 527-mediated regulation.
The kinase activity of p60c-src has been shown to be basically regulated through phosphorylation and dephosphorylation of Y527. We found that catalytic activity of the immunoprecipitated c-Src kinase from NIH3T3 cells was elevated several folds by exposure to 0.5-50 microM of sulfhydryl-reactive Hg2+. Vmax of the kinase was increased whereas Km was decreased. N-acetylcysteine neutralized this Hg2+ effect, suggesting a critical role of the Hg2+-mediated sulfhydryl modification of the kinase in the mechanism. Addition of protein tyrosine phosphatase inhibitor Na3VO4 into the reaction mixture did not inhibit the Hg2+-mediated activation. Further study revealed that Hg2+ was capable of activating the v-Src kinase lacking Y527 and the c-Src kinase from mutant cells defective of the Y527-phosphorylating Csk kinase. Cyanogen bromide cleavage maps of radiolabeled Src proteins showed that Hg2+ selectively promoted the autophosphorylation at Y416 and that the previously in vivo radiolabeled phosphorous on Y527 was not deleted during the promotion of Y416 autophosphorylation by Hg2+. Phosphoamino acid analysis demonstrated selective promotion of phosphorylation at tyrosine but not at serine/threonine. Not like bivalent Hg2+, monovalent p-chloromercuribenzenesulfonic acid was incapable of activating c-Src kinase. These results suggest a novel Y416 phosphorylation-linked activation pathway for Src kinases which is initially triggered independent of Y527-mediated or serine/threonine phosphorylation-linked regulation, possibly through sulfhydryl-based protein structural modification for functional alteration.
Pu
M
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Department of Immunology, Nagoya University School of Medicine, Japan.
Akhand
A A
AA
Kato
M
M
Hamaguchi
M
M
Koike
T
T
Iwata
H
H
Sabe
H
H
Suzuki
H
H
Nakashima
I
I
D016475:3T3 Cells; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D008627:Mercuric Chloride / Q000494:pharmacology*; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010449:Peptide Mapping; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D008627:Mercuric Chloride; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)