Nitric oxide controls src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism.
c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N(2)O(3) scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitro labeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivo labeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na(3)VO(4) did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N(2)O(3)-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N(2)O(3)-provoked S-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.
Akhand
A A
AA
Department of Immunology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Pu
M
M
Senga
T
T
Kato
M
M
Suzuki
H
H
Miyata
T
T
Hamaguchi
M
M
Nakashima
I
I
D016475:3T3 Cells; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D004789:Enzyme Activation; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside; D010396:Penicillamine / Q000031:analogs & derivatives; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D019061:src-Family Kinases
C110881:S-nitro-N-acetylpenicillamine; D013439:Sulfhydryl Reagents; D009599:Nitroprusside; D009569:Nitric Oxide; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D010396:Penicillamine