Active site-adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9.
Caspase-9 (casp-9) is an initiator caspase and plays a central role in activating apoptotic cell death. Control of all caspases is tightly regulated by a series of phosphorylation events enacted by several different kinases. Caspase-9 is the most heavily phosphorylated of all caspases, with phosphorylation of at least 11 distinct residues in all three caspase-9 domains by nine kinases. Caspase-9 phosphorylation by the non-receptor tyrosine kinase c-Abl at Tyr-153 reportedly leads to caspase-9 activation. All other phosphorylation events on caspases have been shown to block proteolytic function by a number of mechanisms, so we sought to unravel the molecular mechanism of the putative caspase-9 activation by phosphorylation. Surprisingly, we observed no evidence for Tyr-153 phosphorylation of caspase-9 in vitro or in cells, suggesting that Tyr-153 is not phosphorylated by c-Abl. Instead, we identified a new site for c-Abl-mediated phosphorylation, Tyr-397. This residue is adjacent to the caspase-9 active site but, as a member of the second shell, not a residue that directly contacts substrate. Our results further indicate that Tyr-397 is the dominant site of c-Abl phosphorylation both in vitro and upon c-Abl activation in cells. Of note, phosphorylation at this site inhibits caspase-9 activity, and the bulk of the added phosphate moiety appeared to directly block substrate binding. c-Abl plays both proapoptotic and prosurvival roles, and our findings suggest that c-Abl's effects on caspase-9 activity promote the prosurvival mode.
['Serrano|Banyuhay P|BP|', 'Szydlo|Hannah S|HS|', 'Alfandari|Dominique|D|', 'Hardy|Jeanne A|JA|']
D017209:Apoptosis D001665:Binding Sites D053453:Caspase 9 D020169:Caspases D020134:Catalytic Domain D018360:Crystallography, X-Ray D006801:Humans D010766:Phosphorylation D010770:Phosphotransferases D059748:Proteolysis D016315:Proto-Oncogene Proteins c-abl D014443:Tyrosine
caspase ABL tyrosine kinase apoptosis phosphocapture phosphoenrichment protease protein phosphorylation substrate-binding groove