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.

Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a.

Adam13/33 is a cell surface metalloprotease critical for cranial neural crest (CNC) cell migration. It can cleave multiple substrates including itself, fibronectin, ephrinB, cadherin-11, pcdh8 and pcdh8l (this work). Cleavage of cadherin-11 produces an extracellular fragment that promotes CNC migration. In addition, the adam13 cytoplasmic domain is cleaved by gamma secretase, translocates into the nucleus and regulates multiple genes. Here, we show that adam13 interacts with the arid3a/dril1/Bright transcription factor. This interaction promotes a proteolytic cleavage of arid3a and its translocation to the nucleus where it regulates another transcription factor: tfap2α. Tfap2α in turn activates multiple genes including the protocadherin pcdh8l (PCNS). The proteolytic activity of adam13 is critical for the release of arid3a from the plasma membrane while the cytoplasmic domain appears critical for the cleavage of arid3a. In addition to this transcriptional control of pcdh8l, adam13 cleaves pcdh8l generating an extracellular fragment that also regulates cell migration.