Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3, PIP, inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP, levels in mixed Lipid vesicles. Caspase 3 binding to PIP, was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KI alpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KI alpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP, Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP2 is further substantiated by our finding that PIP5KI alpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KI alpha in vitro. Mutation of the P-4 position (D279A) of the PIP5KI alpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KI alpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KI alpha inactivation contributes to the progres sion of apoptosis, This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.