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. PIP₂ 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 PIP₂ levels in mixed lipid vesicles. Caspase 3 binding to PIP₂ was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase α (PIP5KIα), which synthesizes PIP₂, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIα 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 PIP5KIα was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIα in vitro. Mutation of the P₄ position (D279A) of the PIP5KIα caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIα 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 PIP5KIα inactivation contributes to the progression 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.