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 alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha 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 PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha 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 PIP5KIalpha 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.