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 phos phatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the ap optotic machinery. We now report that another phosphoinositide, phosphatidy linositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspas es 8 and 9, and their common effector caspase 3, PIP, inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apo ptosome system. It inhibited purified caspase 3 and 8 activity, at physiolo gically attainable PIP, levels in mixed Lipid vesicles. Caspase 3 binding t o PIP, was confirmed by cosedimentation with mixed lipid vesicles. Overexpr ession of phosphatidylinositol phosphate 5-kinase alpha (PIP5KI alpha), whi ch synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutan t did not. Protection by the wild-type PIP5KI alpha was accompanied by decr eases in the generation of activated caspases and of caspase 3-cleaved PARP , Protection was not mediated through PIP3 or Akt activation. An anti-apopt otic role for PIP2 is further substantiated by our finding that PIP5KI alph a was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5K I alpha in vitro. Mutation of the P-4 position (D279A) of the PIP5KI alpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apopto sis in vivo. Significantly, the caspase 3-resistant PIP5KI alpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These r esults show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KI alpha in activation contributes to the progres sion of apoptosis, This novel feedfor ward amplification mechanism for maintaining the balance between life and d eath of a cell works through phosphoinositide regulation of caspases and ca spase regulation of phosphoinositide synthesis.