Apoptosis occurs under a variety of physiological and pathological conditions. It is essential for maintaining the homeostasis of multicellular organisms, and inappropriate apoptosis has been implicated in the pathogenesis of many human diseases, including ischemia and cancer. Cell survival is maintained by a balance between proliferative and apoptotic signals. We found that type I phosphatidylinositol phosphate 5 kinase (PIP5KI), the enzyme which synthesizes a key signaling phosphoinositide lipid, phosphatidylinositol 4,5, bisphosphate (PIP2), sits at the crossroad between these pathways. It synthesizes PIP2 which inhibits caspases, and caspases in turn regulates PIP2 generation by inactivating PIP5KI. We propose that growth factors which activate PIP5KI promote cell survival by generating PIP2 to protect against spontaneous caspase activation, and to establish a high threshold for fiill-fledged caspase activation. This tips the balance towards survival and dampens the response of cells to low level adventitious apoptotic signals. On the other hand, strong apoptotic signals massively activate caspases through self-amplifying cascades, and swamp out the dampening effect of PIP2. Furthermore, activated caspases reduces PIP2 level, by destroying the key enzyme that synthesizes PIP2. This releases all caspases from inhibition, dissipates the pro-survival signal and tips the balance completely towards cell death. To test this hypothesis, we will do the following: First, examine the effects of apoptosis on phosphoinositide homeostasis. If there are changes in PTP2 synthesis, we will determine when and where the changes occur, and how they are generated. The relation to PIP5KI behavior and apoptotic progression will be examined. Second, determine if manipulations of cellular PIP2 alter the progression of apoptosis. PIP2 level will be increased by overexpressing PIP5KI, and PIP2 availability will be reduced by overexpressing the PIP2 binding pleckstrin homology domain of PLCdelta. Effects on the balance between survival and apoptosis will be titrated. Third, characterize the components of the proposed regulatory machinery in vitro, in order to understand the mechanistic and structural basis for their interactions. Specific tools will be developed to analyze the behavior of these players in the complex environment of a cell during apoptosis. Some possibilities are caspase PIP2 binding mutants or PIP5KI dominant negative inhibitors. These studies may suggest potential targets for therapeutic intervention of inappropriate apoptosis.
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