IL-3 promotes hematopoietic cell survival, at least in part, by inducing Bcl2 phosphorylation at ser70. Our recent genetic studies with compound phosphomimetic Bcl2 mutants reveal that mono- and multi-site phosphorylation in the regulatory flexible loop domain (FLD) enhances Bcl2's antiapoptotic function. Dynamic (reversible) Bcl2 phosphorylation is regulated by multiple Bcl2 kinases (i.e. PKC alpha, ERK1/2 and JNK1) and a physiological Bcl2 phosphatase (PP2A). Recent reports and our preliminary data indicate that DNA damage can induce a population of p53 (i.e. 40-50%) to translocate to mitochondria, bind Bcl2 and induce apoptosis independent of its transcription activity. The regulatory mechanism of p53's """"""""extranuclear"""""""" death function is not clear. Preliminary data indicate that Bcl2 phosphorylation may block and dephosphorylation enhance its interaction with p53. We hypothesize that p53 binding to Bcl2 at its FLD may induce a conformational change that inactivates Bcl2's antiapoptotic function and/or functionally activates Bax's proapoptotic function. PP2A-mediated dephosphorylation of Bcl2 may promote Bcl2/p53 binding and result in exposure of Bcl2's BH3 """"""""death domain"""""""" leading to its inactivation. We have developed three specific aims to critically test these hypotheses: (1) Determine the mechanism by which Bcl2 and p53 binding is regulated by Bcl2 phosphorylation;(2) Determine the mechanism by which p53 binding inhibits Bcl2's survival function;(3) Determine the mechanism by which PP2A regulates Bcl2's function and p53 binding. State of the art molecular and biochemical methodologies will be employed including site-directed gene mutagenesis, gene silencing, X-Ray crystallography and yeast two-hybrid system for detecting protein:protein interaction. Results will help fill knowledge gaps about IL-3 mediated survival signaling and may point the way in the development of a novel antineoplastic strategy aimed at inhibiting Bcl2 function by enhancing the p53/Bcl2 interaction in hematologic malignancies and other cancers that express Bcl2.
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