Interleukin-3(IL-3) is a multi potential hematopoietic growth factor (HGF) that mediates growth (i.e. the net effect of proliferation plus inhibition of programmed cell death or apoptosis) and differentiation of target cells by first binding to a specific surface receptor system. The mechanism(s) involved in triggering and sustaining the post-receptor anti- apoptosis (signals) generated by this interaction are central to the issue of understanding how normal and malignant hematopoietic stem cell growth is regulated. While many of the molecular details concerning how IL-3 may regulate proliferative signaling are now understood, little is known about how IL-3 and other HGF-induced signals can potentially inhibit apoptosis and specifically regulate BCL2, the prototypic suppressor of apoptosis in factor-dependent hematopoietic cells. Results from studies on the molecular and biochemical characterization of IL-3 signaling obtained during the current period of funding (9/95-present) have indicated a novel, experimentally testable hypothesis by which IL-3 may regulate BCL2 and survive signaling. The model features the regulation of BCL2 phosphorylation at the evolutionarily conserved serine 70 site (in murine, human and chicken) which we recently discovered to be required for BCL2 function. We hypothesize that IL-3-induced phosphorylation of BCL2 results from the direct action of at least two BCL2 kinases (PKCalpha) and an staurosporine-resistant Bcl2 kinase, which are both co-localized with mitochondrial BCL2. To critically test thy hypothesis we have identified two specific aims: (1) To determine the mechanism for physiologic phosphorylation of BCL2 at ser70 by identifying the relevant BCL2 kinases (including PKCalpha and an SRK), and (2) To determine the functional significance of BCL2 phosphorylation at the ser70 site contained in the putative flexible loop regulatory domain (FLD; aa29-87) State of the art molecular and biochemical methodologies will be employed including mutational analysis with in vivo and in vitro expression studies, interactive cloning strategies to identify potent ser70 binding regulating proteins and creating an S70E BCL2 transgenic mouse to further test this gain-of-function mutation in a whole animal. The results are expected to fill in fundamental gaps in our knowledge regarding the signaling mechanism by which Il-2 regulates BCL2 and apoptosis in factor-dependent cells. From the results it is expected that efficient develop of new and novel anti-cancer strategies that target these mechanisms will occur.
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