BCL-2 Function in Endoplasmic Reticulum

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DESCRIPTION (provided by applicant): Bcl-2 is a fascinating protein that regulates both cell cycle entry and apoptosis through its location on the mitochondria and the endoplasmic reticulum (ER). Although much is known about its role on mitochondria, little is known about its role on the ER. This proposal investigates the novel concept that Bcl-2 regulates intracellular Ca2+ signals involved in cell cycle entry and apoptosis. This concept is based on the recent discovery in this laboratory that Bcl-2 interacts with inositol 1,4,5- trisphosphate (IP3) receptors and reduces IP3-mediated Ca2+ release from the ER. The first two aims of this proposal investigate the molecular mechanism by which Bcl-2 interacts with IP3 receptors and regulates IP3-mediated Ca2+ release. Aim 1 employs the GST-pulldown technique to test for direct interaction in vitro and FRET analysis to test for direct interaction in vivo. Regardless of whether the interaction is direct or indirect, Aim 2 tests the hypothesis that Bcl-2 regulates IP3 mediated Ca2+ release by docking a protein phosphatase to IP3 receptors, thereby reducing their phosphorylation and decreasing their Ca2+ channel activity. A major goal in Aims 1 & 2 is to identify Bcl-2 mutations that abrogate the inhibitory effect of Bcl-2 on IP3-mediated Ca2+ release. Aim 3 will use these Bcl-2 mutants, along with physiological approaches, to determine if the inhibitory effect of Bcl-2 on IP3-mediated Ca2+ release contributes to its ability to inhibit cell cycle entry and apoptosis during T cell activation. The central hypothesis is that by reducing IP3-mediated Ca2+ elevation, Bcl- 2 inhibits two proximal steps in T cell activation, calcineurin-mediated dephosphorylation and nuclear translocation of the transcription factor NFAT, and calpain-mediated degradation of the cyclin dependent kinase inhibitor p27Kip1. Overall, the findings of this proposal will provide novel insight into the mechanism of action of Bcl-2.