The long-term objectives of this project focus on determining how dysregulation of apoptotic pathways confers resistance to chemotherapy and sensitivity to oncogenic transformation. New studies have shown that the expression of an RNA splice variant of caspase 9, termed caspase 9b, confers resistance to many apoptotic stimuli. In novel studies by the PI, the generation of the lipid second messenger, ceramide, and the activation of protein phosphatase-1 (PP1) were defined as major components of the signal transduction pathway that induces the inclusion of the four exon cassette into the mature caspase 9 transcript. Preliminary results by the PI's laboratory disclose that the alternative splicing of caspase 9 is intrinsically linked to the SR protein, SRp30a (ASF/SF2). Our laboratory found that downregulation of SRp30a using RNA interference technology dramatically inhibited the inclusion of the 3, 4, 5, 6 exon cassette in the mature caspase 9 transcript. Furthermore, six possible interaction sites for SRp30a were identified within and downstream of each exon in the 3, 4, 5, and 6 exon cassette of the caspase 9 gene. In other mechanistic studies by the PI's laboratory, the protein kinase, Clk/Sty, was found to regulate the phospho-status of SR proteins in A549 cells (23). Furthermore, sphingosine-1-phosphate, a mitogenic bioactive lipid induces an increase in the phosphorylation of SR proteins. Lastly, direct modulation of the alternative splicing of caspase 9 modulated the sensitivity of cells to chemotherapy and oncogenic transformation. Based on the above findings, we hypothesize that SRpSOa is an important regulator of caspase 9 pre- mRNA processing in response to ceramide via interaction with specific RNA c/s-elements, and that SRp30a regulates the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 via its phospho-status. We also hypothesize that prosurvival agonists (e.g. S-1-P) induce the phosphorylation of SRp30a via activation of Clk/Sty, which in turn increases the expression of caspase 9b. Lastly, we hypothesize that the alternative splicing of caspase 9 can modulate the susceptibility of cells to chemotherapy and oncogenic transformation. To validate our hypotheses, we propose the following specific aims: 1) To determine the role of the alternative splicing of caspase 9 in the sensitivity of cells to chemotherapy and oncogenic transformation by c-Myc/RasV12; 2) To determine the ceramide-responsive c/s-elements that regulate the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; 3) To determine the role of SRp30a in regulating the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; 4) To determine the role of the phospho-status of SRp30a on the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; and 5) To determine the role of CLK/STY in the regulation of the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9 in response to mitogenic agonists. These studies will largely define the signal transduction pathway regulating caspase 9 alternative splicing in response to apoptotic agonists. This cannot be understated because the definition of these signal transduction pathways creates, not one, but many new targets, for anti-cancer therapies. ? ? ? ? ? ?
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