Sphingolipid Regulation of Caspase 9 Alternative Splicing

描述（由申请人提供）：该项目的长期目标是确定细胞凋亡通路的失调如何导致对化疗的耐药性和对致癌转化的敏感性。新的研究表明，caspase 9的RNA剪接变体（称为caspase 9b）的表达可以抵抗许多凋亡刺激。在PI的新研究中，脂质第二信使神经酰胺的产生和蛋白磷酸酶-1 （PP1）的激活被定义为诱导四外显子盒包含到成熟的caspase 9转录物中的信号转导途径的主要组成部分。PI实验室的初步结果表明，caspase 9的选择性剪接与SR蛋白SRp30a （ASF/SF2）具有内在联系。我们的实验室发现，使用RNA干扰技术下调SRp30a可显著抑制成熟caspase 9转录物中3,4,5,6外显子盒的包含。此外，在caspase 9基因的3、4、5和6外显子盒的每个外显子内和下游鉴定了6个SRp30a可能的相互作用位点。在PI实验室的其他机制研究中，发现蛋白激酶Clk/Sty调节A549细胞中SR蛋白的磷酸化状态（23）。此外，鞘氨醇-1-磷酸（一种有丝分裂生物活性脂质）诱导SR蛋白磷酸化增加。最后，直接调节caspase 9的选择性剪接调节了细胞对化疗和致癌转化的敏感性。基于上述发现，我们假设SRpSOa是响应神经酰胺的caspase 9前mRNA加工的重要调节剂，通过与特定RNA c/s元件的相互作用，而SRp30a通过其磷酸化状态调节caspase 9的3、4、5和6外显子盒的包含。我们还假设促生存激动剂（例如S-1-P）通过激活Clk/Sty诱导SRp30a磷酸化，进而增加caspase 9b的表达。最后，我们假设caspase 9的选择性剪接可以调节细胞对化疗和致癌转化的易感性。为了验证我们的假设，我们提出了以下具体目标：1)确定caspase 9的选择性剪接在细胞对化疗的敏感性和c-Myc/RasV12的致癌转化中的作用；2)确定调节caspase 9的3、4、5和6外显子盒对神经酰胺响应的神经酰胺响应c/s元件；3)确定SRp30a在调节caspase 9的3、4、5和6外显子盒在神经酰胺应答中的作用；4)确定SRp30a的磷酸化状态对caspase 9的3、4、5和6外显子盒在神经酰胺应答中的作用；和5)确定CLK/STY在响应有丝分裂激动剂时调节caspase 9的外显子3,4,5和6盒的包含中的作用。这些研究将在很大程度上确定凋亡激动剂作用下调节caspase 9选择性剪接的信号转导途径。这一点不能被低估，因为这些信号转导途径的定义为抗癌治疗创造了，不是一个，而是许多新的靶点。

DESCRIPTION (provided by applicant): 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.