UV regulation of anti-apoptotic co-repressor CtBP

描述（由申请人提供）：CtBP 是一组促凋亡基因的转录共阻遏物。它的降解导致细胞死亡，不依赖于 p53。此外，CtBP 的敲低足以抑制体内肿瘤发生。该提案旨在研究 CtBP 降解机制，希望开发新的癌症治疗方法。具体目标是： 1) 确定 HIPK2 是直接调节 CtBP 还是通过其他激酶途径调节 CtBP。 Ser 422 的磷酸化会触发 CtBP 降解。我们制备了一种针对 Ser 422 磷酸化 CtBP 的抗体，以检查 HIPK2 是否与其他信号通路相互作用以磷酸化 CtBP 并触发 CtBP 降解。 JNK1 特异性磷酸化 Ser 422。我们将研究该途径在 CtBP 降解中的作用。 2) 确定 CtBP 磷酸化如何导致蛋白酶体降解。我们将重点关注负责 CtBP 多泛素化和降解的 E3 连接酶的鉴定。肿瘤抑制因子 Fbw7a 与 CtBP 发生物理相互作用，是我们的第一个候选者。我们已经证明 Ser 422 磷酸肽可阻止 CtBP 降解。我们将使用亲和色谱法和质谱法来鉴定与该磷酸肽相关的蛋白质。靶向蛋白与磷酸肽的相互作用将通过生化测定得到证实，并且它们对 CtBP 降解的贡献将使用 siRNA 进行检查。 3) 鉴定参与转化和凋亡的CtBP靶基因。 CtBP 抑制许多上皮特异性和促凋亡基因。造成这些影响的基因是否是 CtBP 的直接靶点尚不确定。我们将使用染色质占用序列分析（将染色质免疫沉淀与长 SAGE 相结合）来鉴定参与细胞凋亡和转化的 CtBP 靶点（蛋白质编码和非编码，例如 microRNA）。将在导致 CtBP 降解的特定信号通路中研究参与细胞凋亡和转化的靶基因的表达。

DESCRIPTION (provided by applicant): CtBP is a transcriptional co-repressor for a panel of pro-apoptotic genes. Its degradation leads to cell death independent of p53. Furthermore, knockdown of CtBP is sufficient to suppress tumorigenesis in vivo. This proposal is designed to study the mechanisms of CtBP degradation in the hope of developing novel approaches for cancer treatment. The specific aims are to: 1) Determine whether HIPK2 regulates CtBP directly or through other kinase pathways. Phosphorylation of Ser 422 triggers CtBP for degradation. We have raised an antibody to Ser 422-phosphorylated CtBP to examine whether HIPK2 interacts with other signal pathways to phosphorylate CtBP and trigger CtBP degradation. JNK1 specifically phosphorylates Ser 422. We will investigate the involvement of this pathway in CtBP degradation. 2) Determine how phosphorylation of CtBP leads to proteasomal degradation. We will focus on the identification of E3 ligase(s) responsible for CtBP polyubiquitination and degradation. The tumor suppressor Fbw7a physically interacts with CtBP and is our first candidate. We have shown that a Ser 422 phospho-peptide blocks CtBP degradation. We will use affinity chromatography and mass spectrometry to identify proteins associated with this phospho-peptide. The interaction of the targeting proteins with the phospho-peptide will be confirmed by biochemical assays and their contribution to CtBP degradation will be examined using siRNA. 3) Identify CtBP target genes involved in transformation and apoptosis. CtBP represses many epithelial- specific and pro-apoptotic genes. Whether the genes responsible for these effects are direct CtBP targets is uncertain. We will identify CtBP targets (both protein-coding and non-coding such as microRNAs) involved in apoptosis arid transformation using Serial Analysis of Chromatin Occupancy, which combines chromatin immunoprecipitation with long SAGE. Expression of target genes involved in apoptosis and transformation will be studied in specific signaling pathways that cause CtBP degradation.