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，敲低CtBP足以抑制体内肿瘤发生。该方案旨在研究CtBP降解的机制，以期开发新的癌症治疗方法。具体目的是：1）确定HIPK2是否直接或通过其他激酶途径调节CtBP。Ser 422的磷酸化触发CtBP降解。我们已经提出了一个抗体丝氨酸422磷酸化CtBP检查HIPK2是否与其他信号通路相互作用，磷酸化CtBP和触发CtBP降解。JNK1特异性磷酸化Ser 422。我们将研究该途径在CtBP降解中的参与。2)确定CtBP的磷酸化如何导致蛋白酶体降解。我们将集中于鉴定负责CtBP多聚泛素化和降解的E3连接酶。肿瘤抑制因子Fbw7a与CtBP物理相互作用，是我们的第一个候选者。我们已经表明，丝氨酸422磷酸肽块CtBP降解。我们将使用亲和层析和质谱来鉴定与这种磷酸化肽相关的蛋白质。靶向蛋白与磷酸肽的相互作用将通过生物化学测定来证实，并且将使用siRNA来检查它们对CtBP降解的贡献。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。负责这些影响的基因是否是直接的CtBP靶点尚不确定。我们将使用染色质占据序列分析（将染色质免疫沉淀与长SAGE相结合）鉴定参与凋亡和转化的CtBP靶标（蛋白质编码和非编码，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.