UV regulation of anti-apoptotic co-repressor CtBP

描述(申请人提供)：CTBP是一组促凋亡基因的转录共抑制因子。它的降解导致细胞死亡，而不是P53。此外，CtBP的敲除足以抑制体内的肿瘤形成。这项建议旨在研究CtBP的降解机制，以期开发新的癌症治疗方法。其具体目的是：1)确定HIPK2是直接调节CtBP还是通过其他激酶途径调节CtBP。Ser422的磷酸化触发CtBP的降解。我们已经提出了一种针对Ser422磷酸化CtBP的抗体，以检测HIPK2是否与其他信号通路相互作用来磷酸化CtBP并触发CtBP降解。JNK1特异性磷酸化Ser422。我们将研究这一途径在CtBP降解中的作用。2)确定CtBP的磷酸化如何导致蛋白酶体的降解。我们将集中在鉴定负责CtBP多泛素化和降解的E3连接酶(S)。肿瘤抑制基因Fbw7a与CtBP在物理上相互作用，是我们的第一个候选基因。我们已经证明了Ser422磷酸肽可以阻断CtBP的降解。我们将使用亲和层析和质谱法来鉴定与该磷酸肽相关的蛋白质。目标蛋白与磷酸肽的相互作用将通过生化分析得到证实，它们对CtBP降解的贡献将通过siRNA进行检测。3)筛选与转化和细胞凋亡相关的CtBP靶基因。CTBP抑制许多上皮细胞特异性和促凋亡基因。负责这些效应的基因是否是CtBP的直接靶标尚不确定。我们将利用染色质占有率序列分析，结合染色质免疫沉淀和长SAGE，识别参与细胞凋亡和转化的CtBP靶标(包括蛋白质编码和非编码，如microRNAs)。参与细胞凋亡和转化的目标基因的表达将在导致CtBP降解的特定信号途径中进行研究。

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.