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. ? ? ?
