The first goal of this proposal is to study the regulation of DNA repair by the MAP kinase signaling pathway. Using functional proteomics, our laboratory has identified hHR23B, a protein involved in DNA damage recognition, as a novel target of MAPK. Western blot analysis with a specific antibody against hHR23B demonstrated that MAPK directs proteolytic processing of hHR23B. The goal of the first specific aim is to understand how this novel target is regulated by MAPK. First, post-translational modification(s) and site(s) of processing regulated by MAPK will be identified, and the importance of these modifications will be evaluated in in vitro and in vivo repair assays. Preliminary observations indicate that MAPK is also activated in response to UV exposure, therefore MAPK may play a role in modulating DNA repair by modifying hHR23B. The completion of this aim will characterize the regulation of a novel MAPK target and provide new insight into the regulation of DNA repair by MAPK pathways. In the second specific aim, the linearity of signaling through the MAPK pathway will be examined using functional proteomics. Although the MAPK pathway is classically thought of as a linear pathway, (i.e. Raf activates MKK which activates ERK), there is increasing evidence that bifurcations within this pathway exist that result in the regulation of distinct target proteins. I will first identify branch-point targets at the level of Raf and MKK using proteomics. Second, I will identify targets that are selectively regulated by kinases downstream of ERK. The completion of this aim will result in the identification of novel signaling targets and address the mechanism by which the MAPK pathway regulates specific cellular responses.

F32GM065027

2002-02-01

1900-01-01