Integration of the phosphorylation-dependent signaling in the DNA damage response network : implications for cancer

Abstract

The cellular response to DNA damage is an evolutionarily conserved process mediated by Ser/Thr kinases that results in the formation of multiple protein-protein complexes designed to control the cell cycle. The assembly of those multi-protein complexes is often triggered by the phosphorylation of a protein generating a short phospho-motif that is subsequently recognized by a downstream phospho-binding domain. To gain a better understanding of the exact mechanisms by which kinases signal through their substrates to phospho-binding domains in the DNA damage signaling process, we used two complementary proteomic screening approaches to identify novel kinase substrates and new phospho-binding domains. First, to explore the p38/stress-activated protein kinase (SAPK) axis of DNA damage signaling, we determined the optimal phosphorylation motifs of mammalian p38/SAPK and MAPKAP Kinase-2. The optimal substrate phosphorylation motif for MAPKAP Kinase-2 closely matches the 14-3-3-binding site on CDC25B/C, and we show that MAPKAP Kinase-2 is directly responsible for CDC25B/C phosphorylation and 14-3-3-binding both in vitro and in response to UV-induced DNA damage within mammalian cells. Using RNA interference we demonstrate that down- regulation of MAPKAP Kinase-2 eliminates both the G2/M and intra S-phase checkpoints. We show that MAPKAP Kinase-2, more aptly named CHK3, is new member of the DNA damage checkpoint kinase family that functions in parallel with CHK1 and CHK2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells.

(cont.) Second, to identify phospho-binding domains functioning downstream of the kinases ATM/ATR, we developed a high-throughput chemical proteomic screening approach to isolate phospho-binding domains that interact with the [Ser/Thr]-Gln motifs phosphorylated by ATM/ATR. We identified the tandem BRCA1 carboxy-terminal (BRCT) domains as novel phospho-specific binding domains regulating PTIP and BRCA1 function and show that the phospho-binding function of BRCA1 is lost in many BRCA1-BRCT mutations that predispose women to breast and ovarian cancer. Together with our 1.85A X-ray crystal structure data of the phosphopeptide bound BRCA1 BRCT domains, we provide a molecular rational as to how these mutations disrupt some BRCA1 functions. In combination, our discoveries expand the molecular understanding as to how kinases signal to phospho-binding domains in the DNA damage response signaling network, provide insight into cancer development and as well as identify novel targets for the development of chemosensitizing therapeutic agents.