Global phosphoproteomic screening reveals a new function of CDK9 in DNA damage response.

DNA double strand breaks (DSBs) are the most toxic lesions for the genetic material leading severe consequences on cell viability predisposing to cancer transformation. In order to preserve the genome integrity, two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ) are activated to repair DSBs; the characterization of these repair mechanisms can be used as a novel strategy to improve the cancer therapy. The DNA damage response (DDR) is finely regulated by complex machineries in order to promote the correct DNA repair; many post-translational modifications regulate the DDR, among these the protein phosphorylation reveals a central function. The Cell cycle Kinases (CDKs) are the best regulators of the cell cycle but also the DDR; CDK9, one of the principal CDKs, is characterized by the presence of two protein isoforms (CDK9 42kDa and 55kDa). We decided to investigate the possible role of CDK9 in the DNA damage response (DDR) thought a global phosphoproteomic screening in order to detect the putative targets of CDK9 55kDa in HR. We found that CDC23, a component of Anaphase promoting complex/cyclosome (APC/C), it is involved in the DNA repair pathway choice between the NHEJ and HR, in response to CDK9 55 phosphorylation. The generation of the HeLa cells carrying the point mutation of CDC23 S588A showed a deficient HR but proficient NHEJ, in response to S-phase DNA damage, respect to HeLa wt cell. Importantly we demonstrated that CDC23 S588 phosphorylation didn’t interfere with cell cycle related function of APC/C and with its complex assembly, suggesting a possible role in the substrate target detection. Finally, we identified CDK9 as well as CDC23 as new HR players, proposing them as possible pharmacological targets for antitumoral synthetic lethal approaches.