The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38γ-independent pathway

Abstract

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes check-points leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38γ has been implicated in the G2 phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38γ. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G2 checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr68 by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38γ but had no effect on p38α activation, indicating that MRK is a specific activator of p38γ after IR. Inhibition of p38γ by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38γ MAPK pathway.