Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-␣ and MRK-␤, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-␤ preferentially activates ERK6/p38␥ via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G 2 /M phase of the cell cycle, whereas dominant negative MRK attenuates the G 2 arrest caused by ␥-radiation. In addition, exposure of cells to ␥-radiation induces MRK activity. These data suggest that MRK may mediate ␥-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells.

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38␥ has been implicated in the G 2 phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38␥. 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 G 2 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 Thr 68 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 p38␥ but had no effect on p38␣ activation, indicating that MRK is a specific activator of p38␥ after IR. Inhibition of p38␥ 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 p38␥ MAPK pathway.