The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38gamma-independent pathway.
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) p38gamma 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 p38gamma. 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 p38gamma but had no effect on p38alpha activation, indicating that MRK is a specific activator of p38gamma after IR. Inhibition of p38gamma 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 p38gamma MAPK pathway.
Tosti
Elena
E
Center for Oncology and Cell Biology, North Shore-Long Island Jewish Research Institute, Manhasset, New York 11030, USA.
Waldbaum
Linda
L
Warshaw
Gregg
G
Gross
Eleanore A
EA
Ruggieri
Rosamaria
R
D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002460:Cell Line; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D020930:MAP Kinase Kinase Kinases; D048291:Mitogen-Activated Protein Kinase 12 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011839:Radiation, Ionizing; D016415:Sequence Alignment; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; D048291:Mitogen-Activated Protein Kinase 12; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C413047:MAP3K20 protein, human; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases