ERK activity facilitates activation of the S-phase DNA damage checkpoint by modulating ATR function.
Although Erk kinase has been recently reported to function in the DNA damage response, the mechanism governing this process is unknown. We report here that hydroxyurea (HU) activates Erk via MEK1, a process that is sensitized by a constitutively active MEK1 (MEK1Q56P) and attenuated by a dominant-negative MEK1 (MEK1K97M). While ectopic MEK1Q56P sensitized HU-induced S-phase arrest, inhibition of Erk activation via U0126, PD98059, and MEK1K97M attenuated the arrest, and thereby enhanced cells to HU-induced toxicity. Taken together, we demonstrate an important contribution of Erk to the activation of the S-phase DNA damage checkpoint. This can be attributed to Erk's regulatory role in modulating ATR function. Inhibition of Erk activation with U0126/PD98059 and MEK1K97M substantially reduced HU-induced ATR nuclear foci, leading to a dramatic reduction of gammaH2AX and its nuclear foci. Reduction of MEK1 function by a small interference RNA (siRNA) MEK1 and ectopic MEK1K97M significantly decreased HU-induced gammaH2AX. Conversely, ectopic MEK1Q56P enhanced gammaH2AX foci. Furthermore, immunofluorescent and cell fractioning experiments revealed cytosolic and nuclear localization of ATR. HU treatment caused the redistribution of ATR from the cytosol to the nucleus, a process that is inhibited by U0126. Collectively, we show that Erk kinase modulates HU-initiated DNA damage response by regulating ATR function.
Wu
D
D
Father Sean O'Sullivan Research Institute, St Joseph's Hospital, Hamilton, Ontario, Canada.
Chen
B
B
Parihar
K
K
He
L
L
Fan
C
C
Zhang
J
J
Liu
L
L
Gillis
A
A
Bruce
A
A
Kapoor
A
A
Tang
D
D
D016475:3T3 Cells; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005455:Fluorescent Antibody Technique; D005799:Genes, Dominant; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D016196:S Phase
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D006918:Hydroxyurea