Late activation of stress kinases (SAPK/JNK) by genotoxins requires the DNA repair proteins DNA-PKcs and CSB.
Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (< or = 2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PK(cs) compared with wild-type cells. The late response however (> or = 4 h), was drastically reduced in DNA-PK(cs) and Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PK(cs) and CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PK(cs) and CSB, appears to be required. DNA-PK(cs) coprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PK(cs) in SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PK(cs) and CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.
Fritz
Gerhard
G
Department of Toxicology, University of Mainz, D-55131 Mainz, Germany. fritz@mail.uni-mainz.de
Kaina
Bernd
B
D000478:Alkylation; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000502:physiology*; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D004261:DNA Replication / Q000187:drug effects; D051747:DNA-Activated Protein Kinase / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D009153:Mutagens / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction
D004268:DNA-Binding Proteins; D009153:Mutagens; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; D008741:Methyl Methanesulfonate; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D004265:DNA Helicases; C078673:ERCC6 protein, human; C513075:Ercc6 protein, mouse; D045643:DNA Repair Enzymes