Wu D, Chen B, Parihar K, He L, Fan C, Zhang J, Liu L, Gillis A, Bruce A, Kapoor A, Tang D
Father Sean O'Sullivan Research Institute, St Joseph's Hospital, Hamilton, Ontario, Canada.
Oncogene. 2006 Feb 23;25(8):1153-64. doi: 10.1038/sj.onc.1209148.
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.
尽管近期有报道称Erk激酶在DNA损伤反应中发挥作用，但调控这一过程的机制尚不清楚。我们在此报告，羟基脲（HU）通过MEK1激活Erk，组成型活性MEK1（MEK1Q56P）可使该过程敏感化，而显性负性MEK1（MEK1K97M）则使其减弱。异位表达的MEK1Q56P使HU诱导的S期停滞敏感化，而通过U0126、PD98059和MEK1K97M抑制Erk激活则减弱了这种停滞，从而增强了细胞对HU诱导毒性的敏感性。综上所述，我们证明了Erk对S期DNA损伤检查点激活的重要作用。这可归因于Erk在调节ATR功能中的调控作用。用U0126/PD98059和MEK1K97M抑制Erk激活可显著减少HU诱导的ATR核灶，导致γH2AX及其核灶显著减少。通过小干扰RNA（siRNA）MEK1降低MEK1功能以及异位表达MEK1K97M可显著降低HU诱导的γH2AX。相反，异位表达的MEK1Q56P增强了γH2AX灶。此外，免疫荧光和细胞分级实验揭示了ATR的胞质和核定位。HU处理导致ATR从胞质重新分布到细胞核，这一过程被U0126抑制。我们共同表明，Erk激酶通过调节ATR功能来调节HU引发的DNA损伤反应。
