ERK1 and ERK2 kinases activate hydroxyurea-induced S-phase checkpoint in MCF7 cells by mediating ATR activation

Modulation of MEK has been demonstrated to affect hydroxyurea (HU) induced-DNA damage response (DDR), implying the involvement of ERK1 and ERK2 in the process. To directly examine how the ERK kinases function in HU-initiated DDR, we knocked-down either ERK1 or ERK2 in MCF7 cells. This resulted in reduction of HU-induced phosphorylation of CHK1 S345 (serine 345), p53 S15, and H2AX S139. While HU potently induced CDC2 Y15 (tyrosine 15) phosphorylation, an event causing CDC2 inactivation, inhibition of ERK kinases using U0126 (a MEK inhibitor), MEK1K97M (a dominant negative MEK1), and knockdown of either ERK1 or ERK2 significantly attenuated HU-induced CDC2 Y15 phosphorylation. As CDC2 kinase activity is required for mitosis, our observations reveal that ERK1 and ERK2 kinases play important roles in preventing mitotic entry in response to HU. Consistent with ATR being the apical kinase to initiate HU-induced DDR, knockdown of ERK1 or ERK2 significantly inhibited HU-induced ATR recruitment to the stalled replication forks (ATR foci), an event required for ATR activation. Mechanistically, knockdown of ERK1 or ERK2 resulted in relocation of ATR from the nucleoplasm to the nucleolus in response to HU, therefore making ATR unavailable to the sites of DNA damage. Taken together, we demonstrate that ERK kinases sit upstream of ATR to facilitate its activation.