Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent Phosphorylation of Chk1 on Ser-317 in Response to Ionizing Radiation

Abstract

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G 2 /M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G 2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G 2 /M checkpoint.

G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer

“…Ataxia -telangiectasia mutated phosphorylates Chk2 at threonine 68, and ATR phosphorylates Chk1 at serines 317 and 345. Significant crosstalk exists between the ATM/Chk2 and ATR/ Chk1 pathways (Gatei et al, 2003). Although Chk1 and Chk2 have overlapping roles in checkpoint signalling, only Chk1 is indispensable for mammalian survival (Liu et al, 2000).…”

Harnessing the complexity of DNA-damage response pathways to improve cancer treatment outcomes

“…ATM and ATR are Ser/Thr-Gln-directed protein kinases with overlapping substrate specificities. Whereas the ATM pathway is active during all phases of the cell cycle in response to double-strand breaks (DSBs), ATR acts primarily in S and G2 phases of the cell cycle in an ATM-dependent manner (Gatei et al, 2003;Jazayeri et al, 2006). The ATR pathway mainly responds to agents that interfere with the function of DNA replication forks (replication stress), such as ultraviolet light and gemcitabine, although it is now recognized that the ATR pathway can also activate downstream components of the ATM arm following replication fork stalling or ultraviolet treatment (Stiff et al, 2006).…”

Replication stress activates DNA polymerase alpha-associated Chk1

“…25 In addition, a complex consisting of Rad9, Hus1 and Rad1 (the 9-1-1 complex) 25 is recruited by a clamp-loader composed of Rad17 and the small subunits of replication factor C. 26,27 Following antimetabolite exposure, Chk1 is phosphorylated and activated by upstream mediators, such as the ATR and ATM kinases. [28][29][30] Chk1 activity is essential for stabilization of stalled replication forks. 31,32 Chk1 is also essential for normal development and DNA synthesis.…”