Phosphorylation of Chk1 on S317 and/or S345 has been the gold standard in the checkpoint field for monitoring the activation state of Chk1 in vivo (31
). Here we report that S317/S345-phosphorylated Chk1 (the presumably “active” form of Chk1) paradoxically accumulates in cells treated with Chk1 inhibitors. The stabilization and accumulation of Cdc25A were observed in cells treated with each Chk1 inhibitor, confirming that they were effective at blocking Chk1 activity in vivo. Furthermore, we demonstrated that phosphorylation of Chk1 under these conditions requires ATR.
One might predict that Chk1 inhibition would elicit a checkpoint response leading to activation of ATM/ATR, and this, in turn, would lead to enhanced Chk1 phosphorylation on S317 and S345. However, Chk1 phosphorylation in response to Chk1 inhibition was detected within 15 min of treatment with Chk1 inhibitors (Fig. ), yet DNA damage, as measured by the appearance of γH2AX foci, was not detected until after 2 h of Gö6976 treatment (Fig. ). In addition, phosphorylation of ATM/ATR targets was not readily detected in cells treated with Chk1 inhibitors (Fig. ). These results led us to investigate the contribution made by protein phosphatases to Chk1 regulation. Several interesting observations relevant to Chk1 dephosphorylation were made. First, phosphorylated Chk1 accumulated in cells treated with OA while both OA and fostriecin but not I-2 blocked Chk1 dephosphorylation in cell extracts. Second, S317/S345-phosphorylated Chk1 accumulated in cells where PP2A levels were reduced by siRNA treatment and PP2A was able to directly dephosphorylate Chk1 on S317 and S345 in vitro. Taken together, these findings argue that PP2A regulates Chk1 in vivo. Dis2, the fission yeast homolog of PP1, dephosphorylates S345 in fission yeast Chk1 (13
) and PP1 dephosphorylates a human Chk1 S345 phosphopeptide in vitro (32
), suggesting that PP1 might regulate both fission yeast and human Chk1. The following lines of evidence suggest that this is not the case: I-2 failed to block human Chk1 dephosphorylation in vitro (Fig. ), PP1 knockdown resulted in a decrease in levels of phosphorylated Chk1 in vivo (Fig. ), and PP1 was incapable of dephosphorylating full-length human Chk1 on S317 and S345 in vitro (Fig. ). Interestingly, PPM1D (or Wip1) has recently been shown to dephosphorylate Chk1 on S345 and to a lesser extent on S317 (32
). PPM1D is a p53 transcriptional target gene that is insensitve to OA. Thus, in response to DNA damage, p53 induces PPMID, which in turn may function to return Chk1 to a hypophosphorylated state as cells recover from checkpoint arrest. Our study indicates that PP2A has a role distinct from that of PPM1D in that it facilitates Chk1 dephosphorylation in the absence of genotoxic stress.
Given that ATR was not detectably activated in HeLa cells treated with Chk1 inhibitors, another model to explain the accumulation of phosphorylated Chk1 under conditions of Chk1 inhibition would be if Chk1 inhibitors also functioned as PP2A inhibitors. However, unlike OA, Chk1 inhibitors did not block Chk1 dephosphorylation in cell extracts or block the ability of PP2A to dephosphorylate Chk1 in vitro. A third important observation made in this study is that kinase-inactive Chk1 is phosphorylated on S317 and S345 under basal, unstressed conditions and its dephosphorylation occurs more slowly than does that of wild-type Chk1. Taken together, these results suggest a regulatory circuit whereby ATR continually phosphorylates Chk1 on S317 and S345 and phosphorylated “active” Chk1 in turn, either directly or indirectly, stimulates PP2A to dephosphorylate S317 and S345 (Fig. ). In this model, kinase-inactive Chk1 would be unable to stimulate its own dephosphorylation and therefore would be expected to accumulate in a more highly phosphorylated form in vivo. We conclude that Chk1 inhibitors disrupt the ATR/Chk1/PP2A feedback loop by blocking PP2A “activation” by Chk1 allowing the ATR/Chk1 phosphorylation circuit to dominate and likewise, phosphatase inhibitors like OA and fostriecin block the enzymatic activity of PP2A, thereby enabling phosphorylated Chk1 to accumulate. Future studies will focus on how Chk1 kinase activity contributes to Chk1 dephosphorylation in vivo. Possibilities include direct activation of PP2A by Chk1 or enhanced recognition of phosphorylated Chk1 by PP2A. In addition, it will also be important to understand how checkpoint activation pushes the equilibrium in favor of the ATR/Chk1 arm of the pathway and away from the Chk1/PP2A arm of the pathway.
FIG. 7. The ATR-Chkl-PP2A regulatory circuit. During an unperturbed cell division cycle, Chk1 is continually being phosphorylated on S317 and S345 by ATR. However, phosphorylated Chk1 does not accumulate to any significant extent because PP2A continually dephosphorylates (more ...)
Interestingly, treatment of cells with increasing doses of OA (see Fig. S1A in the supplemental material) or with a single dose of OA for increasing periods of time (see Fig. S1B in the supplemental material) caused phosphorylated Chk1 to accumulate to higher levels than in cells treated with Gö6976 and to even higher levels than that of the initial phospho-Chk1 at the start of the reaction. This suggests that, in vivo, OA stimulates both the kinase and phosphatase arms of the pathway (i.e., inhibits dephosphorylation of Chk1 and stimulates phosphorylation of Chk1). In contrast, treatment of cells with increasing doses of Gö6976 (see Fig. S1A in the supplemental material) or with a single dose of Gö6976 for increasing periods of time (see Fig. S1B in the supplemental material) blocked dephosphorylation of Chk1, but did not stimulate Chk1 phosphorylation above the initial phospho-Chk1 level at the start of the reaction. This result coupled to the observation that Gö6976 does not activate ATR, strongly argues that Gö6976 regulates the phosphatase rather than the kinase arm of the pathway. How does OA regulate the ATR-Chk1 arm of the pathway? Chowdhury et al. (9
) demonstrated that DNA DSB repair and checkpoint recovery in vivo require dephosphorylation of γH2AX by PP2A. OA blocks γH2AX dephosphorylation, thereby sustaining/enhancing the DNA damage signaling pathway. These results predict that if HU-treated cells are incubated with OA subsequent to HU removal, these cells would fail to repair DNA DSBs and sustained incubation in the presence of OA might lead to enhanced DNA damage and further activation of checkpoint pathways. If this were the case, phosphorylated Chk1 would be expected to accumulate to higher levels than in cells treated with Gö6976 alone because OA would not only block Chk1 dephosphorylation by PP2A, it would also stimulate Chk1 phosphorylation by ATR.
Recent studies have uncovered important roles for serine/threonine protein phosphatases in regulating checkpoint responses. In addition to Chk1 (this study), PP2A regulates ATM (19
), Chk2 (14
), and γH2AX (9
). PP5 regulates ATM, ATR, and DNA protein kinase signaling (2
). Recently the Pph3 phosphatase complex has been shown to dephosphorylate γH2AX in budding yeast (27
). PPM1D dephosphorylates Chk1, Chk2, and p53 (16
), and PP1 regulates fission yeast Chk1 (13
) and Xenopus
). Interestingly, OA was shown to induce the autophosphorylation of ATM on serine 1981 in unirradiated cells, indicating that PP2A functions during a normal cell division cycle to counteract the autoactivation tendencies of ATM (19
). Thus, PP2A functions to maintain both ATM and Chk1 in a low-activity state during an unperturbed cell division cycle. In the case of ATM, PP2A counteracts ATM autophosphorylation, whereas in the case of Chk1, PP2A counteracts ATR-mediated phosphorylation. The net effect is to prevent ATM and Chk1 from relaying checkpoint signals when cells are not experiencing genotoxic stress.