Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin‐dependent kinase 1 (Cdk1)

Abstract

Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphaseanaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.

Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage

“…Even higher levels were observed in a nocodazole-treated (ND) culture, where approximately 60% of cells were mitotic. Thus, Chk1 S301 phosphorylation increases as cells progress through the cell cycle with the highest levels observed in S and G2/M phase cells, consistent with the idea that it might be catalyzed by both Cdk1 and Cdk2 (Shiromizu et al, 2006;Ikegami et al, 2008).…”

“…Interestingly, it was recently reported that Chk1 itself is also a Cdk target (Shiromizu et al, 2006;Ikegami et al, 2008). During the G2/M transition, Cdk1 phosphorylates Chk1 at Ser286 and Ser301 to promote Chk1 transport from the nucleus to the cytoplasm during prophase and trigger a positive feedback loop that contributes to Cdk1 activation in the nucleus (Kramer et al, 2004;Enomoto et al, 2009).…”

“…Both Cdk1 and Cdk2 contribute to Chk1 S301 phosphorylation in vivo Previous studies have provided evidence that Chk1 can be phosphorylated at S286 and S301 by both Cdk1 and Cdk2 in vitro, and that modification of these residues is observed both during mitosis and under conditions of DNA damage and replication stress (Shiromizu et al, 2006;Ikegami et al, 2008). The potential role of Cdkmediated Chk1 phosphorylation during checkpoint activation in vivo, however, remains unclear.…”

“…Chk1 can be phosphorylated at serines 286 and 301 (S286 and S301) by Cdk1 during mitosis (Shiromizu et al, 2006). Mitotic hyper-phosphorylation of Chk1 seems to promote translocation from the nucleus to the cytoplasm in prophase, thus releasing an inhibitory effect of Chk1 on Cdk1 to promote rapid mitotic entry (Enomoto et al, 2009).…”

Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase

“…If this point precedes the onset of mitosis in late G 2 , it is possible to predict a transition period during which Chk1 could no longer be activated in response to DNA damage but Chk2 would (Figure 8). In fact, there is evidence that Chk1 is refractory to ATR-mediated phosphorylation and activation by DNA damage in mitotic cells (Shiromizu et al, 2006), possibly owing to cell cycle phase-specific degradation of the adaptor protein Claspin (Mailand et al, 2006;Peschiaroli et al, 2006), therefore it is highly plausible that the transition precedes the onset of this phase. We hypothesize that during this transition period Chk2 becomes increasingly, and ultimately solely, responsible for G 2 delay in response to damage in cells which are imminently about to enter mitosis (Figure 8).…”