Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

Abstract

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

GSK‐3β‐dependent destabilization of cyclin D1 mediates replicational stress‐induced arrest of cell cycle

“…1A). No change in the levels of cdc25A -a mediator of cell cycle arrest following ionizing radiation [20] -was observed either (Fig. 1B).…”

Chk1 is required to maintain Claspin stability

“…One possible mechanism is through phosphorylation of Claspin by Chk1. Chk1 exists in an active form during the S-to M-phase of the cell cycle (Kaneko et al, 1999), and it has been shown to phosphorylate substrates even in the absence of DNA damage (Sorensen et al, 2003(Sorensen et al, , 2004. In addition, Chk1 is known to regulate the stability of several proteins through phosphorylation.…”

“…In addition, Chk1 is known to regulate the stability of several proteins through phosphorylation. Chk1 phosphorylates Cdc25A and targets it for ubiquitin-dependent degradation (Zhao et al, 2002;Sorensen et al, 2003). In contrast, the Chk1-dependent phosphorylation of Pds1 inhibits Pds1 ubiquitination and promotes its stabilization (Agarwal et al, 2003).…”

“…Several studies indicate that Chk1 has basal activity and phosphorylates its target proteins, such as Cdc25A, even in the absence of exogenous DNA damage and/or replication stress (Kaneko et al, 1999;Sorensen et al, 2003). This is because Chk1 is required for the maintenance of normal DNA replication.…”

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

“…We also investigated the extent to which Chk1 and Chk2 were activated 1 h after various doses of irradiation and monitored the levels of Cdc25A, a downstream target of checkpoint signalling which can be degraded in response to DNA damage (Sorensen et al, 2003). Perhaps surprisingly, at radiation doses below 2 Gy there was little or no activation of Chk2 in WT cells as judged by the appearance of the slower migrating phosphorylated isoform, nor was activatory phosphorylation of Chk1 at serine 345 (pS345) significantly induced in either WT or Chk2À/À cells (Figures 5b and c).…”