Proc Natl Acad Sci U S A 99(23): 14795-14800

Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G<sub>2</sub> checkpoints

Departments of ^{Cell Biology and Physiology, and}^{Internal Medicine,}^{Howard Hughes Medical Institute, Washington University School of Medicine, Box 8228, 660 South Euclid Avenue, St. Louis, MO 63110-1093}

^{To whom correspondence should be addressed. E-mail:}ude.ltsuw.oibllec@acinwiph.

Communicated by Joan V. Ruderman, Harvard Medical School, Boston, MA

Received 2002 Jun 28; Accepted 2002 Sep 12.

Abstract

Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G₂ checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G₂ checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks.

Abstract

Checkpoint kinase (Chk)1 was first identified in fission yeast as an essential component of the DNA damage checkpoint but was later shown to also function in the DNA replication checkpoint (1–7). Although chk1 is not an essential gene in fission yeast, it is essential in mice (1, 3, 8, 9). The essential function provided by murine Chk1 throughout early embryonic development has not been determined. Mouse embryos and conditional embryonic stem cell lines lacking Chk1 exhibit defective checkpoint responses to replication blocks and DNA-damaging agents establishing a checkpoint function for Chk1 in mice (8, 9). Evidence that Chk1 contributes to G₂ checkpoint control in human cells comes from studies showing that agents such as UCN-01 and SB-218078, which are potent inhibitors of Chk1, abrogate G₂ checkpoint function in human cells (10–12). However, it is clear that Chk1 is not the only kinase targeted by UCN-01 (see below).

Although Chk1 is conserved throughout evolution, the signals that Chk1 responds to have diverged among eukaryotic organisms. In fission yeast, Chk1 responds to DNA damage induced by ionizing radiation (IR) and UV light (UV) (3, 4), as well as to DNA replication inhibitors (2, 5, 6). In Xenopus (Xe), Chk1 is activated by UV irradiation and by agents that block DNA replication (13). In contrast to fission yeast, Xe Chk1 is not activated by double-stranded DNA breaks of the type induced by IR (13). In Xe and humans, Chk1 is phosphorylated and activated by ATR (ATM and Rad3-related protein kinase), and the ATR/Chk1 pathway responds to agents that impair DNA replication, either directly (hydroxyurea, aphidicolin) or indirectly (UV irradiation) (8, 13, 14). Human Chk1 is phosphorylated on serines 317 and 345 by ATR in vitro, and these residues are phosphorylated in vivo in an ATR-dependent manner (8, 14).

The contribution made by Chk1 to the IR-induced DNA damage checkpoint in humans remains controversial. Some studies have reported that the electrophoretic mobility of human Chk1 is retarded after IR (8, 10, 15), whereas other studies have failed to observe changes in the electrophoretic mobility of Chk1 after IR treatment. Human Chk1 is phosphorylated on serine-345 after IR (8), and we consistently observe a 2-fold increase in Chk1 kinase activity after IR that also accompanies S317 phosphorylation (Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org). In humans, fission yeast, and Xe, Chk1 has been proposed to regulate the G₂ checkpoint by phosphorylating the Cdc25C protein phosphatase on residue(s) that facilitate the binding of 14-3-3 proteins (5, 15–18). In addition, human Chk1 has been proposed to regulate the stability of the Cdc25A protein phosphatase in UV-damaged cells (19).

In this study, we investigated the contribution made by human Chk1 to the cell division cycle both in the absence and presence of checkpoint activation. We report that cells deficient in human Chk1 exhibit radio-resistant DNA synthesis and do not delay in G₂ after IR treatment. We provide evidence that Chk1 directly regulates Cdc25A throughout an unperturbed cell cycle, and that the integrity of the Chk1/Cdc25A regulatory pathway is essential for cells to respond appropriately to ionizing radiation.

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Acknowledgments

We thank M.-S. Chen (Washington University School of Medicine) for recombinant adenovirus encoding myc-tagged Cdc25A. We thank A. Ferguson (Washington University School of Medicine) for designing the siRNAs for human Cdc25A, D. Dean (Washington University School of Medicine) for the luciferase-siRNAs, and C. Lovly for comments on the manuscript. This work was supported by a grant from the National Institutes of Health. H.P.W. is an Investigator of the Howard Hughes Medical Institute.

Acknowledgments

Abbreviations

Chk, checkpoint kinase
Cdc, cell division cycle
IR, ionizing radiation
siRNA, small interfering RNAs
UV, UV light

Abbreviations

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