A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Abstract

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

ATM: A mediator of multiple responses to genotoxic stress

“…Phosphorylation of Chk2 does occur in A-T cells at later time points or upon higher doses of IR or radiomimetic drugs (Matsuoka et al, 1998;Brown et al, 1999;Uziel, unpublished). This suggests that in analogy to the induction of p53, ATM is required for the immediate activation of Chk2 upon in¯iction of DSBs, while other kinases such as ATR might be involved at later stages of this response, and/or in response to other types of genotoxic agents.…”

“…In keeping with a defect in the G2 delay, A-T cells do not exhibit a reduction in the activities of the Cdc2 kinase and the Cdc25C phosphatase, shortly after irradiation (Paules et al, 1995;Beamish et al, 1996;Blasina et al, 1999). Accordingly, the rapid modi®cation and activation of Chk2 in response to IR and other DSBs generating agents, but not to UV or HU, is also dependent on ATM (Matsuoka et al, 1998;Brown et al, 1999;Chaturvedi et al, 1999;Uziel, unpublished). The recent ®nding that Chk2 is an in vitro substrate of ATM (Rotman, unpublished) suggests this checkpoint kinase is directly targeted by ATM.…”

“…Inactivation of Cdc25C is mediated by two protein kinases, Chk1 and Chk2 (HuCds1), which are phosphorylated and activated in response to DNA damage. Chk2 also responds to replication blocks such as those caused by treatment with hydroxyurea (HU) (Sanchez et al, 1997;Matsuoka et al, 1998;Blasina et al, 1999;Brown et al, 1999;Chaturvedi et al, 1999).…”

“…Blasina et al (1999) reported that both these kinases directly phosphorylate and inactivate Cdc25C in vitro. However, an alternative explanation suggests that in vivo phosphorylation of Cdc25C on Ser-216, the site phosphorylated by Chk1 and Chk2 in vitro (Sanchez et al, 1997;Matsuoka et al, 1998;Brown et al, 1999), promotes Cdc25C binding to 14-3-3 proteins, leading to its export from the nucleus and sequestration in the cytoplasm. This would result in the inability of this phosphatase to activate the nuclear Cdc2/cyclinB complex (reviewed by Nurse, 1997;Pines, 1999).…”

“…Initial explanations for A-T radiosensitivity were based on a DSB repair defect in A-T cells, but were confronted with lack of clear evidence for such a defect, and replaced by models based on cell cycle checkpoint defects or increased apoptosis (reviewed by Meyn, 1995;Lohrer, 1996). However, these models have been seriously questioned (Murnane and Kapp, 1993;Brown, 1997;Jeggo et al, 1998), and several lines of evidence argue against those assumptions (reviewed by Jeggo et al, 1998;Rotman and Shiloh, 1998): (1) Survival of A-T cells is not increased even when additional time is given for repair before entering the cell cycle; (2) The radiosensitivity and cell cycle defects of A-T are separable phenotypes and (3) No correlation was observed between radiosensitivity and increased IR-induced apoptosis of A-T cells.…”