Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G ₁ /S checkpoint activation in response to DNA damage

Abstract

ATR [ataxia-telangiectasia-mutated (ATM)-and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM-and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser 635 and Ser 645 in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G 1, S, and G2͞M, but not in early-mid G1. Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G 1. Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser 645 was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G 1͞S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells. C ell cycle checkpoints activated by stalled replication forks and DNA damage protect genomic integrity by preventing damaged DNA from being replicated and passed on to new daughter cells (1-5). In Schizosaccharomyces pombe (Sp), conserved checkpoint Rad proteins, including Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, are required for activation of checkpoint signaling pathways in response to stalled replication forks and DNA damage. Inactivation of any one of the checkpoint rad genes abolishes phosphorylation and activation of two downstream kinases, SpChk1 and SpCds1 (6, 7), resulting in defective activation of checkpoints. Human homologues of all of the Sp checkpoint rad genes have been identified, except rad26.The Sprad3 ϩ gene, the Saccharomyces cerevisiae (Sc) MEC1 gene, and the human ATM (ataxia-telangiectasia-mutated) and ATR (ATM-and Rad3-related) genes encode related protein kinases (8, 9). ATR and ATM are involved in the replication and DNA damage-induced checkpoints (10-14). Although ATM has been extensively studied, it has been difficult to ascertain ATR function because ATR-deficient cells and embryos are not viable (15,16). ATM deficiency results in hypersensitivity to ionizing irradiation (IR) in humans and mice (17,18). Similarly, ATRdeficient blastocysts have increased sensitivity to IR that correlates with chromosomal fragmentation (15). By using cells overexpressing kinase-inactive ATR (ATR Ki ) under the regulation of doxycycline, an elevated cellular sensitivity to DNA damage, a defective cell cycle response, and a significant loss of cell viability were observed (13,19). Cellular substrates of ATM͞ATR include p53 (20-23) and BRCA1 (24, 25), but substra...

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

“…Although we cannot exclude the possibility that SFK inhibition perturbs Chk1 dephosphorylation or degradation, SFK inhibition increased and v-Src suppressed Rad17-Ser 645 phosphorylation (Figs. 5, C and D; 6, C and D; and 7, A, B, and E), and the Rad17 phosphorylation regulates ATR-dependent Chk1 phosphorylation (17)(18)(19). Therefore, regulation of ATR-dependent Rad17 phosphorylation is involved in the mechanism by which SFKs regulate Chk1 phosphorylation and activation.…”

“…ATR-dependent checkpoint signaling is mediated by protein complex formation on damaged chromatin (4 -6). Rad17 is phosphorylated by ATR, and the Rad17 phosphorylation is required for interaction with Claspin and activation of Chk1 (17)(18)(19). Therefore, phosphorylation of Rad17 at Ser 645 (Rad17-Ser 645 ) was examined.…”

“…Chk1 activation is essential for the maintenance of G 2 checkpoint arrest in response to DSB induction, and inhibition of Chk1 activity during G 2 checkpoint arrest induces premature mitotic entry even though DNA repair has not been completed (12)(13)(14)(15)(16). Rad17 is another phosphorylation substrate of ATR, and the phosphorylation of Rad17 is required for its interaction with Claspin and Chk1 activation (17)(18)(19). Claspin mediates the ATR-dependent phosphorylation of Chk1 to activate the ATR-Chk1 signaling pathway (20).…”

Cell and tissue responses to genotoxic stress

“…In a 'double whammy', ATM also phosphorylates Mdm-2, further preventing it from degrading p53, and Mdm-2 phosphorylation appears to precede p53 phosphorylation [64,65]. In addition to p53/Mdm-2, ATM and/or ATR phosphorylates a variety of other proteins involved in repair and checkpoint control, such as Rad9, Rad17, p95Nbs1, Chk1, and Chk2 [66][67][68][69]. In turn, activated Chk1 and Chk2 phosphorylate p53 at serine 20, further affecting the stability and activity of p53 [70,71].…”

Human hRad1 but not hRad9 protects hHus1 from ubiquitin–proteasomal degradation

“…Therefore, Rad1, Rad9, Hus1, and Rad17 may serve as DNA damage sensors that form a DNA damage-responsive complex clamped around the damaged DNA to transduce the damage signal to downstream effectors. The nature of the Rad9 checkpoint complex assembly, however, remains to be precisely investigated, although each member of this checkpoint complex (Rad1, Rad9, Hus1, and Rad17) has been shown to be phosphorylated in response to DNA damage or replication inhibition (St Onge et al, 1999Bao et al, 2001;Chen et al, 2001;Post et al, 2001;Yoshida et al, 2002Yoshida et al, , 2003Foray et al, 2003;Roos-Mattjus et al, 2003). In mammalian cells, the nuclear localization of the hRad9-hRad1-hHus1 checkpoint complex is dependent on a nuclear localization signal (NLS) located in the C-terminus of the hRad9 protein (Hirai and Wang, 2002).…”