DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues

Abstract

Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM-and Nbs1-dependent but ataxia telangiectasia-and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G 1 phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2 S3A ) showed impaired dimerization, defective auto-and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2S3A failed to inhibit cell growth and, in response to IR, to arrest G 1 /S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2

“…Moreover, dose-dependent experiments have shown that 5 to 10 Amol/L of VRX0466617 markedly inhibit the intracellular autophosphorylation of Chk2 on Thr 387 . Although the phosphorylation of Thr 68 by ATM is essential for Chk2 activation, two other recently identified phosphoresidues, Ser 19 and Ser 33 -35 , participate in this process (26). We have therefore examined the in vivo effect of VRX0466617 on these phosphoryl residues and showed that it inhibits, in a dose-dependence manner, the induced phosphorylation of Ser 19 seemed to be phosphorylated.…”

Serine/threonine phosphatases in the DNA damage response and cancer

“…Subsequently, activated Chk2 dissociates into a monomer and phosphorylates several key substrates, including Brca1, p53 and so on (reviewed in Ahn et al, 2004;Reinhardt and Yaffe, 2009). Recent studies have identified additional phosphorylation sites in Chk2, including Ser 19, Ser 33 and Ser 35 in the SQ/TQ region that are targeted by ATM, and Ser 516 that is autophosphorylated in cis (Mochan et al, 2003;Schwarz et al, 2003;Wu and Chen, 2003;Kurz et al, 2004;Buscemi et al, 2006). Phosphorylation at all these sites has been shown to contribute to Chk2 activation and function (Schwarz et al, 2003;Wu and Chen, 2003;Buscemi et al, 2006).…”

“…Recent studies have identified additional phosphorylation sites in Chk2, including Ser 19, Ser 33 and Ser 35 in the SQ/TQ region that are targeted by ATM, and Ser 516 that is autophosphorylated in cis (Mochan et al, 2003;Schwarz et al, 2003;Wu and Chen, 2003;Kurz et al, 2004;Buscemi et al, 2006). Phosphorylation at all these sites has been shown to contribute to Chk2 activation and function (Schwarz et al, 2003;Wu and Chen, 2003;Buscemi et al, 2006). Similarly, DNA damage-induced Chk1 phosphorylation at Ser 317 and 345 results in kinase activation and phosphorylation of downstream targets (Zhao and Piwnica-Worms, 2001;Reinhardt and Yaffe, 2009).…”

Chk2 mediates RITA-induced apoptosis

“…13,14 The best-characterized activating phosphorylation site of Chk2 is Thr68, 15 but phosphorylation of residues Ser19, Ser33 and Ser35 has been described to contribute to Chk2 activation. 16 The p53 pathway is strongly affected at multiple levels by DNA damage signaling. ATM, ATR, Chk1 and Chk2 were all reported to directly mediate N-terminal phosphorylation on p53.…”