Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1

Abstract

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Cds1 (Chk2) tumour-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo and can phosphorylate Cds1 in vitro, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

“…38,39 It has also been reported that following DNA damage ATM phosphorylates Chk2 at Thr68, an event critical for the activation of Chk2. [9][10][11][12][13] Here we show that Wip1 dephosphorylates Thr68 in Chk2 both in vivo and in vitro (Figures 2 and 3), and may act as a negative regulator of Chk2 (Figures 4b and 5). It has been reported that Ptc2 and Ptc3, two members of the PP2C family, bind to Rad53 and inactivate Rad53-dependent pathways in S. cerevisiae.…”

“…It had been shown previously that phosphorylation of Chk2 on Thr68 is required for full activation of Chk2 via auto-(trans-or cis)phosphorylation. [9][10][11][12][13][31][32][33][34] We therefore investigated the role of Thr68-phosphorylation in the electrophoretic mobility shift of Chk2. We found that a Chk2 mutant, the alanine68 mutant (HA-Chk2 (T68A)), did not undergo the mobility shift when overexpressed (data not shown), indicating that Thr68 is required.…”

“…[9][10][11][12][13] Hence, we examined Thr68-phosphorylation and mobility shift of HA-Chk2 (WT) coexpressed with Flag-Wip1 (WT) or Flag-Wip1 (D314A) in 293T cells, before or after girradiation, by immunoblotting with antiphospho-Chk2 (Thr68). As shown in Figure 2b (upper panel), ectopic overexpression of HA-Chk2 per se induced some Thr68-autophosphorylation, but this phosphorylation, and that of the endogenous Chk2, was enhanced following g-irradiation, in cells not coexpressing Flag-Wip1 (WT).…”

“…[1][2][3][4][5][6][7][8] Upon DNA damage, Chk2 is activated by phosphorylation of threonine68 (Thr68) by ATM (ataxiatelangiectasia mutated). [9][10][11][12][13] Activated Chk2 then phosphorylates its downstream effectors, including the tumour suppressors p53, BRCA1, PML, E2F-1, and Cdc25 phosphatases, [14][15][16][17][18][19] thereby regulating cellular responses following DNA damage. Although the molecular basis of Chk2 kinase activation and roles of Chk2 in checkpoint activation are rather well understood, it remains largely unknown as to how activated Chk2 is inhibited to release from checkpoint arrest or to prevent cells from undergoing Chk2-mediated apoptosis.…”

The Wip1 phosphatase (PPM1D) antagonizes activation of the Chk2 tumour suppressor kinase

“…Chk2 activation requires its phosphorylation by ATM at Thr68 within an amino-terminal SQ/TQ cluster Melchionna et al, 2000). This ATM-dependent phosphorylation event might be facilitated by the Plk3-mediated phosphorylation of Chk2-Ser73 (Bahassi el et al, 2006).…”