Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

Abstract

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln͞Thr-Gln (SQ͞TQ) cluster domain (SCD) on Chk2, which contains seven SQ͞TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM-and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ͞TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

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.…”

ATM polymorphisms as risk factors for prostate cancer development

“…The proteins encoded by the BRCA1 and BRCA2 genes participate in the maintenance of genomic stability through their involvement in the homologous recombination pathway for the repair of DNA double-strand breaks and transcription coupled repair and the CHEK2 protein is also involved in DNA damage signalling pathways. BRCA1 and CHEK2 are both phosphorylated in response to DNA damage in an ATMdependent fashion (Matsuoka et al, 2000). Thus, we hypothesised that the ATM gene, whose protein functions upstream of these known susceptibility genes, could also be a mutation target in prostate cancer.…”