Phosphorylation of Threonine 68 Promotes Oligomerization and Autophosphorylation of the Chk2 Protein Kinase via the Forkhead-associated Domain

Abstract

Phosphorylation of Thr-68 by the ataxia telangiectasia-mutated is necessary for efficient activation of Chk2 when cells are exposed to ionizing radiation. By an unknown mechanism, this initial event promotes additional autophosphorylation events including modifications of Thr-383 and Thr-387, two amino acid residues located within the activation loop segment within the Chk2 catalytic domain. Chk2 and related kinases possess one or more Forkhead-associated (FHA) domains that are phosphopeptide-binding modules believed to be crucial for their checkpoint control activities. We show that the Chk2 FHA domain is dispensable for Thr-68 phosphorylation but necessary for efficient autophosphorylation in response to ionizing radiation. Phosphorylation of Thr-68 promotes oligomerization of Chk2 by serving as a specific ligand for the FHA domain of another Chk2 molecule. In addition, Chk2 phosphorylates its own FHA domain, and this modification reduces its affinity for Thr-68-phosphorylated Chk2. Thus, activation of Chk2 in irradiated cells may occur through oligomerization of Chk2 via binding of the Thr-68-phosphorylated region of one Chk2 to the FHA domain of another. Oligomerization of Chk2 may therefore increase the efficiency of trans-autophosphorylation resulting in the release of active Chk2 monomers that proceed to enforce checkpoint control in irradiated cells.The maintenance of genomic integrity following DNA damage requires the coordinated actions of DNA repair and cell cycle checkpoint control. The Chk2/hCds1 protein kinase is activated by DNA damage and phosphorylates several known modulators of cell cycle control including the tumor suppressor proteins, p53 and BRCA1, and Cdc25A phosphatase (1-9). Mutations in the CHK2 gene have been identified in human hereditary and sporadic cancers suggesting that post-translational modifications mediated by Chk2 play important roles in altering the activities of these checkpoint control proteins (10 -14). Therefore, exploring the mechanisms by which Chk2 activity is regulated will enhance our understanding of the complex network of signaling pathways that serve to limit tumor development.Chk2 is a direct target and major effector of the ATM 1 kinase, a key regulator of cell cycle checkpoint control in irradiated cells (for review see Ref. 15). In response to ionizing radiation (IR) and double-stranded DNA breaks, ATM phosphorylates Chk2 on Thr-68 within the amino-terminal SQ/TQrich domain, and this event is necessary for efficient activation of the Chk2 kinase (16 -18). Recent evidence suggests that Chk2 is phosphorylated on Thr-383 and Thr-387 in a phosphothreonine 68 (Thr(P)-68)-dependent manner (19). These residues are located within the activation loop segment of Chk2 and therefore may be Chk2 autophosphorylation sites that increase the specific activity of the enzyme when modified (19,20). This model is consistent with our observations that a catalytic inactive Chk2 fails to exhibit phosphorylationdependent mobility shifts upon SDS-PAGE even though clearly phosphoryla...

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

“…The variant protein is clearly overexpressed, but is phosphorylated on Thr68 at markedly reduced levels compared to that of wild-type, endogenous CHK2. Dimerisation is proposed to represent an important step in the regulation of CHK2 activity (Ahn et al, 2002;Xu et al, 2002). However, we were not able to detect dimerisation of variant protein with the wild-type CHK2 in cells that ectopically expressed both proteins (data not shown), indicating that the truncated protein is not likely to affect wild-type CHK2 activity in this way, even if it is expressed at very low levels (undetectable with our methodology).…”

“…In response to ionising radiation (IR), threonine 68 of CHK2 is rapidly phosphorylated by ataxia telangiectasia mutated (ATM) (Matsuoka et al, 2000;Zhou et al, 2000), allowing oligomerisation and transautophosphorylation of CHK2 (Ahn et al, 2002;Xu et al, 2002). Activated CHK2 is involved in maintaining the G1/S and G2/M checkpoints by phosphorylation of CDC25A, CDC25C and p53 (Chaturvedi et al, 1999;Shieh et al, 2000;Falck et al, 2001), and repair of double-strand DNA breaks via homologous recombination (HR) through phosphorylation of BRCA1 (Lee et al, 2000).…”

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

“…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.…”

“…These results are consistent with the idea that Wip1 can dephosphorylate the phosphorylated-Thr68 in Chk2 induced by g-irradiation or ectopic overexpression of Chk2, and that Thr68-phosphorylation of Chk2 is a prerequisite event for (trans-)phosphorylating Thr387 (and possibly Thr383) in Chk2 (see Figure 2c). [31][32][33][34] We also examined whether Wip1 could dephosphorylate Ser19, Ser33/35, Thr432, and Ser516 in Chk2. As shown in Figure 3a, GST-Wip1 (WT), but not GST-Wip1 (D314A), dephosphorylated-Ser19, -Ser33/35, -Thr432, but not Ser516 in Chk2 in vitro.…”

HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

“…We illustrated the scope of the technique by the preparation of phosphorylated CHK2 protein. CHK2 is a serine/threonine kinase which upon activation by phosphorylation on Thr‐68 plays a central role in DNA damage response 27. Furthermore, it is involved in cell cycle checkpoint activation, apoptosis, viral infectivity and other pathways 28.…”