Chk2 Activation and Phosphorylation-Dependent Oligomerization

Abstract

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto-and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

“…It was shown that ectopic overexpression of Chk2 in 293T cells results in its electrophoretic mobility shift (Figure 2a), 13,32 indicating auto-or transphosphorylation. In fact, electrophoretic mobility shift induced by overexpression of Chk2 was abrogated following treatment of cell extracts with CIP or BAP (data not shown).…”

“…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.…”

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

“…This ATM-dependent phosphorylation event might be facilitated by the Plk3-mediated phosphorylation of Chk2-Ser73 (Bahassi el et al, 2006). Intermolecular interactions between one phospho-Thr68 Chk2 with the forkhead-associated (FHA) domain of another Chk2 molecule lead to homodimerization of the kinase and autophosphorylation at Thr383 and Thr387 within the activation loop of the catalytic domain that is required for full Chk2 activation (Xu et al, 2002;Schwarz et al, 2003). Interestingly, Thr68 phosphorylation is not required to maintain Chk2 kinase activity after autophosphorylation of its T-loop .…”

The cell cycle checkpoint kinase CHK2 mediates DNA damage-induced stabilization of TTK/hMps1

“…CHK2 SCD and FHA domains, in addition to the kinase activity, are essential in the induction of TTK/hMps1 To identify the regions in CHK2 that are required to induce TTK/hMps1, several CHK2 mutants were made (Figure 3a), all with reported compromised CHK2 activity in vitro (Xu et al, 2002;Figure 3a). These mutants and WT CHK2 were cotransfected with HA-TTK/hMps1 into 293T cells, and the lysates were analysed by western blotting.…”