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.
Highlights
The maintenance of genomic integrity following DNA damage requires the coordinated actions of DNA repair and cell cycle checkpoint control
This is exemplified in RAD53, where the carboxylterminal FHA domain binds to DNA damage-induced phosphorylated forms of RAD9, and this interaction is required for DNA damage-induced checkpoint functions in budding yeast [21]
It is difficult to interpret data obtained with the R145W mutant because it is unclear whether this mutation solely affects FHA domain function or induces a more drastic conformational change that alters the entire structure of Chk2
Summary
The maintenance of genomic integrity following DNA damage requires the coordinated actions of DNA repair and cell cycle checkpoint control. 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. GST-FHA Binding Assay—Whole cell extracts were prepared from 293T/17 cells or HCT-15 cells expressing wild type or T68A mutant FLAG-tagged Chk2 in NTEN buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM NaF, 1 mM Na3VO4, and protease inhibitors).
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