Interdependent phosphorylation within the kinase domain T-loop regulates CHK2 activity

Abstract

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser19, Ser 33, Ser35, and Thr68. This precedes autophosphorylation of Thr383/Thr387 in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366-406). We report four novel phosphorylation sites at Ser372, Thr378, Thr389, and Tyr390. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr 389 was a prerequisite to phosphorylation at Thr387 but not at Thr383. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser 379, Thr389, and Thr383/Thr389. Phosphorylation at Tyr390 was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/ nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr372, Thr379, Thr383, Thr389, Thr383/Thr387, and Thr383/Thr389. Chk2 hyper-phosphorylated species at Thr383/ Thr387/Thr389 and Thr 383/Thr387/Thr389/ Tyr390 relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.