Interdependent phosphorylation within the kinase domain T-loop Regulates CHK2 activity.
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 Ser(19), Ser(33), Ser(35), and Thr(68). This precedes autophosphorylation of Thr(383)/Thr(387) 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 Ser(372), Thr(378), Thr(389), and Tyr(390). 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 Thr(387) but not at Thr(383). 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), Thr(389), and Thr(383)/Thr(389). Phosphorylation at Tyr(390) 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 Thr(372), Thr(379), Thr(383), Thr(389), Thr(383)/Thr(387), and Thr(383)/Thr(389). Chk2 hyper-phosphorylated species at Thr(383)/Thr(387)/Thr(389) and Thr(383)/Thr(387)/Thr(389)/Tyr(390) 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.
Guo
Xin
X
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507.
Ward
Michael D
MD
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507.
Tiedebohl
Jessica B
JB
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507.
Oden
Yvonne M
YM
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507.
Nyalwidhe
Julius O
JO
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507.
Semmes
O John
OJ
From the Department of Microbiology and Molecular Cell Biology, Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507. Electronic address: semmesoj@evms.edu.
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000528:radiation effects; D019943:Amino Acid Substitution; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D004789:Enzyme Activation / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics / Q000528:radiation effects; D055503:Protein Multimerization / Q000235:genetics / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011839:Radiation, Ionizing; D054875:Ubiquitination / Q000235:genetics / Q000528:radiation effects
D002843:Chromatin; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases