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AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

ABSTRACT: We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR - CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

SUBMITTER: Ta HQ

PROVIDER: S-EPMC7338052 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

Ta Huy Q HQ Dworak Natalia N Ivey Melissa L ML Roller Devin G DG Gioeli Daniel D

eLife 20200624

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR ...[more]

PMID: 32579110

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Project description: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.

Dataset Information

AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

Publications

AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

Similar Datasets

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