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Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1's K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage. [ABSTRACT FROM AUTHOR]

B-cell translocation gene 3 (BTG3) is a member of the antiproliferative BTG/ Transducer of ErbB2 gene family and is induced by genotoxic stress in a p53- and Checkpoint kinase 1 (CHKI)-dependent manner. Down-regulation of BTG3 has been observed in human cancers, suggesting that it plays an important role in tumor suppression, although the underlying mechanisms are unclear. Here, we report that BTG3 interacts with CHK1, a key effector kinase in the cell cycle checkpoint response, and regulates its phosphorylation and activation. Upon interaction, BTG3 mediates K63-linked ubiquitination of CHK1 at Lys132 through the cullin-RING ligase 4Cdt2 E3 complex, thus facilitating CHK1 chromatin association. We show that BTG3-depleted cells phenocopy those CHK1-deficient cells, exhibiting increased cell death after replication block and impaired chromosome alignment and segregation. These defects could be corrected by wild-type BTG3 but not by a mutant impaired in CHK1 interaction. We propose that BTG3-dependent CHK1 ubiquitination contributes to its chromatin localization and activation and that a defect in this regulation may increase genome instability and promote tumorigenesis. [ABSTRACT FROM AUTHOR]