JAK2-CHK2 signaling safeguards the integrity of the mitotic spindle assembly checkpoint and genome stability.

Checkpoint kinase 2 (CHK2) plays an important role in safeguarding the mitotic progression, specifically the spindle assembly, though the mechanism of regulation remains poorly understood. Here, we identified a novel mitotic phosphorylation site on CHK2 Tyr156, and its responsible kinase JAK2. Expression of a phospho-deficient mutant CHK2 Y156F or treatment with JAK2 inhibitor IV compromised mitotic spindle assembly, leading to genome instability. In contrast, a phospho-mimicking mutant CHK2 Y156E restored mitotic normalcy in JAK2-inhibited cells. Mechanistically, we show that this phosphorylation is required for CHK2 interaction with and phosphorylation of the spindle assembly checkpoint (SAC) kinase Mps1, and failure of which results in impaired Mps1 kinetochore localization and defective SAC. Concordantly, analysis of clinical cancer datasets revealed that deletion of JAK2 is associated with increased genome alteration; and alteration in CHEK2 and JAK2 is linked to preferential deletion or amplification of cancer-related genes. Thus, our findings not only reveal a novel JAK2-CHK2 signaling axis that maintains genome integrity through SAC but also highlight the potential impact on genomic stability with clinical JAK2 inhibition.

CHK2-mediated regulation of PARP1 in oxidative DNA damage response.

Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor, which upon activation, recruits downstream proteins by poly(ADP-ribosyl)ation (PARylation). However, it remains largely unclear how PARP1 activity is regulated. Interestingly, the data obtained through this study revealed that PARP1 was co-immunoprecipitated with checkpoint kinase 2 (CHK2), and the interaction was increased after oxidative DNA damage. Moreover, CHK2 depletion resulted in a reduction in overall PARylation. To further explore the functional relationship between PARP1 and CHK2, this study employed H2O2 to induce an oxidative DNA damage response in cells. Here, we showed that CHK2 and PARP1 interact in vitro and in vivo through the CHK2 SCD domain and the PARP1 BRCT domain. Furthermore, CHK2 stimulates the PARylation activity of PARP1 through CHK2-dependent phosphorylation. Consequently, the impaired repair associated with PARP1 depletion could be rescued by re-expression of wild-type PARP1 and the phospho-mimic but not the phospho-deficient mutant. Mechanistically, we showed that CHK2-dependent phosphorylation of PARP1 not only regulates its cellular localization but also promotes its catalytic activity and its interaction with XRCC1. These findings indicate that CHK2 exerts a multifaceted impact on PARP1 in response to oxidative stress to facilitate DNA repair and to maintain cell survival.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

Casein kinase 1α (CK1α), a component of the ß-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by ß-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.