Date Published: January 26, 2017
Publisher: Public Library of Science
Author(s): Yoshimi Endo Greer, Bo Gao, Yingzi Yang, Andre Nussenzweig, Jeffrey S. Rubin, Thomas G Hofmann.
Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1.
Casein kinase 1 delta (CK1δ) is an evolutionarily conserved serine/threonine kinase that participates in diverse cellular processes, including vesicle trafficking, chromosome segregation, circadian rhythm, Wnt signaling, neurite outgrowth and ciliogenesis [1–6]. Several studies have demonstrated an important role for CK1δ in DNA repair and cell cycle regulation . Hrr25, the CK1δ ortholog in budding yeast, was first identified when one of its mutants was associated with a deficiency in DNA repair . Subsequent work indicated that Hrr25 contributes to the transcriptional response to DNA damage , and others reported that mutation of Hrr25 resulted in a mitotic checkpoint defect . CK1 homologs are also required for a mitotic checkpoint in the fission yeast S. pombe .
This study demonstrated that loss of CK1δ expression in mouse cells results in genomic instability and the accumulation of DNA damage. The former was manifested by grossly enlarged nuclei in embryonic retinal tissue and multiple abnormal phenotypes in cultured MEF cells, including multiple centrosomes, micronuclei and aneuploidy documented by flow cytometry. These findings were consistent with earlier work in yeast that linked the disruption of the CK1δ ortholog to genomic instability and aberrant DNA repair [8, 9]. Previous experiments with human trophoblasts involving the use of the CK1δ/ε kinase inhibitor IC261 also implicated CK1δ in mediating normal spindle assembly and function [42, 43]. However, the interpretation of these data was challenged by subsequent reports claiming that IC261 bound directly to tubulin and thereby disrupted the mitotic spindle [44, 45]. Several articles have linked CK1δ to the regulation of p53 turnover via multiple mechanisms in the context of DNA damage [14–18]. Our work suggests there are other mechanisms for CK1δ function in DNA repair that involve the regulation of Chk1 activation and protein stability. In the absence of CK1δ, basal levels of Chk1 protein typically were reduced and activation in response to HU-induced replicative stress was significantly diminished. A corresponding decline of activated Chk1 in the nucleus we presume contributed to a defective DDR.
The loss of CK1δ expression in mammalian cells results in genomic instability and the accumulation of DNA damage. Abrogation of CK1δ expression also increased the vulnerability of cells to genotoxic stress. Inhibition of CK1δ expression was associated with a decline in total and phosphorylated Chk1, an important mediator of DNA damage repair and mitotic checkpoints, suggesting that regulation of Chk1 by CK1δ contributes to the proper functioning of these processes.