Anti-CSNK1D monoclonal antibody, clone 5I9

Circadian rhythms are intrinsic similar to 24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta (CK1 delta, CSNK1D) is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1 delta can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2:: LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1 delta, that regulates CK1 delta activity towards PER2. A mutant CK1 delta T347A was more active in promoting PER2 degradation. This CK1 delta regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1 delta by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1 delta-dependent pathways including the circadian clock.

Hypoxia inducible factor 2 (HIF-2) is a transcriptional activator implicated in the cellular response to hypoxia. Regulation of its inducible subunit, HIF-2 alpha (also known as EPAS1), involves post-translational modifications. Here, we demonstrate that casein kinase 1d (CK1d; also known as CSNK1D) phosphorylates HIF-2 alpha at Ser383 and Thr528 in vitro. We found that disruption of these phosphorylation sites, and silencing or chemical inhibition of CK1 delta, reduced the expression of HIF-2 target genes and the secretion of erythropoietin (EPO) in two hepatic cancer cell lines, Huh7 and HepG2, without affecting the levels of HIF-2 alpha protein expression. Furthermore, when CK1 delta-dependent phosphorylation of HIF-2 alpha was inhibited, we observed substantial cytoplasmic mislocalization of HIF-2 alpha, which was reversed upon the addition of the nuclear protein export inhibitor leptomycin B. Taken together, these data suggest that CK1 delta enhances EPO secretion from liver cancer cells under hypoxia by modifying HIF-2 alpha and promoting its nuclear accumulation. This modification represents a new mechanism of HIF-2 regulation that might allow HIF isoforms to undertake differing functions.