Figure 5. CSNK1-independent phosphorylation and destabilization of PER2 by SIK3.

(a) In vitro kinase assays of Myc-PER2 purified from HEK293T17 cell lysate and recombinant SIK3 catalytic domain. Incubation of PER2 protein with SIK3 increased the up-shifted PER2 band. (b) Purified Myc-PER2 was treated with λPPase prior to a kinase assay with SIK3. No-phosphorylated PER2 was also phosphorylated by recombinant SIK3. (c) Up-shifted band shown in b was confirmed as phosphorylation-form of PER2 by 2nd λPPase assay. Purified Myc-PER2 from cell lysates was incubated with λPPase and then with SIK3 (200 ng). After washing SIK3 away, PER2 was subjected to secondary λPPase treatment. (d) NIH3T3 and HEK293T/17 cells expressing SIK3 were incubated with CSNK1 inhibitor (IC261) for indicated time periods. CSNK1 inhibitor did not affect SIK3-mediated PER2 degradation in both cell lines. (e) NIH3T3 cells were transfected with Sik3-KD constructs as well as Csnk1e-WT or kinase dead form of Csnk1e (Csnk1e-KD). Knockdown of Sik3 did not affect CSNK1-mediated phosphorylation and degradation of PER2. (f) A schematic representation of a potential role for SIK3 compared with that of CSNK1. Although both SIK3 and CSNK1 mediate PER2 phosphorylation and degradation, their roles and degradation pathways appears to be different. Therefore, it may not be the case that SIK3 acts as a priming kinase for CSNK1, but the two kinases regulate PER2 independently. P represents phosphate.