GAPVD1 regulates CSNK1D-dependent PER2 FASP phosphorylation

Abstract

The mammalian circadian clock relies on precisely timed transcriptional and post-translational events to generate [~]24-hour rhythms. A key post-translational mechanism is phosphorylation of PER2 by CSNK1D, governed by a phosphoswitch that integrates stabilizing and destabilizing phosphorylation marks at the FASP and Degron sites to control PER2 stability and circadian period. Here, a novel role for the PER complex protein GAPVD1 in regulating CSNK1D-mediated FASP site phosphorylation is revealed. GAPVD1 associates with PER2 and CSNK1D via a distinct helical bundle domain, weakening PER2-CSNK1D association and attenuating PER2 FASP phosphorylation in cells and in vitro. Mechanistically, this helical bundle interacts with the PER2 casein kinase binding domain and is contacted by the adjacent VPS9 region in GAPVD1, providing a structural explanation for how flanking GAPVD1 domains can control access of binding proteins to the helical bundle. Notably, a minimal GAPVD1 helical bundle fragment is sufficient for binding to PER2 and CSNK1D but not for robust attenuation of PER2 FASP phosphorylation, indicating that additional regions of GAPVD1 are required. GAPVD1-mediated inhibition of PER2 FASP phosphorylation also turned out to be independent of the CSNK1D C-terminal tail, pointing to mechanisms such as displacement of PER2 FASP from the CSNK1D active site or allosteric regulation of CSNK1D substrate engagement. Together, these findings identify GAPVD1 as a PER-complex scaffold that modulates the PER2-CSNK1D control node and introduce an additional regulatory layer into the circadian phosphoswitch. A GAPVD1 helical bundle domain binds the PER2 CKBD and CSNK1D to weaken PER2-CSNK1D interaction and attenuate PER2 FASP phosphorylation.