Regulation of PER proteins phosphorylation and their interaction

The circadian clock is an evolutionary adaptation to the daily lightdark cycle generated by the sun. It allows organisms to organize behavior and physiology over the 24 hr day to adapt and optimize, body function to predictably recurring daily events. Malfunction or disruption of the circadian clock in humans results in various pathologies including obesity, cancer, and neurological disorders.

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus regulated in many cases by kinases.

PER1 gene is important to maintain circadian rhythms in cells. We have identified that Cyclin-dependent kinase 5 (CDK5) interacts with PER2 and phosphorylates it at Ser394 to maintain its stability and transport it into the nucleus. PER1 & PER2 genes are involved in regulating distinct and opposite biological functions. Most of PER proteins phosphorylation sites are not characterized, especially for PER1.

We aim first to find the potential CDK5 dependent phosphorylation site in PER1 & investigate the interactome of PER1 depending on CDK5 phosphorylation site. Our preliminary data indicate that PER1 and CDK5 can interact in vitro in cells. We plan to do invitro kinase essay followed by mass spectrometry to identify PER1 amino acid(s) phosphorylated by CDK5.

The phosphorylation site(s) will be mutagenized followed by a kinase essay. We will produce an appropriate antibody to test CDK5-dependant PER1 phosphorylation, address the molecular meaning of the identified CDK5-dependant phosphorylation site of PER1, the protein stability, its half-life and its period length. We will also see its impact on protein oscillations of clock genes such as CLOCK, BMAL1, PER2, CRY and potential changes in the transcriptional activity.

Finally, we will characterize CDK5 dependent phosphorylation site of PER1 in term of temporality and spatial nature in the suprachiasmatic nuclei (SCN) in vivo in mice.