Abstract
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, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.
Highlights
The circadian clock, prevalent in most organisms, is an evolutionary adaptation to the daily lightdark cycle generated by the sun and the earth’s rotation around its own axis (Rosbash, 2009)
We carried out a synthetic dosage lethality (SDL) screen, which is based on the concept that a high dosage of a given protein (i.e. Period 2 (PER2) in this case) may have negligible effect on growth in wild-type cells, but may compromise growth in mutants that have defects in pathway components or in functionally related processes (Measday et al, 2005; Sopko et al, 2006)
We found that degradation of PER2 was faster when Cdk5 was knocked down compared with unspecific shRNA treatment (Figure 6D), indicating that reduction of Cdk5 accelerated PER2 degradation
Summary
The circadian clock, prevalent in most organisms, is an evolutionary adaptation to the daily lightdark cycle generated by the sun and the earth’s rotation around its own axis (Rosbash, 2009). In order to measure the length of one day, organisms have developed cell-based molecular mechanisms relying on feedback loops involving a set of clock genes. The existence of such loops was suggested by the analysis of Drosophila having various mutations in their period (per) gene (Hardin et al, 1990). After the degradation of the inhibitor complex, the repression is relieved and a new circadian cycle starts
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