Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS)

Abstract

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients' behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbt S and dbt L ) in Drosophila.[ These cell-autonomous oscillations are thought to be established by feedback loops involving transcription of clock genes and their subsequent autoregulatory transcriptional repression. In mammals, the transcription factor heterodimer CLOCK-BMAL1 activates the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes via E-box enhancer elements in their promoters. PER and CRY proteins are believed to form complexes that translocate in the nucleus to inhibit their own transcription by directly interacting with the CLOCK-BMAL1 complex.Critical to the properties of this oscillator is the delay between the production of PER and CRY proteins and their autorepression. Post-translational events such as complex formation among clock proteins, nuclear import and export, regulated degradation, modulation of transcriptional activity, and chromatin modification have all been implicated in the generation of this delay (for a review, see Harms et al. 2004). In many cases, phosphorylation of clock proteins is the key step that both initiates these events and regulates their correct timing. In cyanobacteria, even the core of the circadian oscillator seems to be based on rhythmic phosphorylation and dephosphorylation of clock proteins rather than on a transcriptional-translational feedback loop (Nakajima et al.

Role of Phosphorylation in the Mammalian Circadian Clock

“…Interestingly, the oscillation dynamics of PER2-FASPS expressing cells is very similar to the behavior of FASPS patients: an early phase of entrainment and a short free-running period ( Fig. 1) (Vanselow et al 2006).…”

“…The mass spectrometric approach mentioned above led to the identification of 21 (of 275 theoretically possible) endogenous phosphorylation sites in mPER2 (exclusively serine and threonine residues). Interestingly, we found the serine residue phosphorylated, which is mutated in FASPS (Vanselow et al 2006). This is the first direct evidence that the FASPS site is indeed a target for phosphorylation in living cells.…”

“…However, at least in the case of mPER2, it has been speculated that yet unknown kinases are involved in phosphorylation-dependent subcellular localization (Vanselow et al 2006).…”

Dependence of the period on the rate of protein degradation in minimal models for circadian oscillations

“…It involves interlocked feedback loops (Glossop et al 1999;Lee et al 2000;Ripperger & Schibler 2001). Moreover, experiments showed that multiple phosphorylations of the PER protein have differential effects on the protein degradation and translocation in the nucleus (Vanselow et al 2006;Xu et al 2007). Finally, the Michaelis-Menten term used in this study assumes an enzymatic process but other sources of nonlinearity in the degradation kinetics may also be present, such as the cooperative dimerization of proteins, which was shown to affect the dynamics of genetic circuits (Buchler et al 2005).…”

Circadian genes, rhythms and the biology of mood disorders

“…Even individuals that are genetically predisposed towards "eveningness" (a preference for the evening) versus "morningness" (a preference for the morning) are more likely to develop depression (Drennan et al, 1991;Chelminski et al, 1999). Genetic variations in the circadian genes have been found to associate with these sleep disorders and diurnal preference measures including an association between certain variants of Per2, and CK1δ with FASPS; Per3, CLOCK, and CK1ε with DSPS; and Per1, Per2, Per3 and CLOCK with diurnal preference (Katzenberg et al, 1998;Iwase et al, 2002;Archer et al, 2003;Johansson et al, 2003;Takano et al, 2004;Carpen et al, 2005;Mishima et al, 2005;Xu et al, 2005;Carpen et al, 2006;Vanselow et al, 2006). This suggests a connection between proper mood regulation and a normal functioning circadian clock.…”