title: Casein kinase 1-dependent phosphorylation of familial advanced sleep phase syndrome-associated residues controls PERIOD 2 stability.
abstract: The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.
authors.affiliation: University of Wisconsin School of Medicine and Public Health, the Department of Pharmacology, Madison, WI 53705, USA.
authors.forename: Naval P
authors.initials: NP
authors.lastname: Shanware
authors.forename: John A
authors.initials: JA
authors.lastname: Hutchinson
authors.forename: Sang Hwa
authors.initials: SH
authors.lastname: Kim
authors.forename: Lihong
authors.initials: L
authors.lastname: Zhan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bowler
authors.forename: Randal S
authors.initials: RS
authors.lastname: Tibbetts
mesh_terms: D000818:Animals; D047389:Casein Kinase I / Q000378:metabolism*; D002460:Cell Line; D002940:Circadian Rhythm / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D041681:NIH 3T3 Cells; D056950:Period Circadian Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020178:Sleep Disorders, Circadian Rhythm / Q000378:metabolism*
chemical_list: C421902:PER2 protein, human; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D047389:Casein Kinase I; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases