Casein Kinase 1-dependent Phosphorylation of Familial Advanced Sleep Phase Syndrome-associated Residues Controls PERIOD 2 Stability
Casein Kinase 1-dependent Phosphorylation of Familial Advanced Sleep Phase Syndrome-associated Residues Controls PERIOD 2 Stability
复制标题
DOI：
10.1074/jbc.m111.224014
复制
发表时间：
2011-04-08
影响因子：
4.8
通讯作者：
Tibbetts, Randal S.
中科院分区：
文献类型：
--
作者：
Shanware, Naval P.;Hutchinson, John A.;Tibbetts, Randal S.
哺乳动物生物钟组分PERIOD2（PER2）在昼夜节律的夹带中起关键作用。最近，在患有家族性早睡早起综合征（FASPS）的患者中发现了人PER2（hPER2）一个假定的磷酸化位点丝氨酸 - 662（Ser - 662）的错义突变。患有FASPS的患者表现出异常的睡眠 - 觉醒模式，其特征是终生在傍晚早睡且清晨早起。尽管从功能研究中强烈暗示了PER2的磷酸化，但一直无法研究PER2在Ser - 662位点特异性的磷酸化，并且该残基的生化功能尚不清楚。在此，我们使用磷酸化特异性抗体表明，在体内PER2在Ser - 662以及其侧翼的酪蛋白激酶（CK）位点被磷酸化。PER2的磷酸化是由酪蛋白激酶1δ（CK1δ）和酪蛋白激酶1ε（CK1ε）的联合作用进行的，并被蛋白磷酸酶1拮抗。PER2的磷酸化在哺乳动物细胞系的昼夜节律夹带时迅速诱导，并且在细胞质和细胞核区室中均发生。重要的是，我们发现Ser - 662磷酸化的PER2蛋白池比总PER2分子池更稳定，这意味着FASPS磷酸化簇拮抗PER2的降解。与这一观点一致，一个消除PER2磷酸化的Ser - 662→Ala突变显著降低了其半衰期，而一个模拟磷酸化的Ser - 662→Asp替代导致半衰期升高。我们的综合研究结果为PER2的调控以及FASPS的生化基础提供了新的见解。
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 1 delta (CK1 delta) and casein kinase 1 epsilon (CK1 epsilon) 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.