Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability

The duration of the transcription-repression cycles that give rise to mammalian circadian rhythms is largely determined by the stability of the PERIOD protein, the rate-limiting components of the molecular clock. The degradation of PERs is tightly regulated by multisite phosphorylation by Casein Kinase 1 (CK1δ/ε). In this phosphoswitch, phosphorylation of a PER2 degron (Degron 2, D2) causes degradation, while phosphorylation of the PER2 Familial Advanced Sleep Phase (FASP) domain blocks CK1 activity on the degron, stabilizing PER2. However, this model and many other studies of PER2 degradation do not include the second degron of PER2 that is conserved in PER1, termed Degron 1, D1. We examined how these two degrons contribute to PER2 stability, affect the balance of the phosphoswitch, and how they are differentiated by CK1. Using PER2-luciferase fusions and real-time luminometry, we investigated the contribution of both D2 and of CK1-PER2 binding. We find that D1, like D2, is a substrate of CK1 but that D1 plays only a ‘backup’ role in PER2 degradation. Notably, CK1 bound to a PER1:PER2 dimer protein can phosphorylate PER1 D1 in trans. This scaffolded phosphorylation provides additional levels of control to PER stability and circadian rhythms.