CNOT1 Mediates Phosphorylation Of The Mammalian Clock Through The Protein Kinase A

Protein kinase A(PKA) is a key component in the Neurospora circadian feedback loop, and the NOT1 protein is important for maintaining both WC-1 and WC-2 levels and promotes their phosphorylation. However, it remains unknown whether the related protein CNOT1 is associated with protein kinase A in the eukaryotic clockwork. Our data indicate that CNOT1 could inhibit circadian clock protein BMAL1 and CLOCK transcriptional activity in mammalian cells though luciferase reporting system experiments; It is shown that CNOT1 associates with both CLOCK and BMAL1, promotes their phosphorylation and stability, and inhibits the transcriptional activity. Over-expression of CNOT1,BMAL1,CLOCK shows that they respectively interact with endogenous PKA and CNOT1 promotes the interaction between CLOCK/BMAL1 and PKA. Co-transfection experiments illustrate that PKA can directly phosphorylate CLOCK and BMAL1 and interaction between CLOCK/BMAL1 and PKA is promoted by CNOT1. In order to further clarify the role of PKA in the circadian feedback loop, the genetic targeting of PKA by CRISPR/Cas9 results in longer periods of the circadian rhythm; while overexpression of PKA induces shorter periods. PKA also regulates PER2 phosphorylation and increases its stability. Taken together, our results suggest that CNOT1 and PKA play a critical role in the mammalian circadian clock.