SIK3 isoforms and PKA phosphorylation sites differentially regulate sleep and circadian rhythms

STUDY OBJECTIVES: Sleep is regulated by conserved kinase signaling pathways, yet how specific molecular variants encode sleep need, sleep intensity, and circadian timing remains unclear. We investigated the functional specialization of SIK3 isoforms and protein kinase A (PKA)-phosphorylation sites in mouse sleep/wake and circadian regulation. METHODS: We first generated isoform-selective Sik3 knock-in mice expressing either the 150 kDa or 70 kDa SIK3 isoform. To determine the isoform-specific effects of the Sleepy (Slp) mutation, which causes exon 13 skipping and deletes a region containing S551, we introduced the mutation separately into the 150 kDa-specific or 70 kDa-specific allele. In addition, we generated knock-in mice carrying alanine substitutions at the PKA-phosphorylation sites T469, S551, or S674. Sleep/wake states were quantified by EEG/EMG recording, and circadian behavior was assessed by wheel-running under light-dark and constant-dark conditions. RESULTS: The mouse brain expressed two major SIK3 isoforms: a 150 kDa isoform and a 70 kDa isoform generated by intron 14 retention. Isoform-selective knock-in analyses showed that the 150 kDa isoform was required for normal wakefulness at dark onset and circadian period regulation, whereas the 70 kDa isoform contributed predominantly to NREM sleep EEG delta power. Introducing the Sleepy mutation into either isoform increased NREM sleep time and delta power. Alanine substitution of T469 or S551, but not S674, produced a Sleepy-like hypersomnia phenotype. CONCLUSION: Partially separable molecular features of SIK3, including its isoforms and phosphorylation sites, contribute to sleep and circadian regulation.