Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need

Abstract

Although sleep is one of the most conserved behaviors, the intracellular mechanism regulating sleep/wakefulness remains unknown. We recently identified a protein kinase, SIK3, as a sleep-regulating molecule. Mice that lack a well-conserved protein kinase A (PKA) phosphorylation site, S551, showed longer non-rapid eye movement (NREM) sleep and increased NREMS delta density. S551 of SIK3 is conserved in other members of the SIK family, such as SIK1 (S577) and SIK2 (S587). Here, we examined whether the PKA phosphorylation sites of SIK1 and SIK2 are involved in sleep regulation by generating Sik1S577A and Sik2S587A mice. The homozygous Sik1S577A mice showed a shorter wake time, longer NREMS time, and higher NREMS delta density than the wild-type mice. The heterozygous and homozygous Sik2S587A mice showed increased NREMS delta density. Both the Sik1S577A and Sik2S587A mice exhibited proper homeostatic regulation of sleep need after sleep deprivation. Despite abundant expression of Sik1 in the suprachiasmatic nucleus, the Sik1S577A mice showed normal circadian behavior. Although Sik2 is highly expressed in brown adipose tissue, the male and female Sik2S587A mice that were fed either a chow or high-fat diet showed similar weight gain as the wild-type littermates. These results suggest that PKA-SIK signaling is involved in the regulation of sleep need.

Sleep Need, the Key Regulator of Sleep Homeostasis, Is Indicated and Controlled by Phosphorylation of Threonine 221 in Salt Inducible Kinase 3

“…Results from SIK3 catalytic activity show that SIK3 T221A could be used as an LOF mutant for SIK3, whereas SIK3 T221E could not be used as a GOF mutant for 8 SIK3. We note that, rather than being more active mutants, S to D mutations in SIK3 (S551D), SIK1 (S587D) and SIK2 (S577D) were all less active than their corresponding wt 29,34 with S to D mutants ending up with phenotypes qualitatively similar to those of S to A mutants 29,34 . In the case of these SIK phospho-site mutants, it was not helpful to use E or D mutants to find mutants more active than wt SIK proteins, making it meaningful to examine the in vivo phenotypes of only S or T to A mutants.…”

Nuts and bolts of the salt-inducible kinases (SIKs)

“…This is consistent with the notion that sustained SIK3 catalytic activity drives sleep need via phosphorylation of the SNIPPs. Loss of the PKA-dependent phosphorylation site in SIK1 (Ser577Ala, equivalent to Ser575 in humans) or SIK2 (Ser587Ala) also increased sleep need, but less markedly than in the SIK3 mutant mouse ( Figure 1A and Table 1 ) [ 92 ].…”

Phosphorylation Hypothesis of Sleep

“…Among the multiple phosphorylation sites, S551 appears to be critical for promoting NREM sleep, because the CRISPR-mediated embryonic introduction of S551A point mutation reproduced the effect of sleep promotion ( Honda et al, 2018 ). By using the similar CRISPR-mediated targeted mutagenesis, it was demonstrated that the PKA-targeted site plays a role in the NREM sleep promotion in other SIK family proteins, SIK1 and SIK2 ( Park et al, 2020 ). Interestingly, the role of SIK family in the sleep regulation appears to be conserved in invertebrate animals because the temporal induction of S563A (corresponding to the S551A in mice) SIK3 mutant also increase sleep duration in Drosophila ( Funato et al, 2016 ).…”