Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein
- Research Institute of Environmental Medicine, Nagoya University, Japan
- Japan Science and Technology Agency, Japan
- Kindai University Faculty of Medicine, Japan
- School of Science, The University of Tokyo, Japan
- Ritsumeikan University, Japan
- National Institutes of Biomedical Innovation, Health and Nutrition, Japan
Figures
Figure 1 with 1 supplement
Sik3-/- mice exhibit aberrant circadian rhythms in physiology and behavior.
(a) Oxygen consumption rhythm was measured in Sik3-/- mice and Sik3+/+ controls for 2 days (n = 3 per group), and mean values are shown. The peak in the Sik3-/- mice (blue line) was phase-delayed by …
Figure 1—figure supplement 1
Expression patterns of the Sik3 mRNA in the mouse brain.
(a) Distribution of Sik3 mRNA in the mouse brain was detected by in situ hybridization using a 35S-labeled riboprobe. Note that the mRNA was expressed in almost entire areas of the brain and intense …
Figure 2 with 3 supplements
Varied circadian periods and phases in individual cells of the Sik3-/- suprachiasmatic nucleus (SCN).
Time-lapse single-cell bioluminescence images of ex vivo SCN explant cultures of (a) Sik3+/+; Per2Luc/Luc and (e) Sik3-/-; Per2 Luc/Luc mice were used for the following analyses: Period distribution …
Figure 2—figure supplement 1
Sik3 knockdown cells exhibit shortened and unstable period of circadian Per2 Lucc rhythms.
(a) A representative Bmal1-luc bioluminescence rhythms of NIH3T3 cells transfected with Sik3-KD (sh #1 or sh #4, red) and control shRNA (blue) (n = 4) as shown in Figure 2k and l. (b) Average …
Figure 2—video 1
Time-lapse bioluminescence imaging of a coronal SCN slice of a Sik3+/+; Per2 Luc/Luc mouse.
Exposure time for a single image was 20 min, and time-lapse images were captured for 4 consecutive days. Per2 Luc rhythms are well synchronized in majority of the cells in the SCN.
Figure 2—video 2
Time-lapse bioluminescence imaging of a SCN slice of a Sik3-/-; Per2 Luc/Luc mouse.
Exposure time for a single image was 55 min, and time-lapse images were captured for 3 consecutive days. Note that Per2 Luc rhythms were desynchronized among individual cells in the SCN.
Figure 3 with 3 supplements
SIK3 alters expression levels and phosphorylation states of PER2 protein.
(a) HEK293T17 cells were transfected with SIK3 and PER2 expressing constructs, and the effect on PER2 expression levels was examined by western blotting analysis. Overexpression of SIK3 (SIK3-OX) in …
Figure 3—figure supplement 1
Constitutive active mutant, but not kinase-deficient SIK3, alters PER2 abundance in NIH3T3 cells.
Wild-type (WT) or mutant SIK3 (kinase dead from K37M or constitutive active from T163E) were expressed in NIH3T3 cells (n = 3). Expression levels of co-expressed PER2 were examined using western …
Figure 3—figure supplement 2
Overexpression of SIK3 promotes PER2 degradation.
(a) Upshifted band of PER2 was examined in HEK293T/17 cells expressing myc-PER2 and Flag-SIK3. SIK3-OX induces PER2 upshift. Load volume of cell lysate was adjusted for comparison of phosphorylation …
Figure 3—figure supplement 3
Sik3 knockdown reduces degradation of PER2 and increases its levels.
(a) Another set of experiment (sample set #1) showed the effect of Sik3 knockdown on PER2 levels in NIH3T3 cells. Sample set #2 was also shown in Figure 3e. All four different Sik3 shRNAs increased …
Figure 4
Elevated expression of PER2 in Sik3-/- fibroblasts and liver.
(a) MEFs were established from Sik3+/+ (WT) Per2Luc/+ mice and Sik3-/- (KO); Per2Luc/+ mice. The cellular rhythms were synchronized by dexamethasone (DEX, 100 nM) treatment to examine rhythmic …
Figure 5
CSNK1-independent phosphorylation and destabilization of PER2 by SIK3.
(a) In vitro kinase assays of Myc-PER2 purified from HEK293T17 cell lysate and recombinant SIK3 catalytic domain. Incubation of PER2 protein with SIK3 increased the up-shifted PER2 band. (b) …
Author response image 1
No significant effects of SA mutations at phosphorylation sites of PER2 on its degradation induced by SIK3.
HEK293T17 cells were transfected with SIK3 (or empty vector [–]) and mutant PER2 expression vectors with green-fluorescent protein (GFP) expression vector. The PER2 and GFP protein levels were …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.24779.014
