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. 2019 Nov 5;8:e50925. doi: 10.7554/eLife.50925

Figure 4. PER2 interacts with CDK5 in a temporal fashion in the cytoplasm.

(A) Overexpression of PER2 and CDK5-HA in NIH 3T3 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows 5% of the input and the right panel co-precipitation of PER2 with CDK5. (B) Overexpression of PER2-V5 and CDK5-HA in HEK293 cells in presence or absence of 34 μM roscovitine (CDK5 inhibitor) and DMSO (solvent). Left panel shows 5% of the input and the right panel the immunoprecipitation with anti-CDK5 or without antibody. (C) Immunoprecipitation (IP) of PER2 and CDK5 from total mouse brain extract collected at ZT12. Left panel shows the input. The right panel depicts co-immunoprecipitation of PER2 and CDK5 using either anti-CDK5 antibody or anti-GST antibody for precipitation. The middle lane shows PER2-CDK5 co-immunoprecipitation in control animals (Per2+/+) but not in Per2-/- mice illustrating the specificity of the PER2-CDK5 interaction. The * in the blot indicates unspecific signal. (D) Temporal profile of the PER2-CDK5 interaction in total extracts from SCN tissue around the clock. Input was analyzed by immunoblot using anti-CDK5, anti-PER2, and anti-HSP90 antibodies (left panel). CDK5 co-immunoprecipitated PER2 in a diurnal fashion with a peak between ZT12 and ZT16. The statistical analysis of the PER2/CDK5 signal around the clock is shown below (one-way ANOVA with Bonferroni’s post-test, n = 3, *p<0.0001, values are mean ± SEM). * in the blot indicates unspecific signal. (E) Immunoprecipitation of PER2 with CDK5 from cytoplasmic and nuclear brain extracts collected at ZT12. The left panel shows the input and the right panel co-IP of PER2 and CDK5, which occurs only in the cytoplasm but not in the nucleus. The smaller band detected by the anti-PER2 antibody depicts an unspecific band that is smaller than PER2. * in the blot indicates unspecific signal. (F) Slices from the SCN obtained at ZT12 were immunostained with PER2 antibody (green), CDK5 (red), and nuclei were marked with DAPI (blue). Co-localization of the two proteins results in the yellow color. Scale bar: 10 µm. The z-stacks right and below the micrograph confirm co-localization of PER2 and CDK5 (yellow). (G) Purification of the N-terminal half of PER2 (1–576) or the C-terminal half (577–1256) (left panel, coomassie). CDK5-His was pulled down by both recombinant PER2 attached to the glutathione resin, but only the C-terminal was able to retain CDK5 (immunoblot using anti-His antibody, right panel).

Figure 4.

Figure 4—figure supplement 1. PER2-CDK5 interaction in HEK cells.

Figure 4—figure supplement 1.

Overexpression of PER2 and CDK5 in HEK 293 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows the input and the right panel co-precipitation of PER2 with immunoprecipitated CDK5 when both were overexpressed.
Figure 4—figure supplement 2. PER2-CDK5 interaction at different salt concentrations.

Figure 4—figure supplement 2.

Immunoprecipitation (IP) of PER2 and CDK5 from total mouse brain extract collected at ZT12. Left panel shows the input. The middle and right panels depict co-immunoprecipitation of PER2 and CDK5 at two different NaCl concentrations using either anti-CDK5 antibody or anti-PER2 antibody for precipitation. * in the blot indicates unspecific signal.
Figure 4—figure supplement 3. Co-localization of PER2 and CDK5 in SCN tissue.

Figure 4—figure supplement 3.

Temporal profile of the PER2-CDK5 interaction observed by immunofluorescence at ZT0 and ZT12. SCN slices, obtained from mice perfused at ZT 0 and ZT 12, were stained with anti-PER2 antibody (green) and anti-CDK5 antibody (red). Co-localization of the two proteins results in a yellow color, which was observed only at ZT12. Scale bar: 200 µm. The two panels below show a higher magnification depicting single cells in the SCN. The Z-stacks right and below each image show that PER2 and CDK5 mainly co-localize at ZT12. Scale bar: 25 µm.
Figure 4—figure supplement 4. Deletion of PAS domains had no influence on PER2-CDK5 interaction.

Figure 4—figure supplement 4.

NIH 3T3 cells were transfected with vectors carrying PER2-V5, ΔPasA-PER2-V5, or ΔPasB-PER2-V5, and subsequently, immunoprecipitation (IP) using an anti-CDK5 antibody was performed. The results showed that CDK5 was able to interact with all forms of PER2. None of the PAS domains of PER2 seems to be involved in the interaction with CDK5.
Figure 4—figure supplement 5. Scheme of PER2 fragments used for the pull-down assy.

Figure 4—figure supplement 5.