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

Figure 5. CDK5 phosphorylates PER2 at S394.

(A) An in vitro kinase assay was performed using recombinant CDK5/p35 and either GST-PER2 1–576 or GST-PER2 577–1256 as substrate. The samples were subjected to 10% SDS page (Coomassie, left panel) and the phosphorylation of PER2 was detected by autoradiography in order to visualize 32P-labeled proteins (right panel). CDK5 phosphorylates the N-terminal half (1-576) of a GST-PER2 fusion protein whereas the C-terminal half (577–1257) is not phosphorylated. The signal for CDK5/p35 alone indicates CDK5 auto-phosphorylation seen in all lanes when CDK5 is present. (B) Annotated mass spectrum of the tryptic peptide PER2383-397 ILQAGGQPFDYpSPIR containing the phosphorylated residue S394. The red color depicts the y-ion series (1-12) and blue the b-ion series (2–7, a2); y5-98, y8-98, y11-98 show the de-phosphorylated ions. (C) In vitro kinase assay was performed as in (A). The putative phosphorylation site was mutated to aspartic acid (S394D) or glycine (S394G). Both mutations abrogated the CDK5-mediated phosphorylation. Coomassie staining reveals equal expression of the GST-PER2 fragments. The bar diagram at the right shows the quantification of three experiments. One-way-ANOVA with Bonferroni’s post-test, *: p<0.001 (D) The monoclonal antibody produced against P-S394-PER2 does recognizes PER2 (1–576) S394 phosphorylation mediated by CDK5/p35 in presence but not in absence of the kinase or when CDK5 is inactivated by roscovitine. This antibody does not recognize the S394G mutated form even in presence of CDK5/p35. (E) Temporal profile of P-S394-PER2 and total PER2 in SCN tissue. Upper panels show western blots of the corresponding proteins indicated on the right. Below the quantification of three experiments is shown, in which the value at ZT12 of PER2 has been set to 1. The data were double plotted. Values are the mean ± SEM. Two-way ANOVA with Bonferroni’s multiple comparisons revealed that the two curves are significantly different with p<0.0001, F = 93.65, DFn = 1, DFd = 48. (F) Subcellular localization of P-S394-PER2. Total wild-type mouse brain extracts were separated into cytoplasmic (HSP90 positive) and nuclear (laminB positive) fractions. Phosphorylated PER2 was predominantly detected in the cytoplasm with the P-S394-PER2 antibody, whereas the general PER2 antibody detected PER2 in both compartments with higher amounts in the nuclear fraction.

Figure 5.

Figure 5—figure supplement 1. Scheme of PER2 fragments used for the in vitro kinase assay.

Figure 5—figure supplement 1.

The fragment 1–576 covers the sites that might be phosphorylated by CDK5 on the basis of the conserved consensus (S/T)PX(K/H/R).
Figure 5—figure supplement 2. Additional controls for in vitro kinase assay.

Figure 5—figure supplement 2.

An in vitro kinase assay performed in presence of recombinant CDK5/p35 and using as substrate the GST-PER2 1–576.
Figure 5—figure supplement 3. Testing specificity of the in vitro kinase assay.

Figure 5—figure supplement 3.

The reactions were treated either with LiCl (inhibitor of GSK3β kinase activity) or roscovitine (inhibitor of CDK5 kinase activity) in order to highlight the specificity of the PER2 phosphorylation mediated by CDK5.
Figure 5—figure supplement 4. Characterization of antisera against P-S394-PER2.

Figure 5—figure supplement 4.

Different antisera against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST-PER2 1–576 (in presence or absence of CDK5/p35) followed by WB. Even if at this stage, it was necessary to choose an antiserum that recognized the PER2 peptide regardless of its phosphorylation status, the antiserum one was able to discriminate the two forms and was therefore used for the following amplifications.
Figure 5—figure supplement 5. Characterization of hybridomas against P-S394-PER2.

Figure 5—figure supplement 5.

Different hybridomas producing antibodies against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST PER2 1–576 (in presence or absence of CDK5/p35) followed by WB. From 16 different clones, the positive ones are shown. The clone 10E12 was used to produce the final antibody.
Figure 5—figure supplement 6. Validation of anti-P-S394-PER2 antibody.

Figure 5—figure supplement 6.

Total protein extracts were obtained from wild-type, Per2Brdm1 and Per2-/- mouse brains at ZT12. Western blot was performed in order to validate the specificity of the antibody against the phosphorylated serine 394 on PER2. Only samples obtained from WT tissues showed the phosphorylated form of the protein. Antibody against total PER2 was used as control, which, positively detected the protein only in extracts obtained from wild-type mice.