Cyclin-dependent kinase 5 (Cdk5) regulates the function of CLOCK protein by direct phosphorylation

Abstract

Circadian rhythm is a biological rhythm governing physiology and behavior with a period of ∼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

Keywords: CDK (Cyclin-dependent Kinase); CLOCK; Cdk5; Circadian Rhythms; Clock Genes; Molecular Clock; Neuroscience; Protein Phosphorylation.

FIGURE 1.

Cdk5 phosphorylates Thr-451/461 of CLOCK. A, sequence alignment of CLOCK proteins from various species. Potential Cdk5 phosphorylation sites are indicated in boldface type. B, a schematic diagram of CLOCK protein. Predicted functional domains are indicated by a shaded box, and two fragments of CLOCK protein used for GST fusion protein are indicated as lines. C, panel i, validation of p35 IP-linked in vitro kinase assay. Histone was used for positive control substrate. IP was performed with control IgG or anti-p35 antibody. Arrows and asterisk indicate Histone and phosphorylated p35, respectively. C, panel ii, IP-linked in vitro phosphorylation of CLOCK fragments. IP was performed with control IgG or anti-p35 antibody. Arrows and asterisks indicate GST-fusion CLOCK fragments and phosphorylated p35, respectively. D, panel i, in vitro kinase assay upon treatment of kinase inhibitors. Kinase reactions were carried out with p35 immunoprecipitates preincubated with LiCl (10 and 50 mm) or roscovitine (Rosc.; 20 μm) for 1 h. Arrows and an asterisk indicate the GST-CLOCK^258–477 fragment and phosphorylated p35, respectively. D, panel ii, in vitro kinase assay using purified recombinant Cdk5. Kinase reactions with GST-CLOCK fragments were carried out with the purified recombinant His₆-Cdk5/GST-p25. Arrows and an asterisk indicate GST-CLOCK^258–477 and phosphorylated GST-p25, respectively. E, IP-linked in vitro kinase assay with mutant CLOCK fragments. The gel was stained with Coomassie Brilliant Blue (top) and applied to autoradiography (bottom). Arrows and asterisks indicate GST-fused CLOCK fragments and phosphorylated p35, respectively. Band intensities were measured using ImageJ software and statistical significance was determined by one-way ANOVA (n = 3). Error bars indicate mean ± S.E. ***, p < 0.001; ns, not significant.

FIGURE 2.

Cdk5-mediated phosphorylation CLOCK in cells and brain tissue. A, phosphorylation of CLOCK Thr-451/461 by Cdk5. Immunoprecipitated wild-type and T451F/T461F mutant CLOCK from transfected cells were analyzed by immunoblotting (IB) with the indicated antibodies (n = 3). B, rhythmical phosphorylation of endogenous CLOCK. Left, as a negative control for pThr-Pro signal on CLOCK, anti-CLOCK immunoprecipitates were incubated with alkaline phosphatase (PPase; 10 units) for 1 h at 37 °C prior to immunoblotting. Right, NIH3T3 cells with stable expression of Cdk5/p35 were synchronized by 100 nm dexamethasone (Dex.) and anti-CLOCK immunoprecipitates from each time points were analyzed by immunoblotting (n = 3). C, diminished CLOCK phosphorylation by inhibition of Cdk5. NIH3T3 cells with stable Cdk5/p35 expression were harvested 24 h after dexamethasone treatment. Roscovitine (Rosc.; 20 μm) was treated for 12 h, and anti-CLOCK immunoprecipitates were analyzed by immunoblotting (n = 3). D, CLOCK phosphorylation in heterozygote p35 KO mouse brain. Wild-type and heterozygote p35 knock-out mice were killed at Zeitgeber time 18 h, and anti-CLOCK immunoprecipitates from brain lysates were analyzed by immunoblotting (n = 3). The same membrane was reprobed for pThr-Pro and CLOCK. The pThr-Pro levels were normalized to that of total CLOCK. Band intensities were determined using ImageJ software and statistical significance was determined by one-way ANOVA or two-tailed t test. Error bars indicate mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

FIGURE 3.

Cdk5 physically interacts with CLOCK. A, co-IP of CLOCK and Cdk5/p35. HEK293 cells expressing CLOCK and Cdk5/p35 were incubated with 20 μm MG132 or dimethyl sulfoxide (vehicle) for 5 h, and lysates were prepared 36 h later from transfection. Anti-GFP immunoprecipitates were analyzed by immunoblotting (IB). B, co-localization of CLOCK and Cdk5/p35. The GFP-fused CLOCK (green) and Cdk5/p35 were co-expressed in HEK293 cells and stained with anti-Cdk5 (cyan), anti-p35 antibodies (red) and Hoechst dye (blue). The magnified images are shown in the bottom. C, co-IP of endogenous CLOCK and Cdk5/p35 from mouse brain lysates. IPs were performed with control IgG or anti-p35 antibody and analyzed by immunoblotting. The arrow indicates the band for co-immunoprecipitated CLOCK.

FIGURE 4.

Cdk5 regulates transcriptional activity of CLOCK. A, luciferase assay to assess hPer1 promoter activity. HEK293 cells were transfected as indicated and hPer1 luciferase reporter construct followed by luciferase activity measurement (n = 4). B, increased mPer1 mRNA level by expression of Cdk5. Cdk5/p35 constructs were stably expressed in NIH3T3 cells and incubated with roscovitine (Rosc.; 20 μm) or dimethyl sulfoxide (vehicle) for 12 h and applied to qRT-PCR (n = 3). C, decreased mPer1 mRNA level in cultured cortical neurons by roscovitine treatment. Cultured cortical neurons (days in vitro 7) were incubated with roscovitine for 12 h and subjected to analysis (n = 3). D, decreased mPer1 mRNA level in p35 knock-out neurons. Cortical neurons (days in vitro 7) cultured from wild-type and p35 knock-out mouse embryos were analyzed (n = 6). E, enhanced amplitude of mPer1 mRNA expression by Cdk5. NIH3T3 cells stably expressing Cdk5/p35 were synchronized by dexamethasone (Dex.) and subjected to qRT-PCR at each time points (n = 3). F, increased amplitude of mPer1 mRNA oscillation by phospho-mimic mutant CLOCK (n = 3). G and H, dampened amplitude of mPer1and mPer2 mRNA expression level in heterozygote p35 knock-out mouse brain. Wild-type and p35 heterozygote knock-out mice were entrained in 12-h light/12-h dark cycles for 7 days, and cerebral cortices isolated at each time points were subjected to quantitative RT-PCR analysis. Experiments were performed in duplicate. Each mPer1 or mPer2 mRNA value was normalized to GAPDH. Statistical significance was determined by one-way ANOVA or two-tailed t test. Error bars indicate mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ns, not significant.

FIGURE 5.

Cdk5 regulates the stability of CLOCK. A, decreased CLOCK protein level by co-expression of Cdk5. CLOCK or BMAL1 were expressed with Cdk5/p35 in HEK293 cells, and cell lysates were analyzed by immunoblotting. Desitomtric quantification is also shown (n = 3). B, recovery of CLOCK protein level by inhibition of Cdk5. Transfected cells were incubated with roscovitine (Rosc.; 20 μm) for 12 h and applied to immunoblotting (n = 4). C, enhanced CLOCK degradation by Cdk5 co-expression. The transfected cells were incubated with roscovitine (20 μm) for 12 h followed by 40 μg/ml cycloheximide (CHX) treatment for indicated periods. D, altered CLOCK degradation kinetics by the Thr-451/461 mutations. Band intensities were determined using ImageJ software and normalized to α-tubulin, and statistical significance was determined by one-way ANOVA. Error bars indicate mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ns, not significant. E, co-IP of CLOCK and BMAL1 upon co-expression of Cdk5/p35 or mutation of Thr-451/461. Anti-CLOCK immunoprecipitates from transfected HEK 293 cells were analyzed by immunoblotting. F and G, CLOCK-dependent degradation of BMAL1 upon co-expression of Cdk5/p35. HEK293 cells were transfected and treated as indicated. Cell lysates were analyzed by immunoblotting (n = 3). Band intensities were determined using ImageJ software and normalized to α-tubulin. Statistical significance was determined by one-way ANOVA. Error bars indicate mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ns, not significant.

FIGURE 6.

Altered subcellular distribution of CLOCK by Cdk5 and mutation of Thr-451/461. A, altered cytoplasmic and nuclear distribution of CLOCK by Cdk5. Transfected cells were subjected to nuclear fractionation analysis. Densitometric quantification is shown on the right (n = 3). B, Immunocytochemistry of increased nuclear CLOCK by Cdk5. Wild-type or mutant CLOCK (green) was co-expressed with RFP-BMAL1 (red) and Cdk5 (cyan) in HEK293 cells, and fluorescence intensity was analyzed. Nuclei were stained with Hoechst dye (n > 10). White arrows indicate the cells expressing CLOCK without Cdk5/p35, and yellow arrows indicate the cells expressing CLOCK with Cdk5/p35. C, altered subcellular distribution of Thr-451/451 mutant CLOCKs. Wild-type, T451F/T461F, and T451E/T461E mutant CLOCK proteins were expressed with RFP-BMAL1. Relative (Rel.) fluorescence intensity in individual cells was analyzed across the longest axis of the cell (yellow line). Green indicates GFP-CLOCK, and nuclei stained by Hoechst dye were outlined by white dashed lines (n > 18). D, T451/461F mutant fails to accumulate in the nucleus under leptomycin B treatment. HEK293 cells were transfected as indicated and leptomycin B (LMB; 10 ng/ml) was treated for 5 h. Images were captured using confocal microscopy (n > 25). Measurement of fluorescence intensities was carried out using ImageJ software. Statistical significance was determined by the two-tailed t test or one-way ANOVA. Error bars indicate mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.