Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock

Abstract

The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

Keywords: Arabidopsis; circadian clock; posttranslational regulation; small molecule.

Fig. 1.

The small molecule PHA767491 lengthens circadian period in Arabidopsis. (A) Chemical structure of PHA767491. Circadian luciferase reporter CCA1:LUC (B) and TOC1:LUC (C) activity in Arabidopsis with PHA767491 treatment. Representative traces (Left), and increases in period length relative to untreated (0 µM) control, indicate a dose–response (n = 8 for each concentration, with one-way ANOVA P values showing statistical significance of PHA767491-treated samples compared with untreated, Right).

Fig. 2.

PHA767491 binds to and inhibits CKL family kinases. (A) Structure–activity relationship study of PHA767491 with period lengthening. Clock period change was determined compared with a DMSO treatment control [each concentration was 250 µM, n = 8, except for (1)], with Student’s t test P compared with DMSO control, Upper). Period changes by (5) or (6) (n = 8, with one-way ANOVA P, Lower). (B) Structure of PHA beads (7) and the procedure for screening proteins bound by PHA beads (Left), and binding between PHA beads and recombinant CKLs in vitro (Right). PHA767491 was added as competitor at 50 μM. GST-fusion proteins in PHA-bead fractions were analyzed by Western blotting (WB) with anti-GST antibody. (C) Inhibition of CKL kinase activity by PHA767491. (D) The IC₅₀ of PHA767491 on CKL4 was determined from three separate experiments. (See also SI Appendix, Fig. S3.) (E) Inhibition of CKL4 by PHA767491 analogs. Arrowheads and asterisks indicate phosphorylated GST-fused CKL proteins and phosphorylated casein, respectively, from C to E. Signals around 25 kDa in lanes loaded with GST-fused CKL analyzed by WB were considerable truncated GST-fused CKL from B to E.

Fig. 3.

CKL family is involved in the circadian clock. (A) Relative CKL and MLK expression in cells transfected with CKL-RNAi-1 compared with control cells (mean ± SEM, n = 3). Dots are mean values from three independent biological replicates. Asterisks indicate significant differences between control and CKL-RNAi-1 (Student’s t test P < 0.01, Left). (B) CCA1:LUC reporter activity codelivered with the CKL-RNAi-1 construct. Representative traces (Left), period lengths from three trials with Student’s t test P (Right). (C) Effect of a potent CK1 inhibitor PF-670462 on CKL4 and CKL1 kinase activity in vitro. Arrowheads indicate phosphorylated GST-fused CKL proteins, and asterisks show phosphorylated casein. (D) Effect of PF-670462 on period length of CCA1:LUC with one-way ANOVA P.

Fig. 4.

CKLs control PRR5 and TOC1 proteins. (A) Expression of clock-associated genes with 3-h treatment of PHA767491 (mean ± SEM, n = 3). Only P < 0.01 using Student’s t test are shown. Dots are mean values from three independent biological replicates. (B) PHA767491-sensitive genes in current model of clock TTFL. Genes colored red are PHA767491-sensitive genes, and red lines may be PHA767491-sensitive steps. (C) Phosphorylation of MBP-tagged PRR5 and TOC1 by GST-CKL4 (Left). Effect of PHA767491 on PRR5 and TOC1 phosphorylation by CKL4 (Right). Arrowheads and asterisks indicate substrates and GST-CKL4, respectively. (D) Band shifts of PRR5-FLAG by PHA767491 treatment in vivo with three biological replicates. Black arrows indicate PRR5-FLAG. The asterisks indicate nonspecific binding by anti-FLAG antibody. Intensity profiles of each PRR5-FLAG band are shown in the panel by normalizing the peak value to 1. Red arrowheads indicate electrophoretic mobility shift of PRR5-FLAG by PHA767491 treatment. PP is protein phosphatase treatment for extracted protein. (E) PRR5-FLAG, PRR5-VP, TOC1-VP, and PRR7-VP proteins in seedlings treated with PHA767491. Arrowheads indicate PRR-fusion protein bands. Two additional trials were performed with similar results (SI Appendix, Fig. S12). Upper are CBB-stained gels (D and E).

Fig. 5.

PHA767491 and ZTL additively regulate PRR5 and TOC1 for clock control. (A) Period-lengthening effect of PHA767491 in prr5-11 toc1-2 double mutants. (B) PRR7 and CCA1 expression in prr5-11 toc1-2 treated with PHA767491. Dots indicate mean values from three independent biological replicates. Statistical differences between wild type and prr5-11 toc1-2 were determined by Student’s t test. (C) Period-lengthening effect of PHA767491 in ztl-3. (D) Action mechanisms of PHA767491 for clock control. ZTL as a part of ubiquitin E3 ligase targets TOC1 and PRR5 for degradation. CKLs phosphorylate PRR5 as part of the pathway for degradation of TOC1 and PRR5. PHA767491 targets proteins other than the CKLs (SI Appendix, Table S1), and CKLs phosphorylate other circadian clock-controlling proteins or proteins involve in other biological processes.