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.

Structure–function study of a novel inhibitor of the casein kinase 1 family in Arabidopsis thaliana

“…IC261 and PF‐670462 were purchased (Sigma‐Aldrich catalog numbers I0658 and SML0795, respectively). PHA767491 was synthesized as previously described (Uehara et al, ). All chemical compounds were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solutions.…”

“…Extensive genetic redundancy among multiple members of the CKL subfamily (e.g., the 13 CKLs in Arabidopsis) may make further delineation of the biological processes regulated by the CK1 family challenging because of the difficulty in eliminating kinase function by knocking out or knocking down gene expression of one or combinations of CK1 family genes. To meet this challenge, small molecule inhibitors of CK1 can be employed to determine whether or not CK1 enzymes are involved in a given biological process (Uehara et al, ). The small molecule IC261 has mostly been used for this purpose, and more recent studies used PF‐670462, which is a more potent and specific inhibitor of plant CK1 enzymes (Mizoi et al, ; Uehara et al, ).…”

“…To meet this challenge, small molecule inhibitors of CK1 can be employed to determine whether or not CK1 enzymes are involved in a given biological process (Uehara et al, ). The small molecule IC261 has mostly been used for this purpose, and more recent studies used PF‐670462, which is a more potent and specific inhibitor of plant CK1 enzymes (Mizoi et al, ; Uehara et al, ). Chemical screening combined with target identification of the target molecule indicated that PHA767491, a mammalian CDC7 (Cell division control protein 7) inhibitor, also targets plant CK1 (Uehara et al, ).…”

“…The small molecule IC261 has mostly been used for this purpose, and more recent studies used PF‐670462, which is a more potent and specific inhibitor of plant CK1 enzymes (Mizoi et al, ; Uehara et al, ). Chemical screening combined with target identification of the target molecule indicated that PHA767491, a mammalian CDC7 (Cell division control protein 7) inhibitor, also targets plant CK1 (Uehara et al, ). A combination of PF‐670462 and PHA767491 demonstrated that CK1 is involved in Arabidopsis circadian clock regulation (Uehara et al, ).…”

“…This high concentration may have harmful and confounding physiological effects. PHA767491 binds not only to CK1 family proteins, but also to other kinases, such as SHAGGY‐LIKE KINASEs (ATSKs, also called GSK3s), CALCIUM‐DEPENDENT PROTEIN KINASEs (CPKs), MITOGEN‐ACTIVATED PROTEIN KINASE (MPKs), and other kinases, therefore showing relatively low specificity for CK1 (Uehara et al, ). Reducing the effective concentration and increasing the specificity of CK1 inhibitors would enhance their utility.…”