CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis

Abstract

The circadian clock controls numerous physiological and molecular processes in organisms ranging from fungi to human. In plants, these processes include leaf movement, stomata opening, and expression of a large number of genes. At the core of the circadian clock, the central oscillator consists of a negative autoregulatory feedback loop that is coordinated with the daily environmental changes, and that generates the circadian rhythms of the overt processes. Phosphorylation of some of the central oscillator proteins is necessary for the generation of normal circadian rhythms of Drosophila, humans, and Neurospora, where CK1 and CK2 are emerging as the main protein kinases involved in the phosphorylation of PER and FRQ. We have previously shown that in Arabidopsis, the protein kinase CK2 can phosphorylate the clock-associated protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) in vitro. The overexpression of one of its regulatory subunits, CKB3, affects the regulation of circadian rhythms. Whether the effects of CK2 on the clock were due to its phosphorylation of a clock component had yet to be proven. By examining the effects of constitutively expressing a mutant form of the Arabidopsis clock protein CCA1 that cannot be phosphorylated by CK2, we demonstrate here that CCA1 phosphorylation by CK2 is important for the normal functioning of the central oscillator.

Fig. 1.

Identification of CK2 phosphorylation sites in the CCA1 protein and synthesis of mCCA1, which cannot be phosphorylated by CK2. (A) Putative CK2 phosphorylation sites in CCA1, and mutations of serine to alanine in mCCA1. Black arrowheads indicate putative CK2 phosphorylation sites (S/T XX D/E) in CCA1. White stars indicate the six serine residues that were mutated into alanine residues in mCCA1. (B) Schematic representation of the phosphorylation of CCA1 deletion mutants by CK2 in vitro. Mutants are designated by their corresponding amino acid residue numbers in full-length CCA1. Y, phosphorylated; N, not phosphorylated. (C) Autoradiograph of SDS/PAGE analysis of GST-CCA1 and GST-mCCA1 after in vitro CK2 phosphorylation assays. Increasing amounts of GST-CCA1 or GST-mCCA1 were incubated with recombinant CK2 in the presence of [γ-³²P]ATP. The arrow points to full-length GST-CCA1 or GST-mCCA1. The lower bands indicated by the dash could be degradation products of GST-CCA1.

Fig. 2.

Unlike overexpression of CCA1, overexpression of mCCA1 does not abolish circadian rhythms in the central oscillator. Wild-type (filled triangles), CCA1-ox (filled squares), and mCCA1-ox (open circles, dotted line) plants were entrained in light/dark (LD) conditions (12 h of light/12 h of dark) and transferred to constant light (LL). Samples were collected every 4 h and submitted to RNA and protein extractions. (A) Expression of endogenous CCA1 measured by RT-PCR. The relative levels of endogenous CCA1 mRNA were normalized to the lowest value of the wild-type samples. Each reaction was performed three times from two independent experiments with similar results. (B) LHY protein levels in wild-type, CCA1-ox, and mCCA1-ox plants measured by Western blot. Pyrophosphatase (PPase) was used as a loading control. Experiments were performed two times with similar results. The solid arrows indicate the location of both LHY and PPase proteins. The dotted arrow indicates where LHY protein is expected in CCA1-ox plants. White and black bars indicate light and dark periods, respectively, in LD. The white and the hatched bars indicate light and subjective dark periods, respectively, in LL.

Fig. 3.

Unlike overexpression of CCA1, overexpression of mCCA1 does not abolish circadian rhythms of output genes. Plants were treated as in Fig. 2. The relative levels of CAB2 (A) and CCR2 (B) mRNA were normalized to the lowest value of the wild-type samples. For both panels, the white and the hatched bars indicate light and subjective dark periods in LL, respectively. Each reaction was performed three times from two independent experiments with similar results.

Fig. 4.

Mutations in mCCA1 do not alter interaction with CKB3 but do alter interactions with CCA1. All experiments were done according to the manufacturer's instructions for the Matchmaker kit (BD Clontech). For both A and B, the left side illustrates CCA1 (open bars) or mCCA1 (gray bars) fusions to the B42 transcription activation domain (black boxes) that were constructed in pB42AD and introduced into yeast strain EGY48 with a plasmid expressing full-length CKB3 (A) or full-length CCA1 (B) fused to the LexA DNA-binding domain. CCA1 and mCCA1 N-terminal deletions fused to the B42 transcription activation domain are indicated by numbers. Black crosses indicate mutations in mCCA1. The right side shows the corresponding β-galactosidase activities of CPRG (chlorophenol red-β-d-galactopyranoside) assays, relative to the negative control arbitrarily set to zero. The values are the average of six individual colonies from a representative experiment ±SE.

Fig. 5.

Phosphorylation by CK2 is required for the formation of the DNA-protein complex containing CCA1 in plant extracts. Wild-type, CCA1-ox, and mCCA1-ox plants were treated as in Fig. 2. Plant extracts were incubated in the absence (lanes 1, 4, 5, 6, 9, 12, and 15) or presence of λ-phosphatase (PPase) (lanes 2, 7, 10, and 13) or DG (lanes 3, 8, 11 and 14). The arrowhead indicates the DNA-protein complex containing CCA1.