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.

CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL Function Synergistically in the Circadian Clock of Arabidopsis

“…Surprisingly, we were unable to detect any monomers or heterodimers of CCA1 and LHY. CCA1 and LHY monomers are predicted to be 67 and 70 kD, respectively, but migrate at 75 and 80 kD, respectively, when subject to electrophoresis in denaturing conditions Daniel et al, 2004). We found that both CCA1 and LHY are present in a large complex (.440 kD; Fig.…”

“…The immunoprecipitation was performed as described by Shalitin et al (2002) using either anti-GFP (sc-8334; Santa Cruz Biotechnology) or anti-LHY antibody (Daniel et al, 2004). Immunoblotting was performed as described (Lu and Hrabak, 2002) with the appropriate primary antibody (affinity-purified anti-CCA1 antibody; Wang and Tobin, 1998), affinitypurified anti-LHY antibody (Daniel et al, 2004), anti-GFP (sc-8334; Santa Cruz Biotechnology), anti-MYC (Upstate), or anti-HA (Abcam).…”

Rapid Assessment of Gene Function in the Circadian Clock Using Artificial MicroRNA in Arabidopsis Mesophyll Protoplasts

“…4B). Although the only demonstrated targets of CK2 are CCA1 and LHY (Sugano et al, 1998(Sugano et al, , 1999Daniel et al, 2004), the additive nature of the reduction of these three CKBs suggests that they all contribute similarly to the control of CCA1 and LHY activity, and/or that the activity of other clock components are also affected. The latter case is very likely since ectopic expression of CCA1 or LHY, or loss-of-function CCA1/ LHY alleles result in arrhythmicity or period shortening, respectively.…”

Unraveling the circadian clock inArabidopsis

“…The phosphorylation of CCA1, mediated at least in part by CK2, is required for its function. 53,54 A circadian phosphorylation pattern has also been in the alternative splicing of PRR7 and PRR9 partially contribute to lengthening of the circadian period in the skip-1 mutant. 92 STIPL1 is a homolog of the spliceosomal proteins TUFTELIN-INTERACTING PROTEIN11 (TFIP11) in humans and Ntr1p in yeast, which are involved in spliceosome disassembly.…”

“…15,54 The homodimerization and heterodimerization of CCA1α and LHY are important for the ability of these proteins to regulate circadian rhythms. 54,81,82 The splice variant CCA1β represses CCA1α and LHY activity by competitively forming nonfunctional CCA1α/CCA1β and CCA1β/LHY heterodimers, creating a self-regulatory circuit for CCA1 and LHY through the alternative splicing of CCA1. 80 Thus, transcription factors can auto-regulate their expression by generating competitive inhibitors through alternative splicing.…”