Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein

Abstract

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (␤) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third ␤-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 ␤-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

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

“…To create the 35S-YFP-CCA1 and 35S-MYC-CCA1 constructs, CCA1 cDNA was generated by PCR using the full-length CCA1 cDNA in pGEX-3X (Sugano et al, 1998) as template and the primers CCA1-F, 5#-CACCATGGAGA-CAAATTCGTCTGG-3# and CCA1-R, 5#-TCATGTGGAAGCTTGAGTTTC-3#. Inserts were cloned into the pEarleyGate 104 (35S-YFP-CCA1) and pEarleyGate 203 (35S-MYC-CCA1) vectors (Earley et al, 2006) using the GATEWAY recombination system (Invitrogen).…”

“…GST-CCA1 and GST-LHY fusion proteins were expressed in Escherichia coli and were purified as described previously (Sugano et al, 1998). The GST-CCA1 deletions were generated by deleting the appropriate fragment from pGEX-CCA1 (Sugano et al, 1998) and re-ligating the remaining portion of the construct (e.g.…”

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

“…Neither condition causes the period lengthening seen with reduced CKB levels. Previous reports indicate that CK2-mediated phosphorylation of CCA1 enhances CCA1 DNA binding (Sugano et al, 1998), suggesting that excess CKB might increase the residence time of CCA1 at promoters and possibly mimic CCA1 overexpression (arrhythmicity). Similarly, the converse (reduced CCA1 activity and period shortening) would be expected when CKB levels are reduced, and this is not observed.…”

“…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.…”