Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942
- Taeko Nishiwaki*,†,
- Yoshinori Satomi‡,
- Masato Nakajima*,†,
- Cheolju Lee‡,§,
- Reiko Kiyohara*,
- Hakuto Kageyama*,†,
- Yohko Kitayama*,†,
- Mioko Temamoto*,
- Akihiro Yamaguchi¶,
- Atsushi Hijikata*,
- Mitiko Go¶,
- Hideo Iwasaki*,†,
- Toshifumi Takao‡, and
- Takao Kondo*,†,∥
- *Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; †Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Furo-cho, Chikusa, Nagoya 464-8602, Japan; ‡Institute for Protein Research, Osaka University 3-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan; §Biomedical Research Center, Korea Institute of Science and Technology, Seong-buk-Gu, Seoul 136-791, Korea; and ¶Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama Shiga 526-0829, Japan
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Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved August 2, 2004 (received for review June 2, 2004)
Abstract
In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.
Footnotes
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↵ ∥ To whom correspondence should be addressed: E-mail: kondo{at}bio.nagoya-u.ac.jp.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: LL, continuous light; MS/MS, tandem MS; IPTG, isopropyl β-d-thiogalactopyranoside.
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See Commentary on page 13697.
- Copyright © 2004, The National Academy of Sciences
