Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942

Taeko Wishiwaki, Yoshinori Satomi, Masato Nakajima, Cheolju Lee, Reiko Kiyohara, Hakuto Kageyama, Yohko Kitayama, Mioko Temamoto, Akihiro Yamaguchi, Atsushi Hijikata, Mitiko Go, Hideo Iwasaki, Toshifumi Takao, Takao Kondo

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Abstract

In the cyanobacterium Synechococcus elongates 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.

Original languageEnglish
Pages (from-to)13927-13932
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number38
DOIs
Publication statusPublished - 2004 Sep 21
Externally publishedYes

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    Wishiwaki, T., Satomi, Y., Nakajima, M., Lee, C., Kiyohara, R., Kageyama, H., Kitayama, Y., Temamoto, M., Yamaguchi, A., Hijikata, A., Go, M., Iwasaki, H., Takao, T., & Kondo, T. (2004). Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942. Proceedings of the National Academy of Sciences of the United States of America, 101(38), 13927-13932. https://doi.org/10.1073/pnas.0403906101