Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.
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.
Nishiwaki
Taeko
T
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Satomi
Yoshinori
Y
Nakajima
Masato
M
Lee
Cheolju
C
Kiyohara
Reiko
R
Kageyama
Hakuto
H
Kitayama
Yohko
Y
Temamoto
Mioko
M
Yamaguchi
Akihiro
A
Hijikata
Atsushi
A
Go
Mitiko
M
Iwasaki
Hideo
H
Takao
Toshifumi
T
Kondo
Takao
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001683:Biological Clocks / Q000502:physiology*; D002940:Circadian Rhythm / Q000502:physiology*; D056925:Circadian Rhythm Signaling Peptides and Proteins; D000458:Cyanobacteria / Q000502:physiology*; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; D017931:DNA Primers; C114271:KaiC protein, cyanobacteria