Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942
Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942
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DOI：
10.1073/pnas.0403906101
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发表时间：
2004-09-21
影响因子：
11.1
通讯作者：
Kondo, T
中科院分区：
文献类型：
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作者：
Nishiwaki, T;Satomi, Y;Kondo, T
在蓝细菌细长聚球藻PCC 7942中，KaiA、KaiB和KaiC是产生昼夜节律的必需蛋白质。KaiC被认为是基因组中所有基因的昼夜节律表达的负调节因子，其磷酸化受KaiA的正调节和KaiB的负调节，并在体内显示出昼夜节律。为了研究KaiC磷酸化在生物钟系统中的功能，我们通过质谱（MS）鉴定了两个自磷酸化位点，Ser-431和Thr-432。我们通过定点突变产生了聚球藻突变体，其中这些残基被取代为丙氨酸。KaiC的磷酸化在单突变体中减少，在双突变体中完全消除，表明KaiC在体内也在这些位点磷酸化。这些突变体失去了昼夜节律，这表明在这两个网站的磷酸化是必不可少的控制的昼夜振荡。虽然非磷酸化突变体KaiC能够在体外形成六聚体，但它未能在聚球藻细胞中与KaiA，KaiB和SasA形成时钟蛋白复合物。当非磷酸化KaiC过表达时，kaiBC启动子活性仅被短暂抑制。这些结果表明KaiC磷酸化通过控制其与其他时钟蛋白的结合亲和力来调节其转录抑制活性。
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.