Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.     

蓝藻生物钟分子机理的研究进展

蓝藻是具有内源性生物钟的简单生物.虽然蓝藻生物钟具有跟真核生物同样的基础特征,但其相关基因和蛋白质与真核生物没有同源性.蓝藻生物钟的核心是kai基因簇及其编码的蛋白KaiA,KaiB和KaiC.这三种Kai蛋白相互作用调节KaiC的磷酸化状态,从而产生昼夜节律信息.KaiC的磷酸化循环是昼夜节律的起博器,调控包括kai基因在内的相关基因的节律性表达.组氨酸蛋白激酶的磷酸化传递可将环境信息输入和将节律信息输出生物钟核心.     

Spatial and temporal regulation of mitogen-activated protein kinase phosphorylation in the mouse suprachiasmatic nucleus

Circadian and photic regulation of mitogen-activated protein kinase (MAPK) has been shown to associate closely with the function of the circadian clock in vertebrate clock tissues such as the mouse suprachiasmatic nucleus (SCN). Here we show that, in the central region of the mouse SCN, MAPK exhibited circadian and daily rhythms in phosphorylation with a peak at (subjective) night, and this activation was sustained for at least 8 h. In contrast, in the dorsomedial region of the SCN, MAPK showed an overt rhythm in phosphorylation with a transient peak at early subjective day, which was antiphase to that in the central region. Noticeably, the phospho-MAPK-immunoreactive cells observed in the dorsomedial region were distributed from the rostral to the caudal end of the SCN, whereas those observed in the central region were localized within the middle SCN along the rostral-caudal axis. Furthermore, a 15-min light pulse given at subjective night transiently evoked MAPK phosphorylation throughout the ventrolateral region of the SCN peaking within 15 min after the light onset, whereas nighttime-phosphorylated MAPK signals in the central-middle SCN become undetectable within 60 min after the light onset. Thus, the mode of circadian and photic regulation of MAPK phosphorylation varies remarkably among the three subregions within the SCN, suggesting divergent and cell type-specific roles of MAPK in the clock system of the mouse SCN.     

JNK regulates the photic response of the mammalian circadian clock

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.     

Circadian proteomics of the mouse retina

The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.     

Cyanobacterial circadian clockwork: roles of KaiA,KaiB and the kaiBC promoter in regulating KaiC

Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not. One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter. On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo. The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA. Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC. These observations are integrated into a model proposing rhythmic changes in chromosomal status.     

Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Background

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3β signaling pathway regulates BMAL1 protein stability and activity.

Principal Findings

GSK3β phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3β-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3β pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.

Conclusions

These findings uncover a previously unknown mechanism of circadian clock control. The GSK3β kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock.