CikA Modulates the Effect of KaiA on the Period of the Circadian Oscillation in KaiC Phosphorylation

Kaur, Manpreet; Ng, Amy Ng May Kin; Kim, Pyonghwa; Diekman, Casey O.; Kim, Yong Ick

doi:10.1177/0748730419828068

Cited by 22 publications

(26 citation statements)

References 27 publications

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“…Cloning, purifications, and phosphorylation assays were performed as described previously without modification (Kaur et al, 2019;Kim et al, 2015). CikA was cloned into the pET41a(+) vector, expressed in Escherichia coli (BL21DE3) and purified with a GST affinity column using the same method as for KaiC (Kim et al, 2015).…”

Section: Phosphorylation Assay Of the Circadian Clock In Vitromentioning

confidence: 99%

“…Recently, it was reported that CikA directly interacts with the central oscillator, competing with KaiA for binding to KaiB (Tseng et al, 2017). The possibility of its role in the input pathway arises from CikA's nature of competitive binding, since the addition of CikA in the circadian oscillator mixture affects the phosphorylation state of KaiC and shortens its period in vitro (Kaur et al, 2019).…”

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confidence: 99%

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CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

et al. 2020

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The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation. Among the clock proteins, the pseudo-receiver domain of KaiA and CikA can sense external cues by detecting the oxidation state of quinone, a metabolite that reflects the light/dark cycle, although the molecular mechanism is not fully understood. Here, we show the antagonistic phase shifts produced by the quinone sensing of KaiA and CikA. We introduced a new cyanobacterial circadian clock mixture that includes an input component in vitro. KaiA and CikA cause phase advances and delays, respectively, in this circadian clock mixture in response to the quinone signal. In the entrainment process, oxidized quinone modulates the functions of KaiA and CikA, which dominate alternatively at day and night in the cell. This in turn changes the phosphorylation state of KaiC—the central oscillator in cyanobacteria—ensuring full synchronization of the circadian clock. Moreover, we reemphasize the mechanistic input functionality of CikA, contrary to other reports that focus only on its output action.

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Section: Phosphorylation Assay Of the Circadian Clock In Vitromentioning

confidence: 99%

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confidence: 99%

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

et al. 2020

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“…Therefore, in addition to regulating rhythms of transcriptional output from the Kai-based PTO (20), SasA also works to directly modulate KaiB association with KaiC to control formation of the nighttime repressive state and robustness of the PTO itself. 15 The other circadian output kinase, CikA, also contributes directly to robustness of the PTO by enhancing rhythms under limiting concentrations of KaiA (16). Using the FP-PTO assay, we observed that addition of CikA moderately enhanced low amplitude rhythms under limiting concentrations of KaiA while shortening the period (Fig.…”

Section: Main Textmentioning

confidence: 86%

“…This structural similarity and binding competition raised the possibility that 15 SasA helps to regulate formation of the nighttime repressive complex, which is known to be restricted by (i) the phosphorylation state of the KaiC CII domain (13), (ii) the intrinsically slow rate of ATP hydrolysis by the KaiC CI domain (14,15), and (iii) the rare conversion of KaiB to the its fold-switched form that is competent to bind KaiC (11). Similarly, CikA and KaiA bind to the same site on the KaiBC complex (9), and this competition shortens the period (11) and 20 compensates for diminished concentrations of KaiA in the PTO in vitro (16), suggesting that output kinases fortify robustness of the PTO by modulating Kai protein interactions directly. 4 To explore how the intrinsic structural similarity between fold-switched KaiB and the N-terminal thioredoxin-like domain of SasA might influence the cyanobacterial PTO, we took advantage of a new high-throughput fluorescence polarization-based post-translational oscillator (FP-PTO) assay that reports on complex formation with KaiC in real time (17).…”

Section: Main Textmentioning

confidence: 99%

“…The pseudo-receiver (PsR) domain of CikA binds to the same site on KaiB in the nighttime complex as KaiA does (9), and their 20 competition in vivo is made evident by the phenotypes of a kaiA deletion mutant (30). By competing KaiA out of the repressive complex, CikA promotes the activating potential of KaiA to stimulate KaiC phosphorylation (6,16). Either the isolated PsR domain or full-length CikA caused similar concentration-dependent decreases in period length in the FP-PTO assay, while the isolated thioredoxin-like domain of SasA was much less effective at lengthening the period compared to full-length SasA, likely due to avidity effects ( Fig.…”

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confidence: 99%

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Structural mimicry confers robustness in the cyanobacterial circadian clock

Heisler

Swan

Palacios

et al. 2020

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The histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC. Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness.One Sentence SummarySasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

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Protocols for in vitro reconstitution of the cyanobacterial circadian clock

et al. 2023

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Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24‐h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence‐based readout that enables real‐time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

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CikA Modulates the Effect of KaiA on the Period of the Circadian Oscillation in KaiC Phosphorylation

Cited by 22 publications

References 27 publications

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

Structural mimicry confers robustness in the cyanobacterial circadian clock

Protocols for in vitro reconstitution of the cyanobacterial circadian clock

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