<i>Neurospora</i> casein kinase 1a recruits the circadian clock protein <scp>FRQ</scp><i>via</i> the C‐terminal lobe of its kinase domain

Marzoll, Daniela; Serrano, Fidel E; Diernfellner, Axel; Brunner, Michael

doi:10.1002/1873-3468.14435

Cited by 5 publications

(5 citation statements)

References 48 publications

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“…2020; Marzoll et al . 2022b, 2022a), which is consistent with a large number of phosphorylatable residues identified on it. Although over 100 phosphosites have been identified (Baker et al .…”

Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 87%

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Structure-function analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Wang

Stevenson

Dunlap

2022

Preprint

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In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, how all the phosphorylations on FRQ control its activity is still elusive. In this study, we systematically investigated the role in period determination of all 110 phosphorylated residues reported on FRQ by mutagenetic and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were mostly seen while removal of phosphorylation in the C-terminal tail result in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ does not only result in an extremely short period, but also causes an over-compensated circadian oscillator under a range of physiological temperatures. When different groups of phosphomutations on FRQ were combined intramolecularly, an additive effect was observed as expected; unexpectedly, arrhythmicity resulting from one cluster frq phosphorylation mutants was restored by eliminating phosphorylation at another group of sites, suggesting an epistatic effect between phosphoevents.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 87%

Structure-function analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Wang

Stevenson

Dunlap

2022

Preprint

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“…FRQ has been predicted to be a largely unstructured protein comprising many disordered regions that make most of its residues exposed and accessible by kinases in the cell ( Hurley et al 2013 ; reviewed in Pelham et al 2020 ; Marzoll et al 2022b , 2022a ), which is consistent with a large number of phosphorylatable residues identified on it. Although over 100 phosphosites on FRQ have been unambiguously documented ( Baker et al 2009 ; Tang et al 2009 ) and partially confirmed by a recent publication ( Horta et al 2019 ), and Ala mutations to some of these phosphoresidues have been shown to alter period lengths, their functions are still largely unknown due to lack of systematic mutagenesis analyses to all of them.…”

Section: Discussionmentioning

confidence: 82%

Functional analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Wang

Stevenson

Dunlap

2022

G3: Genes, Genomes, Genetics

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In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an over-compensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.

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“…For instance, CK1 in fungi and animals and CK2 in plants are crucial for the phosphorylation of circadian clock proteins. In eukaryotes, the ubiquitin–proteasome system and autophagy are involved in the modification and degradation of clock proteins [ 47 , 48 ].…”

Section: The Molecular Network Of the Circadian Clockmentioning

confidence: 99%

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species

Zhang,

Zhou,

Guo

2024

IJMS

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Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein–protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.

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Neurospora casein kinase 1a recruits the circadian clock protein FRQvia the C‐terminal lobe of its kinase domain

Cited by 5 publications

References 48 publications

Structure-function analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Structure-function analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Functional analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species

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