Posttranslational Regulation of<i>Neurospora</i>Circadian Clock by CK1a-dependent Phosphorylation

Querfurth, Christina; Diernfellner, Axel; Heise, Fred H.; Lauinger, Linda; Neiss, Andrea; Tataroglu, Ozgur; Brunner, Michael; Schafmeier, Tobias

doi:10.1101/sqb.2007.72.025

Cited by 39 publications

(43 citation statements)

References 32 publications

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“…Indeed FRQ has many different binding partners – CK-1A, WC-1, WC-2, FRH, PRD-4(CHK2) as well as FRQ itself - that associate with specific and distinct regions throughout the protein and these are temporally as well as spatially specific (Baker et al, 2009; Cheng et al, 2001; Denault et al, 2001; Guo et al, 2010; He et al, 2006; Pregueiro et al, 2006; Querfurth et al, 2011). Stability, subcellular localization and function of FRQ all depend on these modifications and binding partners (Baker et al, 2009; Diernfellner et al, 2009; Guo et al, 2010; He and Liu, 2005; Querfurth et al, 2007; Querfurth et al, 2011; Tang et al, 2009) (Figure 5 of this work). PONDR prediction along with a variety of other structural predictors show that roughly 75% of the FRQ amino acid sequence falls into the disordered range (Supplemental Figure 3), a finding consistent with data showing that the longest non-phosphorylated region of mature FRQ is only 129 amino acids out of a total of 998 (Baker et al, 2009; Tang et al, 2009).…”

Section: Discussionmentioning

confidence: 88%

Conserved RNA Helicase FRH Acts Nonenzymatically to Support the Intrinsically Disordered Neurospora Clock Protein FRQ

et al. 2013

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Summary Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed Intrinsically Disordered Proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein Frequency (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to Frequency Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

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Section: Discussionmentioning

confidence: 88%

Conserved RNA Helicase FRH Acts Nonenzymatically to Support the Intrinsically Disordered Neurospora Clock Protein FRQ

et al. 2013

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“…Thus, an extended region of FRQ is not phosphorylated, perhaps allowing a site for uncharacterized protein interaction or higher order structure formation in an otherwise highly modified protein. Removal of phosphorylated residues in the PEST-1 domain stabilizes FRQ, and the central part of the protein, which includes the PEST-1 and FCD, is sufficient to promote rapid degradation of a heterologous protein (Querfurth et al, 2007). One strategy to delay FRQ degradation, and therefore extend circadian period, would be to temporally regulate accessibility of this region to kinases.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Proteomics Reveals a Dynamic Interactome and Phase-Specific Phosphorylation in the Neurospora Circadian Clock

et al. 2009

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Summary Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hour rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct post-translational modifications (PTMs). To better understand how PTMs impact a circadian oscillator we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.

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“…The first suggestion that the model required updating came from a study in Neurospora in which steady-state estimates indicated there was insufficient FRQ to inactivate the WCC in a stoichiometric manner; indeed, WCC phosphorylation81 was associated with decreased DNA binding82. This led to a model in which instead of constitutively binding to WCC, rather FRQ interacts with the WCC transiently and serves as a scaffold for CK1 and CK2 which in turn act enzymatically on the WCC54, 83, 84. More recent data, however, indicate that the feedback loop is closed through dynamic hypophosphorylated-FRQ-mediated export of WCC from the nucleus85.…”

Section: Mechanistic Details Of Negative Feedbackmentioning

confidence: 99%

Post-translational modifications in circadian rhythms

Mehra

Baker

Loros

et al. 2009

Trends in Biochemical Sciences

163

135

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The pace has quickened in circadian biology research. In particular, an abundance of results focused on post-translational modifications (PTMs) is sharpening our view of circadian molecular clockworks. PTMs affect nearly all aspects of clock biology; in some cases they are essential for clock function and in others, they provide layers of regulatory fine-tuning. Our goal is to review recent advances in clock PTMs, help make sense of emerging themes, and spotlight intriguing and perhaps controversial new findings. We focus on PTMs affecting the core functions of eukaryotic clocks, in particular the functionally related oscillators in Neurospora crassa, Drosophila melanogaster, and mammalian cells. Post-translational modifications emerge on the sceneAs our understanding of eukaryotic clocks has shifted focus from organismal behavior to molecular underpinnings, in the broadest description these clocks consist of a positively acting driver that is periodically suppressed by an inhibitory brake (Fig 1). More specifically, heterodimeric positively-acting PAS (named for Per-ARNT-Sim motifs) domain-containing transcription factors (+TFs) activate the transcription of negatively acting factors (−Fs). These, in turn, feed back to inhibit the activity of the +TFs for long periods of time, thus setting the phase of transcription and determining the length of the feedback cycle. [1][2][3] This core circadian circuit is broadly controlled by post-translational modifications (PTMs). Indeed, recent evidence points to a clock based entirely on PTMs in cyanobacteria (Box 1), although this review does not focus on this system. What did we know about PTMs within these circuits circa 2004? Phosphorylation of −Fs was typical, rhythmic, and phase specific 4, 5 and appeared to be the major contribution to determining the very long (circa 24 h) time constant of the feedback loop 6 . More nuanced studies showed that the activity state of +TFs that shuttled between cytoplasm and nucleus 7 was accompanied by changes in histone acetylation 8 and chromatin remodeling 9 , and there were hints that phosphorylation of +TFs 10 increased their transcriptional activity 11 and targeted them for degradation 12 . −Fs were known to dimerize and enter the nucleus [13][14][15][16] and the timing and control of this event was a regulated process 17,18 . Importantly, −Fs were known to bind and inhibit +TFs 10,16,19 . Several reports indicated that the joint activity of kinases and phosphatases (some of whose specific roles in the clock are functionally conserved across phyla, e.g., casein kinase 2 (CK2) [20][21][22] , casein kinase 1 (CK1) [23][24][25] and protein phosphatase 2A (PP2A) 26,27 ) caused net progressive © 2009 Elsevier Ltd. All rights reserved. *Corresponding author: Dunlap, J.C. (Jay.C.Dunlap@dartmouth.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyedi...

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Posttranslational Regulation ofNeurosporaCircadian Clock by CK1a-dependent Phosphorylation

Cited by 39 publications

References 32 publications

Conserved RNA Helicase FRH Acts Nonenzymatically to Support the Intrinsically Disordered Neurospora Clock Protein FRQ

Conserved RNA Helicase FRH Acts Nonenzymatically to Support the Intrinsically Disordered Neurospora Clock Protein FRQ

Quantitative Proteomics Reveals a Dynamic Interactome and Phase-Specific Phosphorylation in the Neurospora Circadian Clock

Post-translational modifications in circadian rhythms

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