Article

CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in drosophila

Howard Hughes Medical Institute.
Genes & development (Impact Factor: 10.8). 11/2012; 26(22):2536-49. DOI: 10.1101/gad.200584.112
Source: PubMed

ABSTRACT

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity. Previous work indicates that repression is due to a direct PER-CLK/CYC interaction as well as CLK/CYC phosphorylation. We describe here the role of ubiquitin-specific protease 8 (USP8) in circadian transcriptional repression as well as the importance of CLK ubiquitylation in CLK/CYC transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances CLK/CYC transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, CLK ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal CLK/CYC transcription. As USP8 interacts with CLK and expression of USP8-DN increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK/CYC transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by CLK/CYC, USP8, like PER and TIM, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms.

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    • "We confirmed that CG14619, which is a CCG[37,38], is the orthologue of mUsp2 by protein sequence alignments (S1 Fig) andthat dUsp2-kd suffer from sub viability ([33]; Fig 2C–2F,S1 and S2 Files). Interestingly, dUsp2 and dUsp8, which is a core clock cogwheel in Drosophila are paralogues and possibly differentially evolved towards output effector and core cogwheel status, respectively[36,48]. We found no significant alteration of the circadian free-running period in both clock-neuron specific knockdown of dUsp2 in Drosophila and in our Usp2-KO mouse (Fig 1, Fig 2A and 2B, Table 1). "
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    • "In addition to DBT, kinases known to be involved in CLK phosphorylation include NEMO (NMO), which destabilizes CLK (Yu et al., 2011), and CK2a that stabilizes CLK and inhibits its transcriptional activity (Szabó et al., 2013). CLK is also regulated by ubiquitylation with the HECT-domain ubiquitin ligase CTRIP destabilizing CLK (Lamaze et al., 2011), and the USP8 ubiquitin protease decreasing its transcriptional activity (Luo et al., 2012). Finally, CLK activity is controlled by repressors/activators such as CLOCKWORK ORANGE (CWO) (Matsumoto et al., 2007; Kadener et al., 2007; Lim et al., 2007; Richier et al., 2008) and the FOS ortholog KAYAK (KAY) (Ling et al., 2012). "
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    ABSTRACT: In the Drosophila circadian oscillator, the CLOCK/CYCLE complex activates transcription of period (per) and timeless (tim) in the evening. PER and TIM proteins then repress CLOCK (CLK) activity during the night. The pace of the oscillator depends upon post-translational regulation that affects both positive and negative components of the transcriptional loop. CLK protein is highly phosphorylated and inactive in the morning, whereas hypophosphorylated active forms are present in the evening. How this critical dephosphorylation step is mediated is unclear. We show here that two components of the STRIPAK complex, the CKA regulatory subunit of the PP2A phosphatase and its interacting protein STRIP, promote CLK dephosphorylation during the daytime. In contrast, the WDB regulatory PP2A subunit stabilizes CLK without affecting its phosphorylation state. Inhibition of the PP2A catalytic subunit and CKA downregulation affect daytime CLK similarly, suggesting that STRIPAK complexes are the main PP2A players in producing transcriptionally active hypophosphorylated CLK. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · May 2015 · Cell Reports
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    • "Instead, USP2 appears to regulate PER1 intracellular localization (Yang et al., 2014). Interestingly, the only other DUB that to our knowledge has been implicated in clock mechanisms, Drosophila USP8, also seems to act in a non-degradative manner: it deubiquitinates CLOCK, thereby inhibiting transcriptional activity of CLOCK/CYCLE (CYCLE is the Drosophila homolog of BMAL1; Luo et al., 2012). "
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