A role for O-GlcNAcylation in setting circadian clock speed

Kim, E.Y.,Jeong, E.H.,Park, S.,Jeong, H.-J.,Edery, I.,Cho, J.W. CSH COLD SPRING HARBOR LABORATORY PRESS 2012 Genes & development Vol.26 No.5

Two Distinct Modes of PERIOD Recruitment onto dCLOCK Reveal a Novel Role for TIMELESS in Circadian Transcription.

Sun, Woo Chul,Jeong, Eun Hee,Jeong, Hyun-Jeong,Ko, Hyuk Wan,Edery, Isaac,Kim, Eun Young The Society 2010 The Journal of neuroscience Vol.30 No.43

<P>Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dCLOCK (dCLK)-CYCLE (CYC) transcriptional activity by dPERIOD (dPER) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPER function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPER interacts with dCLK-CYC to promote repression is not known. Here, we identified a small conserved region on dPER required for binding to dCLK, termed CBD (for dCLK binding domain). In the absence of the CBD, dPER is unable to stably associate with dCLK and inhibit the transcriptional activity of dCLK-CYC in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the DOUBLETIME kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPER missing the CBD (dPER(?CBD)) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPER(?CBD) and dCLK is TIM-dependent and modulated by light, revealing a novel and unanticipated in vivo role for TIM in circadian transcription. Finally, dPER(?CBD) does not provoke the daily hyperphosphorylation of dCLK, indicating that direct interactions between dPER and dCLK are necessary for the dCLK phosphorylation program but are not required for other aspects of dCLK regulation.</P>

A Hierarchical Phosphorylation Cascade That Regulates the Timing of PERIOD Nuclear Entry Reveals Novel Roles for Proline-Directed Kinases and GSK-3{beta}/SGG in Circadian Clocks.

Ko, Hyuk Wan,Kim, Eun Young,Chiu, Joanna,Vanselow, Jens T,Kramer, Achim,Edery, Isaac The Society 2010 The Journal of neuroscience Vol.30 No.38

<P>The daily timing of when PERIOD (PER) proteins translocate from the cytoplasm to the nucleus is a critical step in clock mechanisms underpinning circadian rhythms in animals. Numerous lines of evidence indicate that phosphorylation plays a prominent role in regulating various aspects of PER function and metabolism, including changes in its daily stability and subcellular distribution. In this report, we show that phosphorylation of serine 661 (Ser661) by a proline-directed kinase(s) is a key phospho-signal on the Drosophila PER protein (dPER) that regulates the timing of its nuclear accumulation. Mutations that block phosphorylation at Ser661 do not affect dPER stability but delay its nuclear entry in key pacemaker neurons, yielding longer behavioral rhythms. Intriguingly, abolishing phosphorylation at Ser661 also attenuates the extent of dPER hyperphosphorylation in vivo, suggesting the phosphorylated state of Ser661 regulates phosphorylation at other sites on dPER. Indeed, we identify Ser657 as a site that is phosphorylated by the glycogen synthase kinase GSK-3관 (SHAGGY; SGG) in a manner dependent on priming at Ser661. Although not as dramatic as mutating Ser661, mutations that abolish phosphorylation at Ser657 also lead to longer behavioral periods, suggesting that a multi-kinase hierarchical phosphorylation module regulates the timing of dPER nuclear entry. Together with evidence in mammalian systems, our findings implicate proline-directed kinases in clock mechanisms and suggest that PER proteins are key downstream targets of lithium therapy, a potent inhibitor of GSK-3관 used to treat manic depression, a disorder associated with clock malfunction in humans.</P>

Phosphorylation of a Central Clock Transcription Factor Is Required for Thermal but Not Photic Entrainment

Lee, Euna,Jeong, Eun Hee,Jeong, Hyun-Jeong,Yildirim, Evrim,Vanselow, Jens T.,Ng, Fanny,Liu, Yixiao,Mahesh, Guruswamy,Kramer, Achim,Hardin, Paul E.,Edery, Isaac,Kim, Eun Young Public Library of Science 2014 PLoS genetics Vol.10 No.8

<▼1><P>Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the <I>Drosophila</I> circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a <I>Drosophila</I> tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of <I>Clk</I><SUP>out</SUP> flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.</P></▼1><▼2><P><B>Author Summary</B></P><P>Circadian clocks are synchronized to local time by daily cycles in light-dark and temperature. Although light is generally thought to be the most dominant entraining cue in nature, daily cycles in temperature are sufficient to synchronize clocks in a large range of organisms. In <I>Drosophila</I>, dCLOCK is a master circadian transcription factor that drives cyclical gene expression and is likely the rate-limiting component in the transcriptional/translational feedback loops that underlie the timekeeping mechanism. dCLOCK undergoes temporal changes in phosphorylation throughout a day, which is also observed for mammalian CLOCK. However, the role of CLOCK phosphorylation at the organismal level is still unclear. Using mass-spectrometry, we identified more than a dozen phosphorylation sites on dCLOCK. Blocking global phosphorylation of dCLOCK by mutating phospho-acceptor sites to alanine increases its abundance and transcriptional activity, leading to higher peak values and amplitudes in the mRNA rhythms of core clock genes, which likely explains the accelerated clock speed. Surprisingly, the clock-controlled daily activity rhythm fails to maintain synchrony with daily temperature cycles, although there is no observable defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the effective strengths of entraining modalities by adjusting clock amplitude.</P></▼2>

Phosphorylation of the Transcription Activator CLOCK Regulates Progression through a ∼24-h Feedback Loop to Influence the Circadian Period in <i>Drosophila</i>

Mahesh, Guruswamy,Jeong, EunHee,Ng, Fanny S.,Liu, Yixiao,Gunawardhana, Kushan,Houl, Jerry H.,Yildirim, Evrim,Amunugama, Ravi,Jones, Richard,Allen, David L.,Edery, Isaac,Kim, Eun Young,Hardin, Paul E. American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.28

<P>Circadian (≅24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In <I>Drosophila</I>, these clocks keep circadian time via transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) initiates transcription of <I>period</I> (<I>per</I>) and <I>timeless</I> (<I>tim</I>), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new <I>Clk</I> null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.</P>