A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation

Abstract

The circadian clock regulates biological processes from gene expression to organism behavior in a precise, sustained rhythm that is generated at the unicellular level by coordinated function of interlocked transcriptional feedback loops and post-translational modifications of core clock proteins. CLOCK phosphorylation regulates transcriptional activity, cellular localization and stability; however little is known about the specific residues and enzymes involved. We have identified a conserved cluster of serines that include, Ser431, which is a prerequisite phosphorylation site for the generation of BMAL dependent phospho-primed CLOCK and for the potential GSK-3 phosphorylation at Ser427. Mutational analysis and protein stability assays indicate that this serine cluster functions as a phospho-degron. Through the use of GSK-3 activators/inhibitors and kinase assays, we demonstrate that GSK-3β regulates the degron-site by increasing CLOCK phosphorylation/degradation, which correlates with an increase in the expression of CLOCK responsive promoters. Stabilization of phospho-deficient CLOCK delays the phase of oscillation in synchronized fibroblasts. This investigation begins the characterization of a complex phospho-regulatory site that controls the degradation of CLOCK, a core transcription factor that is essential for circadian behavior.

The Mammalian Circadian Clock Protein Period Counteracts Cryptochrome in Phosphorylation Dynamics of Circadian Locomotor Output Cycles Kaput (CLOCK)

“…First, BMAL1-dependent phosphorylation of CLOCK may be required for nuclear translocation of the BMAL1-CLOCK dimer (4). Second, phosphorylated CLOCK (pCLOCK) may be an active form as a transcription factor and an unstable form that leads to degradation (5). Third, in contrast to the report mentioned above, CLOCK may become transcriptionally inactive by phosphorylation (6).…”

Core circadian protein CLOCK is a positive regulator of NF-κB–mediated transcription

“…At the same time, many functional characteristics of CLOCK are regulated by BMAL1, which is subject to strong circadian regulation at multiple levels. Thus, BMAL1 mediates the nuclear translocation (17,33) and site-specific phosphorylation/degradation of CLOCK (18), both of which are tightly linked to the activation of responsive circadian promoters. These findings allow us to speculate that the sharp peak in CLOCK-mediated upregulation of NF-κB-responsive promoters observed at ZT6 may coincide with the daily peak in BMAL1-dependent shuttling of CLOCK into the nucleus, thereby generating an abundant pool of "active nuclear CLOCK" available for cooperation with other transcriptional regulators on noncircadian promoters.…”

“…3 A and D). This phenomenon may be attributed to BMAL1-induced phosphorylation and proteolytic degradation of CLOCK (17,18), which may underlie the observed repression of the modulation function of CLOCK on κB-responsive promoters in the presence of BMAL1 ( Fig. 2A).…”

“…A β-gal expression plasmid driven by the TNF-nonresponsive RSV promoter was used to normalize transfection efficiency. When cells were treated with TNF-α (Sigma-Aldrich), it was added 18-24 h posttransfection for 5-6 h. Cells were harvested in Promega Reporter Lysis buffer and analyzed for luciferase and β-gal activity as previously described (18). Results are representative of at least three experiments performed in duplicate.…”

“…HA-tagged proteins were detected with mouse anti-HA.11 (Covance Research Products). Anti-CLOCK and anti-BMAL1 were raised in guinea pig as previously described (18). Antibodies against pSer535-p65 and Ac310-p65 were purchased from Cell Signaling.…”