A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation.
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-3beta 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 activity and degradation of CLOCK, a core transcription factor that is essential for circadian behavior.
Spengler
Mary L
ML
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY, USA.
Kuropatwinski
Karen K
KK
Schumer
Molly
M
Antoch
Marina P
MP
D056930:ARNTL Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001683:Biological Clocks / Q000502:physiology; D056926:CLOCK Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002940:Circadian Rhythm / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D012694:Serine / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
D056930:ARNTL Transcription Factors; C538961:Bmal1 protein, mouse; D014157:Transcription Factors; D012694:Serine; D056926:CLOCK Proteins; C538944:Clock protein, mouse; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3