Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.
The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.
Eide
Erik J
EJ
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.
Woolf
Margaret F
MF
Kang
Heeseog
H
Woolf
Peter
P
Hurst
William
W
Camacho
Fernando
F
Vielhaber
Erica L
EL
Giovanni
Andrew
A
Virshup
David M
DM
D000818:Animals; D048149:Casein Kinase 1 epsilon / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002940:Circadian Rhythm / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056950:Period Circadian Proteins; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C421902:PER2 protein, human; C513312:Per2 protein, mouse; C513311:Per2 protein, rat; D056950:Period Circadian Proteins; D014157:Transcription Factors; D044844:beta-Transducin Repeat-Containing Proteins; D044767:Ubiquitin-Protein Ligases; D048149:Casein Kinase 1 epsilon; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease