Protein phosphatase 1 regulates the stability of the circadian protein PER2.
The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKI (casein kinase I ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKI-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.
Gallego
Monica
M
Center for Children, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.
Kang
Heeseog
H
Virshup
David M
DM
D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002940:Circadian Rhythm; D006801:Humans; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D056950:Period Circadian Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D014157:Transcription Factors / Q000378:metabolism*; D014981:Xenopus
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C421902:PER2 protein, human; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D014157:Transcription Factors; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1