Ser-557-phosphorylated mCRY2 is degraded upon synergistic phosphorylation by glycogen synthase kinase-3 beta.
Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.
Harada
Yuko
Y
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
Sakai
Mihoko
M
Kurabayashi
Nobuhiro
N
Hirota
Tsuyoshi
T
Fukada
Yoshitaka
Y
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002940:Circadian Rhythm / Q000502:physiology; D056931:Cryptochromes; D005420:Flavoproteins / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D008099:Liver / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D056931:Cryptochromes; D005420:Flavoproteins; D012694:Serine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3