Phosphorylation of clock protein PER1 regulates its circadian degradation in normal human fibroblasts.
Recent advances suggest that the molecular components of the circadian clock generate a self-sustaining transcriptional-translational feedback loop with a period of approx. 24 h. The precise expression profiles of human clock genes and their products have not been elucidated. We cloned human clock genes, including per1, per2, per3, cry2 and clock, and evaluated their circadian mRNA expression profiles in WI-38 fibroblasts stimulated with serum. Transcripts of hPer1, hPer2, hPer3, hBMAL1 and hCry2 (where h is human) underwent circadian oscillation. Serum-stimulation also caused daily oscillations of hPER1 protein and the apparent molecular mass of hPER1 changed. Inhibitor studies indicated that the CKI (casein kinase I) family, including CKIepsilon and CKIdelta, phosphorylated hPER1 and increased the apparent molecular mass of hPER1. The inhibition of hPER1 phosphorylation by CKI-7 [ N -(2-aminoethyl)-5-chloro-isoquinoline-8-sulphonamide], a CKI inhibitor, disturbed hPER1 degradation, delayed the nuclear entry of hPER1 and allowed it to persist for longer in the nucleus. Furthermore, proteasome inhibitors specifically blocked hPER1 degradation. However leptomycin B, an inhibitor of nuclear export, did not alter the degradation state of hPER1 protein. These findings indicate that circadian hPER1 degradation through a proteasomal pathway can be regulated through phosphorylation by CKI, but not by subcellular localization.
Miyazaki
Koyomi
K
Clock Cell Biology Group, IBRF (Institute for Biological Resource and Function), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba central 6, 1-1-1, Higashi, Tsukuba, 305-8566, Japan.
Nagase
Takahiro
T
Mesaki
Miho
M
Narukawa
Junko
J
Ohara
Osamu
O
Ishida
Norio
N
D056930:ARNTL Transcription Factors; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D047388:Casein Kinases; D018797:Cell Cycle Proteins; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002940:Circadian Rhythm / Q000235:genetics*; D003001:Cloning, Molecular; D056931:Cryptochromes; D016895:Culture Media, Serum-Free; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005420:Flavoproteins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D056950:Period Circadian Proteins; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D047388:Casein Kinases; D018797:Cell Cycle Proteins; D056931:Cryptochromes; D016895:Culture Media, Serum-Free; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D005231:Fatty Acids, Unsaturated; D005420:Flavoproteins; D007546:Isoquinolines; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D046988:Proteasome Endopeptidase Complex; D011494:Protein Kinases; D011506:Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; C538966:CRY2 protein, human; C538967:Cry2 protein, mouse; C539022:PER1 protein, human; C421902:PER2 protein, human; C512644:PER3 protein, human; C539023:Per1 protein, mouse; C512645:Per3 protein, mouse; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; C538943:CLOCK protein, human; C538944:Clock protein, mouse; C038753:leptomycin B