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Molecular and Cellular Biology, July 2009, p. 3675-3686, Vol. 29, No. 13
0270-7306/09/$08.00+0 doi:10.1128/MCB.01864-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Roles of CLOCK Phosphorylation in Suppression of E-Box-Dependent Transcription
Hikari Yoshitane,1
Toshifumi Takao,2
Yoshinori Satomi,2,
Ngoc-Hien Du,1
Toshiyuki Okano,1,
and
Yoshitaka Fukada1*
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan,1 Institute for Protein Research, Osaka University, Yamada-Oka 3-2, Suita, Osaka 565-0871, Japan2
Received 6 December 2008/ Returned for modification 21 January 2009/ Accepted 15 April 2009
In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCK
19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.
* Corresponding author. Mailing address: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-5841-4381. Fax: 81-3-5802-8871. E-mail: sfukada{at}mail.ecc.u-tokyo.ac.jp
Published ahead of print on 4 May 2009.
Present address: Pharmaceutical Research Division, Discovery Research Center, Takeda Pharmaceutical Company Ltd., 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.
Present address: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Japan, and PRESTO, Japan Science and Technology Agency, Saitama, Japan.
Molecular and Cellular Biology, July 2009, p. 3675-3686, Vol. 29, No. 13
0270-7306/09/$08.00+0 doi:10.1128/MCB.01864-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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