Negative Control of Circadian Clock Regulator E4BP4 by Casein Kinase Iϵ-Mediated Phosphorylation

Supplemental Experimental Procedures radish peroxidase-conjugatged anti-mouse immunoglobulin (KirPlasmids and Recombinant Proteins kegaard & Perry Laboratories; 1:5000 dilution). To generate Myc-tagged E4BP4, the open reading frame (ORF) of cE4BP4 was cloned into pCS2 MT [S1]. Mutant forms of Myctagged E4BP4 were created by using the QuickChange site-directed In Vitro Kinase Assay mutagenesis kit (Stratagene). An expression construct for MycMyc-tagged proteins were expressed in LMH cells and immunopretagged mPER2 was a generous gift from Louis J. Ptacek (University cipitated with 2 g of anti-Myc 9E10 antibody and 15 l of Protein of Utah, Salt Lake City) [S2]. Expression constructs for CK1 and its G-Sepharose beads. The beads were washed three times with RIPA kinase negative form, CK1 (KN), were generous gifts from Katsuya buffer and divided equally into three aliquots, which were incubated Nagai (Osaka University, Osaka, Japan) [S3]. Flag-tagged CK1 or for 30 min at 37 C in 20 l of phosphatase buffer in the presence CK1 (KN) was generated by cloning the ORF of CK1 or CK1 (KN) or absence of 5 units of CIP. Then the beads were washed three into pCMV-Tag2B (Stratagene), respectively. Glutathione S-transtimes with kinase buffer (30 mM HEPES-KOH [pH 7.8], 7 mM MgCl2 ferase (GST)-fused CK1 or CK1 (KN) was generated by cloning the 50 g/ml of bovine serum albumin, 25 M ATP, and 0.5 mM dithiORF of CK1 or CK1 (KN) into pGEX-2T (Amersham Biosciences), othreitol), and the washed beads were incubated for 30 min at 37 C respectively. A truncated form of CK1 or CK1 (KN) was created by in 25 l of kinase buffer containing 5 Ci of [ -P]ATP and 25 ng introducing a stop codon after the amino acid 320 by using the of either GST-CK1 320 or GST-CK1 (KN) 320. The truncated form QuickChange site-directed mutagenesis kit (Stratagene). GST-fused of CK1 ( 320: lacking the carboxyl terminal autoinhibitory domain) protein was expressed in Escherichia coli cells (BL21) and purified was utilized for avoiding the kinase autoinhibition otherwise seen on glutathione-Sepharose beads as described [S4]. in vitro [S8, S9]. After the phosphorylation reaction, the beads were washed three times with RIPA buffer, and proteins were eluted with Laemmli sample buffer. Nuclear Protein Preparation and Phosphatase Treatment Animals were treated in accordance with the guidelines of the University of Tokyo. Newly hatched chicks were housed under various light/dark conditions with a light intensity of 300 lux at the level Coimmunoprecipitation of chicks. Pineal glands were isolated from the decapitated chicks, Myc-E4BP4 or Myc-mPER2 was expressed in LMH cells together and the nuclear proteins were extracted as described [S5]. For the with either Flag-CK1 or Flag-CK1 (KN). The cells were lysed with phosphatase treatment, the nuclear extract (10 g proteins) was a hypertonic buffer containing 0.6 M NaCl in IP buffer (50 mM Trisincubated for 60 min at 37 C in 80 l of phosphatase buffer (20 mM HCl [pH 8.0], 10% glycerol, 2 mM EDTA, 1 mM dithiothreitol, 1% Tris-HCl [pH 7.8], 1 mM dithiothreitol, 0.1 mM EDTA, 2 mM MgCl2, Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, 1 g/ml 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 mM phenylmethylsulfonyl leupeptin, 1 g/ml aprotinin and 1 mM phenylmethylsulfonyl fluofluoride) with 5 units of calf intestinal alkaline phosphatase (CIP; ride). The cell lysate was centrifuged at 12,000 g for 30 min, and New England Biolabs) or with heat-treated (at 95 C for 30 min) CIP the supernatant was diluted 4-fold with IP buffer and then subjected in the presence or absence of 50 mM sodium phosphate. The phosto immunopreciptation with anti-Flag M2 antibodies and Protein phatase reaction was terminated by the addition of Laemmli sample G-Sepharose beads. The beads were washed three times with IP buffer [S6]. buffer containing 150 mM NaCl, and the proteins precipitated with the beads were eluted with Laemmli sample buffer. Cell Culture and Transfection LMH chicken hepatoma cells were grown in Waymouth’s MB752/1 Cell Staining medium (Life Technologies) supplemented with 10% fetal bovine LMH cells growing on chamber slides were fixed with 4% formaldeserum. The cells were transfected with various plasmids by using hyde and then permeabilized with 0.05% Triton X-100 in phosphateLipofectamine 2000 (Life Technologies) according to the manufacbuffered saline (PBS, Dulbecco’s; Gibco). After incubated in turer’s protocol and harvested 36 hr after the transfection. For inhibiblocking buffer (5% bovine serum albumin in PBS) for 30 min at tion of the proteasomal protein degradation machinery, MG132 (Cal25 C, the cells on the slide were incubated for 12 hr at 4 C with biochem; final concentration 25 M) was added to the culture anti-Myc 9E10 antibody diluted in the blocking buffer (1:1000 dilumedium 6 hr before the harvest. The harvested cells were lysed in RIPA buffer (50 mM Tris-HCl [pH8.0], 100 mM NaCl, 2 mM EDTA, tion). They were rinsed with PBS and then incubated for 1 hr at 25 C 1 mM dithiothreitol, 1% Nonidet P-40, 1% sodium deoxycholate, with Cy3-conjugated anti-mouse IgG antibody (Jackson Immuno 0.1% sodium dodecyl sulfate (SDS), 1 g/ml leupeptin, 1 g/ml Research Laboratories) diluted in the blocking buffer (1:1000 diluaprotinin, and 1 mM phenylmethylsulfonyl fluoride), and the cell tion). The cells were counterstained with 4 , 6-diamidino-2-phenylinlysates were subjected to immunoblot analyses. dole (DAPI; Boehringer) before microscopic observation.