Activation of PPARγ coactivator-1 through transcription factor docking

Science

Volumn 286, Issue 5443, 1999, Pages 1368-1371

  Puigserver, Pere ^a   Adelmant, Guillaume ^a   Wu, Zhidan ^a   Fan, Melina ^a   Xu, Jianming ^b   O'Malley, Bert ^b   Spiegelman, Bruce M ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

^b BAYLOR COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIC AMP RESPONSIVE ELEMENT BINDING PROTEIN; DOCKING PROTEIN; HISTONE ACETYLTRANSFERASE; STEROID RECEPTOR; TRANSCRIPTION FACTOR; CELL RECEPTOR; DNA BINDING PROTEIN; E1A ASSOCIATED P300 PROTEIN; EP300 PROTEIN, MOUSE; HYBRID PROTEIN; NUCLEAR PROTEIN; NUCLEAR RESPIRATORY FACTOR; PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR GAMMA COACTIVATOR 1; PEROXISOME-PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA COACTIVATOR-1; STEROID RECEPTOR COACTIVATOR 1; TRANSACTIVATOR PROTEIN;

ARTICLE; CONFORMATIONAL TRANSITION; ENZYME ACTIVATION; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN DOMAIN; PROTEIN EXPRESSION; PROTEIN PROTEIN INTERACTION; TRANSCRIPTION REGULATION; ANIMAL; BINDING SITE; CELL STRAIN COS1; CHEMISTRY; GENE EXPRESSION REGULATION; GENETIC TRANSCRIPTION; GENETIC TRANSFECTION; METABOLISM; MOUSE; PROTEIN CONFORMATION;

ANIMALS; BINDING SITES; COS CELLS; DNA-BINDING PROTEINS; E1A-ASSOCIATED P300 PROTEIN; GENE EXPRESSION REGULATION; HISTONE ACETYLTRANSFERASES; MICE; NUCLEAR PROTEINS; NUCLEAR RESPIRATORY FACTORS; PROTEIN BINDING; PROTEIN CONFORMATION; RECEPTORS, CYTOPLASMIC AND NUCLEAR; RECOMBINANT FUSION PROTEINS; TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC; TRANSFECTION;

EID: 0032589689     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5443.1368     Document Type: Article

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23. Whole-cell extracts were prepared by lysing the cells in a buffer containing 100 mM tris (pH 8.5), 250 mM NaCl, 1% NP-40, 1 mM EDTA, protease inhibitors (Boehringer Mannheim), and 0.1 mM phenylmethylsulfonyl fluoride. Whole-cell lysates were incubated with monoclonal antibodies against SRC-1 or CBP/ p300 for 2 hours at 4°C, followed by an overnight incubation with protein A/G Sepharose beads. Immunoprecipitates were extensively washed with the lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and protein immunoblot with specific antibodies against GAL4 DBD monoclonal antibodies.
24. Whole-cell lysates were incubated with polyclonal antibodies against GAL4 DBD for 2 hours at 4°C, followed by an overnight incubation with protein A/G Sepharose beads. Immunoprecipitates were washed three times with the lysis buffer and used for HAT assays in solution with histones as substrates [J. E. Brownell and C. D. Allis, Proc. Natl. Acad. Sci. U.S.A. 92, 6364 (1995)].
25. GST-SRC-1 constructs were made as described [T. E. Spencer et al., Nature 389, 194 (1997)]. GST-p300 constructs were made as described in (8). GST pull-down assays were performed as described in (5).
26. GAL4-PGC-1 deletions were performed by ligating PGC-1 polymerase chain reaction (PCR) fragments into Sal I-Eco RV cloning sites of pCMX expression vector. Expression levels for these different deletions in transfected cells were similar. GAL4-p/CAF was performed by ligating p/CAF PCR full-length fragment into Eco RI and Eco RV.
27. 2, 0.05% NP-40, 2 mM dithiothreitol, and 10% glycerol] and kept at -80°C until further use. In vitro binding assays were performed as described in (5), with the same amount of GST fusion proteins (analyzed by Coomassie staining).
28. 3) in a total volume of 30 μl and incubated at 25°C for 10 min. The reaction was stopped by adding SDS-PAGE sample buffer. Digested fragments were analyzed by immunoblotting with a monoclonal antibody to flag (Sigma).
29. We thank M. Montminy, C. Yoon, and M. Monsalve for critical comments and suggestions on the manuscript and M. Wright and A. Troy for technical assistance. Some of the constructs and reagents used in this work were obtained from D. Livingston, Y. Nakatani, R. Goodman, M. Montminy, M. Brown, and R. Scarputla. Supported by a grant from NIH (DK54477 to B.M.S.).