Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-alpha: regulation by phosphorylation sites in the A/B region depends on other receptor domains.

doi: 10.1210/me.2001-0316

Figure Lengend Snippet: Fig. 4. Amino-Terminal Phosphorylation of Full-Length ER Decreases Its Ligand-Independent, Physical Interactions with p160s and CBP A, CBP and p160s can interact physically with unliganded ER in a mammalian two-hybrid assay. B, Quantitation of the effects of the S104/106/118A mutation in ER on its interactions with p160s and CBP. C, Effects of the S118A and S104/106A mutations alone and in combination on ER interactions with SRC-1e. HeLa cells were transfected with 500 ng of expression vector for VP16 or VP16-ER (wt or mutants), along with 100 ng of GAL4 or GAL4-coactivator, and 1000 ng pG5-Luc. In all experiments, duplicate samples were examined, and luciferase activity in cell extracts was normalized to protein amounts. In A, which shows a representative experiment, the activity of VP16-ER wt in the absence of coactivator was set as 100. In B and C, for each ER mutant the activity was expressed as a percentage of that observed for wt ER with the same coactivator, and data are presented as the average SEM of at least three experiments. D, The S118A and S104/106A mutations do not reduce VP16-ER expression. Extracts from HeLa cells transfected with 500 ng of plasmid for VP16 or VP16-ER (wt or mutants), along with 1000 ng of pG5-Luc were analyzed by Western blot using the H222 antibody. E, ER dephosphorylation reduces its ligand-independent interactions with SRC-1 and CBP in co-IP assays. Recombinant human ER was preincubated in -PPase buffer either in the absence (lanes 3 and 8) or presence of -PPase (lanes 4 and 9) or E2 (lanes 2 and 7), before its mixing with HeLa cell extract and co-IP with antibodies against SRC-1 or CBP. An aliquot of ER preincubated without -PPase or E2 was mixed with HeLa cell extract and -PPase before co-IP (lanes 5 and 10). Note that all IP were carried out in the presence of phosphatase inhibitors. Aliquots of input (1%; top) and IP supernatants (1%; middle) and eluates (15%; bottom) were analyzed together by Western blot with antibodies against total ER (left) or phosphoserine118 ER (ER-118P; right), as indicated. All blots analyzed with the same antibody were exposed for the same amount of time to allow comparison of IP samples with input. A lower exposure is also shown for eluates from IP with the CBP antibody.

Article Snippet: Preincubations were stopped on ice with 9 vol of lysis buffer 1 [50 mM HEPES (pH 7.5), 100 mM KCl, 0.2 mM EDTA, 0.1% Nonidet P-40, protease inhibitor cocktail (Complete, Roche Applied Science, Indianapolis, IN)] supplemented with phosphatase inhibitors (0.1 mM Na vanadate, 10 mM Na molybdate, 20 mM NaF, 0.3 mM 1,10-phenanthroline, 50 mM sodium -glycerophosphate, 3.8 nM sodium p-nitrophenyl phosphate), aliquots were taken for Western blot analysis, and the remainder was mixed with HeLa cell extract (0.5 mg proteins in lysis buffer 1), protein G plus/agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and either anti-SRC-1 (Genetex, San Antonio, TX) or anti-CBP (C-1, Santa Cruz Biotechnology, Inc.) antibody for 1 h at 4 C, in a total volume of 1 ml lysis buffer 1 containing phosphatase inhibitors as described above.

Techniques: Phospho-proteomics, Two Hybrid Assay, Quantitation Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, De-Phosphorylation Assay, Co-Immunoprecipitation Assay, Recombinant, Comparison