1. Volume 3, Issue 4, Pages 521-526 (April 1999)
Phosphorylation of the Nuclear Receptor SF-1 Modulates Cofactor Recruitment 
Gary D. Hammer, Irina Krylova, Yixian Zhang, Beatrice D. Darimont, Kimberly Simpson, Nancy L. Weigel, Holly A. Ingraham 
Molecular Cell 
Volume 3, Issue 4, Pages (April 1999)
DOI: /S (00)

2. Figure 1
Phosphorylation of Ser-203 Is Essential for Maximal SF-1 Activity
(A) Radiolabeled SF-1 was immunoprecipitated from metabolically labeled cells (32P or 35S) and analyzed by SDS-PAGE. In normal culture conditions, SF-1 is constitutively phosphorylated; treatment with 8-Br-cAMP (1 mM, 4 hr) failed to significantly increase levels of SF-1 phosphorylation (arrowhead). Deletion of the AF-1 domain results in loss of in vivo phosphorylation of SF-1.
(B) JEG-3 cells were transiently cotransfected with SF-1 reporters (0.3 μg) and either wild-type SF-1 or the phosphorylation-defective mutant SF-1S203A. Transcriptional activation by the SF-1 phosphorylation-defective mutant (SF-1S203A) was also compared to the single AF-2 mutant (SF-1-AF-2m4) (Crawford et al. 1997) and a double mutant (SF-1S203A/AF-2m4).
(C) Cellular localization of wild-type and mutant SF-1 proteins was determined by immunocytochemistry with an anti-HA antibody, as previously described (Nachtigal et al. 1998). All SF-1 proteins were expressed equivalently as judged by Western blot analysis (data not shown).
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 2 MAP-Dependent Kinase Phosphorylation and Activation of SF-1
(A) SF-1, but not SF-1S203A, can be phosphorylated in vitro by the mitogen-activated protein kinase Erk2. Purified GST-SF-1 fusion proteins contained either the major portion of the LBD (LBD, aa265–462), wild-type AF-1 domain (AF-1 aa180–265), or mutant AF-1 domain (AF-1S203A). Reactions were carried out with equivalent amounts of each protein as determined by Coomassie blue staining and in vitro phosphorylation of the heart muscle PKA kinase site present in all constructs (C-terminal to GST). Note that the mutant GST-AF-1S203A migrates at a slightly different position in SDS-PAGE analysis because of six extra amino acids at the C terminus.
(B) EGF stimulation increases phosphorylation of SF-1, in vivo. COS cells expressing SF-1 were serum starved or treated with EGF (180 ng/ml), followed by metabolic labeling, immunoprecipitation, and SDS-PAGE. Labeled SF-1 is indicated (32P, arrowhead), and the relative levels of SF-1 expression (SF-1) are shown for each experimental condition.
(C) The MAPK-specific phosphatase (MKP-1) significantly attenuates SF-1 activation. JEG3 cells were transfected with 5X-21OH-LUC reporter (0.3 μg), SF-1 or mutant SF-1S203A (40 ng), and pMPK-1 (40 ng).
(D) Ser-203 is essential for in vivo activation of SF-1 by the constitutively active form of c-Raf-1 kinase. JEG-3 cells were transiently transfected with the 2X-MIS-RE reporter (0.3 μg) with SF-1 or mutant SF-1S203A (40 ng) and increasing amounts c-Raf-1 kinase (RSV-Raf-BXB).
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 3 SF-1 Associates with the Cofactor GRIP1 In Vivo
(A) A mammalian two-hybrid assay was used to probe a potential interaction between GRIP1 and SF-1. JEG-3 cells were cotransfected with the 2xLexA-luciferase reporter (0.3 μg), various LexA fusion protein constructs (160 ng) containing various combinations of the SF-1 domains as depicted, together with pSG5-GRIP1 (40 ng). Luciferase activity is reported as expression relative to levels achieved by LexA-SF-1 expression plasmids alone.
(B) HA epitope–tagged SF-1 proteins (SF-1 and SF-1S203A) from COS whole-cell extracts were visualized by Western analysis following incubation with immobilized GST-GRIP1. The upper panel shows the interaction of GST-GRIP with wild-type SF-1 or the mutant SF-1S203A. The lower panel shows the interaction of increasing amounts of purified GST-GRIP1 (∼3 or 9 μg) with wild-type SF-1 protein extracted from cells stimulated with cRaf (SF1+R) or with the mutant SF-1S203A. Ten percent of the SF-1 protein used in each assay is shown (Input).
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 4 Phosphorylation of SF-1 Increases Interaction with Cofactors
(A) In vitro phosphorylation of SF-1 increases its affinity for both GRIP1 and SMRT. Increasing amounts of phosphorylated (+Erk2) and nonphosphorylated (-Erk2) GST fusion protein (2, 5, and 10 μg, gray triangle) containing the entire AF-1 and LBD (GST-SF-1AF-1/LBD, aa178–462) were incubated with 35S-labeled GRIP1 and SMRT and the amount bound is shown (GRIP1, 22% +Erk2, 11% −Erk2; SMRT, 12% +Erk2, 3% −Erk2). The amount bound to GST alone is shown (open triangle). Similar studies were carried out with the isolated AF-1 domain (GST-SF-1AF-1, aa180–265).
(B) In a modified mammalian two-hybrid assay, JEG-3 cells were cotransfected with the 2XLexA-luciferase reporter (0.3 μg), and LexA-SF-1 constructs consisting of wild-type SF-1, SF-1S203A, or SF-1-AF-2m4 (160 ng), together with the coactivator, pSG5-GRIP1, and the corepressor, pSG5-SMRT (40 ng). Luciferase activity is reported as expression relative to levels achieved by LexA fusion constructs, alone.
(C) Functional interaction between SF-1 and GRIP1/SMRT requires a phosphorylated SF-1. JEG-3 cells were transiently cotransfected with the 5X-21OH-LUC reporter (0.3 μg) and wild-type SF-1 or SF-1S203A (100 ng) together with increasing amounts of the pSG5-GRIP1 (left panel) or pSG5-SMRT (right panel) vectors (0, 20, 50, and 100 ng).
Molecular Cell 1999 3, DOI: ( /S (00) )