Advertisement

Phosphorylation and Inhibition of Rat Glucocorticoid Receptor Transcriptional Activation by Glycogen Synthase Kinase-3 (GSK-3)

SPECIES-SPECIFIC DIFFERENCES BETWEEN HUMAN AND RAT GLUCOCORTICOID RECEPTOR SIGNALING AS REVEALED THROUGH GSK-3 PHOSPHORYLATION*
  • Inez Rogatsky
    Footnotes
    Affiliations
    From the Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
    Search for articles by this author
  • Carine L.M. Waase
    Affiliations
    From the Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
    Search for articles by this author
  • Michael J. Garabedian
    Correspondence
    To whom correspondence should be addressed: Dept. of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-7662; Fax: 212-263-8276;
    Affiliations
    From the Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by Army Breast Cancer Research Fund Grants DAMD17-94-J-4454 and DAMD17-96-1-6032 (to M. J. G.) and the Irma T. Hirschl Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a National Institutes of Health Training Grant 5T32AI07180-17.
Open AccessPublished:June 05, 1998DOI:https://doi.org/10.1074/jbc.273.23.14315
      Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3)in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation.
      Glucocorticoid hormones control cellular proliferation and metabolism through their association with the glucocorticoid receptor (GR),
      The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; Cdk, cyclin-dependent kinases; GSK, glycogen synthase kinase; ER, estrogen receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; wt, wild type.
      1The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; Cdk, cyclin-dependent kinases; GSK, glycogen synthase kinase; ER, estrogen receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; wt, wild type.
      a member of the intracellular receptor superfamily of transcriptional regulatory proteins (
      • Yamamoto K.R.
      ). Upon glucocorticoid binding, GR enters the nucleus, associates with specific DNA sequences termed glucocorticoid response elements (GREs), and increases transcriptional initiation from nearby promoters. GR can also repress transcription mediated by the heterodimeric AP1 transcription factor complex (c-Jun and c-Fos) (
      • Miner J.N.
      • Diamond M.I.
      • Yamamoto K.R.
      ). Although glucocorticoids act as the primary signal in activating GR's transcriptional regulatory functions, GR-mediated transcriptional activation is also modulated by phosphorylation (
      • Bodwell J.E.
      • Hu J.M.
      • Orti E.
      • Munck A.
      ,
      • Garabedian M.J.
      • Rogatsky I.
      • Hittelman A.
      • Knoblauch R.
      • Trowbridge J.M.
      • Krstic M.D.
      ,
      • Orti E.
      • Mendel D.B.
      • Munck A.
      ).
      Rat GR isolated from cultured mammalian cells or ectopically expressed in yeast (Saccharomyces cerevisiae) is phosphorylated on four major residues (
      • Krstic M.D.
      Functional Analysis of Glucocorticoid Receptor Phosphorylation.
      ). These sites cluster to the N-terminal transcriptional regulatory domain and include threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246) (Fig.1 A). Each of these residues is followed by a proline, thereby forming a motif phosphorylated by a family of serine/threonine-proline-directed kinases that includes the cyclin-dependent kinases (Cdk), the mitogen-activated protein kinases, and glycogen synthase kinase-3 (GSK-3). Differential phosphorylation at these sites both positively and negatively regulate GR transcriptional activation. Positive regulation is accomplished by cyclin-Cdk complexes: cyclin E-Cdk2 phosphorylates Ser224, while cyclin A-Cdk2 phosphorylates both Ser224 and Ser232. Mutations at these sites, or of particular Cdk genes in yeast, reduce GR-dependent transcriptional activation, suggesting that phosphorylation of Ser224 and Ser232 is required for full GR transcriptional enhancement (
      • Krstic M.D.
      • Rogatsky I.
      • Yamamoto K.R.
      • Garabedian M.J.
      ). In contrast, phosphorylation of Ser246 by c-Jun N-terminal kinase, a member of the mitogen-activated protein kinases family, inhibits GR transcriptional activation (
      • Rogatsky I.
      • Logan S.K.
      • Garabedian M.J.
      ).
      Figure thumbnail gr1
      Figure 1Phosphorylation of the rat GR by GSK-3in vitro. A, schematic representation of the major residues phosphorylated on the rat GR (Thr171, Ser224, Ser232, and Ser246), the sequences surrounding the receptor phosphorylation sites and the protein kinases that target the sites: Cdk phosphorylate Ser224 and Ser232 while the c-Jun N-terminal kinase (JNK) phosphorylates Ser246.B, GST fusion proteins containing rat GR amino acid residues 106 through 318 (GST-GR), c-Jun amino acids 1 through 223 (GST-cJun), and estrogen receptor residues 1–121 (GST-ER) were expressed in E. coli, purified by affinity chromatography and phosphorylated in vitro by purified rabbit GSK-3 as described under “Experimental Procedures.” Products were fractionated by SDS-PAGE, stained with Coomassie Blue to visualize the protein substrates (Coomassie; bottom panel), dried and exposed to film (autoradiogram;top panel).
      The remaining GR phosphorylation site, Thr171, also resides in a motif recognized by serine/threonine-proline-directed kinases. However, our previous studies indicate that neither the Cdks, nor c-Jun N-terminal kinase efficiently phosphorylate Thr171 in vitro. Furthermore, phosphorylation of Thr171 is evident in both serum-deprived quiescent and serum-stimulated proliferating cells (
      • Rogatsky I.
      • Logan S.K.
      • Garabedian M.J.
      ), suggesting that Cdks and c-Jun N-terminal kinase are unlikely to phosphorylate Thr171 in vivo, since these kinases are largely inactive in serum-starved, nonproliferating cells. GSK-3, on the other hand, is active throughout the cell cycle, as well as in serum-deprived cells (
      • Woodgett J.R.
      ). Thus, GSK-3 may represent the GR kinase that phosphorylates Thr171.
      GSK-3 was originally isolated as the kinase that phosphorylates glycogen synthase, the rate-limiting enzyme of glycogen synthesis (
      • Plyte S.E.
      • Hughes K.
      • Nikolakaki E.
      • Pulverer B.J.
      • Woodgett J.R.
      ). Two mammalian GSK-3 isoforms have been identified (GSK-3α and GSK-3β) that are 85% homologous at the level of primary amino acid sequence, and share substrate specificity (
      • Welsh G.I.
      • Wilson C.
      • Proud C.G.
      ). GSK-3 is conserved throughout evolution, with homologues present in yeast (S. cerevisiae and Schizosaccharomyces pombe) (
      • Puziss J.W.
      • Hardy T.A.
      • Johnson R.B.
      • Roach P.J.
      • Hieter P.
      ,
      • Plyte S.E.
      • Feoktistova A.
      • Burke J.D.
      • Woodgett J.R.
      • Gould K.L.
      ),Dictyostelium discoideum (
      • Hughes K.
      • Pulverer B.J.
      • Theocharous P.
      • Woodgett J.R.
      ), Drosophila melanogaster (
      • Ruel L.
      • Bourouis M.
      • Heitzler P.
      • Pantesco V.
      • Simpson P.
      ,
      • Siegfried E.
      • Chou T.B.
      • Perrimon N.
      ), and Xenopus laevis (
      • He X.
      • Saint-Jeannet J.P.
      • Woodgett J.R.
      • Varmus H.E.
      • Dawid I.B.
      ,
      • Dominguez I.
      • Itoh K.
      • Sokol S.Y.
      ,
      • Pierce S.B.
      • Kimelman D.
      ).
      Recent studies in Dictyostelium, Xenopus, andDrosophila have implicated GSK-3 in pathways other than glycogen metabolism. GSK-3 has been implicated in cell fate determination and differentiation through its ability to phosphorylate and regulate factors involved in cellular proliferation including CREB, c-Myc, c-Jun, and β-catenin (
      • Plyte S.E.
      • Hughes K.
      • Nikolakaki E.
      • Pulverer B.J.
      • Woodgett J.R.
      ,
      • Nikolakaki E.
      • Coffer P.J.
      • Hemelsoet R.
      • Woodgett J.R.
      • Defize L.H.
      ,
      • Pulverer B.J.
      • Fisher C.
      • Vousden K.
      • Littlewood T.
      • Evan G.
      • Woodgett J.R.
      ,
      • Fiol C.J.
      • Williams J.S.
      • Chou C.H.
      • Wang Q.M.
      • Roach P.J.
      • Andrisani O.M.
      ,
      • de Groot R.P.
      • Auwerx J.
      • Bourouis M.
      • Sassone-Corsi P.
      ). Although GSK-3 has no known activators, its activity in cultured cells can be increased through overexpression. GSK-3 enzymatic activity is, however, negatively regulated by protein kinase B/Akt, an enzyme that phosphorylates and inhibits GSK-3 (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). Akt is, in turn, activated through an association with lipid products generated by phosphatidylinositol-3 kinase at the cell membrane and through phosphorylation (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Marte B.M.
      • Downward J.
      ). The phosphatidylinositol-3 kinase-Akt pathway is induced in response to insulin, insulin-like growth factor, epidermal growth factor, and other mitogens (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ). Recently, the phosphatidylinositol-3 kinase-Akt pathway has been implicated in cell survival, with a constitutively activated form of Akt leading to a reduction in apoptosis in neuronal cells (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). GSK-3 activity is also inhibited by the Wnt signaling pathway through an unknown mechanism, involving the Dishevelled protein (
      • Cadigan K.M.
      • Nusse R.
      ,
      • Cook D.
      • Fry M.J.
      • Hughes K.
      • Sumathipala R.
      • Woodgett J.R.
      • Dale T.C.
      ). Here we examine whether rat GR is a substrate for GSK-3in vitro and investigate the consequences of GSK-3 activation and inhibition on GR transcriptional regulation in cultured mammalian cells.

      DISCUSSION

      We have demonstrated that GSK-3 phosphorylates the rat GR at Thr171 in vitro. In cultured mammalian cells, overexpression of GSK-3 inhibits GR transcriptional activation, while decreasing GSK-3 activity, through expression of the GSK-3 inhibitor, Akt, increases GR transcriptional enhancement. GR-mediated repression of AP1-dependent transcriptional activity, however, was not affected by GSK-3 overexpression. A threonine to alanine mutation at Thr171, the site of rat GR phosphorylation by GSK-3in vitro, eliminates the effect of GSK-3 on rat GR transcriptional enhancement. Although the effect of GSK-3 overexpression on GR-mediated transcriptional activation was relatively modest (∼50%), this likely represents an underestimate of the impact of GSK-3 on GR, since the studies were performed in cell lines containing active endogenous GSK-3. Our in vitrophosphorylation and mapping studies, coupled with activity assays using GR mutants, strongly suggest that GSK-3 phosphorylates rat GR at Thr171, and as a consequence, reduces GR transcriptional activation.
      The mechanism by which GSK-3 phosphorylation of Thr171decreases GR transcriptional activity is unclear. GSK-3 phosphorylates and inactivates other regulatory factors including c-Myc, c-Jun, NF-ATc, and β-catenin. Although the mechanism of c-Myc and c-Jun inactivation by GSK-3 phosphorylation is unknown, GSK-3 phosphorylation of β-catenin targets it for degradation (
      • Yost C.
      • Torres M.
      • Miller J.R.
      • Huang E.
      • Kimelman D.
      • Moon R.T.
      ,
      • Aberle H.
      • Bauer A.
      • Stappert J.
      • Kispert A.
      • Kemler R.
      ), while GSK-3 phosphorylation of NF-ATc promotes its export from the nucleus (
      • Beals C.R.
      • Sheridan C.M.
      • Turck C.W.
      • Gardner P.
      • Crabtree G.R.
      ). It is doubtful, however, that either of these established mechanisms explain GSK-3 regulation of GR, since 1) neither GSK-3 nor Akt overexpression alter steady state GR protein levels, and 2) increased export of GR from the nucleus would also affect GR-dependent transcriptional repression, which has not been observed in our experiments. Alternatively, GSK-3-mediated phosphorylation of rat GR at Thr171 may disrupt protein-protein interactions that favor GR transcriptional enhancement, or recruit inhibitory proteins that antagonize GR-dependent transcriptional activation, hypotheses that are currently being tested.
      Given the high degree of conservation between GRs from different species, it is particularly striking that the hGR does not contain a site of GSK-3-mediated phosphorylation, thereby making hGR insensitive to GSK-3 overexpression. However, when an alanine residue at the position homologous to Thr171 in rat GR is replaced with a threonine, transcriptional activation by the hGR A150T mutant becomes sensitive to GSK-3 overexpression. Sequence comparison between GRs isolated from different species shows that the primary amino acid sequence surrounding and including rat GR Thr171 (residues 164 through 173) is conserved among rodents, including rat, mouse, and guinea pig (
      • Miesfeld R.
      • Rusconi S.
      • Godowski P.J.
      • Maler B.A.
      • Okret S.
      • Wikstrom A.C.
      • Gustafsson J.A.
      • Yamamoto K.R.
      ,
      • Danielsen M.
      • Northrop J.P.
      • Ringold G.M.
      ,
      • Gao X.
      • Kalkhoven E.
      • Peterson-Maduro J.
      • van der Burg B.
      • Destree O.H.
      ). The equivalent region from human, squirrel monkey, owl monkey, and cotton-top tamarin GR remains conserved among primates, but has diverged from rodents (
      • Hollenberg S.M.
      • Weinberger C.
      • Ong E.S.
      • Cerelli G.
      • Oro A.
      • Lebo R.
      • Thompson E.B.
      • Rosenfeld M.G.
      • Evans R.M.
      ,
      • Reynolds P.D.
      • Pittler S.J.
      • Scammell J.G.
      ,
      • Rogatsky I.
      • Trowbridge J.M.
      • Garabedian M.J.
      ). Why this region of GR has diverged between primates and rodents, while the other major phosphorylation sites (Ser224, Ser232, and Ser246) are conserved remains unclear, but likely reflects alternative strategies adopted by each species to regulate GR action.
      The differences in GR primary amino acid structure and signaling between rodents and humans may contribute to the greater sensitivity of murine lymphocytes to glucocorticoid-induced apoptosis relative to human cells. It is conceivable that GSK-3-mediated inhibition of GR transcriptional activation in rodents results in the reduced expression of a putative survival factor induced by GR. Recently, the cyclin-dependent kinase inhibitor p21Cip1 has been shown to be a GR-responsive gene (
      • Rogatsky I.
      • Trowbridge J.M.
      • Garabedian M.J.
      ,
      • Ramalingam A.
      • Hirai A.
      • Thompson E.A.
      ,
      • Cha H.H.
      • Cram E.J.
      • Wang E.C.
      • Huang A.J.
      • Kasler H.G.
      • Firestone G.L.
      ) and forced expression of p21 can block apoptosis (
      • Wang J.
      • Walsh K.
      ). Thus, p21 expression protects cells from apoptosis, and as such, can be considered a survival factor. It is tempting to speculate that inhibition of GR by GSK-3, and the subsequent lack of p21 induction, may facilitate apoptosis in murine but not human lymphocytes. It would be interesting to replace mouse GR with that of the human GR in vivo and examine whether the glucocorticoid-induced apoptosis of murine lymphocytes expressing hGR still occurs. We speculate further that a threonine at position 150 in hGR would result in greater glucocorticoid sensitivity compared with an alanine at this position.
      Our findings demonstrate species-specific differences in human and rat GR signaling, which suggest that studies on GR function in mice and rats may not fully translate into hGR activity. In addition, our results indicate that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation, which antagonizes hGR-dependent transcriptional activation. It would be informative to examine whether alanine to threonine substitutions at residue 150 in hGR are present in glucocorticoid-sensitive, but absent in glucocorticoid-resistant, malignancies.

      Acknowledgments

      We thank James Woodgett and Thomas Franke for the GSK-3 and Akt expression constructs, respectively. We also thank Roland Knoblauch, Adam Hittelman, Samir Taneja, Ian Mohr, and Angus Wilson for critically reading the manuscript.

      REFERENCES

        • Yamamoto K.R.
        Annu. Rev. Genet. 1985; 19: 209-252
        • Miner J.N.
        • Diamond M.I.
        • Yamamoto K.R.
        Cell Growth Differ. 1991; 2: 525-530
        • Bodwell J.E.
        • Hu J.M.
        • Orti E.
        • Munck A.
        J. Steroid Biochem. Mol. Biol. 1995; 52: 135-140
        • Garabedian M.J.
        • Rogatsky I.
        • Hittelman A.
        • Knoblauch R.
        • Trowbridge J.M.
        • Krstic M.D.
        Freedman L.P. The Molecular Biology of Steroid and Nuclear Hormone Receptors. Birkhauser, Boston1997: 237-260
        • Orti E.
        • Mendel D.B.
        • Munck A.
        J. Biol. Chem. 1989; 264: 231-237
        • Krstic M.D.
        Functional Analysis of Glucocorticoid Receptor Phosphorylation.
        (Ph. D. Thesis) University of California, San Francisco, CA1995
        • Krstic M.D.
        • Rogatsky I.
        • Yamamoto K.R.
        • Garabedian M.J.
        Mol. Cell. Biol. 1997; 17: 3947-3954
        • Rogatsky I.
        • Logan S.K.
        • Garabedian M.J.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2050-2055
        • Woodgett J.R.
        Trends Biochem. Sci. 1991; 16: 177-181
        • Plyte S.E.
        • Hughes K.
        • Nikolakaki E.
        • Pulverer B.J.
        • Woodgett J.R.
        Biochim. Biophys. Acta. 1992; 1114: 147-162
        • Welsh G.I.
        • Wilson C.
        • Proud C.G.
        Trends Cell Biol. 1996; 6: 274-279
        • Puziss J.W.
        • Hardy T.A.
        • Johnson R.B.
        • Roach P.J.
        • Hieter P.
        Mol. Cell. Biol. 1994; 14: 831-839
        • Plyte S.E.
        • Feoktistova A.
        • Burke J.D.
        • Woodgett J.R.
        • Gould K.L.
        Mol. Cell. Biol. 1996; 16: 179-191
        • Hughes K.
        • Pulverer B.J.
        • Theocharous P.
        • Woodgett J.R.
        Eur. J. Biochem. 1992; 203: 305-311
        • Ruel L.
        • Bourouis M.
        • Heitzler P.
        • Pantesco V.
        • Simpson P.
        Nature. 1993; 362: 557-560
        • Siegfried E.
        • Chou T.B.
        • Perrimon N.
        Cell. 1992; 71: 1167-1179
        • He X.
        • Saint-Jeannet J.P.
        • Woodgett J.R.
        • Varmus H.E.
        • Dawid I.B.
        Nature. 1995; 374: 617-622
        • Dominguez I.
        • Itoh K.
        • Sokol S.Y.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8498-8502
        • Pierce S.B.
        • Kimelman D.
        Dev. Biol. 1996; 175: 256-264
        • Nikolakaki E.
        • Coffer P.J.
        • Hemelsoet R.
        • Woodgett J.R.
        • Defize L.H.
        Oncogene. 1993; 8: 833-840
        • Pulverer B.J.
        • Fisher C.
        • Vousden K.
        • Littlewood T.
        • Evan G.
        • Woodgett J.R.
        Oncogene. 1994; 9: 59-70
        • Fiol C.J.
        • Williams J.S.
        • Chou C.H.
        • Wang Q.M.
        • Roach P.J.
        • Andrisani O.M.
        J. Biol. Chem. 1994; 269: 32187-32193
        • de Groot R.P.
        • Auwerx J.
        • Bourouis M.
        • Sassone-Corsi P.
        Oncogene. 1993; 8: 841-847
        • Cross D.A.
        • Alessi D.R.
        • Cohen P.
        • Andjelkovich M.
        • Hemmings B.A.
        Nature. 1995; 378: 785-789
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • Burgering B.M.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Marte B.M.
        • Downward J.
        Trends Biochem. Sci. 1997; 22: 355-358
        • Franke T.F.
        • Yang S.I.
        • Chan T.O.
        • Datta K.
        • Kazlauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Kohn A.D.
        • Kovacina K.S.
        • Roth R.A.
        EMBO J. 1995; 14: 4288-4295
        • Dudek H.
        • Datta S.R.
        • Franke T.F.
        • Birnbaum M.J.
        • Yao R.
        • Cooper G.M.
        • Segal R.A.
        • Kaplan D.R.
        • Greenberg M.E.
        Science. 1997; 275: 661-665
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        Cell. 1997; 88: 435-437
        • Cadigan K.M.
        • Nusse R.
        Genes Dev. 1997; 11: 3286-3305
        • Cook D.
        • Fry M.J.
        • Hughes K.
        • Sumathipala R.
        • Woodgett J.R.
        • Dale T.C.
        EMBO J. 1996; 15: 4526-4536
        • Boyle W.J.
        • van der Geer P.
        • Hunter T.
        Method Enzymol. 1991; 201
        • Ausubel F.M.
        • Brent R.
        • Kingston R.E.
        • Moore D.D.
        • Seidman J.G.
        • Smith J.A.
        • Struhl K.
        Current Protocols in Molecular Biology.
        John Wiley & Sons, New York1996: 9.1.4-9.1.11
        • Alam J.
        • Cook J.L.
        Anal. Biochem. 1990; 188: 245-254
        • Trowbridge J.M.
        • Rogatsky I.
        • Garabedian M.J.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10132-10137
        • Schule R.
        • Rangarajan P.
        • Kliewer S.
        • Ransone L.J.
        • Bolado J.
        • Yang N.
        • Verma I.M.
        • Evans R.M.
        Cell. 1990; 62: 1217-1226
        • Yang-Yen H.-F.
        • Chambard J.-C.
        • Sun Y.-L.
        • Smeal T.
        • Schmidt T.J.
        • Drouin J.
        • Karin M.
        Cell. 1990; 62: 1205-1215
        • Miesfeld R.
        • Rusconi S.
        • Godowski P.J.
        • Maler B.A.
        • Okret S.
        • Wikstrom A.C.
        • Gustafsson J.A.
        • Yamamoto K.R.
        Cell. 1986; 46: 389-399
        • Danielsen M.
        • Northrop J.P.
        • Ringold G.M.
        EMBO J. 1986; 5: 2513-2522
        • Hollenberg S.M.
        • Weinberger C.
        • Ong E.S.
        • Cerelli G.
        • Oro A.
        • Lebo R.
        • Thompson E.B.
        • Rosenfeld M.G.
        • Evans R.M.
        Nature. 1985; 318: 635-641
        • Gao X.
        • Kalkhoven E.
        • Peterson-Maduro J.
        • van der Burg B.
        • Destree O.H.
        Biochim. Biophys. Acta. 1994; 1218: 194-198
        • Keightley M.C.
        • Fuller P.J.
        Mol. Endocrinol. 1994; 8: 431-439
        • Yost C.
        • Torres M.
        • Miller J.R.
        • Huang E.
        • Kimelman D.
        • Moon R.T.
        Genes Dev. 1996; 10: 1443-1454
        • Aberle H.
        • Bauer A.
        • Stappert J.
        • Kispert A.
        • Kemler R.
        EMBO J. 1997; 16: 3797-3804
        • Beals C.R.
        • Sheridan C.M.
        • Turck C.W.
        • Gardner P.
        • Crabtree G.R.
        Science. 1997; 275: 1930-1934
        • Reynolds P.D.
        • Pittler S.J.
        • Scammell J.G.
        J. Clin. Endocrinol. Metab. 1997; 82: 465-472
        • Rogatsky I.
        • Trowbridge J.M.
        • Garabedian M.J.
        Mol. Cell. Biol. 1997; 17: 3181-3193
        • Ramalingam A.
        • Hirai A.
        • Thompson E.A.
        Mol. Endocrinol. 1997; 11: 577-586
        • Cha H.H.
        • Cram E.J.
        • Wang E.C.
        • Huang A.J.
        • Kasler H.G.
        • Firestone G.L.
        J. Biol. Chem. 1998; 273: 1998-2007
        • Wang J.
        • Walsh K.
        Science. 1996; 273: 359-361