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Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase

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Abstract

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [3H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274–279, 1984) to near homogeneity. A 23° C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [γ-32P]ATP, Mg2+ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [γ-32P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn2+ or Ca2+, could not be substituted for Mg2+ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.

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Abbreviations

BUGR2:

anti-GR monoclonal antibody

cAMP-PK:

cAMP-dependent protein kinase

DMSO:

dimethyl sulfoxide

EDTA:

ethylenediamine tetra acetic acid

GR:

glucocorticoid receptor

H-8:

Isoquinoline sulfonamide derivative

hsp-90:

90 kDa heat-shock protein

PMSF:

phenylmethylsulfonyl fluoride

PR:

progesterone receptor

NaF:

sodium fluoride

SDS:

sodium dodecyl sulfate

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

SR:

steroid receptor

TA:

triamcinolone acetonide

References

  1. 1.

    Beato M: Gene regulation by steroid hormones. Cell 56: 335–344, 1989

  2. 2.

    Evans RM: The steroid and the thyroid hormone receptor superfamily. Science 240: 889–895, 1989

  3. 3.

    Moudgil VK: Phosphorylation of steroid hormone receptors. Biochim Biophys Acta 1055: 243–258, 1990

  4. 4.

    Weigel NL, Tash JS, Mean AT, Schrader WT, O'Malley BW: Phosphorylation of hen progesterone receptor by cAMP dependent kinase. Biochem Biophys Res Commun 102: 513–519, 1981

  5. 5.

    Dougherty JJ, Puri PK, Toft DO: Phosphorylationin vivo of chicken progesterone receptor. J Biol Chem 257: 14226–14230, 1982

  6. 6.

    Singh VB, Moudgil VK: Phosphorylation of rat liver glucocorticoid receptor. J Biol Chem 260: 3684–3690, 1985

  7. 7.

    Migliaccio A, Rotondi A, Auricchio F: Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody. EMBO J 5: 2867–2872, 1986

  8. 8.

    Teinrungrij W, Sanchez ER, Housley PR, Harrison RW, Pratt WB: Glucocorticoid receptor phosphorylation, transformation, and DNA binding. J Biol Chem 262: 17342–17349, 1987

  9. 9.

    Yamamoto KR: Steroid receptor regulated transcription of specific genes and gene products. Annu Rev Genetic 19: 209–252, 1985

  10. 10.

    Sorger PK, Pelham HRB: Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54: 855–864, 1988

  11. 11.

    Yamamoto KR, Gonzales GA, Biggs WH III, Montminy MR: Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature (London) 334: 494–498, 1988

  12. 12.

    Amster-Choder O, Hommand F, Wright A: Protein phosphorylation regulates transcription of the β-glucoside utilization operon inE. coli. Cell 58: 847–855, 1989

  13. 13.

    Cherry JR, Johnson TR, Dollard C, Shuster JR, Denis CL: Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADRI. Cell 56: 409–419, 1989

  14. 14.

    Cohen P: Protein phosphorylation and hormone action. Proc Roy Soc Lond 234: 115–144, 1988

  15. 15.

    Cater-Su C, Pratt WB: Receptor phosphorylation. In: PM Conn (ed.) The Receptors, Vol. 1. Academic Press, Orlando, FL, 1984, pp 451–585

  16. 16.

    Sanchez ER, Pratt WB: Phosphorylation of L-cell glucocorticoid receptor in immune complexes: Evidence that the receptor is not a protein kinase. Biochemistry 25: 1378–1382, 1986

  17. 17.

    Gametchu B, Harrison RW: Characterization of a monoclonal antibody to the rat liver glucocorticoid receptor. Endocrinology 114: 274–279, 1984

  18. 18.

    Denner LA, Schrader WT, O'Malley BW, Weigel NL: Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. J Biol Chem 265: 16548–16555, 1990

  19. 19.

    Denner LA, Weigel NL, Maxwell BL, Schrader WT, O'Malley BW: Regulation of progesterone receptor-mediated transcription by phosphorylation. Science 250: 1740–1743, 1990

  20. 20.

    Okarinen J, Hamalainen L, Okarinen A: Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis. Biochim Biophys Acta 799: 158–165, 1984

  21. 21.

    Eisen LP, Reichman ME, Thompson ER, Gametchu B, Harrison RW, Eisen JH: Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain. J Biol Chem 260: 11805–11810, 1985

  22. 22.

    Singh VB, Moudgil VK: Protein kinase activity of purified rat liver glucocorticoid receptor. Biochem Biophys Res Commun 125: 1067–1073, 1984

  23. 23.

    Hidaka H, Inagaki M, Kawamoto S, Sasaki Y: Isoquinoline sulfonamides: novel and potent inhibitors of cyclic nucleotide-dependent protein kinase and protein kinase C. Biochemistry 23: 5036–5041, 1984

  24. 24.

    Nakao M, Mizutani T, Bhakta A, Ribarac-Stepic N, Moudgil VK: Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase. Arch Biochem Biophys 298: 340–348, 1992

  25. 25.

    Wrange O, Carlstead-Duke J, Gustafsson JA: Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA. J Biol Chem 261: 11770–11778, 1986

  26. 26.

    Housley PR, Pratt WB: Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells. J Biol Chem 258: 4630–4635, 1983

  27. 27.

    Kurl RN, Jacob ST: Phosphorylation of purified glucocorticoid receptor from rat liver by an endogenous protein kinase. Biochem Biophys Res Commun 119: 700–705, 1984

  28. 28.

    Grandics P, Miller A, Schmidt TJ, Litwack G: Phosphorylationin vivo of rat hepatic glucocorticoid receptor. Biochem Biophys Res Commun 120: 59–65, 1984

  29. 29.

    Roth RA, Cassell PJ: Insulin receptor: evidence that it is a protein kinase. Science 219: 229–301, 1983

  30. 30.

    Miller-Diener A, Schmidt TJ, Litwack G: Protein kinase activity associated with the purified rat hepatic glucocorticoid receptor. Proc Natl Acad Sci USA 82: 4003–4007, 1985

  31. 31.

    Csermely P, Kahn CR: the 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity. J Biol Chem 266: 4943–4950, 1991

  32. 32.

    Sando JJ, LaForest AC, Pratt WB: ATP-dependent activation of L-cell glucocorticoid receptors to the steroid binding form. J Biol Chem 254: 4772–4778, 1979

  33. 33.

    Garcia T, Tuohimaa P, Mester J, Buchou T, Ronoir JM, Baulieu EE: Protein kinase activity of purified components of the chicken oviduct progesterone receptor. Biochem Biophys Res Commun 113: 960–966, 1983

  34. 34.

    Krebs EG: Protein kinases. Curr Top Cell Regul 5: 99–134, 1972

  35. 35.

    Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM: Primary structure and expression of a functional glucocorticoid receptor cDNA. Nature (London) 318: 635–637, 1985

  36. 36.

    Miesfeld R, Rusconi S, Gurdowski PJ, Maler BA, Okret S, Wikstrom AC, Gustafsson JA, Yamamoto KR: Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell 46: 389–399, 1986

  37. 37.

    Tashima Y, Terui M, Itoh H, Mizunuma H, Kobayashi R, Marumo F: Phosphorylated and dephosphorylated types of nonactivated glucocorticoid receptor. J Biochem 108: 271–277, 1990

  38. 38.

    Burnstein KL, Cidlowski JA: Regulation of gene expression by glucocorticoids. A Rev Physiol 51: 683–699, 1989

  39. 39.

    Wei LL, Sheridan PL, Krett NL, Fransis MD, Toft DO, Edward DP, Horwitz KB: Immunological analysis of human breast cancer progesterone receptors. II. Structure, phosphorylation and processing. Biochemistry 26: 6262–6272, 1987

  40. 40.

    Sheridan PL, Krett NL, Gordon JA, Horwitz KB: Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation. Mol Endocrinol 2: 1329–1342, 1988

  41. 41.

    Bailly A, Page CL, Ruch M, Milgrom E: Sequence-specific DNA binding of the progesterone receptor to the uteroglobin gene: effects of hormone, antihormone and receptor phosphorylation. EMBO J 5: 3235–3241, 1986

  42. 42.

    Nakao M, Moudgil VK: Hormone specific phosphorylation and transformation of chicken oviduct progesterone receptor. Biochem Biophys Res Commun 161: 295–303, 1989

  43. 43.

    Meggio F, Agostinis P, Pinna LA: Casein kinases and their protein substrates in rat liver cytosol: evidence for their participation in multimolecular systems. Biochim Biophys Acta 846: 248–256, 1985

  44. 44.

    Dougherty JJ, Rabideau DA, Iannotti AM, Sullivan WP, Toft DO: Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors. Biochim Biophys Acta 927: 74–80, 1987

  45. 45.

    Logeat F, Cunff ML, Rauch M, Brailly S, Milgrom E: Characterization of a casein kinase which interacts with the rabbit progesterone receptor. Differences with thein vivo hormone-dependent phosphorylation. Eur J Biochem 170: 51–57, 1987

  46. 46.

    Savouret JF, Misrahi M, Loosefelt H, Atger M, Bailly A, Perrot-Applant M, Vu Hai MT, Guiochon-Mentel A, Jolivet A, Lorenzo F, Logeat F, Pichon MF, Buchard P, Milgrom E: Molecular and cellular biology of mammalian progesterone receptors. Rec Prog Horm Res 45: 65–120, 1989

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Correspondence to V. K. Moudgil.

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Haske, T., Nakao, M. & Moudgil, V.K. Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase. Mol Cell Biochem 132, 163–171 (1994). https://doi.org/10.1007/BF00926925

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Key Words

  • steroid receptors
  • glucocorticoid receptor
  • phosphorylation
  • cAMP-dependent protein kinase
  • immunopurification
  • dexamethasone mesylate
  • affinity labeling