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. 1993 Apr 1;291(Pt 1):95–101. doi: 10.1042/bj2910095

Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor.

G G Kuiper 1, P E de Ruiter 1, J Trapman 1, W J Boersma 1, J A Grootegoed 1, A O Brinkmann 1
PMCID: PMC1132486  PMID: 8471057

Abstract

Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  2. Bodwell J. E., Ortí E., Coull J. M., Pappin D. J., Smith L. I., Swift F. Identification of phosphorylated sites in the mouse glucocorticoid receptor. J Biol Chem. 1991 Apr 25;266(12):7549–7555. [PubMed] [Google Scholar]
  3. Brinkmann A. O., Kuiper G. G., Bolt-de Vries J., Mulder E. In situ photolabelling of the human androgen receptor. J Steroid Biochem. 1988;30(1-6):257–261. doi: 10.1016/0022-4731(88)90102-1. [DOI] [PubMed] [Google Scholar]
  4. Brown T. A., DeLuca H. F. Phosphorylation of the 1,25-dihydroxyvitamin D3 receptor. A primary event in 1,25-dihydroxyvitamin D3 action. J Biol Chem. 1990 Jun 15;265(17):10025–10029. [PubMed] [Google Scholar]
  5. Chauchereau A., Loosfelt H., Milgrom E. Phosphorylation of transfected wild type and mutated progesterone receptors. J Biol Chem. 1991 Sep 25;266(27):18280–18286. [PubMed] [Google Scholar]
  6. Dalman F. C., Sanchez E. R., Lin A. L., Perini F., Pratt W. B. Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor. J Biol Chem. 1988 Sep 5;263(25):12259–12267. [PubMed] [Google Scholar]
  7. Denner L. A., Schrader W. T., O'Malley B. W., Weigel N. L. Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. J Biol Chem. 1990 Sep 25;265(27):16548–16555. [PubMed] [Google Scholar]
  8. Denner L. A., Weigel N. L., Maxwell B. L., Schrader W. T., O'Malley B. W. Regulation of progesterone receptor-mediated transcription by phosphorylation. Science. 1990 Dec 21;250(4988):1740–1743. doi: 10.1126/science.2176746. [DOI] [PubMed] [Google Scholar]
  9. Denton R. R., Koszewski N. J., Notides A. C. Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding. J Biol Chem. 1992 Apr 15;267(11):7263–7268. [PubMed] [Google Scholar]
  10. Glineur C., Zenke M., Beug H., Ghysdael J. Phosphorylation of the v-erbA protein is required for its function as an oncogene. Genes Dev. 1990 Oct;4(10):1663–1676. doi: 10.1101/gad.4.10.1663. [DOI] [PubMed] [Google Scholar]
  11. Hoeck W., Groner B. Hormone-dependent phosphorylation of the glucocorticoid receptor occurs mainly in the amino-terminal transactivation domain. J Biol Chem. 1990 Apr 5;265(10):5403–5408. [PubMed] [Google Scholar]
  12. Housley P. R., Pratt W. B. Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells. J Biol Chem. 1983 Apr 10;258(7):4630–4635. [PubMed] [Google Scholar]
  13. Husmann D. A., Wilson C. M., McPhaul M. J., Tilley W. D., Wilson J. D. Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology. 1990 May;126(5):2359–2368. doi: 10.1210/endo-126-5-2359. [DOI] [PubMed] [Google Scholar]
  14. Jackson S. P., MacDonald J. J., Lees-Miller S., Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 1990 Oct 5;63(1):155–165. doi: 10.1016/0092-8674(90)90296-q. [DOI] [PubMed] [Google Scholar]
  15. Jenster G., van der Korput H. A., van Vroonhoven C., van der Kwast T. H., Trapman J., Brinkmann A. O. Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol Endocrinol. 1991 Oct;5(10):1396–1404. doi: 10.1210/mend-5-10-1396. [DOI] [PubMed] [Google Scholar]
  16. Jones B. B., Jurutka P. W., Haussler C. A., Haussler M. R., Whitfield G. K. Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain. Mol Endocrinol. 1991 Aug;5(8):1137–1146. doi: 10.1210/mend-5-8-1137. [DOI] [PubMed] [Google Scholar]
  17. Kemppainen J. A., Lane M. V., Sar M., Wilson E. M. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem. 1992 Jan 15;267(2):968–974. [PubMed] [Google Scholar]
  18. Krongrad A., Wilson C. M., Wilson J. D., Allman D. R., McPhaul M. J. Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells. Mol Cell Endocrinol. 1991 Apr;76(1-3):79–88. doi: 10.1016/0303-7207(91)90262-q. [DOI] [PubMed] [Google Scholar]
  19. Kuiper G. G., de Ruiter P. E., Brinkmann A. O. Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step. J Steroid Biochem Mol Biol. 1992 Mar;41(3-8):697–700. doi: 10.1016/0960-0760(92)90407-a. [DOI] [PubMed] [Google Scholar]
  20. Kuiper G. G., de Ruiter P. E., Grootegoed J. A., Brinkmann A. O. Synthesis and post-translational modification of the androgen receptor in LNCaP cells. Mol Cell Endocrinol. 1991 Sep;80(1-3):65–73. doi: 10.1016/0303-7207(91)90143-g. [DOI] [PubMed] [Google Scholar]
  21. Mendel D. B., Bodwell J. E., Munck A. Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein. J Biol Chem. 1987 Apr 25;262(12):5644–5648. [PubMed] [Google Scholar]
  22. Migliaccio A., Rotondi A., Auricchio F. Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody. EMBO J. 1986 Nov;5(11):2867–2872. doi: 10.1002/j.1460-2075.1986.tb04581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moudgil V. K. Phosphorylation of steroid hormone receptors. Biochim Biophys Acta. 1990 Dec 10;1055(3):243–258. doi: 10.1016/0167-4889(90)90040-k. [DOI] [PubMed] [Google Scholar]
  24. Ortí E., Mendel D. B., Smith L. I., Munck A. Agonist-dependent phosphorylation and nuclear dephosphorylation of glucocorticoid receptors in intact cells. J Biol Chem. 1989 Jun 15;264(17):9728–9731. [PubMed] [Google Scholar]
  25. Pearson R. B., Kemp B. E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. doi: 10.1016/0076-6879(91)00127-i. [DOI] [PubMed] [Google Scholar]
  26. Pike J. W., Sleator N. M. Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts. Biochem Biophys Res Commun. 1985 Aug 30;131(1):378–385. doi: 10.1016/0006-291x(85)91813-3. [DOI] [PubMed] [Google Scholar]
  27. Sheridan P. L., Evans R. M., Horwitz K. B. Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment. J Biol Chem. 1989 Apr 15;264(11):6520–6528. [PubMed] [Google Scholar]
  28. Sheridan P. L., Evans R. M., Horwitz K. B. Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment. J Biol Chem. 1989 Apr 15;264(11):6520–6528. [PubMed] [Google Scholar]
  29. Sheridan P. L., Krett N. L., Gordon J. A., Horwitz K. B. Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation. Mol Endocrinol. 1988 Dec;2(12):1329–1342. doi: 10.1210/mend-2-12-1329. [DOI] [PubMed] [Google Scholar]
  30. Smith L. I., Mendel D. B., Bodwell J. E., Munck A. Phosphorylated sites within the functional domains of the approximately 100-kDa steroid-binding subunit of glucocorticoid receptors. Biochemistry. 1989 May 16;28(10):4490–4498. doi: 10.1021/bi00436a055. [DOI] [PubMed] [Google Scholar]
  31. Sullivan W. P., Madden B. J., McCormick D. J., Toft D. O. Hormone-dependent phosphorylation of the avian progesterone receptor. J Biol Chem. 1988 Oct 15;263(29):14717–14723. [PubMed] [Google Scholar]
  32. Tienrungroj W., Sanchez E. R., Housley P. R., Harrison R. W., Pratt W. B. Glucocorticoid receptor phosphorylation, transformation, and DNA binding. J Biol Chem. 1987 Dec 25;262(36):17342–17349. [PubMed] [Google Scholar]
  33. Zegers N. D., Claassen E., Neelen C., Mulder E., van Laar J. H., Voorhorst M. M., Berrevoets C. A., Brinkmann A. O., van der Kwast T. H., Ruizeveld de Winter J. A. Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy. Biochim Biophys Acta. 1991 Jan 23;1073(1):23–32. doi: 10.1016/0304-4165(91)90178-j. [DOI] [PubMed] [Google Scholar]
  34. de Boer W., Bolt J., Kuiper G. G., Brinkmann A. O., Mulder E. Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis. J Steroid Biochem. 1987 Jul;28(1):9–19. doi: 10.1016/0022-4731(87)90117-8. [DOI] [PubMed] [Google Scholar]
  35. van Laar J. H., Berrevoets C. A., Trapman J., Zegers N. D., Brinkmann A. O. Hormone-dependent androgen receptor phosphorylation is accompanied by receptor transformation in human lymph node carcinoma of the prostate cells. J Biol Chem. 1991 Feb 25;266(6):3734–3738. [PubMed] [Google Scholar]
  36. van Laar J. H., Bolt-de Vries J., Zegers N. D., Trapman J., Brinkmann A. O. Androgen receptor heterogeneity and phosphorylation in human LNCaP cells. Biochem Biophys Res Commun. 1990 Jan 15;166(1):193–200. doi: 10.1016/0006-291x(90)91930-q. [DOI] [PubMed] [Google Scholar]
  37. van Laar J. H., Voorhorst-Ogink M. M., Zegers N. D., Boersma W. J., Claassen E., van der Korput J. A., Ruizeveld de Winter J. A., van der Kwast T. H., Mulder E., Trapman J. Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor. Mol Cell Endocrinol. 1989 Nov;67(1):29–38. doi: 10.1016/0303-7207(89)90227-x. [DOI] [PubMed] [Google Scholar]

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