Abstract
Protein phosphorylation is a versatile posttranslational modification that can regulate nuclear receptor function. Although the precise role of receptor phosphorylation is not fully understood, it appears that it functions to direct or refine receptor activity in response to particular physiological requirements.
Identifying and characterizing specific nuclear receptor phosphorylation sites is an important step in elucidating the role(s) receptor phosphorylation plays in function. Although traditional methods of metabolic labeling and in vitro protein phosphorylation have been informative, receptor phosphorylation site-specific antibodies are simple and reliable tools to study receptor phosphorylation. This chapter will discuss how to develop nuclear receptor phosphorylation site-specific antibodies to elucidate function.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Weigel, N.L. and N.L. Moore (2007) Steroid receptor phosphorylation: a key modulator of multiple receptor functions. Mol Endocrinol, 21(10): p. 2311–9.
Zhang, Y., C.A. Beck, A. Poletti, D.P. Edwards, and N.L. Weigel (1995) Identification of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor. Mol Endocrinol, 9(8): p. 1029–40.
Krstic, M.D., I. Rogatsky, K.R. Yamamoto, and M.J. Garabedian (1997) Mitogen-acti-vated and cyclin-dependent protein kinases selectively and differentially modulate tran-scriptional enhancement by the glucocorticoid receptor. Mol Cell Biol, 17(7): p. 3947–54.
Al-Dhaheri, M.H. and B.G. Rowan (2006) Application of phosphorylation site-specific antibodies to measure nuclear receptor signaling: characterization of novel phosphoantibod-ies for estrogen receptor alpha. Nucl Recept Signal, 4: p. e007.
Clemm, D.L., L. Sherman, V. Boonyara-tanakornkit, W.T. Schrader, N.L. Weigel, and D.P. Edwards (2000) Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phospho-serine site-specific monoclonal antibody. Mol Endocrinol, 14(1): p. 52–65.
Gioeli, D., S.B. Ficarro, J.J. Kwiek, D. Aar-onson, M. Hancock, A.D. Catling, F.M. White, R.E. Christian, R.E. Settlage, J. Shab-anowitz, D.F. Hunt, and M.J. Weber (2002) Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J Biol Chem, 277(32): p. 29304–14.
Taneja, S.S., S. Ha, N.K. Swenson, H.Y. Huang, P. Lee, J. Melamed, E. Shapiro, M.J. Garabedian, and S.K. Logan (2005) Cell-specific regulation of androgen receptor phosphorylation in vivo. J Biol Chem, 280(49): p. 40916–24.
Torra, I.P., N. Ismaili, J.E. Feig, C.F. Xu, C. Cavasotto, R. Pancratov, I. Rogatsky, T.A. Neubert, E.A. Fisher, and M.J. Garabedian (2008) Phosphorylation of LXR alpha Selectively Regulates Target Gene Expression in Macrophages. Mol Cell Biol.
Wang, Z., J. Frederick, and M.J. Garabedian (2002) Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivo. J Biol Chem, 277(29): p. 26573–80.
Yang, C.S., M.J. Vitto, S.A. Busby, B.A. Garcia, C.T. Kesler, D. Gioeli, J. Shabanowitz, D.F. Hunt, K. Rundell, D.L. Brautigan, and B.M. Paschal (2005) Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor. Mol Cell Biol, 25(4): p. 1298–308.
Wang, Z., W. Chen, E. Kono, T. Dang, and M.J. Garabedian (2007) Modulation of glu-cocorticoid receptor phosphorylation and tran-scriptional activity by a C-terminal-associated protein phosphatase. Mol Endocrinol, 21(3): p. 625–34.
Lee, M.J., Z. Wang, H. Yee, Y. Ma, N. Swen-son, L. Yang, S.S. Kadner, R.N. Baergen, S.K. Logan, M.J. Garabedian, and S. Guller (2005) Expression and regulation of glucocorticoid receptor in human placental villous fibroblasts. Endocrinology, 146(11): p. 4619–26.
Blind, R.D. and M.J. Garabedian (2008) Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes. J Steroid Biochem Mol Biol, 109(1–2): p. 150–7.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Torra, I.P., Staverosky, J.A., Ha, S., Logan, S.K., Garabedian, M.J. (2009). Development of Phosphorylation Site-Specific Antibodies to Nuclear Receptors. In: McEwan, I.J. (eds) The Nuclear Receptor Superfamily. Methods in Molecular Biology™, vol 505. Humana Press. https://doi.org/10.1007/978-1-60327-575-0_13
Download citation
DOI: https://doi.org/10.1007/978-1-60327-575-0_13
Publisher Name: Humana Press
Print ISBN: 978-1-60327-574-3
Online ISBN: 978-1-60327-575-0
eBook Packages: Springer Protocols