Application of Phosphorylation Site-Specific Antibodies to Measure Nuclear Receptor Signaling: Characterization of Novel Phosphoantibodies for Estrogen Receptor α
Application of Phosphorylation Site-Specific Antibodies to Measure Nuclear Receptor Signaling: Characterization of Novel Phosphoantibodies for Estrogen Receptor α
An understanding of posttranslational events in nuclear receptor signaling is crucial for drug design and clinical therapeutic strategies. Phosphorylation is a well-characterized posttranslational modification that regulates subcellular localization and function of nuclear receptors and coregulators. Although the role of single phosphorylation sites in nuclear receptor function has been described, the contribution of combinations of multiple phosphorylation sites to receptor function remains unclear. The development of phosphoantibodies to each phosphorylation site in a nuclear receptor is a powerful tool to address the role of phosphorylation in multiply phosphorylated receptors. However, phosphoantibodies must be rigorously validated prior to use. This review describes the general methodology for design, characterization and validation of phosphoantibodies using the example of eight phosphoantibodies raised against phosphorylation sites in estrogen receptor α (ERα).
References
1.
ArnoldS. F., and NotidesA. C. (1995a). An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptorProc Natl Acad Sci U S A92, 7475–9.
2.
ArnoldS. F., ObournJ. D., YudtM. R., CarterT. H., and NotidesA. C. (1995b). in vivo and in vitro phosphorylation of the human estrogen receptorJ Steroid Biochem Mol Biol52, 159–71.
3.
BeckC. A., ZhangY., AltmannM., WeigelN. L., and EdwardsD. P. (1996). Stoichiometry and site-specific phosphorylation of human progesterone receptor in native target cells and in the baculovirus expression systemJ Biol Chem271, 19546–55.
4.
BlackB. E., VittoM. J., GioeliD., SpencerA., AfsharN., ConawayM. R., WeberM. J., and PaschalB. M. (2004). Transient, ligand-dependent arrest of the androgen receptor in subnuclear foci alters phosphorylation and coactivator interactionsMol Endocrinol18, 834–50.
5.
BurnsR. (2005). Immunization strategies for antibody productionMethods Mol Biol295, 1–12.
6.
ChenD., WashbrookE., SarwarN., BatesG. J., PaceP. E., ThirunuvakkarasuV., TaylorJ., EpsteinR. J., Fuller-PaceF. V., EglyJ. M., CoombesR. C., and AliS. (2002). Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antiseraOncogene21, 4921–31.
7.
ChenD., PaceP. E., CoombesR. C., and AliS. (1999). Phosphorylation of human estrogen receptor α by protein kinase A regulates dimerizationMol Cell Biol19, 1002–15.
8.
EiseleF., OwenD. J., and WaldmannH. (1999). Peptide conjugates as tools for the study of biological signal transductionBioorg Med Chem7, 193–224.
9.
FrankA. W. (1984). Synthesis and properties of N-, O-, and S-phospho derivatives of amino acids, peptides, and proteinsCRC Crit Rev Biochem16, 51–101.
10.
GarciaB. A., ShabanowitzJ., and HuntD. F. (2005). Analysis of protein phosphorylation by mass spectrometryMethods35, 256–64.
11.
Cshl PressHarlowE., and LaneD. (1988). Antibodies: A Laboratory Manual (CSHL Press Cold Spring Harbor)
12.
LanniganD. A. (2003). Estrogen receptor phosphorylationSteroids68, 1–9.
13.
LeenaarsM., and HendriksenC. F. (2005). Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendationsIlar J46, 269–79.
14.
LeeH., and BaiW. (2002). Regulation of estrogen receptor nuclear export by ligand-induced and p38-mediated receptor phosphorylationMol Cell Biol22, 5835–45.
15.
LipmanN. S., JacksonL. R., TrudelL. J., and Weis-GarciaF. (2005). Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resourcesIlar J46, 258–68.
16.
MichalidesR., GriekspoorA., BalkenendeA., VerwoerdD., JanssenL., JalinkK., FlooreA., VeldsA., van't VeerL., and NeefjesJ. (2004). Tamoxifen resistance by a conformational arrest of the estrogen receptor α after PKA activation in breast cancerCancer Cell5, 597–605.
17.
MurphyL., CherletT., AdeyinkaA., NiuY., SnellL., and WatsonP. (2004). Phospho-serine-118 estrogen receptor-α detection in human breast tumors in vivoClin Cancer Res10, 1354–9.
18.
NarayananR., EdwardsD. P., and WeigelN. L. (2005). Human progesterone receptor displays cell cycle-dependent changes in transcriptional activityMol Cell Biol25, 2885–98.
19.
OrtiE., BodwellJ. E., and MunckA. (1992). Phosphorylation of steroid hormone receptorsEndocr Rev13, 105–28.
20.
Pierson-MullanyL. K., and LangeC. A. (2004). Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2Mol Cell Biol24, 10542–57.
21.
RogatskyI., TrowbridgeJ. M., and GarabedianM. J. (1999). Potentiation of human estrogen receptor α transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complexJ Biol Chem274, 22296–302.
22.
RowanB. G., NarayananR., and WeigelN. L. (2003). Analysis of receptor phosphorylationMethods Enzymol364, 173–202.
23.
ShahY. M., and RowanB. G. (2005). The Src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor (α) promoter interaction and elevated steroid receptor coactivator 1 activityMol Endocrinol19, 732–48.
24.
WangZ., FrederickJ., and GarabedianM. J. (2002). Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivoJ Biol Chem277, 26573–80.
25.
YamashitaH., NishioM., KobayashiS., AndoY., SugiuraH., ZhangZ., HamaguchiM., MitaK., FujiiY., and IwaseH. (2005). Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancerBreast Cancer Res7, R753–64.
