A novel serine phosphorylation site detected in the n-terminal domain of estrogen receptor isolated from human breast cancer cells
- 1 May 2008
- journal article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 19 (5), 729-740
- https://doi.org/10.1016/j.jasms.2008.02.008
Abstract
Activated estrogen receptor (ERα) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ERα activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERα residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERα isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MSn (Finnigan™ LTQ™, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments—i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS—allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol- and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERα, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer.Keywords
This publication has 49 references indexed in Scilit:
- Unraveling the Mechanisms of Endocrine Resistance in Breast Cancer: New Therapeutic OpportunitiesClinical Cancer Research, 2007
- Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometryProceedings of the National Academy of Sciences of the United States of America, 2007
- Bidirectional cross talk between ERα and EGFR signalling pathways regulates tamoxifen-resistant growthBreast Cancer Research and Treatment, 2005
- Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancerEndocrine-Related Cancer, 2005
- Phosphoproteomic Analysis of the Developing Mouse BrainMolecular & Cellular Proteomics, 2004
- Rsk2 allosterically activates estrogen receptor alpha by docking to the hormone-binding domainThe EMBO Journal, 2001
- Probability-based protein identification by searching sequence databases using mass spectrometry dataElectrophoresis, 1999
- Selective oestrogen receptor modulation: molecular pharmacology for the millenniumEuropean Journal Of Cancer, 1999
- Phosphorylation of Purified Estradiol-Liganded Estrogen Receptor by Casein Kinase II Increases Estrogen Response Element Binding but Does Not Alter Ligand StabilityBiochemical and Biophysical Research Communications, 1996
- An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein databaseJournal of the American Society for Mass Spectrometry, 1994