A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells

Abstract

Activated estrogen receptor (ERalpha) 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 ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha 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 ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS(n) (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 ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer.

Figure 1.

(a) The domain structure of ERα, including the largely unstructured N-terminal domain containing AF-1 (NTD/AF-1), the DNA-binding domain (DBD), and the ligand binding domain containing AF-2 (LBD/AF-2). (b) Detail of the NTD of ERα. Arrows indicate the 14 serine residues within the NTD. Phosphorylation has been reported for the 5 serine residues shown in red (102, 104, 106, 118, and 167). Residue 154 (shown in blue) is identified here as a novel serine phosphorylation site.

Figure 2.

(a) Phospho-ER. Western analysis of total and phosphorylated ERα at serine residues 104/106, 118, and 167 following immunoprecipitation from MCF-7 cells treated with E2 (10 nM), EGF (50 ng/mL), or appropriate control. (b) Immunoprecipitation and one-dimensional SDS-PAGE. Coomassie-stained ERα immunoprecipitated from MCF-7 cellular lysates using the agarose conjugated anti-ERα antibody (F-10), versus commercially obtained recombinant ERα (rERα). (c) Western blot analysis; 10 μL aliquots from the same MCF-7 cell lysate before and after immunoprecipitation, probed with the F-10 antibody.

Figure 3.

(a) ESI-MS/MS spectrum of triply charged monophosphorylated peptide LApS¹⁶⁷TNDKGSM*AM*ESAK (residues 165–180) at m/z 584.9 (M = 1751.72) from tryptic digestion of ERα isolated from MCF-7 cells treated with E2 (M* = mono-oxidized methionine.) (b) three-dimensional plots of ESI-MS representing m/z, retention time, and ion intensity on the x, y, and z axes, respectively, showing increased ion current for phosphorylated peptide m/z = 584.9, eluting at 13.5 min, from cells treated with estradiol compared to untreated control.

Figure 4.

vMALDI-MS/MS and MS³ identification of phosphorylated peptide M*LLHPPPQLpS¹¹⁸PF (residues 109–120) from chymotrypsin-digested endogenous ERα. (a) MS/MS of the putative precursor ion at m/z 1472.60 showing a characteristic loss of 98 Da. (b) MS³ of the neutral loss fragment at m/z 1374.58, confirming the conversion of phospho-Ser-118 to dehydro-alanine.

Figure 5.

vMALDI-MS/MS, MS³ and MS⁴ identification of phosphorylated tryptic peptide EAGPPAFYRPNpS¹⁵⁴DNRR (residues 143–158) from MCF-7 cells treated with E2. (a) The precursor ion [M + H]⁺ at m/z 1927.0 selected for MS/MS analysis. −P represents loss of phosphoric acid, i.e., loss of 98 Da. (b) MS³ spectrum of m/z 1829.0 [M+H−98]⁺ confirming that Ser-154 is now dehydro-alanine. The c₁₁ fragment ion (1217.7 m/z) was fragmented (MS⁴; for additional sequence information see inset). (c) MS/MS of the unmodified peptide at m/z 1847.0 corresponding to EAGPPAFYRPNSDNRR. The c₁₁ ion (1217.7 m/z) was further fragmented (MS³; see inset).

Figure 6.

Relative quantitation of phospho-Ser-154 levels by MS and immunoblotting. (a) vMALDI-LTQ in SRM mode demonstrates levels of Ser-154 phosphorylation after E2 (10 nM) or EGF (50 ng/mL) treatment relative to their respective controls. (b) Western analysis of total and phosphorylated ERα at Ser-154 following immunoprecipitation from MCF-7 cells treated with E2 (10 nM), EGF (50 ng/mL), or appropriate control.