Systematic mapping of posttranslational modifications in human estrogen receptor-alpha with emphasis on novel phosphorylation sites

Abstract

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.

Fig. 1.

A, analytical strategy for affinity purification, digestion, and sequence mapping of PTMs in immunopurified endogenous ERα. rERα was used as a standard for optimization of experimental conditions. B, Coomassie-stained one-dimensional (1-D) gel electrophoresis of affinity-purified MCF-7 ERα showing co-migration with rERα at 66 kDa. C, Western blots of estradiol (E2)-treated MCF-7 cell lysates before and after immunopurification (IP) demonstrate the effectiveness of the enrichment protocol.

Fig. 2.

MALDI-TOF peptide mass fingerprint of chymotrypsin-digested endogenous ERα. All underlined sequences were confirmed by MS/MS.

Fig. 3.

Coverage map for MS/MS identification of amino acid residues within MCF-7 ERα digested with various enzymes. chym, chymotrypsin; tryp, trypsin; LyAs, Lys-C/Asp-N.

Fig. 4.

Acetylation at ERα N terminus and acetylation (or trimethylation) of Lys⁴⁷². A, LC-MS/MS of MH₂²⁺ (m/z 437.23) of peptide Ac²TMTLHTK from endogenous ERα. B, vMALDI-MS/MS of ⁴⁶⁸SLEEKDHIHR (m/z 1263.4). C, the corresponding spectrum of a modified peptide (m/z 1305.4). Inset to B and C, MS³ of m/z 562.4 common to both MS/MS spectra.

Fig. 5.

Identification of novel phosphorylation site Ser²¹² and comparison with a structurally similar phosphopeptide containing Ser(P)¹⁵⁴. A, vMALDI-LTQ-MS/MS of MH⁺ for the Asp-N peptide ²⁰³EGCKAFFKRSIQGHN (m/z 1778.9) from endogenous ERα with expansion of weak fragment peaks at m/z 800–1600. B, the corresponding MH⁺ spectrum from the phosphorylated peptide (m/z 1858.8). C, vMALDI-LTQ-MS/MS of MH⁺ for the Asp-N peptide ¹⁴³EAGPPAFYRPNS (m/z 1305.6) from endogenous ERα. D, the corresponding MH⁺ spectrum from the phosphorylated peptide (m/z 1385.6). pS, phosphoserine.

Fig. 6.

Identification of novel phosphorylation site Ser²⁹⁴ in endogenous MCF-7 ERα. A, vMALDI-LTQ-MS/MS of MH⁺ from chymotryptic peptide ²⁸⁷RAANLWPSPL (m/z 1124.8). B, the corresponding MH⁺ spectrum from the phosphorylated peptide (m/z 1204.5). C, MS³ spectrum of (MH − 98)⁺ (m/z 1106.5) from the phosphopeptide MH⁺. D, LC-MS/MS spectrum of MH₃³⁺ of phosphorylated Lys-C/Asp-N peptide ²⁸⁵DMRAANLWPSPLMIK (m/z 608.15). pS, phosphoserine; dS, dehydroalanine.

Fig. 7.

Identification of novel phosphorylation site Ser⁵⁵⁹ in endogenous MCF-7 ERα. A, LC-MS/MS spectrum of MH₃³⁺ of tryptic peptide ⁵⁵⁶GGASVEETDQSHLATAGSTSSHSLQK (m/z 889.1) eluting at 26.4 min. B, LC-MS/MS spectrum of MH₃³⁺ of the analogous phosphorylated peptide (m/z 889.1) eluting at 27.1 min. C, vMALDI-LTQ-MS/MS spectrum of MH⁺ of the unmodified peptide (m/z 2585.3). D, the corresponding spectrum of MH⁺ of the phosphorylated peptide (m/z 2665.3). E, MS³ spectrum of (MH − 98)⁺ (m/z 2567.2) from the phosphopeptide. pS, phosphoserine; dS, dehydroalanine.

Fig. 8.

LC-MS/MS of tryptic peptides from endogenous MCF-7 ERα. A, spectrum of MH₂²⁺ (m/z 710.9) of peptide ²⁸⁸AANLWPpSPLMIK (where pS is phosphoserine). B, spectrum of MH₂²⁺ (m/z 670.9) of the analogous unphosphorylated peptide. C, MRM/MS analysis of Ser²⁹⁴ induction following estradiol (E2) treatment showing chromatograms of the y₇ Q1/Q3 MRM transitions for the unmodified (670.9/785.5) and phosphorylated (710.9/865.4) peptide from control and estradiol-treated (10 nm; 30 min) cells. D, comparing peak areas for the phosphorylated with unmodified peptides demonstrates that estradiol induces Ser²⁹⁴ oxidation 28-fold. The error bar represents the standard deviation for three biological replicates. XIC, extracted ion chromatogram; cps, counts/s.

Fig. 9.

Summary of ERα modifications observed in this study. ** signifies previously unreported modifications identified in ERα from MCF-7 cells. * signifies a previously unreported modification identified only in rERα. In parentheses previously known modification identified here only in rERα. All others are previously known modifications identified in ERα from MCF-7 cells. LBD, ligand-binding domain; NTD, N-terminal domain; pSer, phosphoserine.