A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells
A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells
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DOI：
10.1016/j.jasms.2008.02.008
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发表时间：
2008-05-01
影响因子：
3.2
通讯作者：
Baldwin, Michael A.
中科院分区：
文献类型：
--
作者：
Britton, David J.;Scott, Gary K.;Baldwin, Michael A.
激活的雌激素受体（ER α）在乳腺癌的发展中起着关键作用，是药物治疗的主要靶点。N-末端结构域（NTD）内的丝氨酸磷酸化有助于ER α活化，也可能导致耐药性。先前对磷酸化ER α残基的生化鉴定仅限于在转染细胞系中人工过表达的蛋白质。我们报告的质谱方法，允许识别一个新的网站内的NTD的ER α从培养的人乳腺癌细胞分离。免疫沉淀、胰蛋白酶消化和通过nano-LC-ESI-MS/MS（Q-STAR，MDS Sciex）和vMALDI-MS”（Finnigan（TM）LTQ（TM），Thermo-Electron）的分析鉴定了在NTD内含有14个丝氨酸残基中的8个的肽，一个是部分磷酸化的Ser-167，已知但先前未通过MS报道。胰凝乳蛋白酶消化揭示了在Ser-102/104/106和118处的其它已知位点。为含Ser-118的肽开发的串联方法和使用假设驱动的实验-即，在vMALDI-MS/MS-中显示无分子离子的完整磷酸化肽可能产生包括磷酸损失的碎片离子的假设允许在Ser-154处鉴定新位点。通过选择反应监测的定量表明，与对照组相比，雌二醇和EGF处理的细胞中Ser-154磷酸化分别增加了6倍和2.5倍，这一点通过用新型兔多克隆抗体进行免疫印迹得到证实。因此，本文所述的蛋白质分离和MS策略可以促进在低丰度、临床重要的癌症靶点（如ER α）内发现新的磷酸化位点，从而有助于我们理解磷酸化在乳腺癌发展中的作用。
Activated estrogen receptor (ER alpha) 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 ER alpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ER alpha 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 ER alpha 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" (Finnigan(TM) LTQ(TM), 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 phosphopepticle 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 ER alpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breastcancer.