A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies - Identification of a novel protein, Frigg, as a protein kinase A substrate
A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies - Identification of a novel protein, Frigg, as a protein kinase A substrate
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DOI：
10.1074/mcp.m200010-mcp200
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发表时间：
2002-07-01
影响因子：
7
通讯作者：
Pandey, A
中科院分区：
文献类型：
--
作者：
Gronborg, M;Kristiansen, TZ;Pandey, A
虽然酪氨酸残基上磷酸化的蛋白质可以通过用抗磷酸酪氨酸抗体免疫沉淀来富集，但由于缺乏免疫沉淀抗体，很难鉴定丝氨酸/苏氨酸残基上磷酸化的蛋白质。在本报告中，我们描述了几种通过蛋白质印迹法识别含磷酸丝氨酸/磷酸苏氨酸蛋白质的抗体。重要的是，这些抗体也可用于通过免疫沉淀富集丝氨酸/苏氨酸残基上磷酸化的蛋白质。使用这些抗体，我们从未处理的细胞或用calyculin A（一种丝氨酸/苏氨酸磷酸酶抑制剂）处理的细胞中免疫沉淀蛋白质。基于质谱的分析，从一维凝胶，具体观察到在calyculin A处理的样品中导致几个已知的丝氨酸/苏氨酸磷酸化蛋白，包括dreplastin 1，α-辅肌动蛋白4，和细丝蛋白-1鉴定。我们还确定了一种蛋白质，聚（A）结合蛋白2，这是以前不知道被磷酸化，除了一种新的蛋白质没有任何明显的结构域，我们指定为Frigg。Frigg广泛表达，并在体外被证明是蛋白激酶A底物。我们确定了几个在体内磷酸化位点的串联质谱使用Frigg蛋白免疫沉淀细胞。我们的方法应适用于作为一个通用的战略富集和鉴定丝氨酸/苏氨酸磷酸化底物的信号转导途径。
Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways.