The elusive phosphotyrosine: pinning down a rare species. Focus on "Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination".
The elusive phosphotyrosine: pinning down a rare species. Focus on "Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination".
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难以捉摸的磷酸酪氨酸：确定一种稀有物种。
DOI：
10.1152/ajpcell.00363.2011
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Ecelbarger,CarolynM
中科院分区：
文献类型：
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作者：
Ecelbarger,CarolynM
酪氨酸残基的磷酸化通常发生在生长因子的信号级联以及与细胞结构和极性的设计、组织和维持相关的途径中（7，8）。经历酪氨酸（Y）磷酸化的蛋白质的鉴定没有得到与丝氨酸或苏氨酸磷酸化相同水平的深入审查，主要是因为“Y”位点相对于其他位点相对罕见。Knepper及其同事（1）在肾内髓集合管（IMCD）细胞（调节体液渗透压的关键区域）中的早期研究表明，肽上2%的磷酸化位点位于酪氨酸残基上。因此，阐明他们的身份需要更大程度的创造力和集中的努力。Zhao、Knepper及其同事修改了从癌症文献中收集的磷酸酪氨酸富集方案（10），使用大规模、全面的磷酸蛋白质组学鉴定了IMCD细胞中大量独特的含磷酸酪氨酸的肽。Zhao等人使用的基本方法涉及在基础条件下从大鼠的内髓质分离富集的IMCD细胞。然后用过钒酸盐（一种蛋白酪氨酸磷酸酶抑制剂）处理IMCD悬浮液以稳定磷酸酪氨酸，然后用三种不同的市售抗磷酸酪氨酸抗体进行免疫沉淀。这是在使用固定化金属亲和层析（IMAC）的磷酸肽富集步骤之后（或在某些情况下在其之前）。然后通过液相色谱-串联质谱法（LC-MS/MS）对肽进行鉴定。使用美国国立卫生研究院（Bethesda，MD）基于NIH的Biowulf Linux集群，使用几种不同的软件程序（应用略微不同的算法），从MS光谱中鉴定肽和现有的酪氨酸磷酸化位点。大约有一半，即990，被鉴定的肽是磷酸肽。其中，超过90%是磷酸酪氨酸，说明富集方案的效率。总体而言，19%的磷酸酪氨酸位点是新发现的，即在PhosphoSitePlus数据库中未报告（3）。发现许多磷酸酪氨酸位点在物种间高度保守，表明可能在细胞生理学中起重要作用。进行了额外的分析以评估和鉴定含有磷酸位点的常规基序（MOTIF-X），以及可能参与磷酸化的激酶（NetworkKIN版本2.0）（5）。这些分析预测了许多与胰岛素和肝配蛋白信号传导相关的激酶途径。需要对这些信号通路进行额外的测试来确认
PHOSPHORYLATION OF TYROSINE residues often occurs in the signaling cascades of growth factors and in pathways relevant to the design, organization, and maintenance of cell structure and polarity (7, 8). Identification of proteins undergoing tyrosine (Y) phosphorylation has not received the same level of intensive scrutiny as that of serine or threonine phosphorylation primarily because “Y” sites are relatively rare in relation to the others. Earlier studies by Knepper and colleagues (1) in renal inner medullary collecting duct (IMCD) cells, a region key to regulation of body fluid osmolality, showed that 2% of phosphorylatable sites on peptides are on tyrosine residues. Thus, elucidating their identity requires a greater degree of creativity and concentrated effort. Modifying a phosphotyrosine enrichment scheme gleaned from the cancer literature (10), Zhao, Knepper, and associates (11) identify a large number of unique phosphotyrosine-containing peptides in IMCD cells using large-scale, comprehensive phosphoproteomics. The basic approach utilized by Zhao et al. involved isolating enriched IMCD cells from the inner medulla of rats under basal conditions. IMCD suspensions were then treated with pervanadate (a protein tyrosine phosphatase inhibitor) to stabilize phosphotyrosines, which were then immunoprecipitated with three different commercially available anti-phospho-tyrosine antibodies. This was following (or preceding, in some cases) a phospho-peptide enrichment step using immobilized metal affinity chromatography (IMAC). Peptides were then run through liquid chromatography-tandem mass spectroscopy (LC-MS/MS) for identification. Peptides and existing tyrosine phosphorylation sites were identified from MS spectra using the NIH-based Biowulf Linux cluster at the National Institutes of Health (Bethesda, MD), with the use of several different software programs applying slightly different algorithms. About half, ie, 990, of the peptides identified were phosphopeptides. Of those, over 90% were phosphotyrosines, illustrating the efficiency of the enrichment protocol. Overall, 19% of the phosphotyrosine sites identified were novel, ie, not reported previously in the PhosphoSitePlus database (3). Many phosphotyrosine sites were found to be highly conserved across species, indicating likely fundamental roles in cell physiology.Additional analyses were done to evaluate and identify conventional motifs (MOTIF-X) containing the phospho-sites, and likely kinases involved in phosphorylation (NetworKIN version 2.0)(5). These analyses predicted a number of kinase pathways related to insulin and ephrin signaling. Additional testing of these signaling pathways would be needed to confirm