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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites
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Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites

机译：血管加压素敏感肾细胞的磷酸化蛋白质组学定量分析：两个部位水通道蛋白2磷酸化的调节

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摘要

Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry(n) neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated short-term with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at 5256 and diphosphorylated AQP2 (pS256/261) increased in abundance, whereas AQP2 monophosphorylated at 5261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromotography-mass spectrometry" neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system.
机译：蛋白磷酸化在肾收集管中的加压素信号传导中起关键作用。为了获得对该过程的全面系统了解，需要对加压素触发的磷酸化事件进行大规模鉴定和定量。我们结合固定化金属亲和色谱法的磷酸肽富集和液相色谱-质谱法（n）的中性损失扫描对磷酸化位点的结合，对大鼠内髓收集管细胞进行了蛋白质组学分析。从用钙调蛋白A或加压素短期处理过的内髓收集管样品中鉴定出223个独特的磷蛋白上总共714个磷酸化位点。确定了许多与细胞骨架重组，囊泡运输和转录调控有关的蛋白质。以前未发现的膜蛋白的磷酸化位点是收集导管生理所必需的膜蛋白，包括aquaporin-2（AQP2），aquaporin-4和尿素转运蛋白同工型A1和A3中的8个位点。通过无标记的磷酸肽定量，我们鉴定了许多对短期加压素治疗有明显改变的磷酸化状态的蛋白质：AQP2，Bclaf1，LRRC47，Rgl3和SAFB2。在存在加压素的情况下，在5256处单磷酸化的AQP2和在5261处单磷酸化的AQP2（pS256 / 261）大量增加，而在5261处单磷酸化的AQP2减少，这增加了两个位点都参与依赖血管加压素的AQP2转运的可能性。这项研究揭示了液相色谱-质谱“中性损失扫描”在天然哺乳动物系统中细胞信号分析中大规模鉴定和定量蛋白质磷酸化的实用性。

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