Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Am J Physiol Renal Physiol. 2019 Oct 1;317(4):F789-F804. doi: 10.1152/ajprenal.00281.2019. Epub 2019 Jul 17.
Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: ) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; ) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; ) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and ) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.
加压素主要通过 PKA 依赖性效应来控制水平衡，从而调节集合管水通道水通道蛋白-2（AQP2）。尽管已经积累了大量关于集合管细胞中水和溶质转运的调节信息，但关于 PKA 与转运反应之间的信号连接的信息却很少。在这里，我们利用蛋白质质谱分析的最新进展，对响应血管加压素 V2 受体选择性激动剂 1-去氨基-8-D-精氨酸血管加压素的大鼠内髓集合管细胞的磷酸蛋白质组进行了全面的、多次重复的分析。在定量的 10738 个磷酸肽中，只有 156 个磷酸肽的丰度显著增加，只有 63 个磷酸肽减少，表明对血管加压素的反应具有高度选择性。上调磷酸化位点的列表显示了几个一般特征：）在相对于磷酸化氨基酸的位置 -2 和 -3 处具有碱性（带正电荷）氨基酸精氨酸（R）和赖氨酸（K）的磷酸化位点占优势，这与 PKA 和/或其他嗜碱性激酶的磷酸化一致；）以前被证明由 PKA 磷酸化的位点的可能性大于随机；）膜蛋白中的位点占优势，与膜结合的调节一致；并且）具有 I 类 COOH 末端 PDZ 配体基序的蛋白质中的位点的可能性大于随机。下调磷酸化位点的列表显示，相对于磷酸化氨基酸，位置+1 处脯氨酸的磷酸化位点占优势，这与脯氨酸定向激酶（例如 MAPK 或细胞周期蛋白依赖性激酶）的下调或一种或多种选择性去磷酸化此类位点的蛋白磷酸酶（例如蛋白磷酸酶 2A）的上调一致。磷酸蛋白质组学数据被用于创建一个网络资源，用于研究 G 蛋白偶联受体信号传导和 AQP2 介导的水转运的调节。
