Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore, India.
Br J Pharmacol. 2021 Mar;178(6):1426-1444. doi: 10.1111/bph.15352. Epub 2021 Feb 14.
The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V receptor, which triggers a cAMP-mediated activation of a PKA-dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins.
We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method).
Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk).
In V receptor-mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web-based data resource for future studies of GPCRs.
肽激素血管升压素通过 V 受体调节肾脏集合管中的水转运，该受体触发 cAMP 介导的 PKA 依赖性信号网络激活。PKA 下游的蛋白激酶尚未被完全鉴定或映射到受调控的磷酸化蛋白上。
我们对大规模磷酸蛋白质组学数据进行了系统水平分析，定量测量了血管升压素诱导的表达水通道蛋白-2 的培养集合管（mpkCCD）细胞中磷酸化的变化。使用稳定同位素标记（SILAC 方法）进行定量。
定量了 644 个磷酸肽。严格的统计分析确定了其中 429 个磷酸肽对血管升压素反应的显著变化。相应的磷酸化蛋白被映射到已知的血管升压素调节的细胞过程。根据磷酸化氨基酸周围的序列对血管升压素调节的位点进行分类，得到 11 个组。在血管升压素调节的磷酸化蛋白中，有 25 种不同的蛋白激酶。其中，加上 PKA，这六种激酶似乎占了 313 个血管升压素调节磷酸化位点中约 81%的磷酸化。这六种下游激酶分别是盐诱导激酶 2（Sik2）、周期蛋白依赖性激酶 18（Cdk18）、钙调蛋白依赖性激酶激酶 2（Camkk2）、蛋白激酶 D2（Prkd2）、丝裂原激活蛋白激酶 3（Mapk3）和肌球蛋白轻链激酶（Mylk）。
在 V 受体介导的信号转导中，PKA 位于一个复杂网络的顶端，该网络至少包括六种下游的血管升压素调节蛋白激酶，它们是未来研究的主要目标。本研究报告的广泛的磷酸蛋白质组学数据作为未来 GPCR 研究的基于网络的数据库提供。
