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Abstract：
The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 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 V2 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.
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 V2 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.
摘要：
肽激素加压素主要通过V2受体调节肾集合管中的水运输，这触发了PKA依赖性信号网络的cAMP介导的激活。PKA下游的蛋白激酶尚未被完全鉴定或定位为受调节的磷蛋白。
我们对大规模磷酸化蛋白质组数据进行了系统水平分析，定量了表达水通道蛋白2的培养收集管（mpkCCD）细胞中血管加压素诱导的磷酸化变化。使用稳定同位素标记(SILAC方法)进行定量。
定量了640个磷酸肽。严格的统计分析确定了这些磷酸肽中的429种对加压素的反应的显着变化。相应的磷蛋白被定位到已知的加压素调节的细胞过程。根据磷酸化氨基酸周围的序列对加压素调节的位点进行分类，得到11组。在加压素调节的磷蛋白中有25种不同的蛋白激酶。其中,在313个加压素调节的磷酸化位点中,6+PKA的磷酸化似乎占81%.六种下游激酶是盐诱导型激酶2（Sik2），细胞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），丝裂原活化激酶3(Mapk3)和肌球蛋白轻链激酶(Mylk)。
在V2受体介导的信号传导中，PKA处于复杂网络的头部，该网络包括至少六种下游加压素调节的蛋白激酶，这些蛋白激酶是未来研究的主要目标。本研究中报道的广泛的磷酸蛋白质组数据作为基于网络的数据资源提供，用于GPCRs的未来研究。
我们对大规模磷酸化蛋白质组数据进行了系统水平分析，定量了表达水通道蛋白2的培养收集管（mpkCCD）细胞中血管加压素诱导的磷酸化变化。使用稳定同位素标记(SILAC方法)进行定量。
定量了640个磷酸肽。严格的统计分析确定了这些磷酸肽中的429种对加压素的反应的显着变化。相应的磷蛋白被定位到已知的加压素调节的细胞过程。根据磷酸化氨基酸周围的序列对加压素调节的位点进行分类，得到11组。在加压素调节的磷蛋白中有25种不同的蛋白激酶。其中,在313个加压素调节的磷酸化位点中,6+PKA的磷酸化似乎占81%.六种下游激酶是盐诱导型激酶2（Sik2），细胞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），丝裂原活化激酶3(Mapk3)和肌球蛋白轻链激酶(Mylk)。
在V2受体介导的信号传导中，PKA处于复杂网络的头部，该网络包括至少六种下游加压素调节的蛋白激酶，这些蛋白激酶是未来研究的主要目标。本研究中报道的广泛的磷酸蛋白质组数据作为基于网络的数据资源提供，用于GPCRs的未来研究。
