Arthur Jonathan W, Sanchez-Perez Angeles, Cook David I
Department of Medicine, University of Sydney Sydney, Australia.
Bioinformatics. 2006 Sep 15;22(18):2192-5. doi: 10.1093/bioinformatics/btl381. Epub 2006 Jul 14.
Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2.
We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest.
上皮钠通道（ENaC）介导钠（Na）在肾脏、肠道和肺部上皮细胞的转运，对血压调节至关重要。它们受到泛素蛋白连接酶如Nedd4-2的抑制。这些连接酶与ENaC亚基C末端存在的富含脯氨酸基序（PY基序）结合。这种抑制作用的丧失会导致高血压。我们之前报道过，ENaC通道通过G蛋白偶联受体激酶GRK2维持在活性状态。该酶与原发性高血压的发生有关[R. D. Feldman（2002年），《分子药理学》，61卷，707 - 709页]。我们实验室的其他研究结果表明GRK2在Nedd4-2的磷酸化和失活中可能发挥作用。
我们通过结合序列分析、同源建模和表面可及性计算预测了Nedd4-2上的GRK2磷酸化位点。通过序列分析共预测了24个潜在的磷酸化位点。其中，16个可以用同源建模进行模拟，并且发现其中6个具有足够的表面暴露，可被负责Nedd4-2磷酸化的GRK2酶识别。该方法提供了Nedd4-2上最可能的GRK2磷酸化位点的有序列表，为未来旨在突变某些Nedd4-2残基以防止被GRK2磷酸化的实验研究提供了宝贵的指导。所开发的方法可应用于涉及一个分子与蛋白质结合的各种生物学应用中。该技术的相对有效性主要取决于为感兴趣的蛋白质构建的同源模型的质量。
