Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity.
Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity.
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DOI：
10.1128/mbio.00242-13
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发表时间：
2013-05-07
期刊：
影响因子：
6.4
通讯作者：
Harwood CS
中科院分区：
文献类型：
--
作者：
Huangyutitham V;Güvener ZT;Harwood CS
WspR 是一种混合响应调节剂-二鸟苷酸环化酶，响应铜绿假单胞菌在表面的生长，由 Wsp 信号转导复合物磷酸化。活性 WspR 产生环状二 GMP (c-di-GMP)，进而刺激生物膜形成。在之前的工作中，我们发现，当被磷酸化激活时，黄色荧光蛋白（YFP）标记的 WspR 会形成通过荧光显微镜在单个细胞中可见的簇。未磷酸化的 WspR 在细胞中扩散且不可见。因此，簇形成是 WspR 信号转导的一种测定。为了了解 WspR 形成亚细胞簇的方式和原因，我们分析了 WspR 的六个单氨基酸变体的簇形成和酶活性。一般来说，簇形成的增加与变体体内和体外二鸟苷酸环化酶活性的增加相关。此外，WspR 的比活性在体外具有强烈的浓度依赖性，并且当用磷类似物氟化铍处理 WspR 时，蛋白质浓度对二鸟苷酸环化酶活性的影响被放大。簇形成似乎是磷酸化 WspR (WspR-P) 的固有特性。这些结果支持这样的模型：体内响应表面刺激而形成 WspR-P 亚细胞簇对于增强 WspR 的二鸟苷酸环化酶活性非常重要。亚细胞簇形成似乎是调节反应调节蛋白活性的另一种手段。当受到环境信号刺激时，细菌传感器蛋白通常会磷酸化同源反应调节蛋白。然后磷酸化反应调节剂介导适当的适应性细胞反应。大约 6% 的反应调节蛋白具有参与产生或降解环二 GMP (c-di-GMP) 的酶结构域，环二 GMP 是一种刺激细菌生物膜形成的分子。在这项工作中，我们检查了反应调节剂二鸟苷酸环化酶 WspR 的体内和体外行为。当响应与表面生长相关的信号而磷酸化时，WspR 倾向于形成在细胞中可见的亚细胞簇寡聚体。我们的结果表明，磷酸化 WspR (WspR-P) 亚细胞簇的形成对于增强 WspR-P 的二鸟苷酸环化酶活性非常重要，使其在 c-di-GMP 生产中更加活跃。我们得出的结论是，可视化为亚细胞簇的寡聚体形成是可以调节反应调节剂二鸟苷酸环化酶活性的另一种机制。
WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.