Phosphorylation-mediated 14-3-3 Protein Binding Regulates the Function of the Rho-specific Guanine Nucleotide Exchange Factor (RhoGEF) Syx
Phosphorylation-mediated 14-3-3 Protein Binding Regulates the Function of the Rho-specific Guanine Nucleotide Exchange Factor (RhoGEF) Syx
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DOI：
10.1074/jbc.m112.432682
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发表时间：
2013-03-01
影响因子：
4.8
通讯作者：
Anastasiadis, Panos Z.
中科院分区：
文献类型：
--
作者：
Ngok, Siu P.;Geyer, Rory;Anastasiadis, Panos Z.
Syx 是一种 Rho 特异性鸟嘌呤核苷酸交换因子 (GEF)，定位于细胞与细胞连接处，并通过激活 RhoA 和下游效应器 Diaphanous 同源物 1 (Dia1) 来促进连接稳定性。之前，我们鉴定了几种分子，包括 14-3-3 蛋白，作为 Syx 相互作用的伙伴。在本研究中，我们表明 14-3-3 亚型以磷酸化依赖性方式在其 N 端和 C 端区域与 Syx 相互作用。我们确定了蛋白激酶 D 介导的 Syx 上丝氨酸 92 的磷酸化以及丝氨酸 938 上的额外磷酸化，作为 14-3-3 关联的关键位点。我们的数据表明 14-3-3 蛋白的结合抑制 Syx 的 GEF 活性。此外，我们发现磷酸化缺陷、14-3-3-解偶联的 Syx 表现出增强的连接靶向性和增强的 GEF 活性，从而增强 Madin-Darby 犬肾细胞中的圆周连接肌动蛋白环。这些发现揭示了一种通过磷酸化诱导的 14-3-3 结合来调节连接 Syx 定位和功能的新方法，并进一步支持 Syx 功能在维持稳定的细胞-细胞接触中的重要性。
Syx is a Rho-specific guanine nucleotide exchange factor (GEF) that localizes at cell-cell junctions and promotes junction stability by activating RhoA and the downstream effector Diaphanous homolog 1 (Dia1). Previously, we identified several molecules, including 14-3-3 proteins, as Syx-interacting partners. In the present study, we show that 14-3-3 isoforms interact with Syx at both its N- and C-terminal regions in a phosphorylation-dependent manner. We identify the protein kinase D-mediated phosphorylation of serine 92 on Syx, and additional phosphorylation at serine 938, as critical sites for 14-3-3 association. Our data indicate that the binding of 14-3-3 proteins inhibits the GEF activity of Syx. Furthermore, we show that phosphorylation-deficient, 14-3-3-uncoupled Syx exhibits increased junctional targeting and increased GEF activity, resulting in the strengthening of the circumferential junctional actin ring in Madin-Darby canine kidney cells. These findings reveal a novel means of regulating junctional Syx localization and function by phosphorylation-induced 14-3-3 binding and further support the importance of Syx function in maintaining stable cell-cell contacts.