Regulation of Dbl and Dock family Rho guanine nucleotide exchange factor activity by His switch-mediated pH-dependent phosphoinositol phosphate binding

通过 His 开关介导的 pH 依赖性磷酸肌醇磷酸盐结合调节 Dbl 和 Dock 家族 Rho 鸟嘌呤核苷酸交换因子活性

• 批准号: 190629931
• 负责人: Dr. André Schönichen
• 金额: --
• 依托单位: Department of Microbiology and Immunology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/190629931?language=en
• 关键词: Regulation; Dbl; Dock; family; Rho

The Rho GTPase Cdc42 is a key regulator of cell polarity and migration and its activity is tightly controlled. Recent work has shown that Cdc42 at the leading edge of a migrating cell is not activated in cells with decreased pH. The Dbl family Rho guanine nucleotide exchange factor (GEF) Dbs specifically activates Cdc42 via its catalytic Dbl homology (DH) domain. Dbl family Rho GEFs also contain an adjacent pleckstrin homology (PH) domain that is important for phosphoinositol phosphate (PIP) binding. Although controversial, PIP binding to the PH domain is suggested to regulate GEF activity allosterically rather than to target GEFs to membranes. Binding of Dbs to PIPs is pH-dependent, which may be due to protonation of a histidine residue in the PH domain, which is referred to as a His switch. In contrast, the newly identified Dock family of Rho GEFs lack DH-PH domains, but like Dbl family Rho GEFs they contain a catalytic domain (DHR2), a PIP binding domain (DHR1), and additional membrane targeting regions. Moreover, certain Dock GEFs possess several histidines that may bind to PIPs directly.I will test a paradigm of His switches for PIP binding and its functional significance by focusing on guanine nucleotide exchange factors (GEFs) that regulate the low molecular weight GTPase Cdc42.To reveal pH-dependence of PIP binding, I will compare binding of Dbl and Dock family Rho GEFs that have or lack histidines at their PIP binding site using surface plasmon resonance. To measure GEF activity at different pH values, I will employ an in vitro system that mimics physiological conditions by immobilizing GTPases on lipid vesicles. I will then study functional significance of pH-dependent PIP binding to Rho GEFs using cells with adjustable intracellular pH. Therefore I will measure Cdc42 activity using a biosensor and F-actin content with Lifeact. Results will reveal a mechanism for His switch regulated PIP binding to Rho GEFs and its consequences for activation of Cdc42.

Rho GTPase Cdc42 是细胞极性和迁移的关键调节因子，其活性受到严格控制。最近的研究表明，迁移细胞前沿的 Cdc42 在 pH 值降低的细胞中不会被激活。 Dbl 家族 Rho 鸟嘌呤核苷酸交换因子 (GEF) Dbs 通过其催化 Dbl 同源 (DH) 结构域特异性激活 Cdc42。 Dbl 家族 Rho GEF 还包含一个相邻的 pleckstrin 同源 (PH) 结构域，该结构域对于磷酸肌醇磷酸 (PIP) 结合非常重要。尽管存在争议，但 PIP 与 PH 结构域的结合被认为是变构调节 GEF 活性，而不是将 GEF 靶向膜。 Dbs 与 PIP 的结合具有 pH 依赖性，这可能是由于 PH 结构域中组氨酸残基（称为 His 开关）的质子化所致。相比之下，新鉴定的 Rho GEF 的 Dock 家族缺乏 DH-PH 结构域，但与 Dbl 家族 Rho GEF 一样，它们包含催化结构域 (DHR2)、PIP 结合结构域 (DHR1) 和其他膜靶向区域。此外，某些 Dock GEF 拥有多个可以直接与 PIP 结合的组氨酸。我将通过关注调节低分子量 GTPase Cdc42 的鸟嘌呤核苷酸交换因子 (GEF) 来测试用于 PIP 结合的 His 开关范例及其功能意义。为了揭示 PIP 结合的 pH 依赖性，我将使用表面等离子共振比较 Dbl 和 Dock 家族 Rho GEF 的结合，这些 GEF 在其 PIP 结合位点具有或缺乏组氨酸。为了测量不同 pH 值下的 GEF 活性，我将采用体外系统，通过将 GTP 酶固定在脂质囊泡上来模拟生理条件。然后，我将使用具有可调节细胞内 pH 值的细胞来研究 pH 依赖性 PIP 与 Rho GEF 结合的功能意义。因此，我将使用生物传感器测量 Cdc42 活性，并使用 Lifeact 测量 F-肌动蛋白含量。结果将揭示 His 开关调节 PIP 与 Rho GEF 结合的机制及其对 Cdc42 激活的影响。