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 GTdR Cdc 42是细胞极性和迁移的关键调节因子，其活性受到严格控制。最近的工作表明，Cdc 42在迁移细胞的前沿不被激活，在细胞的pH值降低。Dbl家族Rho鸟嘌呤核苷酸交换因子（GEF）的Dbs通过其催化Dbl同源（DH）域特异性激活Cdc 42。Dbl家族Rho GEF还含有对磷酸肌醇磷酸（PIP）结合重要的相邻普列克底物蛋白同源（PH）结构域。虽然有争议，PIP结合到PH结构域被认为是调节GEF活性变构，而不是将GEF靶向膜。Db与PIP的结合是pH依赖性的，这可能是由于PH结构域中组氨酸残基的质子化，其被称为His开关。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，它们含有催化结构域（DHR 2）、PIP结合结构域（DHR 1）和另外的膜靶向区域。此外，某些Dock GEF具有几个可以直接与PIP结合的组氨酸，我将通过关注调节低分子量GTdR Cdc 42的鸟嘌呤核苷酸交换因子（GEF）来测试PIP结合的His开关范例及其功能意义。我将使用表面等离子体共振比较在PIP结合位点具有或缺乏组氨酸的Dbl和Dock家族Rho GEF的结合。为了测量GEF在不同pH值下的活性，我将采用体外系统，该系统通过将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的细胞研究pH依赖性PIP与Rho GEFs结合的功能意义。因此，我将使用生物传感器测量Cdc 42活性，并使用Lifeact测量F-actin含量。结果将揭示His开关调节PIP与Rho GEFs结合的机制及其对Cdc 42活化的后果。