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

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，在ph值降低的细胞中，位于迁移细胞前沿的Cdc42不被激活。Dbl家族Rho鸟嘌呤核苷酸交换因子（GEF） Dbs通过其催化Dbl同源性（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，该结构域对磷酸肌醇（PIP）结合很重要。尽管存在争议，但PIP与PH结构域的结合被认为是调节GEF活性的变构性，而不是将GEF靶向膜。Dbs与PIPs的结合是PH依赖性的，这可能是由于PH结构域中的组氨酸残基的质子化，这被称为His开关。相反，新发现的Dock家族Rho gef缺乏DH-PH结构域，但与Dbl家族Rho gef一样，它们含有一个催化结构域（DHR2）、一个PIP结合结构域（DHR1）和额外的膜靶向区域。此外，某些Dock gef具有几种可以直接与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His开关与PIP结合的范例及其功能意义。为了揭示PIP结合的ph依赖性，我将使用表面等离子体共振比较Dbl和Dock家族Rho gef在其PIP结合位点具有或缺乏组氨酸的结合。为了测量GEF在不同pH值下的活性，我将采用一种体外系统，通过将gtp酶固定在脂质囊泡上来模拟生理条件。然后，我将使用细胞内ph可调的细胞研究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开关调节的PIP与Rho gef结合的机制及其对Cdc42激活的影响。

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.