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降低的细胞中，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DBS通过其催化的DBL同源(DH)结构域特异性地激活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)结合非常重要。尽管存在争议，但PIP与PH结构域的结合被认为是为了通过变构方式调节全球环境基金的活性，而不是将GEF靶向于膜。DBS与PIP的结合是pH依赖的，这可能是由于PH域中的组氨酸残基质子化，这被称为His开关。相反，新发现的Dock家族的Rho GEF缺乏DH-PH结构域，但与DBL家族Rho GEF一样，它们包含一个催化结构域(DHR2)、一个PIP结合结构域(DHR1)和额外的膜靶向区。此外，某些Dock GEF具有几个可能直接与PIP结合的组氨酸。我将通过集中在调节低分子GTPase CDc42的鸟核苷酸交换因子(GEF)来测试PIP结合的His开关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共振技术比较在PIP结合位点上有或没有组氨酸的DBL和Dock家族Rho GEF的结合。为了测量不同pH值下的全球环境基金活性，我将使用一种体外系统，通过将GTP酶固定在脂泡上来模拟生理条件。然后，我将使用细胞内pH可调的细胞来研究依赖于pH的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的活性，并使用Lifeact测量F-肌动蛋白的含量。结果将揭示His开关调控的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.