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

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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.