Related Experiment Video
Updated: Sep 18, 2025

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates
Published on: March 31, 2012
Regulation of Rho guanine nucleotide exchange factor 3 by phosphorylation in the PH domain
Jesus F Moreno1, Jae-Sung You1, Carlos C Rodriguez2,3,4
1Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
None:
Although most members of the Rho guanine nucleotide exchange factor (RhoGEF) family are found to be phosphorylated, how phosphorylation regulates RhoGEF function is poorly understood. Here we report the discovery of a mechanism of RhoGEF regulation by phosphorylation. We find that ARHGEF3 is phosphorylated in the pleckstrin homology (PH) domain in a protein kinase C (PKC)-dependent manner. This phosphorylation inhibits ARHGEF3 activation of RhoA and actin stress fiber formation in the cells, and it also disrupts ARHGEF3 binding to PI(3,5)P2 but not to PI(4,5)P2. Guided by molecular dynamics simulation, a mutation in the PH domain is identified to uncouple ARHGEF3 binding to the two lipids, which is used to rule out a role of PI(3,5)P2 in regulating GEF activity. Results of in vitro GEF assays suggest that PH domain phosphorylation diminishes ARHGEF3 catalytic activity, likely through an allosteric mechanism. Our findings reveal a previously unknown type of regulatory mechanism for the family of RhoGEFs.
Related Concept Videos
Small GTPases - Ras and Rho
Three regulatory proteins control their activity:
GTPases and their Regulation
Large G-proteins,...
Amplifying Signals via Enzymatic Cascade
Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Activation and Inactivation of G Proteins
Rab Proteins
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...

