Intramolecular Activation Mechanism of the Dictyostelium LRRK2 Homolog Roco Protein GbpC

Wouter N Van Egmond,Arjan Kortholt,Katarzyna Plak,Leonard Bosgraaf,Sylvia Bosgraaf,Ineke Keizer-Gunnink,Peter J.M Van Haastert

doi:10.1074/jbc.m804265200

Abstract

GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco family of proteins that often share a central core region, consisting of leucine-rich repeats, a Ras domain (Roc), a Cor domain, and a MAPKKKinase domain. In addition to this core, GbpC contains a RasGEF domain and two cGMP-binding domains. Here, we report on an intramolecular signaling cascade of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the GDP/GTP exchange of the Roc domain. Moreover, cGMP binding to GbpC strongly stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated GDP/GTP exchange at the Roc domain. The function of the protein in vivo was investigated by rescue analysis of the chemotactic defect of gbpC null cells. Mutants that lack a functional guanine exchange factor (GEF), Roc, or kinase domain are inactive in vivo. Together, the results suggest a four-step intramolecular activation mechanism of the Roco protein GbpC: cGMP binding to the cyclic nucleotide-binding domains, activation of the GEF domain, GDP/GTP exchange of Roc, and activation of the MAPKKK domain.

Highlights

Lar cAMP to the surface cAMP receptor cAR1 causes a G-proteindependent activation of two guanylyl cyclases, soluble guanylyl cyclase and membrane-bound guanylyl cyclase (GCA)
The results lead to an activation model in which cGMP binding to the cyclic nucleotide-binding domains (cNBDs) causes activation of the guanine exchange factor (GEF) domain, the subsequent GDP/GTP exchange of the Roc domain, and activation of the MAPKKK domain
GbpC contains a C-terminal extension with a RasGEF and two cNBDs that bind cGMP with high affinity

Summary

Introduction

Lar cAMP to the surface cAMP receptor cAR1 causes a G-proteindependent activation of two guanylyl cyclases, soluble guanylyl cyclase (sGC) and membrane-bound guanylyl cyclase (GCA). The mGDP- The RasGEF Domain of GbpC Activates the Roc loaded GTPases were incubated at 25 °C in assay buffer GTP Binding Assay—gbpC null cells expressing GbpC-GFP and incubated with excess GDP in the presence or absence of a were washed with lysis buffer

Results

Conclusion