Oligomerization of Rac1 GTPase Mediated by the Carboxyl-terminal Polybasic Domain

Baolin Zhang,Yuan Gao,Sun Young Moon,Yaqin Zhang,Yi Zheng

doi:10.1074/jbc.m008720200

Baolin Zhang, Yuan Gao + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m008720200

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Abstract

The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.

Highlights

Rho family GTPases Rac1, Cdc42, and RhoA belong to the Ras superfamily of monomeric small GTP-binding proteins
The guanine nucleotide exchange factors (GEFs)1 catalyze the exchange of bound GDP for GTP, resulting in activation of the GTPases, whereas the GTPase-activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis by Rho GTPases, leading to the rapid conversion to the GDP-bound inactive state
The observed oligomerization-elicited self-stimulatory GAP activity of Rac1 provides a rational for the dose-dependent fast rate of intrinsic GTPase activity, and the requirement of the C-terminal domain for this activity can be attributed to the presence of Arg186 in the region, which we have previously shown to constitute a “built-in arginine figure” in forming a transition state of the GTPase-activating reaction of other Rho family GTPases such as Cdc42 and Rac2 [17]

Summary

Introduction

Rho family GTPases Rac1, Cdc42, and RhoA belong to the Ras superfamily of monomeric small GTP-binding proteins. Oligomerization of Rac1-GTP Elicits a Self-stimulatory GTPase-activating Protein Activity—To examine the biochemical properties of different forms of Rac1, the eluent fractions corresponding to the oligomer and monomer from the Superdex 200HR gel filtration column were isolated.

Results

Conclusion