Activity of the Bcr GTPase-activating Domain Is Regulated through Direct Protein/Protein Interaction with the Rho Guanine Nucleotide Dissociation Inhibitor

Soo-Mi Kweon,Young Jin Cho,Parviz Minoo,John Groffen,Nora Heisterkamp

doi:10.1074/jbc.m705513200

Abstract

The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.

Highlights

Bcr Forms a Complex with RhoGDI␣ in Mammalian Cells— To investigate the mechanism by which RhoGDI inhibits the GTPase-activating proteins (GAPs) activity of Bcr, we cotransfected COS-1 cells with constructs encoding full-length Bcr and RhoGDI␣ to test whether these proteins interact in vivo
Bcr Binds to RhoGDI␣ through Its GAP Domain—Bcr was originally identified as part of a Bcr-Abl fusion protein, which is responsible for the development of chronic myelogenous leukemia [32]
In contrast to their results, we found that RhoGDI was able to prevent non-prenylated Rac-GTP conversion to Rac-GDP by the Bcr GAP domain

Summary

Introduction

Bcr Forms a Complex with RhoGDI␣ in Mammalian Cells— To investigate the mechanism by which RhoGDI inhibits the GAP activity of Bcr, we cotransfected COS-1 cells with constructs encoding full-length Bcr and RhoGDI␣ to test whether these proteins interact in vivo. The C-terminal end of RhoGDI␣ is sufficient to mediate the binding to Bcr. RhoGDI␣ Inhibits the GAP Activity of Bcr in Vivo and in Vitro— Hancock and Hall [25] reported that, in vitro, Bcr is unable to act as a GAP toward prenylated Rac-GTP when RhoGDI is present.

Results

Conclusion