Distal Switch II Region of Ras2p Is Required for Interaction with Guanine Nucleotide Exchange Factor

Jean-Bernard Créchet,Alberto Bernardi,Andrea Parmeggiani

doi:10.1074/jbc.271.29.17234

Abstract

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

Highlights

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25Mm, yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type-GEF interaction by forming a stable complex
To characterize the interfering properties of Ras2p S24N in vitro in a quantitative manner, we examined its effect on the dissociation rate of Ras2p wt1⁄7[3H]GDP induced by Sdc25p-C (Fig. 1A), Cdc25p-C (Fig. 1B), and CDC25Mm-C (Fig. 1C) or on p211⁄7[3H]GDP in the presence of CDC25Mm-C (Fig. 1D)
In all cases the response of Ras wt to GEF was inhibited by Ras2p S24N in a concentration-dependent manner

Summary

Introduction

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25Mm, yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. Ras proteins are GTPases that regulate cell growth and differentiation by cycling between the active GTP-bound and the inactive GDP-bound states The level of these two forms is determined by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs) (Boguski & McCormick, 1993). To examine the functional role of this region concerning the response to GEF, the mechanism of interference of dominant negative mutations, and the activation of adenylyl cyclase, we have constructed a Ras2p mutant that combines substitutions of dominant (S24N) and recessive type (R80D/ N81D). Ras2p for the action of GEF action and the specific properties of the mechanism of interference of Ras2p mutants

Methods

Results

Conclusion