The Differential Effects of the Gly-60 to Ala Mutation on the Interaction of H-Ras p21 with Different Downstream Targets

Mo-Chou Chen Hwang,Ying-Ju Sung,Yu-Wen Hwang

doi:10.1074/jbc.271.14.8196

Abstract

We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H-Ras. The significance of our findings is discussed in the light of the GTP-induced conformational change model.

Highlights

Like all other members of the regulatory GTPases, Ras p21 cycles between two states: the active, GTP-bound state and the inactive, GDP-bound state [1, 2]
We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator, factors that interact with GTP-bound form of H-Ras
The cycle is controlled by the balance of guanine nucleotide exchange and GTP hydrolysis, which are in turn regulated by guanine nucleotide exchange factors and factors that possess GAP1 activity [3]

Summary

Introduction

Like all other members of the regulatory GTPases, Ras p21 cycles between two states: the active, GTP-bound state and the inactive, GDP-bound state [1, 2]. In H-Ras, the substitution of the glycine residue by alanine (G60A mutation) was found to perturb the GTP-induced conformation change and to abolish the biological activity [29]. A preliminary NMR study supports the hypothesis that the region affected by the G60A mutation is the switch II domain, whereas the switch I domain appears to be unaltered.. A preliminary NMR study supports the hypothesis that the region affected by the G60A mutation is the switch II domain, whereas the switch I domain appears to be unaltered.2 This finding is in accord with the studies of Gs␣ and EF-Tu harboring the corresponding mutation (26 –28) and supports the crucial functional role of the switch II region for regulatory GTPases. We examined the effects of the G60A mutation on the GTPase activity of H-Ras and on the interaction of H-Ras with four factors that interact with H-Ras1⁄7GTP

Methods

Results

Conclusion