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

doi:10.1074/jbc.271.29.17234

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

^‡Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France, the
^§Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette, and the Institute Jacques Monod, Université Paris 7, F-75251 Paris 05, France

^¶To whom correspondence should be addressed. Tel.: 33-1-6933-4180; Fax: 33-1-6933-4840; E-mail:-andrea{at}poly.polytechnique.fr

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.

Footnotes

↵* This work was supported by Contract BIOTECH BIO2-CT93-00005 of the European Community, by the Ligue Nationale Fran¸aise Contre le Cancer, by Grant 6377 from the Association pour la Recherche sur le Cancer, and by the Fédération Nationale des Centres de Lutte Contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GEF

GDP/GTP exchange factor of Ras proteins

DTT

dithiothreitol

MES

4-morpholineethanesulfonic acid

wt

wild type.
↵² Some of the designations used are: Ras proteins, the products of RAS genes in general including human H-Ras p21 and yeast S. cerevisiae Ras2p; Ras2p, the product of S. cerevisiae RAS2 gene; Ras2p S24N, the product of the RAS2 S24N dominant allele; Ras2p R80D/N81D, the product of the ras2 R80D/N81D recessive allele; Ras2p S24N/R80D/N81D, a construct derived from Ras2p S24N and Ras2p R80D/N81D; CDC25^Mm-C, the catalytic domain of a mouse brain-specific GEF; Cdc25p-C, the catalytic domain of the S. cerevisiae GEF Cdc25p; Sdc25p-C, the catalytic domain of the S. cerevisiae GEF Sdc25p; CDC25^GEF, a general term designing Cdc25p, Sdc25p, and CDC25^Mm.
↵³ J.-B. Créchet and O. Fasano, unpublished observations.
↵⁴ E. Jacquet and J. B. Créchet, unpublished observations.
- Received October 2, 1995.
- Revision received April 23, 1996.