Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor.
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.
Créchet
J B
JB
Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
Bernardi
A
A
Parmeggiani
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D015852:Eukaryotic Initiation Factor-2; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins; D000262:Adenylyl Cyclases