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

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.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) 1 (Boguski & McCormick, 1993). The yeast Saccharomyces cerevisiae Cdc25p (Parmeggiani et al., 1993) was the prototype for Ras GEFs, such as S. cerevisiae Sdc25p (Créchet et al., 1990b and mammalian tissuesspecific brain GEFs Jacquet et al., 1992;Schweighoffer et al., 1993). Due to difficulties in isolating the intact protein, only the catalytic domain of these GEFs has been used to investigate the mechanism of the exchange reaction in vitro so far (Créchet et al., 1990b;Lai et al., 1993;Shou et al., 1992;Schweighoffer et al., 1993;Haney & Broach, 1994;Jacquet et al., 1994Jacquet et al., , 1995Poullet et al., 1995). Several reports have dealt in these past years with the structures of Ras proteins involved in the interaction with GEF. Although no definite conclusions could be drawn concerning the location of the binding site of Ras proteins for GEF, site-directed mutagenesis of Ras proteins has given useful information on the role played by the various regions of the Ras molecule in the action of GEF. Specific substitutions in the phosphoryl binding I region, the switch I and II regions of human c-H-Ras p21 and Ras2p were identified that influence negatively the action of GEF; some of them have little effect on the affinity for GEF (Mistou et al., 1992), whereas others can induce dominant negative interferences on the interaction between wild type Ras and GEF, due to the formation of a stable complex between the mutant and GEF (Powers et al., 1989;Szeberényi et al., 1990;Stacey et al., 1991...

Functional-Structural Analysis of Threonine 25, a Residue Coordinating the Nucleotide-bound Magnesium in Elongation Factor Tu

“…In Ras proteins, mutations of the position equivalent to EF-Tu Thr-25 are known to be associated with dominant negative phenotypes (19 -22). This has been shown in vitro for Ras2p(S24N) to be associated with increased stability of the complex of Ras2p with its exchange factor (22,39), thus leading to sequestration of the guanine nucleotide exchange factor. Interestingly, here we describe two mutants of EF-Tu (EF-Tu(H22Y/T25S) and EF-Tu(T25A)) that have a comparable decrease in affinity for GDP, whereas only one, EF-Tu(T25A), shows dominant negative-like behavior through increased stability of the complex with EF-Ts.…”

Different Domains of Mammalian ADP-ribosylation Factor 1 Mediate Interaction with Selected Target Proteins

“…For example, chimeric analysis demonstrated that amino acids 1-102 of Rab 3a, which encompass both putative switch domains, are sufficient to mediate maximal binding to its exchange factor Mss4 (42). In addition, Ras switch domain II and neighboring residues interact with several exchange factors (43)(44)(45)(46)(47)(48). Regions outside of the switch domains also interact with GEFs, but the important domains are not conserved between different families of small GTPbinding proteins.…”

Sites of Phosphorylation by Protein Kinase A in CDC25Mm/GRF1, a Guanine Nucleotide Exchange Factor for Ras

“…In CDC25Mm, substitution of Ser 1124 has been reported to influence the GEF activity (27). The adjacent Ser 1123 is homologous to SOS Ala 877 , which in the three-dimensional model hydrogen-bonds Gln 70 of Ha-Ras (28), a residue situated on ␣-helix 2 of the switch 2 region that is essential for the exchange reaction (29,30). A phosphorylated residue, Ser 643 , was identified in the REM sequence, a conserved stretch essential for the function and structural integrity of the RasGEF family.…”