Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.

Abstract

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant BudS and Ltel proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-s but inactive in promoting exchange on the Ras-related proteins Yptl and Rsrl. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP-or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.Ras proteins, like trimeric G proteins, are active when bound to GTP and inactive when bound to GDP (7). Activation of Ras proteins in vivo may be mediated by proteins referred to as guanine nucleotide exchange factors (also referred to as guanine nucleotide dissociation stimulators or releasing factors) (7). The first such exchange factor for Ras proteins to be identified was the CDC25 gene product of Saccharomyces cerevisiae (10,28 identified, BUD5 and LTEJ (11,26,35). Neither of these genes appears to function in the RAS pathway, but genetic analysis suggests that the BUD5 gene product acts as a GDP-GTP exchange factor for the yeast Rap homolog encoded by RSR1IBUD1 (1, 11). A much closer homolog of CDC25 exists in S. cerevisiae, the SDC25 gene (8). A C-terminal fragment of SDC25 expressed in Escherichia coli is a potent catalyst of GDP-GTP exchange for yeast Ras2 and mammalian p2lHras proteins, yet genetic analysis of SDC25 has not as yet revealed any cellular function for SDC25 in the control of Ras activity (13,14). Perhaps two distinct CDC25-like exchange factors in budding yeast cells allow Ras proteins to transduce signals from different growth stimuli.We undertook this study to determine more precisely which region of the lengthy CDC25 gene (1,589 codons) is required for essential cellular function and to use this information to test recombinant CDC25 protein fragments for GDP-GTP exchange activity. It has been suggested, on the basis of failure of prior attempts ...

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

“…The failure of the catalytic domain mutant of mRasGRP to induce transformation suggests that the in vivo function of RasGRP requires direct activation of Ras GTPases via guanine nucleotide exchange. The ability of the REM-plus-GEF region of mRasGRP to induce transformation when prenylated indicates that GEF activity is probably the only function of RasGRP required for transformation, as the only known function of the REM box is to increase the efficiency of GEF catalysis in vitro (37). It remains possible that beyond serving as a Ras-specific GEF, RasGRP has an additional biological function that is not apparent via fibroblast transformation assays.…”

Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1)

“…As shown in Figure 2, a fusion protein, GST-C(584 ± 1088), e ciently stimulated the dissociation of prebound [ 3 H]GDP from c-Ha-Ras. This fusion protein contains the entire CDC25H domain (`C') including the`core portion' (residues 793 ± 980) of the CDC25H domain conserved not only in all of the CDC25-type GEFs but also in the Bud5-type GEFs (Boguski and McCormick, 1993), the 48-residue region (the`RasGEF-conserved' region, residues 618 ± 665) conserved within the Rasspeci®c GEFs (Lai et al, 1993), and the N-terminal 34-residue region (the`Sos-conserved' region, residues 584 ± 617) conserved in the mSos, hSos, and dSos proteins, but not in other Ras-speci®c GEFs. Two longer fragments, the GST-P-C fusion protein (residues 429 ± 1088) with the PH domain (`P') in addition to the entire CDC25H domain (`C') and the GST-D-P-C Figure 1 Schematic representation of mSos1 fragments tested in the in vitro guanine nucleotide exchange activity assay.…”

“…A central part of Sos shares homology with a region of about 200 amino acid residues conserved in Ras GEFs and related proteins (Boguski and McCormick, 1993). About 130 ± 180 residues upstream of this highly conserved 200-residue region, most of the Ras GEFs have another 48 signi®cantly conserved amino acid residues (Lai et al, 1993). In the case of the S. cerevisiae CDC25 protein, this 48-residue sequence has been proposed to be important for the guanine nucleotide exchange activity (Lai et al, 1993), although there have been no reports on the function of the N-terminal region of the Sos CDC25H domain.…”

A Model for Dynamin Self-assembly Based on Binding Between Three Different Protein Domains

“…We could not determine whether the assembly domain induces nucleotide release or, alternatively, promotes GTP hydrolysis, because hydrolysis and release occur at very high rates even without assembly domain (data not shown). However, binding between the GTPase and the assembly domain might resemble binding between other GTP-binding proteins and guanine nucleotide release proteins, which is also stronger in the nucleotide-free state or with mutations like K44A and S45N (26,27).…”