Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase

Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF.     

The SH3 domain of the S. cerevisiae Cdc25p binds adenylyl cyclase and facilitates Ras regulation of cAMP signalling

Cdc25 and Ras are two proteins required for cAMP signalling in the budding yeast Saccharomyces cerevisiae. Cdc25 is the guanine nucleotide exchange protein that activates Ras. Ras, in turn, activates adenylyl cyclase. Cdc25 has a Src homology 3 (SH3) domain near the N-terminus and a catalytic domain in the C-terminal region. We find that a point mutation in the SH3 domain attenuates cAMP signalling in response to glucose feeding. Furthermore, we demonstrate, by using recombinant adenylyl cyclase and Cdc25, that the SH3 domain of Cdc25 can bind directly to adenylyl cyclase. Binding was specific, because the SH3 domain of Abp1p (actin-binding protein 1), which binds the 70,000 Mr subunit of adenylyl cyclase, CAP/Srv2, failed to bind adenylyl cyclase. A binding site for Cdc25-SH3 localised to the C-terminal catalytic region of adenylyl cyclase. Finally, pre-incubation with Ras enhanced the SH3-bound adenylyl cyclase activity. These studies suggest that a direct interaction between Cdc25 and adenylyl cyclase promotes efficient assembly of the adenylyl cyclase complex.     

Glutathiolated Ras: Characterization and implications for Ras activation

Ras GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. Similar to the action of guanine-nucleotide exchange factors, the rate of GDP dissociation can be greatly enhanced by the reaction of Ras with small-molecule redox agents, such as nitrogen dioxide, which can promote Ras activation. Nitrogen dioxide is an autoxidation product of nitric oxide and can react with an accessible cysteine of Ras to cause oxidation of the bound guanine nucleotide to facilitate Ras guanine nucleotide dissociation. Glutathione has also been reported to modify Ras and alter its activity. To elucidate the mechanism by which glutathione alters Ras guanine nucleotide binding properties, we performed NMR, top-down and bottom-up mass spectrometry, and biochemical analyses of glutathiolated Ras. We determined that treatment of H-Ras, lacking the nonconserved hypervariable region, with oxidized glutathione results in glutathiolation specifically at cysteine 118. However, glutathiolation does not alter Ras structure or biochemical properties. Rather, changes in guanine nucleotide binding properties and Ras activity occur upon exposure of Ras to free radicals, presumably through the generation of a cysteine 118 thiyl radical. Interestingly, Ras glutathiolation protects Ras from further free radical-mediated activation events. Therefore, glutathiolation does not affect Ras activity unless Ras is modified by glutathione through a radical-mediated mechanism.     

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

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 Bud5 and Lte1 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-ras but inactive in promoting exchange on the Ras-related proteins Ypt1 and Rsr1. 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.     

The large N-terminal domain of Cdc25 protein of the yeast Saccharomyces cerevisiae is required for glucose-induced Ras2 activation

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

Cyclic AMP signalling in Dictyostelium: G-proteins activate separate Ras pathways using specific RasGEFs

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium development, RasC and RasG are activated in response to cyclic AMP, with each regulating different downstream functions: RasG regulates chemotaxis and RasC is responsible for adenylyl cyclase activation. RasC activation was abolished in a gefA- mutant, whereas RasG activation was normal in this strain, indicating that RasGEFA activates RasC but not RasG. Conversely, RasC activation was normal in a gefR- mutant, whereas RasG activation was greatly reduced, indicating that RasGEFR activates RasG. These results were confirmed by the finding that RasGEFA and RasGEFR specifically released GDP from RasC and RasG, respectively, in vitro. This RasGEF target specificity provides a mechanism for one upstream signal to regulate two downstream processes using independent pathways.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.     

pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.     

Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae

The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.     

Mechanism of the spatio-temporal regulation of Ras and Rap1

Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.     

Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.     

A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.     

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary 1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms.2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase δ. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K.3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in ‘rasosomes’, randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.     

A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival

In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks.     

Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.     

Many faces of Ras activation

Ras proteins were originally identified as the products of oncogenes capable of inducing cell transformation. Over the last twenty-five years they have been studied in great detail because mutant Ras proteins are associated with many types of human cancer. Wild type Ras proteins play a central role in the regulation of proliferation and differentiation of various cell types. They alternate between an active GTP-bound state and an inactive GDP-bound state. Their activation is catalysed by a specialized group of enzymes known as guanine nucleotide exchange factors (GEFs). To date, four subfamilies of GEF molecules have been identified. Although all of them are able to activate Ras, their structure, tissue expression and regulation are significantly diverse. In this review we will summarize the various mechanisms by which these exchange factors activate Ras.     

Many faces of Ras activation

Ras proteins were originally identified as the products of oncogenes capable of inducing cell transformation. Over the last twenty-five years they have been studied in great detail because mutant Ras proteins are associated with many types of human cancer. Wild type Ras proteins play a central role in the regulation of proliferation and differentiation of various cell types. They alternate between an active GTP-bound state and an inactive GDP-bound state. Their activation is catalysed by a specialized group of enzymes known as guanine nucleotide exchange factors (GEFs). To date, four subfamilies of GEF molecules have been identified. Although all of them are able to activate Ras, their structure, tissue expression and regulation are significantly diverse. In this review we will summarize the various mechanisms by which these exchange factors activate Ras.