The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1.

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.

RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral.

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.

Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs.

Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.

PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2.

The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.

Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade.

Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade.

The Ras mutant D119N is both dominant negative and activated.

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

Effector recognition by the small GTP-binding proteins Ras and Ral.

The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition.

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.

Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.

Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.

Structure determination of the Ras-binding domain of the Ral-specific guanine nucleotide exchange factor Rlf.

Ral-specific guanine nucleotide exchange factors RalGDS, Rgl, and Rlf have been suggested to function as intermediates between Ras and Ral pathways by being able to bind Ras proteins through their C-terminal Ras-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-1 have been shown to have the same tertiary structure. In contrast to the RBDs of Raf and RalGDS, which bind either Ras or Rap with high affinity, Rlf-RBD has a similar affinity for both GTP-binding proteins. To be able to compare these RBDs on a structural level, we have solved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the betabetaalphabetabetaalphabeta-fold of the ubiquitin superfamily and is very similar to that of RalGDS-RBD. The binding interface of Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode similar to that in the case of Rap.Raf-RBD. However, comparison of the putatively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of Rlf-, RalGDS-, and Raf-RBD.

Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.

Activation of the small GTPase Ral in platelets.

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including alpha-thrombin. In contrast, the platelet antagonist prostaglandin I2 inhibited alpha-thrombin-induced Ral activation. Activation of Ral by alpha-thrombin could be inhibited by depletion of intracellular Ca2+, whereas the induction of intracellular Ca2+ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca2+ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.

Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells.

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.

Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases.

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.

Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands.

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.

Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25Mm originate both loss-of-function and gain-of-function proteins.

The role of two residues within the catalytic domain of CDC25Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25Mm(E1048K) and CDC25Mm(S1122V) were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25Mm(S1122A) showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N).

A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.