Fourier transform infrared spectroscopy on the Rap.RapGAP reaction, GTPase activation without an arginine finger

GTPase activating proteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004) Nature 429, 197-201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP(*) conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras.RasGAP reactions. However, the charge shift toward beta-phosphate observed with RasGAP was also observed for RapGAP. A GDP.P(i) intermediate accumulates in the GAP-catalyzed reaction, because the release of P(i) is eight times slower than the cleavage reaction, and significant GTP synthesis from GDP.P(i) was observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap.RapGAP catalytic machinery compensates for the absence of a cis-Gln by a trans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.     

The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues

The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb.     

Unravelling the mechanism of dual‐specificity GAPs

The molecular mechanism by which dual‐specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G‐proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady‐state kinetic assays and time‐resolved Fourier‐transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1^IP4BP and RASAL is mediated by the extra GTPase‐activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch‐II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re‐orientation requires specific interactions between switch‐II of Rap and helix‐α6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.     

The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.     

Rap-specific GTPase activating protein follows an alternative mechanism.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.     

RapGAP9 regulation of the morphogenesis and development in Dictyostelium

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell–substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis in Dictyostelium.     

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.     

Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by guanine nucleotide-binding proteins (GNBPs) is a fundamental enzyme reaction in living cells that acts as an important timer in a variety of biological processes. This reaction is intrinsically slow but can be stimulated by GTPase-activating proteins (GAPs) by several orders of magnitude. In the present study, we synthesized and characterized a new fluorescent nucleotide, 2'(3')-O-(N-ethylcarbamoyl-(5'-carboxytetramethylrhodamine) amide)-GTP, or tamraGTP, which is sensitive towards conformational changes of certain GNBPs induced by GTP hydrolysis. Unlike other fluorescent nucleotides, tamra-GTP allows real-time monitoring of the kinetics of the intrinsic and GAP-catalyzed GTP hydrolysis reactions of small GNBPs from the Rho family.     

Differential G-alpha interaction capacities of the GoLoco motifs in Rap GTPase activating proteins

GoLoco motif proteins act as guanine nucleotide dissociation inhibitors (GDIs) for G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) class. Rap1GAP2 is a newly identified GoLoco motif- and RapGAP domain-containing protein, and thus is considered a potential integrator of heterotrimeric and monomeric GTPase signaling. Primary sequence analysis indicated that the Rap1GAP2 GoLoco motif contains a lysine (Lys-75), rather than an arginine, at the crucial residue responsible for binding the alpha and beta phosphates of GDP and exerting GDI activity. To determine the functional outcome of this sequence variation we conducted a biophysical analysis of the human Rap1GAP2b/c GoLoco motif. We found that human Rap1GAP2b/c was deficient in GDI activity and Galpha interaction capability. Mutation of lysine-75 to arginine could not regain functional activity of the Rap1GAP2b/c GoLoco motif. Thus, the Rap1GAP2b/c GoLoco motif can be classed as inactive towards Galpha subunits. We also found that the Rap1GAP1a GoLoco motif, which lacks seven N-terminal amino acid residues present in canonical GoLoco motifs, does not interact with Galpha(i1). In contrast, the GoLoco motif of Rap1GAP1b, which is canonical in primary sequence, was found to interact with Galpha(i1).GDP.     

The Ability of GAP1IP4BP To Function as a Rap1 GTPase-Activating Protein (GAP) Requires Its Ras GAP-Related Domain and an Arginine Finger Rather than an Asparagine Thumb

GAP1^IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1^m, CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1^m, possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1^IP4BP can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1^IP4BP/GAP1^m chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1^IP4BP, we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1^IP4BP. In contrast, we present evidence consistent with a model in which the RasGRD of GAP1^IP4BP functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.The Ras-like family of small GTPases are ubiquitously expressed, evolutionarily conserved proteins that, by undergoing conformational changes in response to the alternate binding of GDP and GTP, function as binary switches (28, 31, 35). The GDP-bound “off” state and the GTP-bound “on” state recognize distinct effector proteins, thereby allowing the regulation of a variety of downstream signaling events (28, 31, 35). While Ras is the best-known and best-studied Ras-like GTPase, Rap1 has recently attracted considerable attention (reviewed in reference 20).Rap1 was originally identified through its ability, when overexpressed, to reverse the phenotype of K-Ras-transformed NIH 3T3 cells (19). As Ras and Rap1 have very similar effector regions, the ability of Rap1 to reverse the transformed phenotype appeared to arise through an ability to compete with K-Ras effectors. For example, Rap1 binds the Ras effector Raf1 but this does not lead to its activation (11). This is consistent with a simple model in which Rap1 functions as a Ras antagonist (6, 37). However, recent work has challenged this view. Increasing evidence points to Rap1 interacting with its own panel of effectors through which it controls cell-cell adhesion and cell-matrix interactions (reviewed in reference 20).Like that of other GTPases, the activation of Ras and Rap1 is regulated through guanine nucleotide exchange factors, which control activation by stimulating the exchange of GDP for GTP. Inactivation is driven by GTPase-activating proteins (GAPs). These enhance the intrinsic GTPase activity of Ras and Rap1, thereby leading to GTP hydrolysis. A wide variety of guanine nucleotide exchange factors and GAPs specific for these GTPases have been identified (14). Through the arrangement of different modular domains, these proteins are regulated following the activation of cell surface receptors. This occurs either through direct association with the activated receptor or indirectly through second messengers (4, 5, 14, 41).Mammalian proteins capable of functioning as Ras GAPs include NF1 (3, 27, 40), p120^GAP (38), the semaphorin 4D receptor plexin-B1 (29), and members of the GAP1 (reviewed in reference 41) and SynGAP (DAB2IP, nGAP, and SynGAP) families (10, 18, 39). These function as Ras GAPs by supplying a catalytic arginine residue—the arginine finger—into the active site of Ras. This stabilizes the transition state of the GTPase reaction, increasing the reaction rate by more than 1,000-fold (1, 33, 34).Rap1 GAPs include Rap GAPs I and II, the SPA-1 family (SPA-1, SPAR, SPAL, and E6TP1), and tuberin (16, 17, 26, 32). Unlike Ras, Rap1 does not possess the catalytic glutamine residue that is critical for GTP hydrolysis in Ras. This fundamental difference means that the mechanisms by which Ras and Rap1 GAPs function are distinct. Rap1 GAPs do not employ a catalytic arginine residue (8, 9); instead, they provide a catalytic asparagine—the asparagine thumb—to stimulate GTP hydrolysis (15). Here the asparagine carboxamide side chain has a function similar to that of the glutamine residue in Ras, stabilizing the position of the nucleophilic water and γ-phosphate in the transition complex (15, 36).Given such distinct catalytic mechanisms, surprisingly, some Ras GAPs, while having no detectable sequence homology with any Rap1 GAPs, are capable of stimulating the GTPase activity of Rap1. The first protein found to display such dual activities was GAP1^IP4BP (13) (also known as RASA3, GAPIII, and R-Ras GAP). This is a member of the GAP1 family, which also comprises GAP1^m, CAPRI, and RASAL (2, 23-25). These proteins are characterized by a domain architecture comprising amino-terminal tandem C2 domains, a highly conserved central Ras GAP-related domain (RasGRD), and a carboxy-terminal pleckstrin homology (PH) domain that is associated with a Bruton''s tyrosine kinase (Btk) motif (41). Consistent with the presence of the RasGRD, all proteins display Ras GAP activity, although each is differentially regulated following receptor stimulation (41). With the notable exception of GAP1^m, all GAP1 proteins also possess efficient Rap1 GAP activity (22). Such dual specificity is not restricted solely to GAP1 proteins. Recently, C2 domain-containing SynGAP—a neuronal Ras GAP—has also been shown to display Rap1 GAP activity (21), an activity that appears to require, alongside the RasGRD, the presence of a single C2 domain (30).Here we have examined the mechanism behind the dual Ras and Rap1 GAP activities of GAP1^IP4BP. Through the generation of a series of GAP1^IP4BP/GAP1^m chimeras, we have established that while the C2 domains of GAP1^IP4BP are required to stabilize the RasGRD, these domains do not supply catalytic residues required for Rap1 GAP activity. Rather, the Rap1 GAP catalytic machinery appears to reside solely within the RasGRD. By the site-directed mutagenesis of selected asparagine and glutamine residues within this domain—selected following the generation of a predicted molecular model of the GAP1^IP4BP RasGRD-Ras(Rap1) complex—we establish that the ability of GAP1^IP4BP to function as a Rap1 GAP does not occur via a mechanism that utilizes a classic asparagine thumb. Rather, we suggest that the GAP1^IP4BP RasGRD functions to stabilize the switch II region of Rap1 in a manner that allows a catalytic arginine finger from GAP1^IP4BP to drive the hydrolysis of GTP.     

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.     

Regulation of Rap1 activity by RapGAP1 controls cell adhesion at the front of chemotaxing cells

Spatial and temporal regulation of Rap1 is required for proper myosin assembly and cell adhesion during cell migration in Dictyostelium discoideum. Here, we identify a Rap1 guanosine triphosphatase–activating protein (GAP; RapGAP1) that helps mediate cell adhesion by negatively regulating Rap1 at the leading edge. Defects in spatial regulation of the cell attachment at the leading edge in rapGAP1⁻ (null) cells or cells overexpressing RapGAP1 (RapGAP1^OE) lead to defective chemotaxis. rapGAP1⁻ cells have extended chemoattractant-mediated Rap1 activation kinetics and decreased MyoII assembly, whereas RapGAP1^OE cells show reciprocal phenotypes. We see that RapGAP1 translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of chemotaxing cells via an F-actin–dependent pathway. RapGAP1 localization is negatively regulated by Ctx, an F-actin bundling protein that functions during cytokinesis. Loss of Ctx leads to constitutive and uniform RapGAP1 cortical localization. We suggest that RapGAP1 functions in the spatial and temporal regulation of attachment sites through MyoII assembly via regulation of Rap1–guanosine triphosphate.     

Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk

G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown. In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z). One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling. Like G(z), the precise role of Rap1 in cell physiology is poorly understood. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase. Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction. Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex. These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells.     

Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling

The ADP-ribosylation factor (Arf) Arf GTPase-activating proteins (GAPs) are a family of proteins that induce hydrolysis of GTP bound to Arf. A conserved domain containing a zinc finger motif mediates catalysis. The substrate, Arf.GTP, affects membrane trafficking and actin remodelling. Consistent with activity as an Arf regulator, the Arf GAPs affect both of these pathways. However, the Arf GAPs are likely to have Arf-independent activities that contribute to their cellular functions. Structures of the Arf GAPs are diverse containing catalytic, protein-protein interaction and lipid interaction domains in addition to the Arf GAP domain. Some Arf GAPs have been identified and characterized on the basis of activities other than Arf GAP. Here, we describe the Arf GAP family, enzymology of some members of the Arf GAP family and known functions of the proteins. The results discussed illustrate roles for both Arf-dependent and -independent activities in the regulation of cellular architecture.     

Insight into catalysis of a unique GTPase reaction by a combined biochemical and FTIR approach

Rap1 and Rap2 are the only small guanine nucleotide-binding proteins of the Ras superfamily that do not use glutamine for GTP hydrolysis. Moreover, Rap1GAP, which stimulates the GTPase reaction of Rap1 10(5)-fold, does not have the classical "arginine finger" like RasGAP but presumably, introduces an asparagine residue into the active site. Here, we address the requirements of this unique reaction in detail by combining various biochemical methods, such as fluorescence spectroscopy, stopped-flow and time-resolved Fourier transform infrared spectroscopy (FTIR). The fluorescence spectroscopic assay monitors primarily protein-protein interaction steps, while FTIR resolves simultaneously the elementary steps of functional groups labor-free, but it is less sensitive and needs higher concentrations. Combining both methods allows us to distinguish weather mechanistic defects caused by mutation are due to affinity or due to functionality. We show that several mutations of Asn290 block catalysis. Some of the mutants, however, still form a complex with Rap1*GDP in the presence of BeF(x) but not AlF(x), supporting the notion that fluoride complexes are indicators of the ground versus transition state. Mutational analysis also shows that Thr61 is not required for catalysis. While replacement of Thr61 of Rap1 by Leu eliminates GTPase activation by Rap1GAP, the T61A and T61Q mutants have only a minor effect on catalysis, but change the relative rates of cleavage and (P(i)(-)) release. While Rap1GAP(N290A) is completely inactive on wild-type Rap1, it can act on Rap1(T61Q), arguing that Asn290 in trans has a role in catalysis similar to that of the intrinsic Gln in Ras and Rho. Finally, since FTIR works at high, and thus mostly saturating, concentrations, it can clearly separate effects on affinity from purely catalytic modifications, showing that Arg388, conserved between RapGAPs and mutated in the homologous RheBGAP Tuberin, affects binding affinity severely but has no effect on the cleavage reaction itself.     

Tyr39 of Ran Preserves the Ran·GTP Gradient by Inhibiting GTP Hydrolysis

Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound “on” and a GDP-bound “off” state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran·GTP within the nucleoplasm and excess Ran·GDP within the cytoplasm, the hydrolysis of Ran·GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran·GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized.     

Is there a rate-limiting step before GTP cleavage by H-ras p21?

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.     

The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins

We have recently identified E6TP1 (E6-targeted protein 1) as a novel high-risk human papillomavirus type 16 (HPV16) E6-binding protein. Importantly, mutational analysis of E6 revealed a strong correlation between the transforming activity and its abilities to bind and target E6TP1 for ubiquitin-mediated degradation. As a region within E6TP1 has high homology with GAP domains of known and putative Rap GTPase-activating proteins (GAPs), these results raised the possibility that HPV E6 may alter the Rap small-G-protein signaling pathway. Using two different approaches, we now demonstrate that human E6TP1 exhibits GAP activity for Rap1 and Rap2, confirming recent findings that a closely related rat homologue exhibits Rap-specific GAP activity. Using mutational analysis, we localize the GAP activity to residues 240 to 945 of E6TP1. Significantly, we demonstrate that coexpression of HPV16 E6, by promoting the degradation of E6TP1, enhances the GTP loading of Rap. These results support a role of Rap small-G-protein pathway in E6-mediated oncogenesis.     

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.