Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.     

Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras

Ras-related GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states. GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras x GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.     

A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.     

Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP

The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AIF3 molecule, as was seen for the corresponding Ras-p120RasGAP complex, but in contrast to what has been reported for the Rho-Cdc42GAP complex or for heterotrimeric G protein alpha subunits, where AIF4- was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

The mammalian rasGAPs constitute a group of widely expressed proteins involved in the negative regulation of ras-mediated signaling. In this study we have isolated a novel human gene, RASAL (Ras GTPase-activating-like) and its murine ortholog, MRASAL which are most similar to the GAP1 family of rasGAP proteins, based upon the presence and organization of specific conserved domains. Full-length human and murine mRNA sequences are predicted to encode 804 and 799 amino acid polypeptides, respectively. Sequence analysis of these two proteins revealed the presence of two N-terminal calcium-dependent phospholipid binding C2 domains, a conserved GAP related domain (GRD) and a C-terminal pleckstrin homology (PH) domain. Northern blot and mRNA in situ hybridization analyses indicate that RASAL, in contrast to other mammalian rasGAP proteins, has a limited expression pattern; RASAL is highly expressed in the follicular cells of the thyroid and the adrenal medulla and expressed at lower levels in brain, spinal cord and trachea. Human RASAL has been localized by radiation hybrid mapping to chromosome 12q23-24.     

The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors

p190 is a Tyr-phosphorylatable G protein of M(r) 190,000 that binds NH2-terminal SH2 domains of GAP1, a Ras GAP of M(r) 120,000. p190 contains at least two functional domains: a GTPase domain at the NH2 terminus and a GAP domain at the COOH terminus that can attenuate signal-transducing activity of three distinct G proteins (Rac, Rho, and CDC42). Here, we demonstrate that overexpression of either an antisense p190 RNA or a dominant negative mutant (Asn36) of p190 GTPase domain (residues 1-251) but not the wild-type p190 GTPase domain is able to transform normal NIH/3T3 fibroblasts. Furthermore, overexpression of either the wild-type p190 GTPase domain or the COOH-terminal GAP domain can suppress v-Ha-Ras-induced malignant transformation. These results indicate that p190 contains at least two distinct anti-Ras tumor suppressor domains, the GTPase and GAP domains, and suggest that one of the mechanisms underlying the suppression of Ras-transformation by p190 is the attenuation by p190 GAP domain of Rac/Rho/CDC42 signalings, which are essential for Ras-transformation. In fact, the p190 GAP domain alone suppresses the expression of the c-Fos gene, which is mediated by Rac/Rho/CDC42 and is required for oncogenicity of Ras.     

Structural analysis of the GAP-related domain from neurofibromin and its implications

Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most frequent genetic disorders in man. It acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which despite very limited sequence homology is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed which is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions.     

GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein

The majority of the GTP-binding proteins of the Ras superfamily hydrolyze GTP to GDP very slowly. A notable exception to this are the Rac proteins, which have intrinsic GTPase rates at least 50-fold those of Ras or Rho. A protein (or proteins) capable of inhibiting this GTPase activity exists in human neutrophil cytosol. Since Rac appears to exist normally in neutrophils as a cytosolic protein complexed to (Rho)GDI, we examined the ability of (Rho)GDI to inhibit GTP hydrolysis by Rac. (Rho)GDI produced a concentration-dependent inhibition of GTP hydrolysis by Rac1 that paralleled its ability to inhibit GDP dissociation from the Rac protein. Maximal inhibition occurred at or near equimolar concentrations of the GDI and the Rac substrate. The ability of two molecules exhibiting GTPase activating protein (GAP) activity toward Rac to stimulate GTP hydrolysis was also inhibited by the presence of (Rho)GDI. The inhibitory effect of the GDI could be overcome by increasing the GAP concentration to levels equal to that of the GDI. (Rho)GDI weakly, but consistently, inhibited GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) dissociation from Rac1, confirming an interaction of (Rho)GDI with the GTP-bound form of the protein. These data describe an additional activity of (Rho)GDI and suggest a mechanism by which Rac might be maintained in an active form in vivo in the presence of regulatory GAPs.     

Effect of metal cations on the conformation of myosin subfragment-1-ADP-phosphate analog complexes: a near-UV circular dichroism study

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power stroke, suggest new strategies for perturbing S1 structure in the continuous efforts directed toward the elucidation of the mechanism of muscle contraction.     

Requirement for both the amino-terminal catalytic domain and a noncatalytic domain for in vivo activity of ADP-ribosylation factor GTPase-activating protein

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulates intracellular transport by modulating the interaction of coat proteins with the Golgi complex. Coat protein association with Golgi membranes requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Golgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316). In this study, we used this phenotype to define domains in GAP that are required for its function in vivo. As expected, mutations in the amino-terminal part of GAP that were previously found to abolish ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in vivo. Significantly, truncations at the carboxyl-terminal part of GAP that did not affect GAP catalytic activity in vitro also diminished ARF1 deactivation. Thus, a noncatalytic domain is required for GAP activity in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.     

K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors

The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.     

NF1 (neurofibromatosis type 1)

Several distinct Ras GTPase activating proteins (GAPs) from mammals, including Ras GAP of 120 kDa (GAP1) and NF1, stimulate the intrinsic GTPase activity of normal Ras, but not oncogenic Ras mutants (Trahey and McCormick, 1987). That is the reason why normal Ras remains predominantly in the inactive GDP-bound form (D-Ras), whereas oncogenic Ras remains constitutively in the active GTP-bound form (T-Ras). NF1 is a tumor suppressor of 2818 amino acids whose disruption or deletion causes brain tumors called neurofibromatosis type 1 by elevating the T-Ras level. T-Ras activates several distinct oncogenic effectors, including Ser/Thr kinase Raf, GAP1, P1-3 kinase, PKC-zeta and Ra1 GDS. Interestingly, the binding of T-Ras to either GAPs or these oncogenic effectors requires the same effector domain I (residues 32-40) of T-Ras molecule. In other words, these GAPs and effectors compete for binding to T-Ras. Using a series of N- and C-terminal deletion mutants of NF1, we identified a 78 amino acid fragment (NF78, residues 1441-1518) as the minimum GAP domain, and a 56 amino acid fragment (NF 56, residues 1441-1496) as the minimum Ras-binding domain. Furthermore, we identified the Raf fragment of 81 amino acids (Raf81, residues, 51-131) as the minimum Ras-binding domain with a high affinity. We found that (i) these NF1 fragments and Raf81 compete for binding to T-Ras, and that (ii) over-expression of these NF1 or Raf fragments strongly suppresses the malignant transformation caused by oncogenic Ras mutants. Thus, these agents offer a unique opportunity to control the proliferation of T-Ras-associated tumors that represent more than 30% of all human carcinomas including neurofibromatosis type 1.     

Activation of R-Ras GTPase by GTPase-activating proteins for Ras, Gap1(m), and p120GAP

The enzymatic properties of Gap1(m) were characterized using three Ras and R-Ras proteins as substrates and were compared with those of p120GAP. Gap1(m) stimulated the GTPase of Ras better than that of R-Ras, in contrast to p120GAP which promoted the GTPase of R-Ras better than that of Ras. The EC50 values of Gap1(m) for Ha-Ras and R-Ras were 0.48 +/- 0.02 and 1.13 +/- 0.12 nM, respectively, whereas the EC50 values of p120GAP for Ha-Ras and R-Ras were 23.1 +/- 1.9 and 3.86 +/- 0.38 nM, respectively. The affinities of Gap1(m) and p120GAP to the substrates determined by competitive inhibition by using Ha-Ras.GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)) or R-Ras.GTPgammaS as a competitor agreed well with the substrate specificities of these GTPase-activating proteins. The Km values of Gap1(m) for Ha-Ras and R-Ras were 1.53 +/- 0.27 and 3.38 +/- 0.53 microM, respectively, which were lower than that of p120GAP for Ha-Ras (145 +/- 11 microM) by almost 2 orders of magnitude. The high affinity of Gap1(m) to the substrates and its membrane localization suggest that Gap1(m) may act as a regulator of the basal activity of Ha-Ras and R-Ras.     

All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral

Ras, Rap1 and Ral are related small GTPases. While the function of Ras in signal transduction is well established, it has been recognized only recently that Rap1 and Ral also are activated rapidly in response to a large variety of extracellular signals. Between the three GTPase an intriguing interconnectivity exists, in that guanine nucleotide exchange factors for Ral associate with the GTP-bound form of both Ras and Rap1. Furthermore, Rap1 is considered to function as an antagonist of Ras signalling by trapping Ras effectors in an inactive complex. Here, I summarize the recent developments in understanding the functional relationship between these three GTPase and argue that Rap1 functions in a signalling pathway distinct from Ras, while using similar or identical effectors.     

Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum

Small GTPases interact with a variety of proteins that affect nucleotide binding and cleavage. GTPase activating proteins (GAPs) are one class of these proteins that act by accelerating the intrinsic GTPase rate resulting in the formation of the biologically inactive GDP-bound form of the GTPase. For the Rho subfamily of GTPases, there is a growing number of proteins with rhoGAP activity that are identifiable by a homologous region of about 150 amino acids. We have exploited this homology using the polymerase chain reaction to clone the first rhoGAP homolog, called DdRacGAP, from the slime mold Dictyostelium discoideum. The GAP domain of DdRacGAP (amino acids 1-212), when expressed and purified from Escherichia coli, is active on both Dictyostelium and human Rho family GTPases but not human Ras. The full-length protein is 1356 amino acids in length and has several interesting homologies in addition to the GAP domain, including an SH3 domain, a dbl homology domain, and a pleckstrin homology domain.     

RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.     

Evidence for linkage between the development of asthma in childhood and the T-cell receptor beta chain gene in Japanese

GTPase-activating proteins (GAPs) stimulate the hydrolysis of GTP bound to small G-proteins and regulate the signal transduction pathway. Changes in the expression of p21-Ras p120-GAP induced by growth factor treatment were examined in cultured Chinese hamster ovary (CHO) and human choriocarcinoma (BeWo) cells. Expression of p120-GAP and GAP activity were measured. Fetal bovine serum induced a significant increased level of GAP in CHO cells, but did not increase GAP in BeWo cells. The results suggest that growth factors affect Ras GAP expression in CHO cells, while they do not in other cells such as BeWo cells.     

Catalytic domain of the p120 Ras GAP binds to RAb5 and stimulates its GTPase activity

Ras is a master GTPase switch controlling multiple signal transduction cascades in the regulation of cell proliferation and differentiation. Rab5 is a local GTPase switch that is localized on early endosomes and controls early endosome fusion. This study demonstrates that the catalytic domain of p120 GTPase-activating protein (GAP), a well known Ras GAP, is able to interact physically with Rab5 and stimulate its GTPase activity. This GAP activity toward Rab5, however, cannot be extended to other Rab GTPases such as Rab3, Rab4, and Rab6, indicating that it is not a generic GAP for the Rab family of GTPases that regulate intracellular membrane fusion during endocytosis and exocytosis. The findings indicate a level of structural similarity between Ras and Rab5 unexpected from their primary sequences. They also suggest a possible signal transduction regulation of the Rab5-dependent endosome fusion via the Ras GAP.     

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.     

Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling

Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways. GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals. A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling. A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes. The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP. Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone. These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.