Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation.

A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex.

The low molecular weight GTP binding protein Rac is essential to the activation of the NADPH oxidase complex, involved in pathogen killing during phagocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist between Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisoprene of the GTPase, as well as protein-protein interactions. Using the two-hybrid system, we show that nonprenylated Rac1 interacts very weakly with Rho-GDI, pointing to the predominant role of protein-isoprene interaction in complex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), His103(Rac), and the C-terminal polybasic region Arg183(Rac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenylated by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction except in the complex in which Rac1 has a deletion of the polybasic region (Arg183(Rac)-Lys188(Rac)). This complex is, most likely, held together through protein-lipid interaction only. Although able to function as GTPases, the mutants of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119(Rac)Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Rac1/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases were bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins.

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.

The Rac GTPase-activating protein RotundRacGAP interferes with Drac1 and Dcdc42 signalling in Drosophila melanogaster.

RhoGTPases are negatively regulated by GTPase-activating proteins (GAPs). Here we demonstrate that Drosophila RotundRacGAP is active in vitro on Drac1 and Dcdc42 but not Drho1. Similarly, in yeast, RotundRacGAP interacts specifically with Drac1 and Dcdc42, as well as with their activated V12 forms, showing a particularly strong interaction with Dcdc42V12. In the fly, lowering RotundRacGAP dosage specifically modifies eye defects induced by expressing Drac1 or Dcdc42 but not Drho1, confirming that Drac1 and Dcdc42 are indeed in vivo targets of RotundRacGAP. Furthermore, embryonic-directed expression of either RotundRacGAP, or dominant negative Drac1N17, transgenes induces similar defects in dorsal closure and inhibits Drac1-dependent cytoskeleton assembly at the leading edge. Expression of truncated forms of RotundRacGAP shows that the GAP domain of RotundRacGAP is essential for its function. Unexpectedly, transgenes encoding Drac1N17, Dcdc42N17, or RotundRacGAP do not affect the c-Jun N-terminal kinase-dependent gene expression of decapentaplegic and puckered, indicating that another Drac1-independent signal redundantly activates this pathway. Finally, in a situation where Drac1 is constitutively activated, RotundRacGAP greatly reduces the ectopic expression of decapentaplegic, possibly by negatively regulating Dcdc42.

Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI).

The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67(RhoA)-Arg68(RhoA)-Leu69(RhoA) as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.

Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox.

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).

The active N-terminal region of p67phox. Structure at 1.8 A resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease.

Upon activation, the NADPH oxidase from neutrophils produces superoxide anions in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67(phox), p47(phox), and the small G protein Rac with a membrane-associated flavocytochrome b(558). Here we report the crystal structure of the active N-terminal fragment of p67(phox) at 1.8 A resolution, as well as functional studies of p67(phox) mutants. This N-terminal region (residues 1-213) consists mainly of four TPR (tetratricopeptide repeat) motifs in which the C terminus folds back into a hydrophobic groove formed by the TPR domain. The structure is very similar to that of the inactive truncated form of p67(phox) bound to the small G protein Rac previously reported, but differs by the presence of a short C-terminal helix (residues 187-193) that might be part of the activation domain. All p67(phox) mutants responsible for Chronic Granulomatous Disease (CGD), a severe defect of NADPH oxidase function, are localized in the N-terminal region. We investigated two CGD mutations, G78E and A128V. Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro at 25 degrees C. However, this point mutation represents a temperature-sensitive defect in p67(phox) that explains its phenotype at physiological temperature.

Cloning of Rac and Rho-GDI from tobacco using an heterologous two-hybrid screen.

To examine whether molecular similarities exist between the animal and plant Rho GTPase signaling pathways, we have developed a heterologous two-hybrid screening method. By this technique, we have cloned a cDNA encoding a tobacco Rac-like protein able to interact with a mammalian Rho-GDI. In a second screen this tobacco Rac was used as a bait and a tobacco homologue of Rho-GDI was identified. These results show that some components of the animal and plant Rac signaling pathways are similar enough to allow their interaction in an heterologous approach. Moreover these data suggest a similar regulation of Rho GTPases in animals and plants.

Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex.

Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [alpha32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.

The Ku70 autoantigen interacts with p40phox in B lymphocytes.

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

Making hybrids of two-hybrid systems.

Two-hybrid systems are powerful tools to find new partners for a protein of interest. However, exchange of material between two-hybrid users has been handicapped by the various versions of two-hybrid systems available and by the widely accepted idea that they are not compatible. In the present paper we show that, contrary to the dogma, the most often used two-hybrid systems may be combined by either transformation or mating assays. The protocol to be followed in each case is provided. This will greatly increase the prospects of the growing network of interacting proteins, by reconciling the "two-hybrid systems" and the "interaction trap".

In vitro activation of the NADPH oxidase by fluoride. Possible involvement of a factor activating GTP hydrolysis on Rac (Rac-GAP).

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell-free system. It is shown that the stimulatory effect of fluoride is inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP[S]) and potentiated by GTP. The effect of fluoride is not additive with GTP[S]. Fluoride activation requires the presence of Mg2+ in millimolar concentration but is independent of Al3+. The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP-binding proteins by immunoadsorption. Fluoride has no direct action either on the nucleotide exchange of GTP hydrolysis of the isolated Rac protein. In contrast, fluoride effectively inhibits Rac-GTPase activity enhanced by a membrane component. In this way, fluoride could prolong the prevalence of Rac in the GTP-bound state and, as a consequence, activate NADPH oxidase. The possibility of the involvement of a membrane-bound Rac GTPase-activating protein activity in the physiological regulation of the enzyme is raised.

Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase. Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system.

Activation of the superoxide-generating NADPH-oxidase in phagocytic cells requires the assembly of a membrane-bound flavocytochrome b and cytosolic factors p47phox and p67phox under the control of the GTP-binding protein, Rac. A novel cytosolic component p40phox was recently identified. Most of the components of the complex contain SH3 domains and/or polyproline motifs which are likely to mediate protein-protein interactions occurring in the formation of the active NADPH-complex. The two-hybrid system was used to explore associations between the cytosolic factors. Various constructs of p47phox, p67phox and p40phox cDNAs coding for functional domains were inserted into two-hybrid system vectors, expressing fusion proteins either with the DNA binding protein Lex A or with the activation domain of Gal 4. The site of interaction of p67phox with p47phox was restricted to the C-terminal SH3 domain of p67phox and to the polyproline motif of p47phox. The polyproline motif of p47phox was also found to mediate interaction with the SH3 domain of p40phox, as well as intramolecular interaction within p47phox. The site of interation of p67phox with p40phox was found to be in the 150 amino acid stretch between the two SH3 domains of p67phox. As the C-terminal tail of p40phox which interacts with p67phox contains neither a SH3 domain nor a polyproline consensus site, it is concluded that a novel type of interaction occurs between p40phox and p67phox. Taken together, the results of the two-hybrid experiments led us to formulate a model for oxidase activation, induced by phosphorylation, in which p40phox tends to prevent spontaneous activation.

Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system.

The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67phox, p47phox and p40phox as well as a small G protein Rac (for review, see Refs. 1-3). A truncated form of the p40phox cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47phox as target. This truncated form of p40phox consisting of the Src Homology 3 (SH3) domain to the 3' stop codon was also shown to interact with p67phox in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47phox or p67phox. Results show that the SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C terminus of p40phox but not its SH3 domain is involved in the interaction with p67phox.

Small G proteins and the neutrophil NADPH oxidase.

The NADPH oxidase of phagocytic cells is a multimeric enzyme complex activated during phagocytosis. It catalyzes the production of the superoxide anion, precursor of many toxic oxygen metabolites involved in the defense against microorganisms. The enzyme becomes active after assembly on a membrane bound flavocytochrome b of cytosolic factors p47 phox, p67 phox and p40 phox and of low molecular mass GTP binding proteins. This paper reviews recent results concerning the role of two small G proteins, Rac and Rap 1A in oxidase activation. Native prenylated small G proteins are either in the form of a complex in which the GDP bound G protein is associated with a guanine nucleotide dissociation inhibitor, GDI, or in an active GTP bound form able to trigger the activity of its effector. Rac and Rho share a common GDI. As chemotaxis, under Rho control, and oxidase activation, under Rac control, show mutually exclusive signalling pathways, we propose a model where the GDI would switch from one pathway to the other by sequestering either Rac or Rho.

Activation of the O2(-)-generating NADPH oxidase in a semi-recombinant cell-free system. Assessment of the function of Rac in the activation process.

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotide-binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.

Immunoaffinity purification of an oxidase-activating cytosolic complex from bovine neutrophils.

An oxidase activating complex from the cytosol of bovine neutrophils was purified by immunoaffinity using a monoclonal antibody specific for the 67 kDa cytosolic factor of oxidase activation (p67) and assayed for production of superoxide O2- in a cell-free system. The complex comprised not only p67, but also the second cytosolic factor of 47 kDa (p47) in equivalent amounts. The p47-p67 complex showed a good oxidase activating potency when added to neutrophil membranes in the presence of GTP-gamma-S and arachidonic acid. A ras-related small G protein could not be immunodetected in the p47-p67 activating complex, indicating that the GTP required for oxidase activation in the cell free system bound to a protein that was either present in catalytic amounts in the cytosolic complex or present in sufficient amount in the membrane fraction.

A generally applicable ELISA for the detection and quantitation of the cytosolic factors of NADPH-oxidase activation in neutrophils.

An enzyme-linked immunosorbent assay (ELISA) has been developed using polyclonal antibodies raised against two cytosolic proteins of 47 kDa (p47) and 67 kDa (p67) which behave as activation factors for the superoxide-generating NADPH oxidase of neutrophils at the onset of phagocytosis. These two proteins become associated with the NADPH oxidase complex during activation. This immunological technique has been used to follow the purification steps of p47 and p67. It allows the detection of very small amounts of cytosolic factors in a crude neutrophil extract. It is straightforward and much more sensitive (about 1000 times more) than the classical assay based on the use of a cell-free system of oxidase activation and production of superoxide anion. The percentages of p47 and p67 assessed by ELISA with respect to total cytosolic protein were estimated to amount to 0.13 and 0.20%, respectively. The described method has potential applications for the titration of the cytosolic factors of oxidase activation in autosomal recessive forms of chronic granulomatous disease.

Purification and properties of a functional 47-kilodalton cytosolic factor required for NADPH-oxidase activation in bovine neutrophils.

A cytosolic factor of 47 kDa required for activation of the NADPH oxidase, and referred to as p47, has been purified in its functional form from the cytosol of resting bovine neutrophils. The purification was monitored by the determination of the activating potency of p47 in a cell-free system of oxidase activation. The recovery was around 10% and the purification factor greater than 1000. P47 was phosphorylated in vitro by protein kinase A and protein kinase C. [32P] labeled p47 was resolved by isoelectric focusing into two major labeled bands of pI 7.0 and 8.5. Polyclonal antibodies were used to demonstrate that p47 is localized specifically in the cytosol of resting neutrophils, and that, upon activation of neutrophils, p47 is translocated from the cytosol to the membrane.