The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

"THE EMPEROR'S NEW CLOTHES"*

Natalia SigalDagger §, Yara GorzalczanyDagger §, Rive SarfsteinDagger §, Carolyn Weinbaum, Yi Zheng||, and Edgar PickDagger **

From the Dagger  Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, the || Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, and the  Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, October 28, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b559 and four cytosolic components as follows: p47phox, p67phox, p40phox, and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b559 and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67phox but in the absence of amphiphile and p47phox. We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67phox-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg2+ from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phagocytes utilize oxygen radicals for the killing of engulfed microorganisms (reviewed in Ref. 1). Oxygen radicals also serve as signal transduction messengers in a variety of nonphagocytic cells (reviewed in Ref. 2). In phagocytes, the primordial oxygen radical, superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>)1 is produced by the NADPH-driven reduction of molecular oxygen catalyzed by a membrane-localized flavocytochrome (cytochrome b559), comprising two subunits, gp91phox and p22phox (reviewed in Ref. 3). All redox stations involved in electron flow from NADPH to O2 are found on gp91phox, and it is assumed that the redox cascade is initiated by a conformational change induced in gp91phox by interaction with one or more cytosolic proteins. These are p47phox, p67phox, p40phox, and the small GTPase Rac (Rac1 or -2). Upon stimulation of the phagocyte, they translocate to the membrane, resulting in the assembly of what is known as the NADPH oxidase complex (referred to as "oxidase") (reviewed in Ref. 4). The identity of the cytosolic component(s) responsible for causing the conformational change in gp91phox is controversial. The principal candidate is p67phox, based on the identification of an "activation domain" in p67phox, consisting of residues 199-210 (5), and on direct evidence of binding of p67phox to gp91phox, an interaction enhanced by Rac1 (6). This hypothesis also implies that p47phox and Rac serve either as "carriers" for p67phox from the cytosol to the membrane or, following their own translocation, as membrane "anchors" for the correct positioning of p67phox in the assembled complex.

Oxidase assembly can be elicited in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47phox, p67phox, and Rac, exposed to an anionic amphiphile (7, 8). The role of the amphiphile is to induce a conformational change in p47phox, resulting in its binding to p22phox and the passive translocation of p67phox, by virtue of its affinity for p47phox, to the vicinity of gp91phox. The in vivo equivalent of amphiphile action is the phosphorylation of critical serines in p47phox, a process representing the starting point of a "p47phox-initiated" pathway of oxidase activation.

Under certain conditions, oxidase activation in vitro is also possible in the absence of p47phox. Originally, this was achieved in an amphiphile-dependent cell-free system by having recombinant p67phox and Rac present at micromolar concentrations (9, 10). A characteristic of earlier versions of the amphiphile-activated cell-free system is the use of bacterially expressed recombinant Rac, which did not undergo C-terminal geranylgeranylation (prenylation). We recently described oxidase activation in vitro in a system consisting of phagocyte membranes, p67phox and prenylated Rac1, in the absence of amphiphile and p47phox (11). Amphiphile- and p47phox-independent oxidase activation could also be achieved in mixtures of membrane and a chimeric [p67phox-Rac1] construct prenylated at the C terminus of the Rac1 segment (12). These findings support the existence of a second "Rac-initiated" pathway of oxidase activation (13-16), and evidence is accumulating in support of a guanine nucleotide exchange factor (GEF), responsible for GDP to GTP exchange on Rac, being the key link in this pathway. In the present report we show that a minimal functional module of Trio, a Rac-specific GEF (17), markedly potentiates oxidase activation by prenylated Rac1 in vitro in the absence of an activator and of p47phox. Surprisingly, the effect of Trio on Rac does not involve GDP to GTP exchange.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- The following nucleotides were purchased from Sigma (product numbers appear in parentheses following the description of the degree of purity): GTP (lithium salt, 97%, G5884); GDP (sodium salt, type I, 98%, G7127); GMP (disodium salt, 99%, G8377); ATP (disodium salt, 99%, A7699); ADP (sodium salt, 95-99%, A2754), and AMP (sodium salt, 99%, A1752). The hydrolysis-resistant GTP analog, GTPgamma S (lithium salt, 97%, 100 mM solution), was purchased from Roche Molecular Biochemicals. The fluorescent GTP analog mant-GTP was obtained from Molecular Probes, Eugene, OR. Lithium dodecyl sulfate (LiDS, >99% pure) was purchased from Merck. Potassium dihydrogen orthophosphate and potassium chloride, both 99.5% pure, used for preparing the buffers used in the separation of nucleotides by high pressure liquid chromatography (HPLC) were obtained from BDH, Poole, United Kingdom (Aristar® grade).

Preparation of Phagocyte Membrane Vesicles-- Phagocyte membranes were prepared from guinea pig peritoneal macrophages, as described (7). The membranes were solubilized by 40 mM n-octyl-beta -D-glucopyranoside, and membrane vesicles were prepared by extensive dialysis against detergent-free buffer, as described (18).

Preparation of Recombinant Proteins-- p47phox and p67phox were prepared in baculovirus-infected Sf9 cells, as described before (19). p67phox, truncated at residue 212 (p67phox-(1-212)), was produced in Escherichia coli, as described before (11). Nonprenylated Rac1 and Cdc42Hs were expressed in E. coli, as described earlier (19). Rac1 point mutants were generated and expressed in E. coli, as recently described (20). The N-terminal fragment of Trio (residues 1225-1537) and the N1406A/D1407A mutant of this protein were expressed in E. coli as N-terminal His6-tagged fusion proteins and purified by metal affinity chromatography, as described before (21), except for the fact that a Co2+-based resin (TalonTM; BD Biosciences) was used instead of Ni2+-agarose. Chimera [p67phox-(1-212)-Rac1-(1-192)], referred to as chimera 3 in Ref. 22, was expressed in E. coli and purified as described (22). Recombinant geranylgeranyltransferase type I was produced in baculovirus-infected Sf9 cells (23).

Identification of Nucleotides Present on Recombinant Rac1-- Bound nucleotides were liberated from recombinant Rac1 preparations as described before (24). They were identified and quantified by HPLC on a Partisil 10 SAX anion exchange column (250 × 4.6 mm) (Whatman), as described in Ref. 25. Briefly, nucleotide standards or nucleotides liberated from recombinant or other proteins were injected into the column in a volume of 0.5 ml. This was followed by delivering 0.007 M KH2PO4, pH 4.0, at 1.5 ml/min for 15 min, followed by a linear gradient from 0.007 M KH2PO4, pH 4.0, to 0.25 M KH2PO4 and 0.5 M KCl, pH 4.5, at 1.5 ml/min over a period of 45 min, and continuing the high molarity buffer for an additional 40 min at the same flow rate. The column eluate was monitored by passage through a diode array detector, set at 220-450 nm (MD-1510, Jasco, Easton, MD). The amounts of nucleotides were determined by peak integration (based on absorbance at 254 nm) and related to 5-nmol amounts of the following nucleotide standards: AMP, GMP, ADP, GDP, ATP, and GTP.

Enzymatic Prenylation of GTPases-- Nonprenylated Rac1, Cdc42Hs, and [p67phox-Rac1] chimera 3 were prenylated in vitro by recombinant geranylgeranyltransferase type I, as recently described (12).

Nucleotide Exchange-- Prenylated Rac1 was subjected to nucleotide exchange to GTPgamma S, as described before (11).

Cell-free NADPH Oxidase Assay-- Activation of oxidase in vitro supported by prenylated Rac1 was assessed by measuring NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in a semi-recombinant cell-free system, in the absence of an amphiphilic activator and of p47phox, essentially as described earlier (11, 26). The components of the assay were added to 96-well microplates, in the following order: TrioN or buffer, prenylated Rac1, a mixture of solubilized membrane and p67phox, and assay buffer in a total volume of 200 µl. The mixture was incubated for 90 s at 25 °C, and 240 µM NADPH was added to initiate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production. The concentration of membrane was constant throughout all the experiments and corresponded to 5 nM cytochrome b559 heme; the concentrations of TrioN, prenylated Rac1, and p67phox were varied and are indicated under "Results." Most experiments were performed in the assay buffer used by this laboratory before (18), which contains 1 mM MgCl2. In some experiments, assay buffers containing 0.4 µM and 4 mM free Mg2+ were used; these were prepared by adding EDTA and MgCl2 to the basic assay buffer in amounts calculated as described in Ref. 27.

Guanine Nucleotide Exchange Assay-- The ability of TrioN to perform guanine nucleotide exchange on Rac1 and Rac1 mutants was assayed by the increase in fluorescence (excitation = 361 nm; emission = 440 nm), consequent to the uptake of the fluorescent GTP analog mant-GTP by Rac1, as described in Ref. 28. Briefly, 1 µM Rac1 or Rac1 mutants were incubated with 0.375 µM mant-GTP at 20 °C in a magnetically stirred thermostated cuvette in a model FP-750 spectrofluorometer (Jasco, Easton, MD) in 20 mM Tris-HCl buffer, pH 7.4, also containing 150 mM NaCl and 4 mM MgCl2. This was followed by the addition of 0.5 µM TrioN, and the kinetics of change in fluorescence was recorded over time.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nucleotide Content of Native Rac1-- It is generally assumed that recombinant small GTPases produced in E. coli are in the GDP-bound form. We put this hypothesis to a direct test by determining the nature and amount of nucleotide bound to native recombinant Rac1. As illustrated in Fig. 1B, the only nucleotide identified on Rac1 was GDP, and it was present at a ratio of 1.046 ± 0.064 mol GDP/mol Rac1 (mean ± S.E. of five experiments). We have shown before that the procedure employed to liberate the nucleotides bound to Rac did not lead to degradation of GTP to GDP (24). Further proof for the reliability of the procedure was offered by the finding that Rac1 mutant Q61L, known to hydrolyze GTP poorly (29), was found indeed to contain 0.8 mol of nucleotide/mol of Rac1, of which 83.5% was GTP and 16.5%, GDP (mean of two determinations) (Fig. 1C). The fact that a major part of mutant Q61L shall remain in the GTP-bound state throughout protein purification was predicted by Xu et al. (30) and is now proven unequivocally. All other recombinant Rac1 mutants, used in the experiments to be described, were found to contain exclusively bound GDP (results not shown).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   HPLC analysis on a Partisil 10 SAX column of nucleotides liberated from recombinant Rac1. The methodology is described under "Experimental Procedures." Chromatograms represent analysis as follows: A, a mixture of GMP, GDP, and GTP (5 nmol, each) in 0.5 ml. The nucleotides were eluted at 26.13 (GMP), 50.61 (GDP), and 86.34 min (GTP). B, nucleotides liberated from 23 nmol of recombinant Rac1 in 0.8 ml. A single nucleotide peak was detected, eluting at 50.56 min (corresponding to GDP). C, nucleotides liberated from 19 nmol of recombinant Rac1 mutant Q61L in 0.6 ml. Two nucleotide peaks were detected, eluting at 50.29 (corresponding to GDP) and 87.94 min (corresponding to GTP). Results illustrate single characteristic chromatograms, out of five experiments performed with standards and Rac1 and two experiments performed with Rac1 Q61L mutant. The materials eluting between 20 and 40 min do not possess a spectrum characteristic for nucleotides and are of unknown nature.

Effect of Trio on Oxidase Activation by Prenylated Rac1-GDP-- We found earlier that prenylated Rac1 exchanged to either GTPgamma S or GDPbeta S supported oxidase activation in the amphiphile- and p47phox-independent cell-free system with equal potency (11). In those experiments, Rac1 was subject to nucleotide exchange at low Mg2+ concentration achieved by metal chelation by EDTA. The original purpose of the experiments, having led to the findings described in this report, was to study the effect of "enzymatic" as opposed to "chemical" nucleotide exchange on prenylated Rac1 to GTP on its ability to support oxidase activation. We first examined the ability of prenylated native Rac1 (found to contain only GDP and, therefore, to be referred to as Rac1-GDP) to activate the oxidase in the amphiphile- and p47phox-independent cell-free system. As seen in Fig. 2A, only a modest, dose-dependent, activation was found.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   TrioN enhances the activation of NADPH oxidase by prenylated Rac1-GDP in the absence of amphiphile, p47phox, and exogenous GTP. In the assays illustrated in A-C, NADPH oxidase activation was measured in an in vitro system consisting of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox, or p67phox-(1-212) (500 nM) and prenylated Rac1-GDP (10-500 nM) in the absence or presence of TrioN (300 nM); no LiDS and p47phox were present. A, oxidase activation by prenylated Rac1-GDP in the absence (filled circles) or presence (open circles) of TrioN and in the presence of TrioN and free GTPgamma S (5 µM) (open squares). B, oxidase activation by prenylated Rac1-GDP in the absence (filled circles) or presence (open circles) of TrioN, but p67phox was replaced by p67phox-(1-212). C, oxidase activation by prenylated Rac1, exchanged to GTPgamma S, and separated from free GTPgamma S on a HiTrap desalting column, in the absence (filled circles) or presence (open circles) of TrioN. D, oxidase activation by nonprenylated Rac1-GDP assayed in an in vitro system consisting of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox (500 nM), p47phox (500 nM), nonprenylated Rac1-GDP (10-500 nM), and LiDS (130 µM) in the absence (filled squares) or presence (open squares) of TrioN (300 nM) or of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox (500 nM), and nonprenylated Rac1-GDP (10-500 nM) in the absence of LiDS and p47phox, in the absence (filled circles), or presence (open circles) of TrioN (300 nM). E, oxidase activation in an in vitro system consisting of membrane (equivalent of 5 nM cytochrome b559 heme) and prenylated chimera [p67phox-(1-212)-Rac1-(1-192)] in the GDP-bound form (10-300 nM), in the absence of LiDS and p47phox and in the absence (filled circles) or presence (open circles) of TrioN (300 nM). F, TrioN-enhanced oxidase activation by prenylated Rac1-GDP in relation to the concentration of TrioN. The in vitro system consisted of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox (300 nM), prenylated Rac1-GDP (300 nM), and TrioN (10-500 nM); no LiDS and p47phox were present. The results in all panels are means ± S.E. of three experiments.

We next assessed the effect of inducing nucleotide exchange on Rac1 to GTP, in a manner closest to the physiological mechanism, by using a GEF. For this purpose, we chose Trio, a GEF containing two distinct guanine nucleotide exchange domains, specific for Rac1 and Rho (17, 31). A recombinant N-terminal segment of Trio (residues 1225-1537), including the Rac1-specific N-terminal Dbl homology (DH) and pleckstrin homology (PH) domains, to be referred to as TrioN, was found to stimulate GDP to GTP exchange on Rac1 (21). The ability of TrioN to modulate oxidase activation by prenylated Rac1-GDP was assayed in the presence of exogenous GTPgamma S and, as a control, in its absence. Surprisingly, TrioN exhibited a pronounced potentiating effect on oxidase activation by prenylated Rac1-GDP in the absence of added GTPgamma S (Fig. 2A). Oxidase activation by prenylated Rac1-GDP, in the presence of TrioN and in the absence of exogenous GTPgamma S, reached a Vmax of 69.5 ± 2.9 mol O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/s/mol cytochrome b559 heme, compared with 31.3 ± 1.4, in the absence of TrioN; EC50 values were 5.1 ± 2.1 nM Rac1, in the presence of TrioN, and 69.8 ± 12.1, in its absence (means ± S.E. of three experiments for each group).

Paradoxically, in the presence of exogenous GTPgamma S (5 µM), TrioN did not potentiate oxidase activation by prenylated Rac1-GDP (Fig. 2A). Oxidase activation by prenylated Rac1-GDP and TrioN, in the presence of added GTPgamma S, was not significantly different from that in the absence of TrioN and GTPgamma S (Vmax = 37.7 ± 2.4 mol O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/s/mol cytochrome b559 heme; EC50 = 168.7 ± 28.3 nM Rac1, representing means ± S.E. of three experiments). Dose-response studies indicated that inhibition of TrioN-enhanced oxidase activation by GTPgamma S was evident at 0.5 µM GTPgamma S and was complete at 5 µM (results not shown).

The enhancing effect of TrioN was seen only in the presence of both prenylated Rac1 and p67phox (Table I) and was related to the relative concentrations of Rac1 and p67phox present in the reaction. Thus, significantly higher activities were obtained when p67phox was in excess over Rac1 than when the two components were present in equimolar amounts (results not shown). TrioN was inactive when heat-denatured and had no effect on prenylated Cdc42Hs-GDP, a Rho subfamily GTPase lacking oxidase-activating ability (Table I). p67phox could be replaced by p67phox truncated at residue 212, although both the basal and the TrioN-enhanced activity were lower than in the presence of non-truncated p67phox (Fig. 2B). This suggests that the tetratricopeptide repeat region, involved in binding of p67phox to Rac (32), and the activation domain (5) play important but not exclusive roles in TrioN-dependent activation.

                              
View this table:
[in this window]
[in a new window]
 
Table I
TrioN enhances NADPH oxidase activation by prenylated Rac1
Cell-free NADPH oxidase activation was performed as described under "Experimental Procedures." The concentrations of components were as follows: membrane, equivalent of 5 nM cytochrome b559 heme; p67phox, 300 nM; prenylated Rac1-GDP (or Cdc42Hs-GDP), 300 nM; TrioN (or TrioN mutant), 300 nM. Results represent means ± S.E. of three to seven experiments for each combination of NADPH oxidase components.

TrioN exhibited no enhancing effect on oxidase activation by prenylated Rac1, exchanged to GTPgamma S and freed of unbound nucleotide on a desalting column (Fig. 2C). It is of interest that although the level of oxidase activation achieved with prenylated Rac1-GTPgamma S was higher than that measured with native Rac1 (Rac1-GDP), it was considerably lower than that found with Rac1-GDP combined with TrioN, in the absence of free GTPgamma S. This suggests that the conformational change(s) occurring in Rac1 as a consequence of nucleotide exchange to GTPgamma S might, at least in part, be different from those taking place under the influence of TrioN, although they both lead to an enhancement in the oxidase activating capacity of Rac1.

TrioN had only a minor enhancing effect on oxidase activation by nonprenylated Rac1-GDP in the canonical amphiphile- and p47phox-dependent system and did not convey an activating potential to nonprenylated Rac1-GDP, known to be inactive in the absence of amphiphile and p47phox (both situations are shown in Fig. 2D). This was not due to a requirement for prenylation for interaction of Rac1 with TrioN to take place because nonprenylated Rac1 was found to bind TrioN (20).

We recently showed that a chimera consisting of the N-terminal 212 residues of p67phox and full-length Rac1, when prenylated and in the GDP-bound form, was capable of moderate oxidase activation in the absence of an amphiphile and p47phox (12). In marked contrast to its enhancing effect on a combination of prenylated Rac1-GDP and p67phox-(1-212) (Fig. 2B), TrioN did not potentiate the activity of a [p67phox-(1-212)-Rac1-(1-192)] chimera in GDP-bound form (Fig. 2E). It is possible that Rac1 chimerized to p67phox is less accessible for interaction with TrioN or less "flexible" to respond to such an interaction by a change in conformation.

We performed dose-response experiments in which the concentration of either prenylated Rac1-GDP or TrioN was varied from 10 to 500 nM, although the concentration of the counterpart component was kept constant at 300 nM. As seen in Fig. 2, A and F, in both situations, plateaus were reached when the concentration of the component added in varying amounts was close to 300 nM. This is suggestive of the possibility that [Rac1-TrioN] complexes with a 1:1 stoichiometry are involved in activation, but more rigorous evidence is required to sustain such a proposal.

Enhancement of Oxidase Activation by TrioN Occurs in the Absence of Nucleoside Triphosphates-- Because of the finding that TrioN acts on Rac1 in the absence of exogenous GTP, it was essential to ensure that GTP was not introduced inadvertently into the assay as a contaminant of one of the components, or generated enzymatically by ATP-dependent conversion of GMP or GDP to GTP (33). Consequently, we examined the solubilized membrane preparation, recombinant baculovirus-derived p67phox, and recombinant TrioN for a possible content of ATP, GTP, GDP, or GMP. 10-mg amounts of solubilized and dialyzed membrane (corresponding to an amount 2000 times higher than that present in an individual oxidase assay mixture) and 20-nmol amounts of p67phox and TrioN (corresponding to amounts 210 times higher than the maximal amounts present in the assay mixture) were subjected to the nucleotide extraction procedure applied to Rac1. The extracts were examined for the presence and quantity of nucleotides by chromatography on a Partisil 10 SAX anion exchange column, in relation to established nucleotide standards. No adenine or guanine nucleotides were detected in any of the samples examined. We conclude that, even if the presence of GTP-generating enzymes in the only non-recombinant component present in the assay (the membrane) is suspected, no nucleotides with the potential to serve as phosphate donors or acceptors were detected. This establishes with certainty that TrioN exerts it action on Rac1-GDP independently of nucleotide exchange to GTP.

Rac1-TrioN Interaction and a Catalytic Event Are Required for Enhancement of Oxidase Activation by TrioN-- It was demonstrated that specific residues on Rac1, located in the switch I and II regions, are essential for either binding of TrioN or the subsequent catalytic event leading to nucleotide dissociation (20). We examined the importance of these residues in the oxidase activation enhancing effect of TrioN by using the Rac1 mutants Y32A (switch I) and W56F, Q61L, and Y64A (switch II). Rac1 Y32A was shown to retain the ability to bind TrioN but to lose its ability to respond to TrioN by GDP dissociation. The switch II mutants were found to be unable to bind TrioN; of special interest is mutant W56F because Trp56 appears to be the critical residue determining the Rac specificity of GEFs, including Trio (20). Among switch II mutants, Rac1 Q61L stands out because it was found to be predominantly in the GTP-bound form (Fig. 1C), unlike all the other mutants that contained only GDP. It was also reported to have a higher affinity for a yet unidentified oxidase component, most likely p67phox (29). We also examined the responsiveness of the negative dominant mutant T17N, known to possess a markedly lower affinity for GTP (34) but an unimpaired ability to bind GEFs (reviewed in Ref. 35).

As a preliminary to examining their responsiveness to TrioN in the oxidase assay, we assessed the ability of the Rac1 mutants to respond to TrioN by nucleotide exchange from GDP to mant-GTP. As seen in Fig. 3, wild type Rac1-GDP reacted to TrioN by a vigorous change in the slope of mant-GTP uptake. All Rac1 mutants, in GDP-bound form, were unresponsive to TrioN, as evident in the lack of change in the slope of mant-GTP uptake, following the addition of TrioN (Fig. 3). It is of interest that mutants T17N and Q61L also evidenced the lowest spontaneous uptake of mant-GTP, in accordance with the reported impairment in GTP binding of mutant T17N (34) and the slow nucleotide exchange rate of mutant Q61L (30).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   TrioN stimulates uptake of mant-GTP by wild type Rac1 but not by several Rac1 mutants. Wild type Rac1-GDP or Rac1 mutants in the GDP-bound form were added to cuvettes, containing mant-GTP, at the points of time indicated. After a few minutes, TrioN was added. Fluorescence was recorded continuously. The effect of TrioN is shown by the acute change in the slope of fluorescence increase or by the lack of such change. For methodological details, see "Experimental Procedures." The panels illustrate representative experiments out of three performed with each Rac1 preparation.

We next examined the effect of the mutations on the capacity of nonprenylated Rac1 to support oxidase activation in the cell-free system. Mutants T17N, Y32A, W56F, Q61L, and Y64A (all at a concentration of 300 nM) were assayed in an amphiphile-dependent cell-free system consisting of membrane (5 nM cytochrome b559 heme), p47phox (300 nM), p67phox (300 nM), nonprenylated native (unexchanged) wild type or mutant Rac1 (300 nM), and LiDS (130 µM). All Rac1 mutants, with the exception of T17N and Q61L, exhibited unchanged oxidase activating ability, in the range of 103-114% that of wild type Rac1. Rac1 T17N was only 41% as active as wild type Rac1, whereas Rac1 Q61L was more active (128%) than wild type Rac1 (results represent means of two experiments). Finally, the influence of mutations on the ability of the prenylated form of Rac1 to support oxidase activation in the amphiphile and p47phox-independent cell-free system was examined. To achieve a maximal effect, prenylated wild type Rac1 and mutants were exchanged to GTPgamma S and assayed in a system consisting of membrane (5 nM cytochrome b559 heme), p67phox (300 nM), and prenylated wild type or mutant Rac1- GTPgamma S (300 nM) in the absence of LiDS. The activating abilities of the mutants were 39 (Y32A), 52 (W56F), and 72% (Y64A) that of wild type Rac1 (results represent means of two experiments). Mutant Q61L was 3.75 times more active than wild type Rac1, in support of the hypothesis that it has a higher affinity for p67phox.

As seen in Fig. 4, A and B, TrioN was incapable of enhancing oxidase activation supported by prenylated Rac1 mutants T17N and Y32A. Because both mutants were expected to bind TrioN normally but did not respond to TrioN by mant-GTP uptake, it appears that an event, subsequent to binding and related to the catalytic action of TrioN, is required for the enhancement of oxidase activation. TrioN had little or no oxidase activation potentiating effect on Rac1 mutants W56, Q61L, and Y64A, known to be unable to bind TrioN (a minor effect was evident on mutant Y64A) (Fig. 4, C-E). It appears that the basal oxidase activating ability of the prenylated mutants, especially T17N and Y32A, was also lower than that of wild type Rac1 (with the notable exception of Q61L), as already noted in the course of preliminary testing of the mutants in GTP-bound form at a single concentration. The reason for this is not obvious.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of TrioN on oxidase activation supported by Rac1 mutants. Oxidase activation was assayed in an in vitro system consisting of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox (500 nM), prenylated Rac1 mutants in GDP-bound form (10-500 nM), in the absence (open triangles) or presence (open circles) of TrioN (300 nM); no LiDS and p47phox were present. The Rac1 mutants tested were T17N (A), Y32A (B), W56 (C), Q61L (D), and Y64A (E). The results in all panels are the means ± S.E. of three experiments.

A special case is mutant Q61L, which was found to contain predominantly GTP. Its basal oxidase activating ability (Fig. 4D) was found to exceed significantly that of wild type Rac1 in both native (GDP-bound) and GTPgamma S-bound (exchanged to GTPgamma S) forms (Fig. 2, A and C), and TrioN exerted no further enhancing effect. Thus, Vmax was 58.1 ± 0.7 and 57.3 ± 1.4 mol O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/s/mol cytochrome b559 heme, in the absence and presence of TrioN, respectively, and the corresponding EC50 values were 36.1 ± 2.3 and 34.8 ± 4.3 nM Rac1 mutant Q61L (means ± S.E. of three experiments for each group).

Recently, a double mutant of TrioN was described, the mutations being located at the C terminus of the DH domain (N1406A/D1407A).2 This mutant binds normally to Rac1 but fails to stimulate nucleotide exchange. We tested the ability of TrioN mutant N1406A/D1407A to enhance oxidase activation by prenylated Rac1-GDP in the presence of p67phox and in the absence of amphiphile and p47phox. As seen in Table I, the TrioN mutant failed to enhance oxidase activation. This finding offers additional support to the conclusion derived from the results obtained with Rac1 mutants T17N and Y32A that enhancement of oxidase activation by prenylated Rac1 requires, in addition to Rac1-TrioN interaction, yet another step associated with the catalytic action of TrioN.

Effect of TrioN Is Mediated by Mg2+ Displacement-- It was shown that nucleotide exchange by Trio is effected principally through the displacement of bound Mg2+ (21). Therefore, we reasoned that the effect of TrioN on Rac1 could be mimicked by removal of Mg2+ from Rac1 by the divalent cation chelator EDTA. As shown in Fig. 5 and Table I, lowering the free Mg2+ concentration to 0.4 µM in a mixture of phagocyte membrane, prenylated Rac1-GDP and p67phox, led to oxidase activation (Vmax = 72.6 ± 2.8 mol O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/s/mol cytochrome b559 heme; EC50 = 41.1 ± 7.7 nM Rac1; means ± S.E. of three experiments). Raising the concentration of free Mg2+ to 4 mM eliminated oxidase activation by prenylated Rac1-GDP, reaching a level (Vmax = 16.3 ± 2.6 mol O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/s/mol cytochrome b559 heme; means ± S.E. of three experiments) lower than that found under standard assay conditions, in which the concentration of free Mg2+ is 1 mM. In control experiments, in which EDTA was replaced by an equal concentration of the Ca2+-specific chelator EGTA, no enhancement of oxidase activation was seen (results not shown). Enhancement of oxidase activation was also obtained when prenylated Rac1-GDP was treated with EDTA for 10 min at 30 °C, to bring the concentration of free Mg2+ to 0.27 µM, and then freed of EDTA and chelated Mg2+ by passage through a desalting column (results not shown). This indicates that enhancement of oxidase activation is mediated by displacement of Mg2+ from Rac1 and not from another component present in the cell-free assay. Cross et al. (36) first described "spontaneous" oxidase activation in the absence of Mg2+ in a cell-free system, consisting of purified cytochrome b559, recombinant p47phox, p67phox, and prenylated Rac2, in the absence of amphiphile.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Lowering of free Mg2+ concentration enhances the activation of NADPH oxidase by prenylated Rac1-GDP, in the absence of amphiphile and p47phox. Oxidase activation was measured in an in vitro system consisting of membrane (equivalent of 5 nM cytochrome b559 heme), p67phox (500 nM), and prenylated Rac1-GDP (10-500 nM), in the absence of LiDS and p47phox, in the presence of 0.4 µM (open circles) or 4 mM (closed circles) free Mg2+ concentrations in the reaction buffer. Results are means ± S.E. of three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These results point toward a novel mechanism of Rac activation, the essence of which is the induction of a conformational change in Rac consequent to its interaction with a GEF. Based on findings made with Rac1 mutants W56F and Y64A, binding of TrioN to Rac1 is an absolute requirement for oxidase activation. The lack of effect of TrioN on Rac1 mutants T17N and Y32A demonstrates that binding to TrioN, although required, is not sufficient for activation and that an event normally related to the nucleotide exchange promoting effect of TrioN is involved. However, the fact that TrioN was fully effective when added to Rac1-GDP in the absence of exogenous GTP indicates that the effect of TrioN occurs independently of actual nucleotide exchange. Based on a model of GEF action (37), it is likely that, under these conditions, a [TrioN-Rac1-GDP] complex is formed. It was found that in the complex of the DH-PH module of the GEF Tiam1 with Rac1, the conformation of the switch I and II regions and their vicinities are altered (28). We suggest that TrioN induces a conformational change in Rac1-GDP, normally related to nucleotide exchange to GTP but now taking place in the absence of such an exchange. This conformational change appears to be related to the displacement by TrioN of Mg2+ bound to Rac1 (21), in agreement with the TrioN-mimicking effect of Mg2+ depletion by EDTA. It was, indeed, reported that a conformational change, represented by the opening of the switch I region, took place in RhoA-GDP as the direct result of Mg2+ dissociation induced by Li2SO4 (38). Less pronounced conformational changes were also found to be induced by Mg2+ depletion in the switch II and insert regions. We propose that the conformational change in Rac1, consequent to interaction with TrioN, in the absence of exogenous GTP, results in an increased affinity of Rac1 for another oxidase component. The principal candidates are p67phox (change in switch I) (39) or cytochrome b559 (change in the insert region) (40). We favor the hypothesis that a [TrioN-Rac1-GDP- p67phox] complex is formed that translocates to the membrane. Once there, p67phox interacts with cytochrome b559 and activates the oxidase. Support for this proposal is offered by the finding that conditions leading to high affinity binding of Rac to p67phox, such as a Q61L mutation in Rac (29) or chimerization of Rac with p67phox (12, 22), are also the situations in which TrioN is incapable of further enhancement of oxidase activation. Support for the proposal that a [TrioN-Rac1-GDP-p67phox] ternary complex is formed, is offered by the finding of a similar [GEF-GTPase-effector] ternary complex, consisting of the minimal functional domains of Dbl, Cdc42Hs, and p21-activated kinase 1 (PAK1), in which PAK1 was activated.3 An alternative to the [TrioN-Rac1-GDP-p67phox] complex model is the causation by TrioN of a conformational change in Rac1, in the absence of complex formation with TrioN, leading to an increased affinity of Rac1 for another oxidase component. Further work is required for proving the veracity of one of the two models.

TrioN is a sequence module consisting of the N-terminal DH and PH domains of Trio. An issue to be clarified is the relative importance of the DH and PH domains in the effect of TrioN on Rac1. The finding that the TrioN mutant N1406A/D1407A is inactive indicates that the DH domain is involved in the oxidase activation enhancing activity of TrioN. It has been shown that the N-terminal PH domain of Trio binds to acidic phospholipids and might serve as a membrane localizing signal (41). We cannot yet establish the possible involvement of the PH domain in the potentiating effect of Trio, but it is likely that a [TrioN-Rac1-GDP] complex will express a high affinity for the membrane because of the presence of two groups binding to acidic phospholipids, the prenylated polybasic C terminus of Rac1 and the PH domain of Trio.

Finally, it remains to be established whether activation of Rac1 by Trio, in the absence of nucleotide exchange, represents but a particular example of a property shared by other Rac-specific GEFs and whether such a mechanism is at work in the intact cell. Recently, a concept is emerging looking upon Rac GEFs not merely as mediators of nucleotide exchange on Rac but also as factors directing Rac toward specific effector pathways (42).

    ACKNOWLEDGEMENTS

We thank T. L. Leto (National Institutes of Health) for providing baculoviruses carrying cDNA of p67phox and p47phox and the GST-Rac1 and GST-Rac1 mutant Q61L expression plasmids in E. coli; D. Manor (Cornell University) for providing GST-Cdc42Hs expression plasmid in E. coli; F. Wientjes (University College, London, UK) for providing GST-p67phox expression plasmid in E. coli; and M. Hirshberg (University of Cambridge, UK) for fruitful discussions.

    FOOTNOTES

* This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 128/01 (to E. P.), and National Institutes of Health Grants GM60523 (to Y. Z.) and GM46372 (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence and should be addressed: Dept. of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-640-7872; Fax: 972-3-642-9119; E-mail: epick@post.tau.ac.il.

Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M211011200

2 B. Debreceni and Y. Zheng, submitted for publication.

3 K. Zhu and Y. Zheng, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, superoxide; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; GEF, guanine nucleotide exchange factor; mant-GTP, 2'-(or 3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate; LiDS, lithium dodecyl sulfate; GST, glutathione S-transferase; TrioN, the N-terminal fragment of Trio representing residues 1225-1537; HPLC, high pressure liquid chromatography; DH, Dbl homology; PH, pleckstrin homology.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Klebanoff, S. J. (1999) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I. , and Snyderman, R., eds), 3rd Ed. , pp. 721-768, J. B. Lippincott, Philadelphia
2. Irani, K., and Goldschmidt-Clermont, P. J. (1998) Biochem. Pharmacol. 55, 1339-1346[CrossRef][Medline] [Order article via Infotrieve]
3. Leto, T. L. (1999) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I. , and Snyderman, R., eds), 3rd Ed. , pp. 769-786, J. B. Lippincott, Philadelphia
4. DeLeo, F. R., and Quinn, M. T. (1996) J. Leukocyte Biol. 60, 677-691[Abstract]
5. Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668[Abstract/Free Full Text]
6. Dang, P. M.-C., Cross, A. R., and Babior, B. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3001-3005[Abstract/Free Full Text]
7. Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221[Medline] [Order article via Infotrieve]
8. Abo, A., Boyhan, A., West, I., Thrasher, A., and Segal, A. W. (1992) J. Biol. Chem. 267, 16767-16770[Abstract/Free Full Text]
9. Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582[Abstract/Free Full Text]
10. Koshkin, V., Lotan, O., and Pick, E. (1996) J. Biol. Chem. 271, 30326-30329[Abstract/Free Full Text]
11. Gorzalczany, Y., Sigal, N., Itan, M., Lotan, O., and Pick, E. (2000) J. Biol. Chem. 275, 40073-40081[Abstract/Free Full Text]
12. Gorzalczany, Y., Alloul, N., Sigal, N., Weinbaum, C., and Pick, E. (2002) J. Biol. Chem. 277, 18605-18610[Abstract/Free Full Text]
13. Bokoch, G. M., Bohl, B. P., and Chuang, T.-H. (1994) J. Biol. Chem. 269, 31674-31679[Abstract/Free Full Text]
14. Akasaki, T., Koga, H., and Sumimoto, H. (1999) J. Biol. Chem. 274, 18055-18059[Abstract/Free Full Text]
15. Price, M. O., Atkinson, S. J., Knaus, U. G., and Dinauer, M. C. (2002) J. Biol. Chem. 277, 19220-19228[Abstract/Free Full Text]
16. Welch, H. C. E., Coadwell, W. J., Ellson, C. D., Ferguson, G. J., Andrews, S. R., Erdjument-Bromage, H., Tempst, P., Hawkins, P. T., and Stephens, L. R. (2002) Cell 108, 809-821[Medline] [Order article via Infotrieve]
17. Debant, A., Sierra-Pages, C., Seipel, K., O'Brien, S., Tang, M., Park, S.-H., and Streuli, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5466-5471[Abstract/Free Full Text]
18. Shpungin, S., Dotan, I., Abo, A., and Pick, E. (1989) J. Biol. Chem. 264, 9195-9203[Abstract/Free Full Text]
19. Koshkin, V., Lotan, O., and Pick, E. (1997) Biochim. Biophys. Acta 1319, 139-146[Medline] [Order article via Infotrieve]
20. Gao, Y., Xing, J., Streuli, M., Leto, T. L., and Zheng, Y. (2001) J. Biol. Chem. 276, 47530-47541[Abstract/Free Full Text]
21. Zhang, B., Zhang, Y., Wang, Z., and Zheng, Y. (2000) J. Biol. Chem. 275, 25299-25307[Abstract/Free Full Text]
22. Alloul, N., Gorzalczany, Y., Itan, M., Sigal, N., and Pick, E. (2001) Biochemistry 40, 14557-14566[CrossRef][Medline] [Order article via Infotrieve]
23. Zhang, F. L., Moomaw, J. F., and Casey, P. J. (1994) J. Biol. Chem. 269, 23465-23470[Abstract/Free Full Text]
24. Bromberg, Y., Shani, E., Joseph, G., Gorzalczany, Y., Sperling, O., and Pick, E. (1994) J. Biol. Chem. 269, 7055-7058[Abstract/Free Full Text]
25. Hartwick, R. A., and Brown, P. R. (1975) J. Chromatogr. 112, 651-662[CrossRef]
26. Toporik, A., Gorzalczany, Y., Hirshberg, M., Pick, E., and Lotan, O. (1998) Biochemistry 37, 7147-7156[CrossRef][Medline] [Order article via Infotrieve]
27. Sunyer, T., Codina, J., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 15447-15451[Abstract/Free Full Text]
28. Worthylake, D. K., Rossman, K. L., and Sondek, J. (2000) Nature 408, 682-688[CrossRef][Medline] [Order article via Infotrieve]
29. Xu, X., Barry, D. C., Settleman, J., Schwartz, M. A., and Bokoch, G. M. (1994) J. Biol. Chem. 269, 23569-23574[Abstract/Free Full Text]
30. Xu, X., Wang, Y., Barry, D. C., Chanock, S. J., and Bokoch, G. M. (1997) Biochemistry 36, 626-632[CrossRef][Medline] [Order article via Infotrieve]
31. Bellanger, J.-M., Lazaro, J.-B., Diriong, S., Fernandez, A., Lamb, N., and Debant, A. (1998) Oncogene 16, 147-152[CrossRef][Medline] [Order article via Infotrieve]
32. Koga, H., Terasawa, H., Nunoi, H., Takeshige, K., Inagaki, F., and Sumimoto, H. (1999) J. Biol. Chem. 274, 25051-25060[Abstract/Free Full Text]
33. Peveri, P., Heyworth, P. G., and Curnutte, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2494-2498[Abstract]
34. Feig, L. A., and Cooper, G. M. (1988) Mol. Cell. Biol. 8, 3235-3243[Medline] [Order article via Infotrieve]
35. Zheng, Y. (2001) Trends Biochem. Sci. 26, 724-732[CrossRef][Medline] [Order article via Infotrieve]
36. Cross, A. R., Erickson, R. W., Ellis, B. A., and Curnutte, J. T. (1999) Biochem. J. 338, 229-233[CrossRef][Medline] [Order article via Infotrieve]
37. Cherfils, J., and Chardin, P. (1999) Trends Biochem. Sci. 24, 306-311[CrossRef][Medline] [Order article via Infotrieve]
38. Shimizu, T., Ihara, K., Maesaki, R., Kuroda, S., Kaibuchi, K., and Hakoshima, T. (2000) J. Biol. Chem. 275, 18311-18317[Abstract/Free Full Text]
39. Lapouge, K., Smith, S. J. M., Walker, P. A., Gamblin, S. J., Smerdon, S. J., and Rittinger, K. (2000) Mol. Cell 6, 899-907[Medline] [Order article via Infotrieve]
40. Diebold, B. A., and Bokoch, G M. (2001) Nat. Immunol. 2, 211-215[CrossRef][Medline] [Order article via Infotrieve]
41. Liu, X., Wang, H., Eberstadt, M., Schnuchel, A., Olejniczak, E. T., Meadows, R. P., Schkeryantz, J. M., Janowick, D. A., Harlan, J. E., Harris, E. A. S., Staunton, D. E., and Fesik, S. W. (1998) Cell 95, 269-277[Medline] [Order article via Infotrieve]
42. Scita, G., Tenca, P., Frittoli, E., Tocchetti, A., Innocenti, M., Giardina, G., and Di Fiore, P. (2000) EMBO J. 19, 2393-2398[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.