From the 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
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ABSTRACT |
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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.
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 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.
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,
GTP 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- 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 GTP Cell-free NADPH Oxidase Assay--
Activation of oxidase
in vitro supported by prenylated Rac1 was assessed by
measuring NADPH-dependent O 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.
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).
Effect of Trio on Oxidase Activation by Prenylated
Rac1-GDP--
We found earlier that prenylated Rac1 exchanged to
either GTP
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 GTP
Paradoxically, in the presence of exogenous GTP
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.
TrioN exhibited no enhancing effect on oxidase activation by prenylated
Rac1, exchanged to GTP
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).
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 GTP
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.
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 GTP
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 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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
-D-glucopyranoside, and membrane
vesicles were prepared by extensive dialysis against detergent-free
buffer, as described (18).
S, as described before (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
S or GDP
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.
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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 GTP 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 GTP
S, and
separated from free GTP
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.
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
GTP
S (Fig. 2A). Oxidase activation by prenylated
Rac1-GDP, in the presence of TrioN and in the absence of exogenous
GTP
S, reached a Vmax of 69.5 ± 2.9 mol
O
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 GTP
S, was
not significantly different from that in the absence of TrioN and
GTP
S (Vmax = 37.7 ± 2.4 mol
O
S was evident at 0.5 µM GTP
S and was complete at
5 µM (results not shown).
TrioN enhances NADPH oxidase activation by prenylated Rac1
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-GTP
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 GTP
S. This suggests that the conformational
change(s) occurring in Rac1 as a consequence of nucleotide exchange to
GTP
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.
View larger version (23K):
[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.
S and assayed in a
system consisting of membrane (5 nM cytochrome b559 heme), p67phox (300 nM), and prenylated wild type or mutant Rac1- GTP
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.
View larger version (30K):
[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.
S-bound (exchanged to GTP
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
View larger version (19K):
[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
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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.
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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.
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ABBREVIATIONS |
---|
The abbreviations used are:
OS, 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.
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