(Received for publication, September 6, 1995; and in revised form, October 6, 1995 )
From the
NADPH oxidase is a plasma membrane enzyme of phagocytes
generating superoxide anions which serve as bactericidal agents.
Activation of this multimolecular enzyme minimally requires assembly at
the membrane with flavocytochrome b of cytosolic
components p47
, p67
,
and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small
GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH
oxidase activation in vitro; however, Rac2 is largely
predominant in human phagocytes. Here, using the yeast two-hybrid
system, we provide data demonstrating in vivo interactions
between human p47
, p67
,
and Rac proteins. Rac proteins interact with p67
in a GTP-dependent manner, but do not interact with
p47
. Moreover, Rac effector site mutants, which
are known to be inactive in NADPH oxidase, lose their interaction with
p67
; Rac2L61 mutant, which has an increased
NADPH oxidase affinity, shows an increased affinity for
p67
. Finally, we observe that p67
interacts 6-fold better with Rac2 than with Rac1. We also
show a strong intracellular interaction between p47
and p67
. These results indicate that
activated Rac can regulate NADPH oxidase by interacting with
p67
and that Rac2 is the main
p67
-interacting GTPase in human cells.
NADPH oxidase is a multicomponent plasma membrane-bound enzyme used by phagocytes to produce superoxide anions in response to stimuli from microbial infections(1, 2, 3) . This massive production of oxygen metabolites, known as the respiratory burst, is an efficient bactericidal mechanism as underscored by severe infections in case of enzyme deficiency in chronic granulomatous disease(4) .
Early biochemical and genetic studies (5, 6) in combination with reconstitution of enzyme
activity in a cell-free system, characterized the minimal structure of
the enzyme(7, 8) . This includes the transmembrane
flavocytochrome b, consisting of subunits
gp91
and p22
, and three
regulatory cytosolic proteins, p47
,
p67
(9, 10) , and the more
recently isolated small GTPase Rac1 or Rac2(11, 12) .
Rac1 and Rac2 are 92% homologous Ras-like proteins, initially isolated
from cell cytosol, Rac1 from guinea pig macrophages, and Rac2 from
human neutrophils, as the GTP-binding protein enhancing superoxide
generation in the cell-free assay(11, 12) . The
physiological requirement for Rac proteins in NADPH oxidase has then
been demonstrated in a whole cell assay, using rac antisense
oligonucleotides or a dominant-negative
mutant(13, 14) .
In addition, the small GTPase
Rap1a, found tightly associated with membranous cytochrome b(15) , although dispensable in the
cell-free system, is required for enzyme activity in a whole cell
environment(14, 16) . The recently isolated cytosolic
protein p40
(17, 18) has been
reported to interact with p47
and
p67
components, and may play the role of a
chaperone in the activated complex(19) .
How is Rac regulating the NADPH oxidase? Clearly the nucleotide state of Rac proteins controls the activity of NADPH oxidase: there is an absolute requirement for GTP in the cell-free system, GTP-preloaded Rac activates the enzyme, and factors which modulate the GTP/GDP state of Rac also modulate the activation of the enzyme(1) . It has been shown that Bcr, a complex protein whose GTPase activating function acts on Rac in vitro, regulates the activation of the enzyme in vivo(20) . An intact Rac effector domain is also required for enzyme activation, as shown by the inefficiency of Rac effector mutants in cell-free assays(21, 22, 23) . It has been reported that Rac2L61, a GTPase-deficient mutant (therefore mainly in the GTP-bound form) has at least a 3-fold enhanced affinity for the enzyme, as compared to wild type or RacV12 proteins(22) . Both Rac1 and Rac2 proteins are able to activate the enzyme and are roughly equivalent for activation of the recombinant cell-free system(24, 25) . However, in the presence of human neutrophil cytosol in the assay, Rac2 was a more potent activator than Rac1(24) .
Upon cell stimulation, cytosolic regulatory
proteins translocate to the plasma membrane and/or membrane-associated
cytoskeleton to form the active enzyme
complex(26, 27, 28) . Components,
p47 and p67
, tightly
associated as a cytosolic complex(18, 29) ,
translocate to the membrane where p47
interacts
with cytochrome b
-subunit
p22
, probably through SH3 and proline-rich
domain interactions(30, 31) . Although contradictory
data have been published about Rac recruitment at the
membrane(32) , overall it appears that Rac-GTP activates NADPH
oxidase at the membrane level (33) and that in whole cells, Rac
translocation seems to correlate with enzyme
activation(27, 28, 34, 35) .
Interactions between p47 and
p67
have been widely documented
recently(30, 31, 36, 37, 38, 39) ,
but direct interactions of Rac proteins with NADPH oxidase components
are so far poorly described. After we began this study, Diekmann et
al.(21) reported, using glutathione S-transferase fusion proteins, that Rac1-GTP was able to
interact with p67
through its effector domain,
suggesting that p67
was a molecular target for
Rac proteins. However, several points can be raised about this result:
a 100 times molar excess of p67
over Rac
recombinant proteins was necessary to observe a rather weak
interaction; only Rac1 has been studied even though Rac2, mainly
expressed in NADPH oxidase expressing cells, is highly predominant over
Rac1 in human phagocytes(34, 40) ; and in a more
biological environment, Sf9 cells overexpressing
p47
, p67
, and p21Rac1,
Rac1-p67
complex was undetectable(33) .
Another study (41) also very recently reported a weak
interaction in vitro between Rac1-GTP and p67
recombinants proteins.
Here, we have used a two-hybrid
system in yeast (42, 43, 44) to study in an
intracellular environment, interactions between the three regulatory
cytosolic factors, p47,
p67
, and p21Rac. Our results provide clear
evidence showing that p47
and p67
interact in a stable complex, that p67
is an in vivo target for Rac-GTP, and that Rac2 in
a whole cell system, interacts much better than Rac1 with
p67
.
Proteins fused to the GAL4 DNA binding domain
were expressed from plasmid pGBT10(45) . Proteins fused to the
GAL4 activation domain (GAL4 AD) ()were expressed from
plasmid pGAD-GH(47) . Proteins fused to the LexA protein were
expressed from either plasmid pBTM116 (46) or from pVJL10, a
derivative of pBTM116 with modified polylinker. (
)HF7c or
L40 cells were cotransformed with pairs of two-hybrid plasmids and
selected by growth on medium lacking tryptophane and leucine.
Cotransformed colonies were patched on the same selective medium and
subsequently replicated to test for protein interaction, using
-galactosidase activity assay on filter with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside or using
growth in the absence of histidine in the medium, see indications in
figure legends(44, 48) .
Figure 4:
Comparison between Rac1 and Rac2 proteins
for interaction with p67. In yeast strain L40,
Rac1V12 and Rac2V12 fused to LexA, were tested for interaction with
p67
fused to the GAL4 activation domain.
Protein interactions are demonstrated by histidine-independent growth
or
-galactosidase activity. A, growth in the absence of
histidine is shown in the right column (-His), observations at
72 h. Control growth of cotransformed yeast in presence of histidine is
shown on left column (+His). B,
-galactosidase activity on a filter assay, observations at 24
h. C,
-galactosidase activity quantified by a liquid
assay.
Since previous evidence suggested an interaction between
p67 and p47
(18, 29) , we
first tested the ability of these two proteins to interact in the
two-hybrid system. A plasmid expressing p47
fused to the
GAL4 activation domain and a plasmid expressing p67
fused to the GAL4 DNA binding domain were introduced into yeast
reporter strain HF7c(45) . Transformants were tested for their
ability to grow in the absence of histidine. The interaction between a
hybrid of SNF1 and a hybrid of SNF4, two yeast proteins known to
interact with each other, provided a positive control(42) . All
transformants grew at similar rates in the presence of histidine, but
only cells containing GAL4 AD-p47
and GAL4 DNA binding
domain-p67
hybrids maintained their growth in the
absence of histidine (Fig. 1). The reciprocal combination GAL4
AD-p67
/GAL4 DNA binding domain-p47
had
the same effect. Growth capacity on medium lacking histidine was not
induced when p47
or p67
were replaced by
SNF1 or SNF4 proteins (Fig. 1). Therefore, we conclude that
expression of the reporter gene HIS3 results from the interaction
between p47
and p67
. This interaction was
initially suspected from observations showing that a large protein
complex found in phagocyte cytosol contained a fraction of both
proteins(18, 29) , and that p67
translocation to the membrane depended on the presence of
p47
(52) . A direct interaction between both
proteins in cell cytosol, involving SH3 and proline-rich domains of
each protein has been
observed(30, 33, 36, 37) .
Consistent with these data, the results of our two-hybrid study provide
evidence for the formation of a complex between p47
and
p67
proteins in the intact cell. They further indicate
that this complex is independent of Rac.
p47
-p67
interactions in the two-hybrid
system provide a means for investigating in an intracellular
environment how this interaction could be modulated by biological
signals. It should be feasible, for example, to stimulate or inhibit in
yeast, pathways known to activate NADPH oxidase in phagocytes (protein
kinase C activation or arachidonate synthesis) and to analyze their
effect on the p47
-p67
complex.
Figure 1:
Interaction between p47 and p67
. Interactions between
proteins are tested in yeast strain HF7c. Cells were cotransformed with
pairs of recombinant plasmids, one expressing the indicated protein
fused to the GAL4 DNA binding domain (column I) and the other
expressing the indicated protein fused to GAL4 activation domain (column II). Cotransformed cells, growing on selective plates
lacking tryptophan and leucine but in the presence of histidine, are
shown in the left lane. Interactions between two-hybrid
proteins are indicated by histidine-independent growth as shown in the right lane, observations at 36 h. Yeast proteins SNF1 and SNF4
were used as positive control.
Interactions between p21 and p47
or
p67
were first investigated using the same two-hybrid
system based on GAL4 reconstitution in reporter yeast HF7c. Different
alleles of Rac1 were used: Rac1 wild type; Rac1V12, an activated mutant
of Rac1 blocked in GTP-bound form; and Rac1V12N17, a dominant-negative
mutant of Rac1 blocked in the GDP-bound form(49) . Rac proteins
undergo an isoprenylation of their C-terminal part, at cysteine 189,
which probably allows this cytosolic protein to bind
membrane(53) . To avoid decreasing detection sensitivity due to
partial membrane localization of proteins in yeast, as already reported
in two-hybrid study of Ras-Raf interactions(54) , we tested
Rac1 alleles with a serine 189 mutation, thus preventing C-terminal
isoprenylation and membrane localization. In the first set of
experiments (data not shown) cotransformed colonies expressing the
different Rac alleles with p47
or p67
proteins were checked for their ability to grow in the absence of
histidine. Only transformants expressing Rac1V12 in combination with
p67
showed a significant histidine-independent growth.
Although the signal was rather weak, it was significant and suggested
that an activated form of Rac1 was required for Rac1-p67
interaction. To increase signal intensity, we decided to use
another yeast two-hybrid system, which in our hands appeared more
sensitive.
This system, based on LexA DNA binding protein and yeast
strain L40 (46) , which carries HIS3 and LacZ as reporter
genes, allowed us to make a more detailed analysis of Rac-p67 interactions. The same Rac1-serine 189 alleles were used as
fusion protein with LexA, to look at interactions with p67
or p47
fused to the GAL4 activation domain (Fig. 2). The interaction between RasV12 hybrid and c-Raf1
hybrid provided a positive control in L40 cells (Fig. 2B). The various cotransformed cells were able to
grow at similar rates in the presence of histidine, indicating no
particular toxicity of expressed hybrid proteins. The ability to grow
in the absence of histidine was clearly induced when Rac1V12 and
p67
hybrid proteins were coexpressed (Fig. 2A). The ``deactivation'' of Rac1V12 by
the Asn-17 mutation resulted in a dramatic decrease of
histidine-independent growth of Rac1V12NI7-p67
transformants (Fig. 2A). By contrast,
Rac1V12-p47
or Rac1V12NI7-p47
combinations were completely inefficient in promoting
histidine-independent growth (Fig. 2A). These results
therefore indicate that only the activated GTP-bound form of Rac1 is
capable of interacting with cytosolic regulator of NADPH oxidase,
p67
, but not with p47
. We also conclude
that isoprenylation of the C-terminal part of Rac proteins is not
essential for Rac-p67
interactions, consistent with
previous studies of Rac activity in a cell-free system(24) .
Figure 2:
Interaction between Rac1V12 and
p67. Proteins expressed as fusion with GAL4
activation domain (column II) were tested for interaction with
various small GTPases fused to LexA protein (column I), in
yeast strain L40. Growth of cotransformed yeast, in the presence of
histidine, is shown in the left lane. Interactions between
proteins are demonstrated by histidine-independent growth in the right lane (-His), observations at 72 h. A,
interactions between Rac1V12 or Rac1V12N17 with p47
or p67
. B, interactions
between p67
and Rac effector mutants or various
small GTPases. Ras and Raf proteins were used as a positive control. C, immunoblot showing expression of the various LexA fusion
proteins, from yeast used in A and B. 1, LexA without fusion; 2, LexA-Rac1V12; 3, LexA-RhoBV14; 4, LexA-RhoGV12; 5, LexA-Rac1V12A35; 6, LexA-Rac1V12A38; 7, LexA-RasV12; 8, LexA-Rac1V12; 9, LexA-Rac1V12N17.
To check for specificity of the interaction, various activated small
GTPase proteins were also tested for interaction with p67 in L40 cells. RasV12, an activated form of Ras, did not interact
with p67
in the assay. Similarly, RhoGV12
CAAX, an
activated form of RhoG lacking the C-terminal CAAX box, and an
activated RhoB mutant, RhoBV14, showed no interaction with p67
able to stimulate growth in the absence of histidine (Fig. 2B). Activated forms of Rap2 (Rap2V12) and RalA
(RalAV23 with 18 C-terminal amino acids deleted) were also negative in
a two-hybrid assay with p67
(not shown). The various
GTPases used were able to interact with their own partners (unpublished
two-hybrid assays). We conclude that p67
interacts
specifically with the NADPH oxidase activator Rac.
To further
investigate whether p67 is a functional target for Rac,
we used Rac proteins mutated in the putative effector region (amino
acids 32 to 40). Substitutions of conservative residues at codon 35
(Thr
Ala) or codon 38 (Asp
Ala) have demonstrated in Ras
studies that the effector domain is crucial for biological effects of
Ras and for direct interaction with Raf(55, 56) .
Identical Rac mutants have been shown unable to activate NADPH oxidase
in cell-free assays (21, 22) . Here we cotransformed
cells with p67
and Rac effector mutants, specifically
Rac1V12 with the additional mutation Ala-35 or Ala-38. We observed that
the resulting cells almost entirely lose their ability to grow in the
absence of histidine (Fig. 2B), showing that
interaction of Rac1 with p67
was strongly impaired by
both mutations. It thus indicates that Rac effector domain plays an
important role in the interaction with p67
.
In all
cases, hybrids of the various GTPases tested were correctly expressed
and at similar levels (Fig. 2C). Histidine-independent
growth has been reported here as an indication of interactions;
however, equivalent induction of the other reporter gene, LacZ, was
also detected by -galactosidase filter assay (not shown).
Altogether, these experiments clearly demonstrate that interaction
between Rac and p67 is specific and Rac-GTP dependent
and, in addition, involves the Rac effector domain. All these
observations correlate directly with functional characteristics of Rac
in the NADPH oxidase cell-free system, and thus, bring convincing
evidence for p67
being the Rac-specific effector in
NADPH oxidase complex. Our two-hybrid data obtained in an intracellular
environment are in full agreement with in vitro results
obtained with recombinant proteins and recently published by Diekmann et al.(21) and Prigmore et al.(41) .
Therefore, both sets of results strongly support the view that
p67
is a direct target for Rac in phagocyte NADPH
oxidase.
To complement our study of Rac-p67 interaction, we wondered if activated RacL61 mutant, known to
have a higher affinity for NADPH oxidase than RacV12 or Rac wild
type(22) , also had a higher affinity for the p67
component. L40 cells were cotransformed with p67
encoding plasmid and plasmids encoding Rac1L61 or Rac1V12 fused
to LexA. The Rac1L61 mutant appeared highly toxic in yeast cells.
Therefore, although Rac1L61-induced signals appeared stronger than
those from Rac1V12 (data not shown), accurate quantification of the
difference could not be obtained.
Because Rac2 is the predominant
Rac protein in human phagocytes (34, 40) and because
the functional ``L61'' data were obtained using Rac2 (22) , we decided to use Rac2 alleles to further investigate
effects of L61 mutation. Activated Rac2V12 (GTP-bound), deactivated
Rac2N17 (GDP-bound), or activated Rac2L61 (GTP-bound) mutants, fused to
LexA, were coexpressed in L40 cells with p67. We
observed that the Rac2V12-p67
combination induced a
clear histidine-independent growth (Fig. 3A). Rac2N17
allele expressed concomitantly with p67
allowed no
growth in the absence of histidine as compared to control cells
expressing Rac2N17 alone. On the contrary, Rac2L61 mutant induced a
highly efficient growth which is clearly stronger than the growth
induced by the Rac2V12 mutant. Identical observations were made using
the second reporter gene, LacZ, as shown by
-galactosidase filter
assays (Fig. 3B). Quantification of interactions by
measurement of
-galactosidase activity in a liquid assay showed
that Rac2L61-p67
induced a 6-fold higher signal than
Rac2V12-p67
complex (Fig. 3C).
Interactions between Rac2 mutants and p47
were also
checked and have not been detected (data not shown). In all cases the
different Rac2 alleles were checked for their expression in yeast and
were found to be expressed at similar levels. These observations
indicate that Rac2, like Rac1, interacts with p67
in a
GTP-dependent manner, but does not seem to interact with
p47
. They also show that Rac2L61, in an intracellular
environment, has a 6-fold higher affinity for p67
than
Rac2V12. This is in agreement with a recently published report showing
the higher potency of Rac1L61 recombinant protein to interact with
p67
(41) , and correlates directly with
observations on Rac2L61 activity in the cell-free system(22) .
Figure 3:
Effect of the L61 mutation on
Rac-p67 interaction. In yeast strain L40,
various Rac2 mutants, as fusion with LexA, were tested for interaction
with p67
fused to the GAL4 activation domain.
Interactions between the indicated proteins are demonstrated by
histidine-independent growth or
-galactosidase activity. A, growth in the absence of histidine are shown in the right column (-His), observations at 48 h. Control growth of
cotransformed yeast in presence of histidine is shown in the left
column (+His). B,
-galactosidase activity on a
filter assay, observations at 12 h. C,
-galactosidase
activity quantified by a liquid assay.
We observed that Rac2-p67 interactions were strong
and comparable to positive controls but that Rac1-p67
interactions appeared relatively weak. A more precise comparison
of Rac1 and Rac2 affinity for p67
, using Rac V12 or L61
activated mutants fused to LexA proteins, was made directly on the same
experiment. Cells coexpressing Rac1V12 or Rac2V12 and p67
grew at normal rates in the presence of histidine and were tested
for their capacity to grow in the absence of histidine. In several
independent experiments, Rac2V12 allowed a much better rate of growth
than Rac1V12 (Fig. 4A). Similar observations were made
using the other reporter gene, LacZ, as shown by a stronger
-galactosidase activity (Fig. 4B). Quantification
of Rac1- and Rac2-p67
interactions was obtained by
measurement of
-galactosidase activity in a liquid assay from the
exact same number of cells. We observed that Rac2V12 induced around
6-fold more enzyme activity than Rac1V12 (Fig. 4C). It
shows that LacZ reporter gene, as well as HIS3 reporter gene, are both
stimulated to higher levels by the Rac2V12-p67
complex
than by the Rac1V12-p67
complex. Expression of both Rac
mutant proteins were checked by Western blot and were found to be
similar. We tried further to compare Rac1 and Rac2 affinity for
p67
using the Rac L61 mutants. Again Rac2L61-induced
signals appeared clearly stronger; however, due to the high toxicity of
Rac1L61 mutant we cannot provide a reliable estimation of this
difference. In all, these data indicate that p67
, in an
intracellular environment, has a much better affinity for Rac2 protein
than for Rac1 protein.
NADPH oxidase activation by small GTPase led
to isolation of Rac1 from guinea pig macrophages (11) and to
Rac2 from human phagocytes(12) . Whether this difference
reflects a species specificity remains to be determined. However, the
predominant interaction of p67 with Rac2 that we
observed provide new information relevant to this question. Our data,
in combination with the fact that human phagocytes contain
predominantly Rac2 rather than Rac1(34, 40) , show
that the Rac-p67
interaction in a whole cell environment
is almost specific for Rac2 protein.
This raises the question of the
molecular basis for the difference between Rac2 and Rac1. We showed
that the Rac-p67 interaction involved the Rac
GTP-binding site and required the Rac effector domain. Rac2 and Rac1
are almost identical in these areas; however, the C-terminal portions
of the two proteins are divergent. This suggests that the basis for the
p67
higher interaction efficiency with Rac2 probably
resides in that C-terminal part of Rac2.
In the recombinant
cell-free system, Rac1 and Rac2 are roughly equipotent in NADPH oxidase
activation. However, it has been previously observed that Rac2 was a
more potent activator than Rac1 when neutrophil cytosol was present in
the assay(24) , thus indicating involvement of additional
intracellular factor(s). The yeast cells could also provide such an
environment and allow Rac2 to interact more efficiently with
p67 than Rac1.
Our data are in favor of p67 being the Rac target in NADPH oxidase. However, we cannot exclude
that, in addition, Rac could directly act on cytochrome b
(23, 40, 57) or, as
suggested very recently(58) , that Rac could act through
activation of p21-activated kinases (PAK1 and PAK2). Our data in
combination with current knowledge on Rac1 function could also suggest
that in human neutrophils, Rac2 could have a different function than
Rac1: Rac2 regulating NADPH oxidase activity and Rac1 perhaps
regulating cytoskeleton modifications.
Overall, we conclude that
Rac2 can regulate the human NADPH oxidase by interacting in a
GTP-dependent manner directly with the p67 component.
The Rac2-p67
interaction, probably occurring at the
plasma membrane level(1, 33) , would not promote
assembly at the membrane of cytosolic components(35) , but
would rather directly activate the assembled membranous complex, thus
stimulating superoxide production.