(Received for publication, April 24, 1995; and in revised form, June 23, 1995)
From the
Production of microbicidal oxidants by phagocytic leukocytes
requires activation of a latent NADPH oxidase by the coordinated
assembly of a membrane-associated flavocytochrome b, with three cytosolic components,
p47
, p67
, and the low
molecular weight GTP-binding protein Rac. Rac1 and Rac2 have 92%
sequence identity and are both active in supporting the oxidase, while
CDC42Hs, the closest relative to Rac with 70% sequence identity, only
weakly supports oxidase activation in vitro. We have used
CDC42Hs as a foil to identify residues in Rac that are critical for
oxidase activation. Most of the divergent sequences of CDC42Hs could be
incorporated into Rac-CDC42Hs chimeric proteins without affecting
cell-free NADPH oxidase activity. However, incorporation of the
amino-terminal segment of CDC42Hs (residues 1-40), which differs
from Rac1 by only four residues (positions 3, 27, 30, and 33), resulted
in a marked loss of oxidase activation capacity. Point mutagenesis
studies showed that this was due to changes at residues 27 and 30, but
not residues 3 and 33. Conversely, incorporation of the amino terminus
of Rac1 (residues 1-40) into CDC42Hs increased its activity to
that of Rac1, indicating that this terminus contains the
effector-specifying domain of Rac. Taken together, these studies show
that the difference in the activity between CDC42Hs and Rac1 is due
entirely to differences in amino acids at position 27 and 30.
In response to a variety of stimuli, phagocytic white blood
cells produce superoxide and a variety of microbicidal
oxidants(1) . The superoxide-generating NADPH oxidase of
phagocytes consists of both membrane and cytosolic proteins. Cytochrome b is the sole membrane component required for
superoxide generation (2, 3, 4) . It is an
integral membrane protein complex composed of a 22-kDa protein
(
-subunit) and a 91-kDa glycoprotein (
-subunit) that
catalyzes the one-electron reduction of oxygen to produce superoxide
anion. Studies by Rotrosen et al.(3) and Segal et
al.(4) showed that cytochrome b
bound FAD and that structural similarities exist between the
-subunit and the putative NADPH- and FAD-binding sites of the
ferredoxin-NADP
reductase family. Two of the cytosolic
proteins, p47
and p67
,
were identified based on their deficiencies in most patients with
autosomal, cytochrome b
-positive forms of
chronic granulomatous disease, an inherited disease characterized by
the inability of phagocytes to produce
superoxide(5, 6, 7, 8, 9) .
Both cytosolic proteins become tightly associated with membranes upon
cell activation(10, 11) . Several studies have
indicated that aside from p47
and
p67
, at least one other cytosolic protein is
required to complement membranes for cell-free production of
superoxide(6, 12, 13) . This component was
identified as Rac1 in guinea pig macrophages (14) and Rac2 in
human neutrophils(15, 16) . These two proteins belong
to the Rho subfamily of Ras-related GTP binding proteins and are 95%
identical to one another in their amino acid sequences. Rac1 and Rac2
were purified as complexes with Rho-GDI, (
)although
Rho-GDI is not required for in vitro activation of the
oxidase(14, 16) . Upon activation of phagocytes,
p47
, p67
, and Rac, but
not Rho-GDI migrate to the
membrane(10, 11, 17, 18) . Although
interactions between Rac and p67
have been
observed(19) , studies with chronic granulomatous disease
neutrophils suggest that Rac membrane translocation is independent of
p47
and p67
, and that
cytochrome b
may stabilize this membrane
interaction (20) .
Cytochrome b alone, reconstituted with certain lipids, is capable of
superoxide generation in vitro(21) , indicating that
it is the sole electron carrier of the oxidase. However, optimal
superoxide production also requires the presence of p47
and p67
and
Rac(3, 22) . p47
and
p67
appear to play distinct roles in the
regulation of electron flow in NADPH oxidase(23) . A role for
Rac in whole cell oxidase activation is supported by studies in intact
cells where inhibition of Rac synthesis by antisense DNA in lymphocytes (24) and the expression of a dominant-negative mutant of
Rac(N17) in HL-60 cells diminished oxidase activity(25) . Like
Ras, Rac1 and Rac2 are active in their GTP-bound states, and their
effects on oxidase activation can be regulated by proteins that
modulate guanine nucleotide exchange and
hydrolysis(26, 27, 28) .
Rac and CDC42Hs have been implicated in various aspects of reorganization of the actin-based cytoskeleton. Rac participates in growth factor-induced membrane ruffling in fibroblasts(29) , and CDC42Hs functions in the polarization of actin filaments preceding bud site assembly in yeast(30) . CDC42Hs, with 72% homology to Rac1, has a low capacity for oxidase activation(16) . Confirmation that CDC42Hs and Rac have distinct effectors is shown by the fact that human CDC42Hs, but neither Rac1 nor Rac2, complements the lethal mutation of CDC42Sc in Saccharomyces cerevisiae(31) . Alignment of the amino acid sequences of Rac1 and Rac2 with that of CDC42Hs revealed three regions containing most of the structural divergence between these proteins, suggesting that one or more of these sequences may determine the functional specificity of these Ras relatives(16) .
To date, most studies addressing structure-function relationships of Rac in oxidase activation have been based on analogy to Ras, which exhibits only 30% homology to Rac and other Rho subfamily members. Consistent with the Ras paradigm, point mutations within the amino terminus of Rac reduced its oxidase-activating capacity, although these experiments have provided only indirect evidence that the effector-specifying domain of Rac is located within the amino terminus (19, 32) . In the present study, we systematically compared a series of chimeras produced from Rac1 and CDC42Hs in order to determine the domain of Rac that functionally distinguishes it from CDC42Hs. This domain was anticipated to confer a Rac-like oxidase activating capacity to CDC42Hs. Analysis of these chimeras has allowed us to assess the impact of mutational changes throughout various regions of Rac.
In our previous report(16) , we identified three
regions (residues 27-52, 130-150, and 174-190) that
are relatively conserved between Rac1 and Rac2, but divergent in
CDC42Hs (Fig. 1). Rac and CDC42 clearly have distinct effectors,
since human CDC42 but not Rac is capable of complementing a lethal
mutation of CDC42 in S. cerevisiae, and Rac but not CDC42
supports oxidase activation. Our working hypothesis is that the domain
of Rac that confers functional specificity is conserved between Rac1
and Rac2 but is structurally divergent in CDC42Hs. In order to assess
the contributions of various regions of Rac1 in their interaction with
the NADPH oxidase, selected regions of Rac1 were substituted
sequentially with the corresponding regions of CDC42Hs (Fig. 2A). Substitution of residues 41-52 of Rac1
(Chimera B), residues 53-173 (Chimera C), and residues
41-192 (Chimera E) with the corresponding residues of CDC42Hs had
no discernible effect on its ability to activate the oxidase in a
cell-free assay (Fig. 2B). Together, these three active
chimeras incorporated a majority of the sequence differences found in
CDC42Hs. In contrast, substitution of the NH-terminal 40
amino acid segment of Rac1 with that of CDC42Hs resulted in a protein
(Chimera A) that had activity reduced to that of CDC42Hs. The lower
activity of Chimera D raised the possibility that the terminal 17 amino
acids of Rac represent another functional domain. However, this
possibility was not supported by the observation that Chimera E, which
lacks an even more extensive COOH-terminal Rac sequence, had activity
comparable to that of wild type Rac1. The reduced activity of Chimera D
may reflect how well sequence difference at the COOH terminus are
complemented by the rest of protein molecule for proper folding and
function.
Figure 1: Comparison of amino acid sequence of Rac1 with that of CDC42Hs. Identical amino acids are represented as soliddots. A gap that optimized the alignment of CDC42Hs is denoted by a dash.
Figure 2:
A, schematic depiction of chimeric
Ras-related GTP-binding proteins constructed from Rac1 and CDC42Hs. Shadedsegment, CDC42Hs; solidsegments, Rac1. B, comparison of
oxidase-activating capacities of chimeric Rac1/CDC42Hs proteins.
Proteins studied were: Rac1 (), CDC42Hs (
), Chimera A
(
), Chimera B (
), Chimera C (
), Chimera D
(
), and Chimera E (
). The data are representative of a
minimum of three experiments and are averages of
duplicates.
To further explore the role of the amino terminus, we
replaced the NH-terminal 40 amino acids of CDC42Hs with
those of Rac1. The resulting protein (Chimera E) had activity
equivalent to that of Rac1 (Fig. 2B), indicating that
this segment of Rac1 contains the effector domain of Rac specifying
oxidase activation. This unique Rac segment may interact directly with
its effector, or alternatively, may act intramolecularly to affect the
interaction of other region(s) of Rac with its effector. Our study did
not distinguish between these two possibilities. In this connection, it
is interesting to note that a Rac peptide spanning residues
18-40, at a concentration of 100 µM, did not inhibit
cytochrome b
dependent-NADPH oxidase activity in
a cell-free oxidase assay containing neutrophil membranes and cytosol
(data not shown). The amino terminus of Rac1 (residues 1-40) is
identical to that of Rac2 and differs from CDC42Hs by only four amino
acids (residues 3, 27, 30, and 33). Point mutants of Rac1 were made by
substituting each of these residues with the corresponding residues of
CDC42Hs, resulting in Rac point mutants A3T (alanine to threonine),
A27K (alanine to lysine), G30S (glycine to serine), and I33V
(isoleucine to valine). The activities of A3T and I33V were comparable
to that of wild type Rac1 (Fig. 3), indicating that these amino
acid substitutions were conservative with respect to their effect on
oxidase activation. In contrast, mutations at either residue 27 (A27K)
or residue 30 (G30S) reduced the activity of the point mutants to that
of CDC42Hs (Fig. 3).
Figure 3:
Activity of point mutants of Rac1.
Proteins studied were: Rac1 (), CDC42Hs (
), A3T (
,
with substitution of alanine at residue 3 of Rac1 by threonine), I33V
(
, with substitution of isoleucine at residue 33 by valine),
G30S (
, with substitution of glycine at residue 30 by serine),
and A27K (
, with substitution of alanine at residue 27 by
lysine). Data are representative of three experiments and are averages
of duplicates.
Until now, only a limited number of
point mutants of Rac have been studied with regard to its oxidase
effector function. Based on the characterization of the Ras effector
region, Diekmann et al.(19) showed that point
mutations at residue 35, 38, or 40 of Rac inhibited cell-free oxidase
activation and block the interaction of Rac with
p67, its putative target. Similarly, Xu et
al.(32) showed that point mutations at residue 28, 35,
36, or 38 diminished its activity in oxidase activation. In the present
study, Chimeras A-E contain extensive mutations throughout Rac
that had not been studied previously. Chimera E contains most of the
sequence divergence between Rac and CDC42Hs, yet this protein retained
the oxidase-activating capacity of Rac. This study showed that the
Rac-like oxidase regulating activity could be conferred to CDC42Hs when
only four amino acids within the amino terminus of CDC42Hs were
substituted by the corresponding residues in Rac (Chimera E). Of these,
only two residues (27 and 30) account for the difference in oxidase
activation between Rac and CDC42Hs; these were located within a region
analogous to the Ras effector domain.
As necessary controls for loss
of function observed in some of these proteins, the integrity of the
thrombin-released proteins was assessed by SDS-polyacrylamide gel
electrophoresis, which confirmed that the recombinant proteins were
generated with the predicted size (approximately 21-22 kDa) and
of high purity (>90%; Fig. 4). Furthermore, to rule out the
possibility that the reduced activities of Chimera A and point mutants
G30S and A27K were due to impairment of guanine nucleotide binding, we
compared their abilities to bind [S]GTP
S.
The results given in Fig. 5showed that these mutations of Rac1
did not affect the amount of guanine nucleotide binding. These studies
also showed that the specific activity of Rac1 in oxidase activation is
actually much higher than what is shown in Fig. 2B,
since only 11% of the recombinant Rac1 bound
[
S]GTP
S, whereas 93% of recombinant CDC42Hs
had binding activity (data not shown).
Figure 4: Coomassie Blue-stained electrophoretogram (8-16% Tris-glycine-SDS-polyacrylamide gel electrophoresis) of recombinant Rac1/CDC42Hs proteins. Lane1, Rac1; lane2, CDC42Hs; lanes 3-7, Chimeras A-E; lane8, A3T; lane9, A27K; lane10, G30S; lane11, I33V.
Figure 5:
Comparison of
[S]
GTP
S binding activity
of 0.5 µg of Rac1, Chimera A, and Rac point mutants. dpm,
protein-bound radioactivity of
[
S]
GTP
S, expressed as
disintegrations/minute. Each bar represents the mean of data
from two experiments. The interval represents the range. Nonspecific
binding, as determined in the presence of 100-fold excess of cold
GTP
S, accounted for <3% of the total
binding.
The identification of the amino terminus of Rac as being essential for optimal oxidase activation in the present studies is analogous to studies that mapped effector sites within Ras, where the amino terminus (residues 21-31 and 45-54) was shown to be essential for Ras transforming activity(34) . Consistent with this, transforming activity could be conferred to Rho (a protein with only 30% homology) when residues 23-46 were substituted with the corresponding sequence of Ras, indicating that this region contains the effector-specifying domain of Ras(35) . Furthermore, a peptide containing residues 17-44 of Ras inhibits its interaction with its effector, Raf(36) .
At this time it is unclear to what extent the large body of structural and functional information available on Ras can be applied to members of the Rho subfamily, including Rac. The identification of critical Rac effector domain residues in this report and others (residues 28, 35, 36, 38, 40) (19, 32) is consistent with some aspects of the Ras model. Two regions in Ras undergo significant changes in conformation upon binding of a non-hydrolyzable GTP analog to Ras. These are residues 30-38 (switch I region) and residues 60-76 (switch II region)(37) . Both regions form a continuous strip on the surface most likely to be recognized by effectors. The recent report by Xu et al.(32) showed that a mutation at residue 61 of Rac (substitution of glutamine by leucine) can overcome an inactivating mutation at residue 38 (aspartic acid to alanine), raising the possibility that the effector-specifying domain of Rac includes both switch regions. Due to the extensive structural homology between Rac and CDC42Hs around residue 61(switch II region), our studies do not exclude this possibility. One important distinction between Ras and members of the Rho subfamily concerns their sites for interaction with their respective GTPase-activating proteins (GAPs). The binding site of RasGAP to Ras appears to overlap with its amino-terminal ``effector domain,'' while mutations in the effector-specifying region of Rac do not affect its responsiveness to its GAPs(32) .
The roles of the other regions of Rac that
are divergent from CDC42Hs remain to be determined. Substitution of the
region spanning residues 41-52 of Rac with the corresponding
divergent sequence of CDC42Hs did not diminish cell-free oxidase
activity (Chimera B, Fig. 2B). The divergent region at
the carboxyl terminus (residues 174-192), which also exhibits
significant sequence differences between Rac1 and Rac2, is modified by
geranylgeranylation, a post-translational process required for
Rac's association with both membranes and GDP/GTP exchange
proteins(38) . However, this processing is not required for
cell-free oxidase reconstitution, since the unmodified forms of both
recombinant Rac1 and Rac2 support oxidase
activity(14, 15, 16, 39, 40) .
This region nonetheless does serve an important oxidase-related
function even in the absence of geranylgeranylation, since the
truncated form of Rac lacking residues 175-195 does not support
cell-free oxidase reconstitution (data not shown). One or more of these
divergent regions between Rac and CDC42Hs may specify interactions with
several regulator proteins, such as p120, RhoGAP, and Bcr. p120
is a protein kinase that binds to
CDC42Hs, but not to Rac(41) . RhoGAP is a GTPase-activating
protein exhibiting a striking preference for CDC42(42) , while Bcr is another GAP implicated as a down-regulator of the
neutrophil respiratory burst(43) .
In conclusion, we present direct evidence in this study that the effector-specifying domain of Rac in NADPH oxidase activation is located in the amino terminus. Furthermore, we demonstrate that residues 27 and 30 alone account for the difference in activity between Rac1 and CDC42Hs in oxidase activation.