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INTRODUCTION |
Rho family GTPases Rac1, Cdc42, and RhoA belong to the Ras
superfamily of monomeric small GTP-binding proteins. They regulate a
wide spectrum of cellular functions, ranging from cell growth and
cytoskeletal organization to secretion (1-4). The physiological significance of Rho family GTPases is highlighted by increasing evidence for their involvement in human diseases such as cancer, hypertension, and inflammation (5-7).
The multiple biological functions of Rho family proteins are mediated
through a tightly regulated GTP-binding/GTP-hydrolyzing cycle (8, 9).
Three classes of regulatory proteins are known to be involved in the
regulatory process. The guanine nucleotide exchange factors
(GEFs)1 catalyze the exchange
of bound GDP for GTP, resulting in activation of the GTPases, whereas
the GTPase-activating proteins (GAPs) stimulate the intrinsic GTP
hydrolysis by Rho GTPases, leading to the rapid conversion to the
GDP-bound inactive state. The Rho guanine nucleotide dissociation
inhibitors (RhoGDIs) preferentially bind to the GDP-bound form of Rho
proteins and prevent both spontaneous and GEF-catalyzed release of the
nucleotide. In addition, RhoGDI recognizes the isoprenoid moiety of the
GTPases and is capable of solubilizing the membrane-associated
proteins. In the GTP-bound form, the Rho proteins interact with their
specific effector or target molecules to trigger diverse cellular
responses. Among a growing panel of the small G-protein
targets, the serine/threonine protein kinases
p21cdc42/rac-activated kinases (PAKs) were found to be
activated upon binding to the GTP-bound form of Rac1 and Cdc42 (10,
36), and they have been implicated in a number of signaling pathways
downstream of Rac1 and Cdc42, including c-Jun N-terminal kinase
activation and cytoskeletal reorganization (36).
Among the Rac subfamily, the Rac1 GTPase shares >90% amino acid
identity with its closest relatives, Rac2 and Rac3, and much functional
redundancy has been expected among the three Rho GTPase members (11).
For example, both Rac1 and Rac2 can be substituted in vitro
effectively as a component of the NADPH oxidase complex. In addition to
differences in tissue distributions (Rac1 and Rac3 are ubiquitous,
whereas Rac2 expression is restricted to hematopoietic cells) (11, 12),
however, two lines of evidence suggest that Rac1 may play a distinct
role in cells. Neutrophils from Rac2 knockout mice are defective in
many actin-based functions as well as in cell proliferation and
survival even though Rac1 expression remains high (13). In
vitro, Rac1 is able to bind to and stimulates the kinase activity
of PAK1 ~4-5-fold better than Rac2 (14). These functional
differences are reflected in their structures, which differ
significantly only at the C termini. Most notably, Rac1 contains a
unique C-terminal domain consisting of six consecutive basic amino
acids, whereas this domain is disrupted by three neutral amino acids in
the Rac2 and Rac3 sequences. This region of the molecule is not
structured in the tertiary model of available Rho GTPases (28-30) and
has been suggested to have a role in the membrane localization of Rac1
(15) and in interaction with effector proteins (14). How the unique
feature of this region in Rac1 might contribute to the distinct
function of Rac1 remains unclear.
Besides the interaction with regulatory proteins and effector
molecules, a few Rho GTPases have been found to interact homophilically among themselves. We have previously shown that Cdc42 and Rac2 may form
homodimers and that the dimerization event elicits a self-stimulatory
GTPase-activating activity of the GTPases similar to the RhoGAP effect
(16). An arginine residue in the C-terminal domain of these GTPases was
shown to confer the catalytic GAP activity (17). These observations
suggest that certain small GTPases may involve self-association as an
additional mode of regulation. Similar suggestions have been made for
other small and large G-proteins. For example, ADP-ribosylation
factor-1 has been shown to exist in a functional dimeric or tetrameric
form (18). A recent study with Ras suggests that dimerization at the
plasma membrane is essential for Raf-1 activation (19). In
analogy, homodimerization or oligomerization in large GTPases of the Mx
family, which are interferon-induced GTPases (76 kDa) responsive to
viral infections, has been known to regulate their GTP-binding and
GTP-hydrolyzing cycle, yielding a highly efficient self-regulatory
large GTPase complex (20, 21). Whether a similar homophilic interaction
occurs between Rac1 GTPases and how such an interaction would affect
the regulatory cycle of Rac1 are of particular interest to us.
In this study, we present evidence that Rac1 forms a reversible monomer
and oligomer under physiologically relevant conditions and that its
C-terminal polybasic domain is an essential determinant for oligomer
formation. The biochemical results reported here, combined with our
previous knowledge of Rac1 regulation, lead to a model in which the
Rac1 oligomeric state may contribute to the generation of a transient
and augmented signal.
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EXPERIMENTAL PROCEDURES |
Materials--
GDP, GTP, GTP
S, and bacterial
purine-nucleotide phosphorylase were purchased from Sigma. All
radiolabeled nucleotides were obtained from PerkinElmer Life Sciences.
2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG) was synthesized
as described previously (22). The polypeptides corresponding to the
C-terminal 12 amino acids of Rac1 and Cdc42 (PVKKRKRKCLLL and
PEPKKSRRCVLL, respectively) were custom-synthesized by
Bio-Synthesis, Inc. (Lewisville, TX). The anti-Myc and anti-HA
monoclonal antibodies were obtained from Sigma and Roche Molecular
Biochemicals, IN), respectively.
DNA Constructs and Recombinant Protein Preparations--
The
Rac1 C-terminal mutants were generated by polymerase chain
reaction-directed mutagenesis using internally derived oligonucleotides encoding the desired sequences. Rac1
8 represents a truncated form of
Rac1 with deletion of the last eight residues of the C terminus. The
Rac1-5Q mutant contains five Gln residues substituted for the polybasic
residues (amino acids 183-187) of Rac1. The Rac1-Cdc42 mutant contains
amino acids 183-188 of Cdc42 substituted for the corresponding
residues of Rac1 in the Rac1 backbone. The mutant cDNAs were
sequence-proofed and subcloned into the pGEX-KG vector, pET28a vector,
pVL1392-His6, pCMV6-Myc vector, or pKH3 vector to be
expressed as the GST, His6, Myc, or (HA)3
fusions in Escherichia coli cells, Sf9 insect cells,
or mammalian cells. The pKH3-Cdc42 and pKH3-RhoA constructs were as
previously described (46). The pCMV6-Myc-PAK1 construct was a kind gift
from Dr. J. Chernoff (Fox Chase Cancer Center).
The preparation of recombinant GTPases was performed as described (23).
The E. coli-expressed proteins were in GST- or
His6-tagged form and were purified by affinity
chromatography on glutathione-agarose or Ni2+-agarose
beads. The insect cell expression of His6-Rac1 was carried out by previously described procedures (24). The N-terminal GST- or
His6-tagged sequences were cleaved by thrombin digestion when necessary. The post-translationally modified form of Rac1 was
purified from the membrane fraction of the Sf9 cells infected with the recombinant baculovirus encoding His6-Rac1. RhoGDI
and the GAP domain of p50RhoGAP containing amino acids
205-439 were expressed as GST fusions in E. coli as
described. The quality of the proteins used in all assays was judged by
SDS-polyacrylamide gel electrophoresis. Protein concentrations were
estimated from Coomassie Blue-stained gels and/or by using BCA protein
assay reagents (Pierce).
For transient expression in COS-7 cells, the pCMV6-Myc and pKH3
constructs were transfected into COS-7 cells grown to ~80% confluence in Dulbecco's modified Eagle's medium containing 10% fetal calf serum using LipofectAMINE regent (Life Technologies, Inc.).
48 h post-transfection, the cells were harvested for the complex
formation assays.
Gel Filtration--
A Superdex 200HR 10/30 column (10 × 30 cm; Amersham Pharmacia Biotech) was used to analyze the homophilic
interactions of the small GTPases in combination with a Bio-Rad
biologic liquid chromatography system as previously described (16). The
column was equilibrated with 50 mM HEPES (pH 7.6), 1 mM DTT, 100 mM NaCl, and 2 mM
MgCl2 and was calibrated with molecular mass standards containing thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), chymotrypsinogen A (25 kDa), and ribonuclease
A (13.7 kDa) (Amersham Pharmacia Biotech). The samples were loaded in a
volume of 0.1 ml, and elutions were performed at the indicated buffer conditions.
GTPase Activity Assays--
The radioactive filter binding
assays measuring the retention of [
-32P]GTP-bound Rac1
were carried out as described (25). Briefly, recombinant Rac1 was
preloaded with [
-32P]GTP in 100 µl of buffer
containing 50 mM HEPES (pH 7.6) and 0.1 mM EDTA
for 10 min at ambient temperature before the addition of
MgCl2 to a final concentration of 1 mM. An
aliquot of [
-32P]GTP-loaded Rac1 was mixed with
reaction buffer containing 50 mM HEPES (pH 7.6), 0.2 mg/ml
bovine serum albumin, 1 mM DTT, and 1 mM
MgCl2. At different time points, the reaction was
terminated by filtering the reaction mixture through nitrocellulose
filters, followed by washing with 10 ml of ice-cold buffer containing
50 mM HEPES (pH 7.6) and 10 mM
MgCl2. The radioactivity retained on the filters was then
subjected to scintillation counting.
The MESG/phosphorylase system monitoring the free
-Pi
released from the GTP-bound proteins was based on the method described by Webb and Hunter (26) and has been applied to the measurement of Rho
GTPases (17, 23, 27). Briefly, a 0.8-ml solution containing 50 mM HEPES (pH 7.6), 1 mM MgCl2, 0.2 mM MESG, 10 units of purine-nucleotide phosphorylase, 200 µM GTP, and the indicated amount of GTP-preloaded
recombinant GTPase was mixed in a 4-mm width, 10-mm path length
cuvette, and the time courses of the absorbance change at 360 nm were
recorded. The concentration of Pi in the solution released
from the G-protein-bound GTP is proportional to the net absorbance
change by a factor of the extinction coefficient
360 nm = 11,000 M
1
cm
1 at pH 7.6 (26). The kinetic data analysis
was carried out as described previously (23).
Immunoprecipitation and Kinase Activity Assay--
COS-7 cells
transfected with the Myc-Rac1, HA-Rac1, HA-Cdc42, HA-RhoA, or Myc-PAK1
construct alone or in combination were harvested 48 h
post-transfection. Cells were washed with ice-cold phosphate-buffered
saline; lysed in 20 mM Tris-HCl (pH 7.4), 0.5% Triton-100,
100 mM NaCl, 1 mM DTT, 1 mM
MgCl2, 10 µg/ml leupeptin, and 10 µg/ml aprotinin; and
centrifuged at 14,000 × g for 10 min at 4 °C.
Protein expression was confirmed by Western blotting of the cell
lysates. For the immunoprecipitation assay, HA-tagged Rac1 was
precipitated with anti-HA monoclonal antibody immobilized on protein
A-Sepharose (Roche Molecular Biochemicals) from the lysates after a 2-h
incubation under constant agitation. The immunoprecipitates were washed
three times with the lysis buffer and subjected to anti-Myc or anti-HA
Western blotting. To carry out the PAK1 kinase assay (14), the
anti-Myc-PAK1 immunoprecipitates were incubated in a 60-µl reaction
mixture containing 50 mM HEPES (pH 7.5), 2 mM
MgCl2, 2 mM MnCl2, 0.2 mM DTT, and 20 µM [
-32P]ATP
in the absence or presence of various Rac1 mutants preloaded with
GTP
S for 20 min at 30 °C. The reaction was terminated by the
addition of an equal volume of 2× Laemmli buffer and subjected to gel
electrophoresis and autoradiography/phosphorimaging analysis.
RhoGDI Competition Assay--
To examine the effect of RhoGDI on
the homophilic interaction of Rac1, the isoprenylated forms of
His6-Rac1 and HA-Rac1 were generated from the membrane
fraction of Sf9 insect cells and COS-7 cells, respectively, by
extraction with 0.5% CHAPS. Immobilized His6-Rac1 was
incubated with HA-Rac1 in a buffer containing 20 mM
Tris-HCl (pH 7.5), 1 mM MgCl2, 50 mM NaCl, 0.02% CHAPS, and various doses of GST-RhoGDI.
After three washes with the incubation buffer, the
His6-Rac1 coprecipitates were then subjected to anti-HA Western blot analysis.
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RESULTS |
Rac1 Exists in Reversible Monomeric and Oligomeric Forms--
We
have previously described the dimerization properties of a few Rho
family GTPase members, including RhoA, Rac2, and Cdc42 (16, 17). These
Rho proteins contain two or more positively charged basic residues in
the C-terminal region that were found to be essential for dimer
formation (17). As a distinct member of the Rho family, Rac1 contains
six consecutive basic residues (183KKRKRK188)
in this region. Initially, we were interested to test whether Rac1
would conform to the similar dimer-forming property of Rac2 and Cdc42.
The gel filtration profile of purified Rac1 shows that Rac1 was eluted
in two peak fractions that correspond to estimated molecular masses of
~25 and ~600 kDa, respectively (Fig.
1A), suggesting that Rac1
could exist in a mixture consisting of a monomer and higher order
oligomer under the physiologically relevant buffer conditions. Both the
GDP- and GTP
S-bound Rac1 proteins yielded similar gel filtration
profiles, indicating that the nucleotide-binding state of Rac1 does not
affect the monomer-oligomer distribution. When the gel filtration
pattern of Rac1 was compared with that of other small GTPases such as
Ras and Cdc42, it became clear that the oligomerization property is
unique to Rac1 since both Ras and Cdc42 (Fig. 1A), as well
as the Rho GTPases including RhoA, RhoB, Rac2, and TC10 (data not
shown), were detected exclusively in the monomeric form or in a mixture
of monomer and dimer. To rule out the trivial explanation that oligomer
formation was due to irreversible aggregation of Rac1, we performed
further gel filtration analysis at varying salt and/or Mg2+
concentrations. Increasing the NaCl concentration from 50 to 300 mM resulted in a gradual shift of the overall population of Rac1 from oligomer to monomer, whereas the divalent Mg2+
ion also appeared to have a dramatic effect on the monomer-oligomer distribution such that >70% Rac1 oligomer was disassembled into monomer when the Mg2+ concentration was raised from 0.2 to
30 mM as exemplified in two such conditions in Fig.
1A. Moreover, the monomer-to-oligomer transition was
dependent on the initial Rac1 concentrations (submicromolar) (data not
shown). These results indicate that the oligomer and monomer of Rac1
exist in a reversible equilibrium.

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Fig. 1.
Rac1 forms reversible oligomers.
A, gel filtration profiles of Ras, Cdc42, and Rac1. A
200-µl sample of the respective GTPases in the GTP S- or GDP-bound
state at an initial concentration of 5 µM was applied to
a Superdex 200HR column as described under "Experimental
Procedures." The standard elution buffer contained 50 mM
HEPES (pH 7.6), 1 mM DTT, 100 mM NaCl, and 2 mM MgCl2. Two additional elution conditions are
shown for Rac1 in which the salt and Mg2+ concentrations
were varied. Arrows indicate the elution positions of 25, 67, and 669 kDa for the gel filtration standards. B, complex
formation between GST-Rac1 and His6-Rac1. 5 µg of
immobilized GST-Rac1 was preloaded with GTP S before incubation with
the purified His6-fused GTPases (5 µM) in the
GTP S-bound state for 30 min at 4 °C in buffer containing 50 mM HEPES (pH 7.6), 1 mM DTT, and the NaCl or
Mg2+ concentrations indicated. After extensive washes,
GST-Rac1 and the associated His6-tagged proteins were
visualized by electrophoresis, followed by Coomassie Blue staining.
C, Rac1 is capable of forming a stable homo-oligomer in
cells. HA-Rac1, HA-Cdc42, HA-RhoA, or the HA-Rac1 8 mutant was
transiently coexpressed with Myc-Rac1 or Myc-Rac1 8 in COS-7 cells.
The cell lysates were immunoprecipitated (IP) with an
immobilized anti-HA antibody and subjected to Western analysis. The
blots were probed using anti-Myc antibody to visualize Myc-Rac1 or
Myc-Rac1 8 that coprecipitated with the respective HA-tagged GTPases.
Additionally, cell lysates were blotted with anti-HA or anti-Myc
antibody to assess the expression levels of the GTPases. The results
shown are representative of three independent experiments.
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To further establish that Rac1 is capable of oligomerization, a complex
formation assay was performed using immobilized GST-Rac1-GTP
S in an
incubation with purified His6-tagged Rac1-GTP
S,
Cdc42-GTP
S, or RhoA-GTP
S. SDS-polyacrylamide gel electrophoresis
analysis of GST-Rac1 coprecipitates revealed that whereas GST-Rac1
bound to Cdc42 or RhoA at a barely detectable level, it was capable of
a tight interaction with His6-Rac1 (Fig. 1B).
The binding affinity between GST-Rac1 and His6-Rac1 was
sensitive to the buffer ionic strength since the amount of
His6-Rac1 coprecipitates was markedly reduced at increasing
concentrations of NaCl or MgCl2. These results confirm that
Rac1 is capable of forming an oligomer and further suggest that
oligomerization is mostly homophilic in nature.
To determine whether Rac1 oligomerization could occur in a cellular
background, we coexpressed two N-terminal tagged forms of Rac1, HA-Rac1
and Myc-Rac1, in COS-7 cells. As a comparison, HA-Cdc42 or HA-RhoA was
also coexpressed with Myc-Rac1 in these cells. Cellular HA-Rac1,
HA-Cdc42, or HA-RhoA was immunoprecipitated from the cell lysates using
an anti-HA monoclonal antibody immobilized on protein A-Sepharose
beads, and the presence of associated Myc-Rac1 was detected by anti-Myc
Western immunoblotting. As shown in Fig. 1C, the HA-Rac1
immunoprecipitates readily associated with Myc-Rac1 under the condition
that no nonspecific binding of Myc-Rac1 to the anti-HA antibody complex
was visible. In contrast, HA-RhoA did not appear to associate with
Myc-Rac1 under similar conditions, whereas HA-Cdc42 displayed weaker
binding to Myc-Rac1 (Fig. 1C). Thus, cellular Rac1 may form
a stable homo-oligomer, raising the possibility that this feature of
Rac1 may be involved in its regulation.
The C-terminal Polybasic Domain of Rac1 Is Involved in
Oligomerization--
The polybasic motif located immediately upstream
of the isoprenylation CAAX box at the C terminus appears to
be conserved among the Rho family GTPases (Fig.
2A). The recently available three-dimensional structures of RhoA, Rac1, and Cdc42, however, do not
provide clues as to how this region is folded with regard to the
overall structural backbone because truncation of this region was
necessary for the crystallization conditions or NMR data collection
process (28-30). Our previous sequence analysis revealed that the
presence of two or more Lys or Arg residues in this region correlates
with the homophilic interaction of the Rho family members RhoA and
Cdc42 (16). To examine whether this region is involved in Rac1
oligomerization, two different experimental approaches were taken.
First, a pair of polypeptides corresponding to the C-terminal region of
Rac1 and Cdc42 (amino acids 180-191), respectively, were synthesized
and used in a competition assay. Incubation of the Rac1-derived
polypeptide (PVKKRKRKCLLL) with Rac1 caused a
concentration-dependent disassembly of the Rac1 oligomer, and
at 300 µM, the oligomer was completely shifted to monomer
(Fig. 2B). In contrast, a peptide corresponding to the C-terminal residues of Cdc42 (PEPKKSSRRCVLL) displayed only a marginal
effect on the oligomer-monomer distribution of Rac1 at the
similar concentrations (data not shown), suggesting that the Rac1
polypeptide effect is specific for the Rac1 homophilic interaction. Second, a set of C-terminal mutants of Rac1 were constructed and tested
for their effects on oligomerization (Fig. 2A). The
Rac1/Cdc42 chimera (Rac1-Cdc42 mutant), which contains the Rac1
backbone with the C-terminal eight residues substituted with those of
Cdc42, suffered a partial loss in the ability to form an oligomer,
whereas the removal of the last eight residues from Rac1 (Rac1
8) or
the substitution of the C-terminal five basic residues of Rac1 with Gln
(Rac1-5Q) completely abolished oligomer formation (Fig. 2C). The combined results from these two independent approaches indicate that the C-terminal polybasic domain of Rac1 directly participates in
oligomer formation.

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Fig. 2.
The C-terminal polybasic domain mediates the
oligomerization of Rac1. A, amino acid sequence
alignments of the C-terminal polybasic domains of human N-Ras, Cdc42,
Rac1, and the Rac1 mutants. The Rac1-Cdc42 mutant contains amino acids
183-191 of Cdc42 substituted for the corresponding residues of Rac1.
The Rac1 8 mutant represents a truncated form of Rac1 with deletion of
the last eight residues (positions 184-191) from the C terminus. The
Rac1-5Q mutant contains five neutral Gln residues substituted for basic
amino acids 184-188 of Rac1. The basic residues lysine and arginine
are underlined. B, a C-terminal polypeptide
derived from Rac1 corresponding to residues 181-191 induces
disassembly of the Rac1 oligomer in a dose-dependent
manner. The gel filtration conditions were similar to those described
in the legend to Fig. 1A. C, the Rac1 mutants
(Rac1-Cdc42, Rac1-5Q, and Rac1 8) show gel filtration profiles
distinct from that of wild-type Rac1. Standard elution conditions as
described in the legend to Fig. 1A were applied.
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The requirement of the C-terminal domain for Rac1 oligomerization was
also tested in vivo. Although Myc-tagged Rac1 was readily co-immunoprecipitated with HA-Rac1 from the lysates of COS-7 cells coexpressing the two Rac1 species, Myc-Rac1
8 failed to associate with
HA-Rac1
8 (Fig. 1C). Moreover, Myc-Rac1 was found to remain capable of forming a stable complex with HA-Rac1
8 in the
co-immunoprecipitation assay (Fig. 1C), suggesting that the
oligomerization is likely to adopt an asymmetric configuration. These
results confirm that the C-terminal domain of Rac1 is intimately
involved in the oligomerization.
Oligomerization of Rac1-GTP Elicits a Self-stimulatory
GTPase-activating Protein Activity--
To examine the biochemical
properties of different forms of Rac1, the eluent fractions
corresponding to the oligomer and monomer from the Superdex 200HR gel
filtration column were isolated. We first compared the intrinsic GTP
hydrolysis activities of the monomeric and oligomeric forms of Rac1. In
a filter binding assay, each of the separated species at 2 µM was loaded with [
-32P]GTP in a buffer
containing 20 mM HEPES (pH 7.6), 100 mM NaCl, and 1 mM EDTA. Single turnover GTP hydrolysis was initiated
by the addition of 10 mM MgCl2. As shown in
Fig. 3A, the Rac1 oligomer was
active in binding and hydrolyzing GTP and exhibited a significantly higher intrinsic GTP hydrolysis activity compared with the monomer. The
apparent reaction rates were determined to be 0.16 and 0.02 min
1 for the oligomer and monomer pools,
respectively. Similar rate constants were obtained by assaying the
respective GTPase activities using the MESG/phosphorylase coupling
reaction to continuously monitor Pi release from bound GTP
(data not shown). The Rac1 oligomer therefore contains ~8-fold higher
intrinsic GTPase activity than the monomer.

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Fig. 3.
The Rac1 oligomer displays significantly
higher intrinsic GTP hydrolysis activity compared with the
monomer. A, time courses of [ -32P]GTP
hydrolysis are compared between the isolated Rac1 oligomer and monomer
at ~2 µM at 20 °C. The Rac1 monomer and oligomer
species were collected from the corresponding gel filtration fractions
as shown in Fig. 1A. The GTPase reactions were performed in
buffer containing 50 mM HEPES (pH 7.6), 1 mM
DTT, 50 mM NaCl, and 1 mM MgCl2.
The kinetic data fit best into a single exponential equation to yield
intrinsic rate constants. B, the C-terminal mutants of Rac1
(20 µM) display distinct GTPase reaction rates as
determined by the MESG/phosphorylase assay. The concentration of
released -Pi in the reaction solution was calculated by
a factor of the extinction coefficient 360 nm = 11,000 M 1 cm 1
from the absorbance change of the phosphorylase coupling reactions
(26). C, dose-dependent GTP hydrolysis time
courses of Rac1 measured by the MESG/phosphorylase assay. D,
concentration dependence of the intrinsic GTPase rate of Rac1 compared
with that of the Rac1 8 mutant. The apparent GTP hydrolysis rates of
Rac1 at various concentrations were derived by the best fit of the
kinetic data from C.
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Next we compared the intrinsic GTPase activities of the C-terminal
mutants of Rac1 with that of Rac1 (Fig. 3B). Both Rac1
8 and Rac1-5Q, which exist only in the monomeric form, behaved similarly to the Rac1 monomer in a MESG/phosphorylase coupled GTPase assay, showing a slow intrinsic GTPase reaction rate of 0.021 and 0.028 min
1, respectively. These rates are similar
to those determined previously for monomeric Ras and RhoB (17). The
Rac1-Cdc42 mutant, which retained some of the Rac1 oligomerization
ability, on the other hand, partially maintained the higher GTP
hydrolysis rate at 0.071 min
1 (Fig.
3B). When the GTPase reaction rates were measured at
increasing GTPase concentrations, the rates for Rac1 were found to
increase significantly with increasing concentrations of Rac1-GTP,
whereas the intrinsic GTP hydrolysis activity of Rac1
8 appeared to be dose-independent over the range of 1-40 µM (Fig. 3,
C and D). Thus, the abilities of Rac1 mutants to
hydrolyze GTP correlate well with their observed differences in
oligomerization states. The dose-dependent intrinsic GTPase
activity of Rac1 and the dose-independent GTPase activity of Rac1
8
further suggest that the C terminus-mediated oligomerization plays a
regulatory role in controlling the basal GTPase state of Rac1.
As demonstrated above, the oligomerization state of Rac1 is sensitive
to the ionic strength of the buffer conditions. The distribution of
Rac1 isoforms at different concentrations of NaCl was revealed by the
gel filtration profiles, with the percentage of oligomer steadily
decreasing at increasing NaCl concentrations (Fig.
4A). In parallel, the GTP
hydrolysis rate of Rac1 was found to decrease accordingly with
increasing NaCl concentrations (Fig. 4B). These results
further suggest that the oligomerization state of Rac1 could account
for the enhanced ability to hydrolyze GTP.

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Fig. 4.
The intrinsic GTPase activity of Rac1
correlates with its oligomerization state in a salt
concentration-dependent manner. A, the
distribution of the Rac1 oligomer and monomer at various NaCl
concentrations. Aside from the specified NaCl concentrations, the gel
filtration conditions were similar to those described in the legend to
Fig. 1A. The ratios of oligomer versus monomer
were determined by measuring the areas of the corresponding elution
peaks of the respective gel filtration profiles. B, salt
concentration dependence of the intrinsic rates of GTP hydrolysis by
Rac1. The GTPase reactions were measured by the MESG/phosphorylase
coupling reactions and by the [ -32P]GTP filter binding
assays. The apparent rate constants were derived by the best fit of the
time courses of Rac1-GTP hydrolysis into a single exponential
equation.
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To test directly the intrinsic GTPase-activating potential of Rac1, the
GDP-bound or GTP
S-bound Rac1 and Rac1 mutants were titrated into
Rac1-GTP, and the GTPase reactions were monitored by measuring
-Pi release from Rac1-GTP. The addition of Rac1-GDP did
not cause any change in Pi release, whereas the addition of Rac1-GTP
S resulted in a significant enhancement of
-Pi release, similar to that seen upon the addition of
p50RhoGAP under these conditions (Fig.
5). The GTP
S-bound Rac1-Cdc42 mutant produced a partial enhancement compared with the effect brought about
by Rac1-GTP
S, and the Rac1
8 and Rac1-5Q mutants had no detectable
effect (Fig. 5). These results indicate that the activated form of Rac1
presents a GAP activity for Rac1-GTP, and this self-stimulatory GAP
activity is dependent upon the homophilic binding ability of Rac1. The
apparent GTPase-activating effect of Rac1-GTP
S was not affected by
isoprenylation since the post-translationally modified form of Rac1
purified from Sf9 insect cells (Fig. 5, Rac1*)
behaved similarly to E. coli-produced Rac1. The observed oligomerization-elicited self-stimulatory GAP activity of Rac1 provides
a rational for the dose-dependent fast rate of intrinsic GTPase activity, and the requirement of the C-terminal domain for this
activity can be attributed to the presence of Arg186 in the
region, which we have previously shown to constitute a "built-in
arginine figure" in forming a transition state of the GTPase-activating reaction of other Rho family GTPases such as Cdc42
and Rac2 (17).

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Fig. 5.
Rac1 possesses a self-stimulatory GAP
activity mediated by the C-terminal domain. -Pi
release by 5 µM Rac1-GTP were determined at the 5-min
time point in the absence or presence of GDP- or GTP S-loaded Rac1 (5 µM), various Rac1 mutants (5 µM), or
p50RhoGAP (50 nM) in buffer containing 50 mM HEPES (pH 7.6), 1 mM DTT, 100 mM
NaCl, and 2 mM MgCl2. Rac1*
indicates the isoprenylated form of Rac1.
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The Rac1 Oligomer Is Insensitive to RhoGAP
Stimulation--
p50RhoGAP has been shown to potently
catalyze GTP hydrolysis by Rho family GTPases including Rac1 (23, 27).
To characterize the interaction of the Rac1 oligomer and monomer with
RhoGAP, we measured the rate of
-Pi release from
Rac1-GTP under low salt conditions, which favor the formation of the
Rac1 oligomer, and from Rac1
8, which maintains in the monomeric state
under these conditions, in the absence or presence of
p50RhoGAP. Although p50RhoGAP effectively
stimulated the rate of GTP hydrolysis of Rac1
8 by >10-fold, it was
unable to significantly increase the GTPase activity of Rac1, which
displayed a higher intrinsic GTPase rate due to the self-stimulatory
GAP activity (Fig. 6A),
indicating that the oligomeric form of Rac1 is insensitive to
p50RhoGAP. The apparent preference of RhoGAP for
monomeric Rac1 was further examined quantitatively by measuring the
initial rate of
-Pi release from the GTP-bound GTPases
as a function of the GTPase concentrations in the presence of a
catalytic amount of RhoGAP (Fig. 6B). Fitting of the data by
a modified Michaelis-Menten equation (23) yielded the kinetic
parameters Km and kcat (Table
I). The catalytic efficiencies
(kcat/Km) of
p50RhoGAP for Rac1
8 were determined to be ~8-fold
higher than for Rac1 at 21.4 ± 2.7 min
1
µM
1 compared with 2.6 ± 0.4 min
1
µM
1, respectively, and the
difference in Km seems to be a major factor for the
varied ability of RhoGAP to elicit the GAP activity for Rac1. We
suspect that the detected residue RhoGAP activity for Rac1 under the
assay conditions was due to a partial dissociation of oligomer to
monomer. Interestingly, although poorly responsive to the RhoGAP
stimulation, Rac1, under similar low salt conditions, or the isolated
Rac1 oligomer from the gel filtration column remained fully reactive
with a Rac-specific GEF, TrioN (data not shown). These results suggest
that the Rac1 oligomer may rely primarily on the self-stimulatory GAP
activity rather than the interaction with a RhoGAP to return to the
GDP-bound basal state and indicate that oligomeric Rac1-GDP can be
subject to reactivation upon GEF catalysis.

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Fig. 6.
The Rac1 oligomer is insensitive to RhoGAP
stimulation. A, time courses of GTPase reactions of
Rac1 and Rac1 8 in the absence or presence of p50RhoGAP
(100 nM) under low salt conditions. The reaction buffer
contained 50 mM HEPES (pH 7.6), 1 mM DTT, 50 mM NaCl, 1 mM MgCl2, 0.2 mM MESG, 200 µM GTP, and 10 units of
purine-nucleotide phosphorylase. B, reaction rates of GTP
hydrolysis by Rac1 or Rac1 8 upon RhoGAP catalysis under low salt
conditions determined as a function of the concentration of the
GTPases. The RhoGAP-catalyzed GTPase reactions were analyzed by the
modified Michaelis-Menten equation (23) to yield the
Km and kcat values.
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Table I
Kinetic parameters of p50RhoGAP that regulate Rac1 and Rac1 8
The p50RhoGAP-catalyzed GTPase reactions of Rac1 and Rac1 8 as
analyzed in Fig. 6 by nonlinear regression yielded the
Km and kcat values listed. GAP
reactions were carried out at 20 °C in 50 mM HEPES (pH
7.6), 50 mM NaCl, 1 mM DTT, 0.1 mM
EDTA, 1 mM MgCl2, 0.2 mM MESG, 200 µM GTP, and 10 units of purine-nucleotide phosphorylase.
The results are representative of three independent measurements.
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Oligomerization of Rac1 Potentiates the Effector PAK1
Activation--
Within a broad range of biochemical contexts,
autophosphorylation stimulated by Rac1 or Cdc42 has been consistently
reported as a common trait of PAK activation (10). To assess the effect of Rac1 oligomerization on effector interaction, we have examined the
ability of Rac1 and its mutants to activate PAK1 by probing the
autophosphorylation state of PAK1 under the low salt kinase reaction
conditions. As shown in Fig. 7, optimal
stimulation of PAK1 autophosphorylation was observed when wild-type
Rac1 loaded with GTP
S was used as a stimulant, leading to a high
level of 32P incorporation into PAK1. Mutations of the
polybasic residues of Rac1 by truncation (Rac1
8) or substitution with
Gln (Rac1-5Q) greatly reduced (~3-fold) the kinase-activating
capability, and the replacement of the C-terminal domain of Rac1 with
the corresponding domain of Cdc42 (Rac1-Cdc42) also resulted in an
~1.5-fold decrease in PAK1 autophosphorylation. Thus, the
oligomerization states of Rac1 and its mutants closely correlated with
their abilities to activate PAK1. Although similar observations of PAK1
activation using various Rac1 C-terminal mutants and Rac1/Rac2 chimeras
were reported before (14), in light of our data on the oligomerization states of Rac1 and its mutants under the low salt conditions that were
employed in the PAK1 kinase assays, we interpret these results as more
likely an indication of a synergistic effect of Rac1 oligomerization on
the effector activation rather than that the C-terminal region of Rac1
constitutes an additional PAK1 effector-binding site as proposed
previously (14).

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Fig. 7.
The C-terminal domain of Rac1 is required for
full activation of PAK1. Myc-PAK1 was expressed in COS-7 cells and
immunoprecipitated from the cell lysates using immobilized anti-Myc
antibody. The immunoprecipitates were incubated with similar amounts of
wild-type or mutant Rac1 proteins (~5 µM) preloaded
with GTP S in kinase reaction buffer containing 50 mM
HEPES (pH .5), 2 mM MgCl2, 2 mM
MnCl2, 0.2 mM DTT, and 20 µM
[ -32P]ATP for 20 min. Upper panel, PAK1
autophosphorylation was visualized by autoradiography after
SDS-polyacrylamide gel electrophoresis. Lower panel, the
bar graph indicates the relative kinase activity of PAK1
stimulated by wild-type Rac1 and its mutants. The results shown are
representative of three separate experiments.
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RhoGDI Induces Disassembly of the Rac1-GDP Oligomer--
Aside
from RhoGAP and GEF, RhoGDI represents another major class of
regulators for Rac1 function. One of the major roles of RhoGDI is
thought to be the regulation of the distribution of Rho GTPases between
the membrane compartments and cytosol, where at the latter location
each Rho family member is in complex with RhoGDI at a 1:1 stoichiometry
(8, 9). It was therefore of interest to determine whether RhoGDI
contributes to the regulation of the oligomerization state of Rac1. To
this end, we took an Ni2+-agarose affinity-based approach
to test directly the effect of RhoGDI on the complex formation between
immobilized His6-Rac1 purified from the Sf9 insect
cell membranes and HA-tagged Rac1 isolated from the membrane fraction
of COS-7 cells transiently transfected with the HA-Rac1 construct. Both
of these Rac1 species were therefore in isoprenylated form and were
capable of interacting with RhoGDI. In agreement with the above
co-immunoprecipitation results (Fig. 1C),
His6-Rac1 formed a stable complex with HA-Rac1 (Fig.
8, lane 2). The addition of
increasing amounts of GST-RhoGDI to the incubation mixtures resulted in
a dose-dependent inhibition of the complex formation
between His6-Rac1 and HA-Rac1 (Fig. 8, lanes
3-5) such that at 10 µM GST-RhoGDI, the homophilic
interaction between the two populations of Rac1-GDP became
undetectable. In parallel, the sample containing 10 µM
GST instead of GST-RhoGDI did not affect the binding interaction
between the Rac1 species (Fig. 8, lane 6). Thus, RhoGDI may
be involved in the disassembly process of the Rac1 oligomer and
actively participate in the oligomer-to-monomer transition as well as
the membrane-to-cytosol translocation of Rac1.

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Fig. 8.
RhoGDI induces the disassembly of Rac1
oligomers. Immobilized His6-Rac1-GDP (~3.0 µg) was
incubated with the lysates of COS-7 cells expressing HA-Rac1 in the
absence (lane 2) or presence of increasing amounts of
GST-RhoGDI (2, 4, and 10 µM (lanes 3-5,
respectively)) or 10 µM GST (lane 6). The
His6-Rac1-GDP coprecipitates were subjected to anti-HA
Western blot analysis after extensive washes.
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DISCUSSION |
In this study, we have characterized a unique biochemical property
of the Rac1 GTPase, self-assembly into large oligomers. We found that
Rac1 exists reversibly in a monomeric state and an oligomeric state,
with a dissociation constant in the submicromolar range. Although the
Rac1 responsiveness to RhoGAP is diminished in the oligomeric state,
the Rac1 oligomer remains fully functional in the interaction with its
GEF, TrioN. The oligomerization process brings on the self-stimulatory
GAP activity, which is mostly responsible for Rac1 down-regulation, and
appears to optimize the effector (PAK1)-activating potential.
Interaction with RhoGDI results in the disassembly of the Rac1 oligomer
and cycles Rac1 to the cytosol in the RhoGDI·Rac1 complex. These
results suggest a novel mode of regulation for Rac1 function.
Oligomerization of Rac1 and the Structural Determinants
Involved--
Our gel filtration and complex formation results clearly
show that Rac1 may exist in an oligomeric form under physiologically relevant buffer conditions. The immunoprecipitation experiments in
cells coexpressing two distinct populations of Rac1 demonstrate that the oligomerization occurs in vivo (Fig. 1). The
observation that the Rac1 monomer and oligomer are in a reversible
equilibrium further suggests that the oligomeric form of Rac1 may bear
physiological significance.
There is a growing body of evidence suggesting that certain GTP-binding
proteins may undergo an oligomeric state in carrying out their cellular
functions. For example, it has been recognized that many members of the
large GTPase family including Mx, dynamin, and yeast Vps1p exist
natively in large oligomers and aggregates (20). Among the members of
the Ras small GTPase superfamily, ADP-ribosylation factor-1 has been
found in functional dimeric or tetrameric forms (18, 20). Ras itself
has recently been suggested to form a dimer at the plasma membrane that
appears to be essential for Raf-1 activation (19). Our previous studies have demonstrated that the Rho GTPases Cdc42, RhoA, and Rac2 are capable of forming homodimers, and the dimerization may play a role in
the negative regulation of the GTPases (16, 17). Interestingly, Rac2
homodimer formation was recently detected in the neutrophils of a
patient with Kostmann syndrome, suggesting that the homophilic interaction of the GTPase may be involved in the functional regulation (7).
Most members of the Rho protein family contain a stretch of two to six
polylysine and/or arginine residues immediately N-terminal of the
isoprenylation motif, the CAAX box. This polybasic domain has been implicated in the dimer formation of Cdc42 and RhoA (16, 17).
Here we present evidence that the corresponding region of Rac1
consisting of six consecutive basic residues is essential for its
oligomerization. This was first supported by a peptide competition
assay in which the polypeptide corresponding to the C-terminal
polybasic domain of Rac1 was able to effective disassemble the Rac1
oligomer to the monomer (Fig. 2B). Mutational analysis of
the polybasic residues revealed that removal of or substitutions in the
polybasic domain led to the exclusive monomeric form of Rac1 or a
gradual shift from the oligomer to monomer, providing the secondary
support for the involvement of this region in the oligomerization.
Combined with the previous observations, it appears that the Rho
proteins containing multiple basic residues (Lys or Arg) in the
C-terminal domain, including Cdc42, RhoA, Rac2, RhoC, and Rac1, may
form either a dimer or higher oligomer and that other Rho proteins
lacking the consecutive basic residues in this region, including TC10
and RhoB, are exclusively monomeric. The homophilic binding
interaction among these proteins seems to correlate with the number of
charged residues in the C-terminal domain. In the absence of available
information on the tertiary structure of this domain, we speculate that
the positively charged nature of the polybasic domain could provide the
contact sites that stabilize the dimer or oligomer conformation by
interacting with a negatively charged region of the neighboring
molecules. Given that the homophilic interactions are guanine
nucleotide state-specific, it is possible that the binding sites of the
polybasic motif of one GTPase would include the switch regions of the
adjacent molecules, joining together two or more molecules in a linear, asymmetric configuration. Such a model is consistent with the self-stimulatory GAP activity associated with the oligomerization process of Rac1 and is in line with our previously proposed model of
Cdc42 dimer configuration in which Arg186 of the C-terminal
domain functions effectively as an "arginine finger" to stabilize
the transition state of the GTP hydrolytic core of the immediately
adjacent molecule (17). Such an oligomer configuration would be
distinct from the proposed oligomer structure of the high molecular
mass G-protein Mx, which depends on the homolytic interaction
through several regions of the molecule that include a conserved
self-assembly motif in the amino-terminal moiety and two amphipathic
helices at the C-terminal end (20, 21, 31) and would be different from
the dimer configuration of Ras, in which case a lipid moiety was
expected to be involved in the dimerization (19). Further studies by
mutagenesis and by the structural biology approach are needed to
resolve the self-associated state of Rac1 and other Rho GTPases.
Self-stimulatory GAP Activity of Rac1-GTP Elicited by
Oligomerization--
Although sharing a high degree of sequence
homology in the GTP-binding core and switch regions, Rho family
proteins behave differently in their ability to hydrolyze GTP. Rac1
appears to display the highest intrinsic GTPase activity among the Rho
GTPases examined, including RhoA, Rac2, and Cdc42; and its
GTP-hydrolyzing potential is dependent upon its concentrations.
However, in the monomeric state, Rac1 behaves like Ras and RhoA, with a
slow intrinsic GTPase reaction rate at 0.02 min
1, whereas the isolated Rac1 oligomer
shows an 8-fold higher basal activity than the monomer. The C-terminal
truncation or substitution mutants of Rac1, known to form the monomer
only (Fig. 2C), displayed a turnover number for GTP
hydrolysis that is indistinguishable from that of the Rac1 monomer.
These results indicate that the oligomeric state of Rac1 correlates
with the GTPase activity and suggest that the enhanced GTP-hydrolyzing
ability may be attributed to the self-association ability of Rac1.
Indeed, direct demonstration of GTPase-activating activity was provided
by the Rac1-GTP
S-elicited GTPase-activating potential, which
resulted in a significant stimulation of
-Pi release
from Rac1-GTP, similar to the effect of RhoGAP, indicating that the
active form of Rac1 presents a specific GAP activity for its own form.
The self-stimulatory GAP activity is apparently dependent on the
oligomerization ability of Rac1 since the mutant forms of Rac1 that are
no longer capable of oligomerization or that have a weaker tendency to
oligomerize suffered complete or partial loss of the self-stimulatory
GAP activity. Furthermore, the isoprenylation modification of the
CAAX sequences does not interfere with either the oligomer
formation or the GTPase-activating activity of Rac1 because a similar
extent of stimulation of Rac1 GTPase activity was observed when the
post-translationally modified form of Rac1 was examined (Fig. 5). Thus,
similar to Cdc42 and Rac2, Rac1 may utilize the
oligomerization-associated self-stimulatory GAP activity for its
negative regulation.
A conserved, surface-exposed arginine residue in RhoGAPs, termed the
arginine finger, has been implicated in the GAP-catalyzed GTPase
reaction of Rho GTPases (32, 33). The arginine residue from GAP protein
appears to contribute to the stabilization of the transition state of
the GTPase reaction and therefore is directly involved in catalyzing
the cleavage of
-Pi from bound GTP. We have previously
observed that an arginine residue in the polybasic domain of Cdc42,
Arg186, functions as a built-in arginine finger to elicit
the self-stimulatory GAP activity. This arginine residue is also
present at the corresponding position of Rac1 protein. We expect that
an arginine residue of the polybasic domain, either Arg184
or Arg186, may undertake the task, similar to
Arg186 in Cdc42, to participate in the GTPase reaction of
an adjacent Rac1 molecule and to stimulate its GTPase activity. The
fact that the Rac1-GTP oligomer is insensitive to RhoGAP stimulation is also consistent with the possibility that the critical arginine residue
of the attacking Rac1-GTP would compete with the arginine finger
provided by RhoGAP for access to the Rac1-GTP substrate and further
supports that the Rac1-GTP oligomer employs mostly the self-stimulatory
GAP activity, rather than depending on a RhoGAP, to arrive at the
basal, GDP-bound state efficiently. Interestingly, such a
self-stimulatory GTPase-activating activity has been a common feature
found in the large GTPase (67 kDa) family including Mx, dynamin, and
yeast Vps1p, the endogenous GTPase activities of which were
significantly enhanced upon oligomerization (34, 35).
Oligomerization of Rac1 Contributes to the Effector PAK1
Activation--
The PAK1 serine/threonine kinase represents one of the
best characterized Rac1 effectors (10, 36). Rac1 binding to the p21-binding domain of PAK1 results in the release of the
auto-inhibition of the kinase domain, leading to enhanced
autophosphorylation and subsequent activation of PAK1. Previous
structure-function studies of Rac1 have mapped the regions containing
residues 26-45 and 143-175 as two primary PAK1-interactive sites
(37). Peptide competition studies have also implicated the C-terminal
domain of Rac1 as a potential site for NADPH oxidase activation (38, 39). A recent structure complex between Cdc42 and the polybasic domain
of PAK1 depicts multiple contact sites involving the switch I, switch
II,
1, and
5 regions of Cdc42, but a direct involvement of the
C-terminal domain could not be observed since this region of Cdc42 was
deleted in the Cdc42·PAK1 complex (40). Using a Rac1 mutation and the
Rac1/Rac2 chimera approach, Knaus et al. (14) found that the
C-terminal polybasic domain of Rac1 is also important for PAK1
activation. The difference at the C termini of Rac1 and Rac2, in
particular, could account for the up to 5-fold variation in
PAK1-activating ability (14). Here we have made a similar observation
that the polybasic domain is required for the full potential of Rac1 to
activate PAK1. Optimal activation of PAK1 was observed with the
full-length Rac1 stimulation, whereas the mutations or truncation of
the C-terminal polybasic residues led to a decreased activity of Rac1
in PAK1 activation (Fig. 7). Under the PAK1 kinase assay conditions
(low salt), we expect that Rac1 mostly exists in the oligomeric form.
Therefore, we interpret the effect of various Rac1 C-terminal mutations
on PAK1 activity to be in good correlation with their ability to form
an oligomer, and the oligomeric state of Rac1 is optimal for PAK1
activation. The recently available PAK1 tertiary structure reveals that
a homodimer conformation of the Rac1/Cdc42-interactive binding domain is involved in maintaining PAK1 in the auto-inhibited state (41). Our
results suggest that the oligomeric form of Rac1 may have an advantage
in breaking open the PAK1 dimer to relieve the auto-inhibition and
raise the possibility that the self-assembled Rac1-GTP oligomer may be
an addition to the Rac1-GTP monomer in activation of a selective subset
of Rac1 downstream effectors.
Recent studies by del Pozo et al. (42) showed that
expression of a constitutively active mutant of Rac1 that lacks a
membrane-targeting sequence fails to activate PAK1 in adherent cells,
suggesting that membrane co-localization of Rac1 and PAK1 is essential
for PAK1 activation. Upon serum stimulation, Rac1 was found to be enriched in membrane microdomains made of rafts and caveolae (43). This
process could lead to an increase in the local concentration of Rac1
(44). Accumulation of active Rac1 on the membrane would then initiate a
chain reaction among Rac1 molecules that results in the formation of
oligomers, which in turn results in an enhanced binding of Rac1 to the
plasma membrane. Our biochemical results described here support the
possibility that self-assembly of Rac1 into an oligomer is an important
event in the plasma membrane for the increased coupling of activated
Rac1 to PAK1. Alternatively, the oligomerization of Rac1 may provide
multiple effector interaction sites that could function cooperatively
in effector activation. It remains a challenge to demonstrate that Rac1
oligomerization occurs at a specific plasma membrane site and that the
Rac1 oligomer plays a role in the effector recruitment and/or
activation in vivo. The recently described in
vivo fluorescence energy transfer method (45) that allows
monitoring of small G-protein interactions in live cells may prove
useful to address such issues.
RhoGDI Drives the Oligomer-to-Monomer Transition of Rac1--
The
function of RhoGDI is 2-fold: countering the GEF activity to inhibit
GDP dissociation from the GTPases and solubilizing the GDP-bound Rho
proteins from the membrane compartment to cycle to the cytosol (8). The
cytoplasmic pool of Rac1 is found in complex with RhoGDI, indicating
that RhoGDI is a critical regulator of the subcellular localization of
Rac1. The Rac1 oligomer remained reactive with RhoGDI, but the presence
of excess RhoGDI led to the inhibition of oligomerization and the
disassembly of the Rac1 oligomer (Fig. 8). Such a mode of RhoGDI
interaction with the Rac1 oligomer can be rationalized by the recently
available structure of RhoGDI in complex with Cdc42, in which the
hydrophobic pocket of RhoGDI engulfs the isoprenoid moiety of Cdc42,
and the lid of the lipid-binding pocket provides extended contacts with
the C-terminal polybasic residues of Cdc42 (9). Applying the
RhoGDI-Cdc42 interaction to the Rac1 situation, one would predict that
the high affinity interaction of RhoGDI with Rac1 initiated through hydrophobic lipid binding could efficiently compete with the
hydrophilic binding of a neighboring Rac1 polybasic domain, which is
essential for maintaining the oligomer configuration. The resulting
product of the RhoGDI intervention would be the solubilized Rac1 from a
membrane environment, and meanwhile, the oligomer configuration would
be broken open by the formation of a RhoGDI·Rac1 complex at a 1:1
molar ratio. An additional function of RhoGDI therefore might be to
allow Rac1 to cycle between the oligomeric and monomeric states.