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J Biol Chem. 2008 December 19; 283(51): 35954–35965. doi: 10.1074/jbc.M804717200. | PMCID: PMC2602895 |
Copyright © 2008, The American Society for Biochemistry
and Molecular Biology, Inc. The Polybasic Region of Rac1 Modulates Bacterial Uptake Independently of
Self-association and Membrane
Targeting *Ka-Wing Wong,‡1,2 Sina Mohammadi,‡1 and Ralph R. Isberg§3 §Howard Hughes Medical Institute, ‡Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 Received June 20, 2008; Revised September 2, 2008. The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase
member, is required for Rac1 self-association, membrane localization, nuclear
translocation, and interaction with downstream effectors. We previously
demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the
effectors that requires the polybasic region for interaction, is necessary for
efficient invasin-promoted uptake of Yersinia pseudotuberculosis by
nonphagocytic cells. Here we further examined the role of this region in
invasin-promoted uptake. Using fluorescence resonance energy transfer
experiments (FRET), we determined that engagement of integrin receptors by
invasin caused elevated levels of Rac1 self-association at the site of
bacterial adhesion in a PBR-dependent fashion. Self-association could be
disrupted using several strategies: translocation of the Yersinia
YopT prenylcysteine protease into host cells, inactivation of the Rac1
isoprenylation signal that is required for membrane localization, and
elimination of the PBR. Disruption in each case impaired invasin-promoted
uptake. To determine if there is a role for the PBR in Rac1 effector signaling
that was independent of its role in membrane localization or multimerization,
we examined the effect of the PBR in the context of a Rac1 derivative that was
targeted to the membrane via an NH2-terminal lipid tail. The
membrane-targeted Rac1 derivative restored significant invasin-promoted
bacterial uptake in a PBR-dependent manner and yet displayed no detectable
self-association. This study indicates that, in addition to its role in
promoting membrane localization, the PBR exerts a positive effect on
Rac1-controlled bacterial uptake that is independent of Rac1 self-association,
most likely due to signaling to downstream effectors. Uptake of pathogenic bacteria by normally nonphagocytic cells is uniformly
regulated by members of the Rho GTPase family, including Cdc42, Rac1, and RhoA
( 1). In the case of the
Gram-negative enteropathogenic bacterium Yersinia pseudotuberculosis,
Rac1 is required for uptake, whereas Cdc42 and RhoA play either no role or a
negative role, respectively
( 2- 4).
Rac1 can facilitate bacterial uptake by remodeling the actin cytoskeleton
through one of three mechanisms: 1) inducing actin filament nucleation and
branching by activating the Arp2/3 complex via WAVE family members
( 5); 2) increasing
phosphoinositol 4,5-bisphosphate concentrations in the plasma membrane,
resulting in uncapping of actin filaments
( 6,
7); or 3) inhibiting actin
depolymerization by activating LIM kinases, which deactivate cofilin
( 8). Activation of Rac1 requires GTP loading by guanine nucleotide exchange
factors (RacGEFs), which show specificity for subclasses of Rho family members
( 9). Exchange takes place
simultaneously with release of Rac1 from RhoGDI proteins, which maintain Rho
family GTPases in an inactive state in the cell cytoplasm
( 10). Release allows insertion
of Rac1 in a target membrane via a prenyl group linked to the carboxyl
terminus of the protein ( 11).
After exchange and insertion into the membrane, active Rac1 is able to bind
downstream effectors, many of which modulate the actin dynamics associated
with bacterial uptake ( 12).
The activation observed is often a response to engagement of cell surface
receptors, resulting in interaction with downstream effectors
( 13,
14). One example of a group of
host cell surface molecules that activate Rac1 in response to substrate
engagement is the β1 integrin receptor family, the members of which bind
envelope proteins encoded by a wide range of pathogenic microorganisms
( 15,
16). Y. pseudotuberculosis undergoes high efficiency bacterial uptake
after engagement of β1 integrin receptors by the bacterial cell surface
protein invasin ( 17). Invasin
binds integrins with a much higher affinity than natural ligands, such as
fibronectin and laminin ( 18).
Invasin is also able to form multimers, which is predicted to allow receptor
clustering, thought to be a prerequisite for triggering intracellular
signaling processes required for bacterial uptake
( 19). The combined activities
of high affinity binding and multimerization by invasin are critical for high
efficiency invasin-mediated bacterial uptake that is regulated by activated
Rac1 ( 19,
20). Engagement of β1 integrins by Y. pseudotuberculosis triggers
efficient recruitment of Rac1 to nascent phagosomal membranes, resulting in
localized accumulation of the activated GTPase, as determined by
FRET 4 analysis
( 2). Although the most
attractive model for Rac1 function at the phagocytic cup is that localized
activation of Rac1 occurs at sites of receptor engagement, it is possible that
active Rac1 is simply delivered to these sites by release of the GTP-loaded
form from their soluble RhoGDI-bound complexes in the host cell cytoplasm. The
latter possibility was suggested from a study in which fibronectin-coated
beads were used to challenge cultured cells
( 14). This raises the
possibility that Rac1-GTP can be sequestered by cytosolic RhoGDI and then
directly delivered to the site of receptor clustering without a
membrane-dependent activation step. An additional mechanism for regulating the activity of Rac1 has been
proposed. Gel filtration studies and co-immunoprecipitation experiments
indicated that the polybasic region (PBR) at the COOH terminus of Rac1
mediates self-association of Rac1
( 21). This self-association is
independent of the nucleotide status of Rac1. It has been suggested that
PBR-mediated self-association potentiates Rac1-GTP to activate effectors,
based on the observation that Rac1 derivatives lacking the PBR are defective
for activation of the serine/threonine kinase PAK1
( 21). If local engagement of
β1 integrin receptors indeed triggers a localized RhoGDI release from
Rac1, there should also be an induction of Rac1 self-association at the sites
of integrin engagement. In this report, we investigate the role of the PBR in supporting
invasin-mediated uptake of Y. pseudotuberculosis and identify
sequence elements that are important for Rac1 self-association. Using
derivatives that allow membrane localization of Rac1 without the presence of
the PBR, we provide evidence that the role of this sequence in the uptake
process appears to be independent of its role in self-association, presumably
because the PBR is necessary to interact with downstream effectors or guanine
nucleotide exchange factors. Cell Culture, Transfection, and Plasmid Constructs—Culture
and transfection of COS1 cells were performed as previously described
( 2). Mammalian expression
plasmids pmCFP-Rac1 and pmYFP-Rac1 encoding the NH 2-terminal fusion
of monomeric cyan fluorescence protein (CFP) or yellow fluorescence protein
(YFP) to Rac1 as well as Rac1 derivatives having the G12V, R66A, C189S, or 6Q
( 183KKRKRK → 183QQQQQQ) mutations have been
described ( 22,
23). The K186E mutation in
Rac1 was generated using the Stratagene (La Jolla, CA) QuikChange
site-directed mutagenesis kit. pLyn-mCFP consists of a 10-amino acid
myristoylation/palmitoylation sequence of Lyn kinase fused to the 5′-end
of the mCFP gene ( 6). pLyn-mYFP
was generated by replacing mCFP with mYFP. Rac1(C189S) or Rac1(6Q/C189S)
devoid of the geranylgeranylation signal were inserted in frame into the COOH
terminus of pLyn-mCFP to generate pLyn-mCFP-Rac1(C189S) or
pLyn-mCFP-Rac1(6Q/C189S). mCFP-GerGer and mYFP-GerGer containing fluorescence
protein fused to the CAA X geranylgeranylation signals, without the
upstream polybasic region, were kindly provided by Dr. R. Tsien (University of
California, San Diego)
( 24). Plasmids encoding HA-mYFP, Lyn-HA-mYFP, Myc-mCFP, and Lyn-Myc-mCFP fusions
were constructed by replacing the enhanced green fluorescent protein gene in
pEGFP-C1 (Clontech) with each tag-encoded gene indicated. Various Rac1 alleles
(WT, R66A, 6Q, C189S, and 6Q/C189S) were then cloned into all four plasmids.
All plasmids were verified by sequencing. Oligonucleotide sequences are
available upon request. Rac1 derivatives used in this study and their
properties are described in Table
1. | TABLE 1Rac1 derivatives used in this study |
Culture of Y. pseudotuberculosis Infection of Mammalian Cells and
Immunofluorescence Protection Assay of Bacterial Uptake—Conditions
for growth of virulence plasmid-cured Y. pseudotuberculosis
YPIII(p -) with or without YopE or YopT and infection of COS1 have
been described ( 22). The
plasmid-cured Y. pseudotuberculosis strain lacks the virulence
plasmid (pYV) that encodes YopE and YopT and is efficiently internalized into
host cells. Strains that harbor the YopE Rho family GAP
( 22) or the YopT family
CAA X protease (kind gift of Dr. James Bliska, SUNY Stony Brook)
contain plasmids encoding these proteins as well as the plasmid pYV
( yopT-deficient yopE:: kan yopH:: cam;
referred to as strain YP17) to allow translocation of these proteins via the
bacterial type III secretion system
( 22). For bacteria lacking the virulence plasmid, the YPIII(p -) strain
was grown logarithmically in Luria Bertani broth at 26 °C until an
A600 of 0.7, prior to inoculation onto cultured mammalian
cells ( 22). For strains
harboring the pYV plasmid and Yop-encoding plasmids, bacteria were grown with
aeration at 26 °C overnight in broth supplemented with 2.5 m m
CaCl 2 and 100 μg/ml ampicillin and then subcultured and grown at
26 °C until A600 of 0.2. At this point, the cultures
were shifted to 37 °C and aerated for 1 h. A multiplicity of infection of
50:1 was used for YPIII(p -) incubations, and a multiplicity of
infection of 25:1 was used for other derivatives. For the pYopE-expressing
plasmid, 0.1 m m isopropyl-β- d-thiogalactopyranoside
was supplemented during infection to induce YopE expression. Bacterial uptake was assayed using immunofluorescence protection as
described ( 6). Briefly,
bacteria appropriately cultured were incubated with transfected cells for 30
min at a multiplicity of infection of 50 at 37 °C. After incubation, the
adherent cells were analyzed for internalized or surface-bound bacteria as
described previously ( 6). The
monolayers were fixed in 3% paraformaldehyde and probed with primary antibody
directed against Y. pseudotuberculosis, followed by a fluorescent
secondary antibody (anti-IgG conjugated to either Alexa Fluor 594 or Cascade
Blue) to detect extracellular bacteria. The cells were then permeabilized
( 2) and probed with antibodies
directed against the bacteria to allow detection of both intracellular and
extracellular bacteria. The coverslips were then probed with appropriate
secondary antibodies to detect intracellular bacteria. FRET Measurements—The basis of the assay is that association
between Rac1 derivatives fused to either monomeric CFP or monomeric YFP should
be detected as a FRET readout. COS1 cells were first transfected with a
combination of various derivatives of mCFP-Rac1 and mYFP-Rac1 and then
challenged with bacteria. Infections were stopped by fixation after 30 min.
The cells were then imaged and analyzed for sensitized FRET from CFP to YFP
essentially as described ( 22),
using correction factors for CFP (0.32) and YFP (0.18) for bleed-through from
CFP emission and cross-YFP excitation by the FRET filter set. To measure FRET,
images from YFP, CFP, and FRET filter sets
( 22) were captured, choosing
regions of interest about nascent phagosomes. Sensitized FRET was then
calculated from these regions, by subtracting the CFP and YFP correction
factors, using exactly the same procedure as described previously
( 22). FRET signals were
normalized by combination of mCFP and mYFP emissions calculated from the CFP
and YFP filter sets using the following formula as described
( 25).
Determination of mYFP-Rac1 Expression Levels Relative to Endogenous
Rac1—COS1 cells were transfected with mYFP-Rac1 and cultured
overnight. Transfected cells were lifted and subjected to flow cytometry using
YFP fluorescence to sort cells into four fractions according to levels of
fluorescence (YFP negative, low, medium, and high). A portion of the sorted
cells were plated onto fibronectin-coated coverslips, allowed to adhere for
~3 h, fixed in 4% paraformaldehyde, and imaged to quantify YFP
fluorescence. YFP fluorescence quantification was performed as described above
for FRET experiments. The remaining sorted cells were lysed in sample buffer;
lysates were resolved by SDS-PAGE, blotted, and probed for Rac1 using a
monoclonal anti-Rac1 antibody (clone 23A8; Sigma). Immunoprecipitation of Myc/HA-tagged mCFP/mYFP Fusions—293T
cells were transfected with 0.5 μg of each plasmid in 6-well dishes. Bait
constructs were Myc-tagged, and prey constructs were HA-tagged. Cells were
lysed 24 h post-transfection in 500 μl of lysis buffer (20 mm
Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and complete protease
inhibitor mixture (Roche Applied Science)). Lysates were then incubated at 4
°C (with agitation) for 10 min and spun to remove debris. 50 μl of the
cleared lysates was saved as the input fraction, and 400 μl was applied to
washed anti-HA epitope affinity resin (monoclonal anti-HA; Sigma) and
incubated at 4 °C (with agitation) for 1 h. The resin was washed three
times in wash buffer (25 mm Tris, pH 7.5, 30 mm
MgCl2, 40 mm NaCl, 0.1% Nonidet P-40), and bound
proteins were eluted in 100 μl of sample buffer. 10 μl of eluted
proteins (immunoprecipitate) as well as 10 μl of cleared lysates (input)
were analyzed by SDS-PAGE and Western blotting. Input and immunoprecipitated
proteins were detected using an anti-Myc epitope antibody (rabbit polyclonal;
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as well as an anti-HA epitope
antibody (rabbit polyclonal; Santa Cruz Biotechnology). Rac1 Self-associates in Cultured Cells— Y.
pseudotuberculosis employs a variety of proteins to activate and
misregulate Rac1 in host cells
( 22). In this report, we
examined whether Y. pseudotuberculosis could also control
self-association of Rac1 ( 21).
Previously, self-association was demonstrated in vitro by gel
filtration chromatography and immunoprecipitation of differentially tagged
Rac1 derivatives in an event requiring the COOH-terminal PBR
()
( 21). To determine if
self-association occurs in cells and can be detected at spatially distinct
sites within the cell, Rac1 self-association was analyzed by a FRET-based
system, using monomeric CFP and YFP derivatives (mCFP-Rac1 and mYFP-Rac1)
(). When
coexpressed in the same cells, mCFP-Rac1(WT) and mYFP-Rac1(WT) produced FRET
throughout the cells in all regions except for the nucleus
(). When normalized to the concentration of Rac1 at
individual sites in the cell, FRET signals (normalized FRET) were notably
stronger at membrane ruffles, indicating enhanced Rac1 self-association
(,
arrows). These elevated FRET levels were specific to these sites,
because at regions where no ruffles were evident, the lower levels of
normalized FRET were independent of Rac1 concentrations. Consistent with biochemical data, self-association of Rac1, as detected by
FRET, required the presence of the PBR on both partners. Co-transfection of
mCFP-Rac1(WT) with mYFP-Rac1(6Q), which has each of the basic residues in the
PBR replaced with Gln ( 26),
produced a lower FRET signal (; 6Q), confirming the previous
published findings that the PBR mediates Rac1 self-association in
vitro ( 21). Self-association Is Not Due to Overexpression of Rac1
Constructs—Since any self-association observed by FRET could result
from aggregation of overexpressed protein, the concentration of Rac1 expressed
from the transfected plasmids relative to endogenous levels of Rac1 was
determined. COS1 cells were transfected with mYFP-Rac1 and sorted by flow
cytometry to determine the concentration of Rac1 relative to the amount of YFP
fluorescence observed by microscopy. The transfected cells showed a broad
distribution of fluorescence (), with a peak of untransfected cells and a shoulder of
cells having increasing amounts of fluorescence. The total cell population was
collected into four separate fractions consisting of cells with increasing
amounts of YFP fluorescence (neg, lo, med, hi;
), and each
population was analyzed to determine the amount of mYFP-Rac1 expression as
well as the level of fluorescence using our standard microscopic
techniques. The expression of mYFP-Rac1 in the YFP-lo fraction was lower than that of
endogenous Rac1, based on Western blotting with anti-Rac1 antibody. The levels
of mYFP-Rac1 were about 50% of the levels of endogenous Rac1 in this
population (;
YFP lo). In contrast, the other sorted fractions
(; YFP
med and YFP hi) expressed much higher levels of YFP-Rac1
compared with endogenous Rac1. Transfectants plated onto coverslips from the
YFP-lo population clearly had fluorescence levels similar to those being used
for FRET analysis (; compare lo versus med). When the fluorescence
was determined on individual cells from the mYFP-lo fraction, the average
intensity of individual cells was ~500 fluorescence units
(), which is
about the mean intensity of all the cells used for FRET
(;
mYFP-Rac1(FRET)). In fact, the cell populations that showed
overexpression of YFP-Rac1 gave fluorescence intensities that were well beyond
what was analyzed by FRET (; med). Therefore, a large proportion of the
cells analyzed by FRET expressed amounts of mYFP-Rac1 that are at or below
endogenous levels of Rac1. We conclude that self-association is not due to
overexpression of the fluorescence derivatives. Plasma Membrane Localization of Rac1 Is Not Sufficient to Promote
Self-association—The 6Q mutation that replaces the PBR has the
secondary effect of interfering with plasma membrane localization of Rac1
( 14)
(, compare A
with E). We therefore tested whether blocking plasma membrane
localization would affect the ability of Rac1 to self-associate. The C189S
mutation, immediately downstream of PBR, prevents geranylgeranylation of Rac1
at the carboxyl-terminal CAA X box and resulted in a large pool of the
protein localizing in the nucleus, as previously described
( 27)
(). This lipid
modification is essential for the Ras superfamily of small GTPases to anchor
onto the plasma membrane ( 28).
Based on the FRET assay, mCFP-Rac1(C189S) showed no ability to interact with
mYFP-Rac1(WT) (; C189S), indicating the importance of plasma membrane
localization for Rac1 self-association. The C189S mutation also blocks RhoGDI
binding ( 29), raising the
possibility that RhoGDI could play some role in Rac1 self-association. To rule
out this possibility, the interaction of the Rac1(R66A) mutant was analyzed,
which is unable to bind RhoGDI but maintains plasma membrane localization
( 22,
30). mCFP-Rac1(R66A) produced
FRET with mYFP-Rac1(WT) as efficiently as was observed for the interaction
between the fluorescent wild type derivatives of Rac
(; R66A). Thus, plasma membrane localization,
but not RhoGDI binding via the CAA X geranylgeranylation site, was
necessary for Rac1 self-association. To determine whether plasma membrane localization was sufficient to cause
intermolecular FRET in this particular assay, we targeted mCFP/mYFP onto the
plasma membrane by either an NH 2-terminal myristoylation signal
from the Lyn protein ( 24) or
the COOH-terminal geranylgeranylation site from Rac1 (see
for Lyn-mYFP
localization). Neither of the two tags could enable the CFP/YFP pair to
generate FRET (, compare
A with B and C; see
) even at high concentrations
(). This lowered self-association was observed, in
addition, with cells that were challenged with Y. pseudotuberculosis,
since no FRET signal could be detected at regions of membrane aggregation
resulting from invasin binding or at regions of membrane ruffling (see below;
). We conclude that plasma membrane localization was not
sufficient to cause protein self-association. Forcing plasma membrane localization of proteins that do not interact also
was not sufficient to cause protein self-association. The addition of the Lyn
myristoylation signal to either Rac1(C189S)
(, compare C
with D) or Rac1(6Q/C189S) (, compare E with F) resulted in plasma membrane
localization of these derivatives. Neither Lyn-modified derivative generated a
significant FRET signal for Rac1-Rac1 interaction
(, and ). Therefore, the aberrant localization of
these Rac1 derivatives to sites such as the nucleus and perinuclear locales
() does not explain the
inability of these proteins to self-associate, since even when they are plasma
membrane-localized, they could not self-associate. Additionally, GTPase
activation does not cause self-association under these conditions, because
constitutively active membrane-targeted Rac1 variants lacking the appropriate
carboxyl-terminal sequences fail to generate a FRET signal
(). Lowering Affinity for Effectors Reduces Rac1
Self-association—The PBR has been characterized to have a role in
binding effectors, such as phosphatidylinositol-4-phosphate 5-kinase-α
(PIP5Kα) and protein kinase C-related kinase 1
( 7,
31). To determine whether
disruption of effector recognition reduces Rac1 self-association, we
introduced the K186E mutation into Rac1, which has been demonstrated to block
the binding of PIP5Kα without affecting plasma membrane localization of
Rac1 ( 7)
(). A lower
level of mCFP-Rac1(K186E) self-association was observed relative to wild type
(Figs. and
).
Therefore, maximum Rac1 self-association depended on sequence determinants
that are also important for effector binding (residue Lys 186) as
well as geranylgeranylation (residue Cys 189). Formation of a Stable Rac1-Rac1 Complex Requires the Presence of the
PBR and Prenylation Signal—To support the data from the FRET assay,
an independent test of Rac1 self-association was performed using a
coimmunoprecipitation procedure (). The power of the FRET readout is that it provides
spatial information and may allow the detection of transient interactions. If
the observed self-association is transient, then it may not survive other
detection strategies, such as immunoaffinity techniques, that require slow
dissociation rates. To determine if interaction could be observed using more
restrictive conditions, several of the constructs used in the microscopic
assay were tagged with either Myc or HA epitopes and expressed in mammalian
cells (). For
each condition, two constructs were co-transfected into 293T cells, each
having different tags to allow detection of self-association. Extracts from
the transfectants were then subjected to immunoprecipitation with anti-HA,
followed by immunoblotting with anti-Myc, to determine if a fraction of the
Myc-tagged partner was found in the precipitate (see “Materials and
Methods”). When HA-tagged CFP-Rac1(WT) was immunoprecipitated, it was
able to pellet either Myc-tagged YFP-Rac1(WT) or Myc-tagged YFP-Rac1(R66A)
based on immunoblotting, consistent with the FRET observations
(). On the
other hand, any disruption of the prenylation site (Rac1(C189S)) or the PBR
(Rac1(6Q)) was sufficient to interfere with co-immunoprecipitation
(). This
loss of association was true even if only one of the partners was lacking the
critical carboxyl-terminal sequences (; wt + 6Q and
wt + C189S) or if membrane localization was forced by adding
a Lyn myristoylation site (; Lyn constructs). Therefore, there is
concordance between the two assays for detecting interaction, since Rac1
self-association is sufficiently long lived to survive detergent extraction
and immunoprecipitation. Furthermore, these results provide direct evidence
that Rac1 self-association requires the PBR and COOH-terminal prenylation
signal. Invasin Binding Results in Localized Rac1
Self-association—Engagement of β1 integrin receptors by the
Y. pseudotuberculosis outer membrane protein invasin triggers the
recruitment of Rac1 onto the phagosomal membrane
( 6). To examine the degree of
self-association of Rac1 in response to invasin binding, we visualized Rac1
clustered about Y. pseudotuberculosis associated with COS1 after a
30-min incubation with bacteria. To analyze self-association, cells were
challenged with a bacterial strain that expressed invasin as the only adhesin
and lacks the ability to translocate Rac1-inactivating Yops
( 33). FRET analysis showed
that the mCFP-Rac1 recruited around nascent phagosomes had elevated levels of
self-association in comparison with the nearby Rac1 located at regions distal
from the phagosomes (). Rac1 on membrane ruffles displayed similar higher
efficiency of self-association (data not shown;
). This enhanced
self-association was not due to simple recruitment of mCFP-Rac1 and mYFP-Rac1
to the active sites, because the FRET levels were still elevated after the
FRET signals were normalized against the concentration of mCFP-Rac1,
indicating that the fraction of Rac1 undergoing self-interaction is higher at
the site of bacterial adhesion than at other sites
(, compare A and
B; see arrows). In addition, the increased FRET signal was
dependent on the presence of Rac1 in the fusion constructs as well as the
CAA X motif. When the simple mCFP-GerGer/mYFP-GerGer pair or the
Lyn-mCFP-Rac1(C189S)/Lyn-mYFP-Rac1(C189S) pair was analyzed, neither was able
to generate a FRET signal at any site in the host cells even on nascent
phagosomes associated with sites of bacterial adhesion
(; quantified in ). This is despite the fact that the
CAA X-containing constructs showed evidence of concentration about the
phagosomal cups (). Therefore, enhanced Rac1 self-association was not a
result of increased membrane density resulting from invasin engagement of
integrins but was a property of the presence of an intact Rac1 protein. Removal of the CAAX Motif by Yersinia YopT Protease Destroys Rac1
Self-association—The above data argue that the CAA X
prenylation motif and the PBR collaborate to promote Rac1 self-association.
One possibility for how this occurs is that membrane insertion resulting from
prenylation facilitates close contact between Rac1 monomers, stimulating PBR
self-association. The other possibility is that insertion in the membrane is
required to maintain self-association. To determine if membrane insertion was
required for maintenance of the interaction, we analyzed the consequences of
translocating the Y. pseudotuberculosis YopT protein into host cells
on Rac1 self-association. The YopT protease activity targets
membrane-localized Rac1 ( 13),
cleaving just upstream of the CAA X box, releasing Rac1 from the
membrane. If the role of the prenyl group is limited to initiating Rac1
association, then protein released from the membrane should continue to
self-associate. To test this model, transfectants expressing the
mCFP-Rac1/mYFP-Rac1 pair were challenged with a YopT-expressing Y.
pseudotuberculosis strain, and the amount of FRET between the Rac1 pairs
was determined ( 22). Within 1
h of incubation with Y. pseudotuberculosis, YopT had reduced Rac1
self-association, since no FRET signal could be detected between the Rac1
pairs ().
Therefore, because Rac1 rapidly lost self-association after release from the
membrane, localization of the protein in the membrane via the prenyl groups
was required to maintain this relationship. Furthermore, the loss of
self-association after release from the membrane could not be due to
competition for binding by RhoGDI, because removal of the prenyl group
prevents recognition by RhoGDI
( 32). Loss of Self-association Is Not the Cause of Defective Signaling That
Results from Alterations in the Polybasic Region—To investigate the
function of the Rac1 COOH terminus in invasin-promoted uptake, a strategy was
pursued in which mutated plasmid-encoded Rac1 genes were introduced into cell
lines that expressed endogenous Rac1. To this end, we took advantage of the
Y. pseudotuberculosis YopE RhoGAP protein. YopE is translocated into
mammalian cells and subsequently inactivates endogenous Rac1, thus inhibiting
the phagocytosis of the bacterium. This uptake inhibition can be overcome by
the exogenous expression of a GAP-insensitive (constitutively active) Rac1
allele. A number of Rac1 PBR mutants were introduced onto the constitutively
active Rac1V12 allele and were transfected into COS1 cells. Cells were
challenged with YopE-expressing Y. pseudotuberculosis (strain
YP17/pYopE), and the extent of bacterial uptake was quantified. As demonstrated previously, Rac1V12 restored significant bacterial uptake
in the presence of YopE, allowing the effects of mutations to be compared
(,
mCFP-Rac1V12). Not surprisingly, the Rac1V12(C189S) derivative that
lacks gerenylgerenylation, and therefore could not target to the membrane, was
unable to bypass YopE activity (; mCFP-Rac1V12(C189S) versus Rac1V12; p ≤
0.0004). Similarly defective was the RacV12(6Q) mutant, which also could not
restore uptake in the presence of YopE
(; mCFP-Rac1V12(6Q)
versus mCFP-Rac1V12; p ≤ 0.004). Thus, invasin-mediated
uptake required the PBR. To determine if the defect in uptake seen in these
mutants was due to an absence of self-association or due to loss of effector
interactions, the K186E effector binding mutation
( 34) that also lowers Rac1
self-association (Figs. and ) was analyzed. The effect of this mutation on
bacterial uptake was similar to that seen with the 6Q mutation, since the two
derivatives showed no statistical difference in being able to promote uptake
(;
mCFP-Rac1V12(K186E) versus Rac1V12(6Q); p = 0.47). Since both the 6Q and C189S mutations reduce plasma membrane localization
of Rac1, we could not determine if lowered uptake was a result of lowered
self-association, effector interactions, or plasma membrane localization
( 14). To address this issue,
uptake was analyzed under conditions in which plasma membrane localization of
the mutant Rac1 proteins was forced, using the constructs that have a Lyn
NH 2-terminal myristoylation site
( 24). The addition of the Lyn
myristoylation site significantly suppressed the defect observed in the C189S
mutant (;
mCFP-RacV12(C189S) versus Lyn-mCFP-RacV12(C189S); p <
0.004). Therefore, the loss of uptake that results from lack of prenylation
appears to be almost totally due to defective membrane localization, because
the Lyn construct lacking the prenylation site showed no evidence of
self-association (Figs.
,,
and ) yet still could promote
uptake (). On the other
hand, if the 6Q mutation was introduced into the Lyn construct
(Lyn-Rac1V12(6Q/C189S)), then uptake was as poor as observed in the construct
lacking the Lyn site (;
Rac1V12(C189S) versus Lyn-Rac1V12(6Q/C189S); p < 0.6).
The defect in uptake resulting from the 6Q mutation was unlikely to be due to
poor plasma membrane localization, because the myristoylation site was intact
(). Since self-association of Rac1 does not appear to be essential for
promoting invasin-mediated uptake in this system, and the 6Q mutation that
replaces the PBR is defective even when plasma membrane localization is
forced, the PBR must be required either for recognition by downstream
effectors or RacGEF proteins. The importance of the latter could not be the
cause of the defect in this assay, because Rac1V12 derivatives were used,
which do not require guanine nucleotide exchange factors for activation. The
importance of PBR recognition by effectors for invasin-dependent uptake is
supported by results using the K186E mutant. Therefore, it appears that in
this system, the PBR was required for downstream effector recognition. In this report, we used FRET technology to demonstrate that Rac1 is able to
self-associate in live cells and that the highest levels of this interaction
occurred at sites documented to contain the highest concentrations of actin
remodeling ( 14,
22), such as regions of cell
surface ruffling and nascent phagosome formation during bacterial uptake.
These are also the regions of the cell that have the highest levels of
activated Rac1 ( 14,
22). This result supports the
model that the Rac1 PBR is an important determinant of self-association
( 21). Our data indicate that
the PBR is not sufficient for this interaction within host cells, however,
since insertion of Rac1 into the membrane via its CAA X prenylation
motif was required for detectable self-association. Using an assay for the
ability of Rac1 to support uptake of Y. pseudotuberculosis, we
demonstrated that Rac1 self-association was not required for driving actin
polymerization events that lead to internalization of the microorganism. The
PBR, however, was important for efficient uptake in this system under
conditions in which uptake did not require self-association of Rac1,
indicating that the PBR was probably required for interaction with downstream
effectors. The nature of the assay used to analyze uptake involved using
constitutive active derivatives of Rac1 and did not rule out the possibility
that Rac1 self-association may be important to stimulate its activation. Rac1 self-association was originally demonstrated using gel filtration
experiments, which showed that the COOH-terminal PBR was required for the
formation of high molecular weight species of Rac1 that had been purified as a
nonprenylated protein ( 21).
Our observations are consistent with the importance of this region in
promoting self-association within mammalian cells
(), but unlike
this biochemical assay, we could obtain no evidence for self-association in
the absence of membrane localization. In particular, the orientation of the
protein relative to the membrane was extremely important, since forcing
membrane localization by an NH 2-terminal myristoylation tag was not
sufficient to generate a Rac1:Rac1 FRET signal (Figs.
and
). It may be that an important
nucleation event that initiates self-interaction requires a particular
orientation of the protein in the membrane. Alternatively, prenylation could
target Rac1 into microdomains on the plasma membrane that are required for
close association of Rac1 monomers. The second possibility has support from a
pair of observations. First, active Rac1 is preferentially localized on
cholesterol-rich lipid raft domains, which could stimulate self-interaction
( 35). Second, Rac1
preferentially self-associated at the sites of actin polymerization observed
in lamellipodia and nascent phagosomes. After recruitment to these sites,
there could be back-signaling to membrane-localized Rac1 that stimulates
self-association. In fact, the presence of a high concentration of active Rac1
at these sites ( 13,
14) could act as a further
important stimulus of Rac1 self-interaction, perhaps by initiating cycles of
signaling from membrane-localized Rac1 to downstream effectors and back to
membrane-localized Rac1. The self-association initiated at the host cell membrane is stable in the
presence of gentle detergent, but it may have a limited lifetime within a host
cell once Rac1 is liberated from the membrane. The FRET data obtained in this
study could be reproduced using immunoprecipitation analysis, in which
epitope-tagged Rac1 variants were used to identify self-interaction complexes
(). Only Rac1 derivatives
that showed self-association by FRET analysis were positive in the
immunoprecipitation assay, so the FRET analysis was a good predictor of
whether multimeric Rac1 complexes could survive the detergent extraction
procedure. Release of Rac1 from the membrane by the Yersinia YopT
protease, which removes the CAA X prenylation signal, disrupted
self-interaction (). This
could be due to binding of soluble proteins within the cell after release or
the introduction of the protein into a microenvironment within the cell that
stimulates dissociation. RhoGDI is the most obvious candidate for a protein
that could stimulate dissociation of Rac1 monomers; however, the proteolytic
product of YopT, nonprenenylated Rac1, is not a good substrate for RhoGDI
( 36). Release of Rac1 from the
membrane by YopT results in PBR-dependent translocation of Rac1 into the
nucleus, where at least a subpopulation of the protein is active
( 22). Although it is not clear
what downstream effectors interact with the protein in this compartment,
either the biochemical environment of the nucleus or its interacting partners
could interfere with self-association. The close correlation between results of the FRET assay and the
co-immunoprecipitation of Rac1 derivatives indicated that the FRET analysis
could be reproduced using traditional methods of detecting interaction. This
connection was not predicted from previously published work using
membrane-targeted CFP/YFP derivatives, which indicated that acylated proteins
inserted into membranes associate with each other in the absence of any
evidence of stable protein interactions
( 24). Using live cell imaging,
Zacharias et al. ( 24)
showed that the geranylgeranylated mYFP/mCFP pair generated a
concentration-independent FRET signal. The FRET by proximity observed in that
work, in the absence of any protein domains that promote interaction, is
thought to be due to the formation of microdomains within membranes, resulting
in close congregation of proteins
( 24). Although not
investigated in that work, it is unlikely that such proximity interactions
could have survived detergent extraction and allowed co-immunoprecipitation of
protein pairs. Presumably, our fixation procedures prior to FRET (see
“Materials and Methods”) prevented fluorophores that are located
on closely oriented but noninteracting proteins from generating a signal,
indicating that only strong protein-protein interactions can be detected. A
recent study indicates that fixation can reduce the yield of FRET in selected
circumstances, particularly when both fluorophores are fused on a single
polypeptide. Mathematical modeling indicates that a likely explanation for
this reduction is that with some FRET pairs, fixation can limit protein
flexibility and lock the fluorophores in conformations that interfere with
energy transfer ( 38). Although
such interference appears irrelevant in the case of two physically unlinked
proteins that have interacting regions
( 38), it is possible that FRET
caused by proximally localized but noninteracting proteins requires a level of
flexibility of the fluorophore that is blocked by fixation. It has been suggested that PBR-mediated self-association of Rac1 might
drive optimal effector signaling, based on the observation that a
PBR-defective Rac1 derivative was highly defective in activating one of its
downstream effectors, the serine/threonine kinase PAK1
( 21). Arguing against this
hypothesis were results from another study showing that
NH 2-terminal myristoylation could fully restore the ability of the
PBR-defective Rac1 to activate the kinase activity of PAK1
( 13). From our studies, we
think it likely that the restoration of PAK1 signaling was due to the
myristoyl group forcing membrane localization of the Rac1 mutant rather than
due to self-association. Our results support the idea that the loss of
membrane targeting that results from the disruption of the PBR is a more
profound consequence than loss of self-association, because membrane targeting
of Rac1 in the absence of self-association appeared sufficient to promote
uptake of Y. pseudotuberculosis. A membrane-targeted myristoylated
Rac1V12(C189S) did not self-associate, yet this activated Rac1 mutant was able
to restore bacterial uptake under conditions in which the endogenous Rac1 was
inactive, as long as the PBR was intact in such constructs. In addition to promoting self-association and membrane targeting, the PBR
is known to facilitate nuclear localization of Rac1 and binding to
PIP5Kα as well as other downstream effectors of Rac1
( 7,
31,
37,
39). Phosphatidylinositol
4,5-bisphosphate, the product of PIP5K, stimulates invasin-promoted uptake
( 6). Therefore, the importance
of PBR could be explained by its ability to stimulate localized
phosphoinositol 4,5-bisphosphate accumulation at the phagocytic cup. The fact
that we could not bypass the requirement for the PBR in bacterial uptake by
simply forcing localization of the protein into the membrane
() was very different
from the result showing that PAK1 signaling was PBR-independent after
localization of Rac1 in the plasma membrane
( 14). Therefore, stimulation
of PAK1 is probably not sufficient to bypass the requirement of this sequence
determinant for bacterial uptake. Consistent with this hypothesis was the
finding that a less drastic change in the PBR that interferes with
PIP5Kα binding (the Rac1(K186E) allele), was sufficient to interfere
with the ability of a membrane-localized and constitutively active Rac1
variant to promote uptake. Although the evidence presented here argue that loss of multimerization
resulting from alterations in the PBR cannot explain the observed defects in
invasin-mediated uptake, it should be pointed out that the Rac1 variants used
to test uptake were activated forms of the protein
(). It is possible that
this approach bypasses a requirement for multimerization. At least for the
integrin receptor, multimerization is important, although this does not mean
that downstream signaling molecules require multimerization. Artificially
dimerizing invasin greatly reduces the concentration of the protein required
to promote uptake of particles
( 19). The evidence that
downstream multimerization my play a role in regulating this event is that
many GTPases are known to form oligomers, an event associated with
self-stimulatory GAP activity
( 21). Although this latter
observation argues that multimerization plays a negative role in signaling,
self-inhibition of Rac1 may provide a mechanism to limit Rac1 activation that
is important for successful completion of cytoskeletal activities. Such
negative feedback may be critical at sites of local Rac1 activity, especially
when GAP and RhoGDI activities are expected to be down-regulated. Since
downstream signaling from Rac1 to other GTPases has complex consequences on
invasin-mediated uptake ( 23),
localized down-modulation of Rac1 signaling could play an important role in
completing phagocytic events. Future critical tests of the importance of multimerization in either
down-modulation of Rac1 activity or stimulating downstream signaling will
require the development of straightforward systems to analyze mutants at
limiting and highly defined protein concentrations. Even so, this work
demonstrates that multimerization and membrane localization promoted by the
PBR do not encompass the entire spectrum of events that are modulated by this
small region of the protein. We thank Drs. Matt Heidtman, Matthias Machner, Molly Bergman, and Vicki
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