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INTRODUCTION |
The activity of phospholipase C-
(PLC
)1 enzymes that
hydrolyze phosphatidylinositol 4,5-bisphosphate
(PtdInsP2) is stimulated with different orders
of efficacy by G protein
q subunits and by G protein

dimers (1-3). In addition, the PLC
2 isozyme is
specifically activated in vitro by the Rho GTPases Rac and Cdc42, but not by RhoA (4-6). As for all PLC
isozymes, activation by
q requires the C-terminal region of
PLC
2, and mutants carrying deletions in this region,
such as the mutant PLC
2
that lacks the
Phe819-Glu1166 segment, are resistant to
stimulation by
q but are susceptible to activation by
Rho GTPases and G protein 
subunits (3, 4, 7). Recent
studies (4-6) show that 
dimers and Rho GTPases activate
PLC
2 by interacting with different regions of the
effector enzyme. Thus, the PLC
2 catalytic subdomains X
and Y are sufficient for efficient stimulation by 
, whereas the putative pleckstrin homology (PH) domain of PLC
2 is
absolutely required for stimulation by Rho GTPases (6). Among the Rho GTPases, Rac1 and Rac2 are more potent stimulators than Cdc42 (6).
Evidence for a tight connection between PLC
2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLC
2 and Rac1, Rac2 and
Cdc42 (8-11). Moreover, in accord with our in vitro studies
on PLC
2 activation by Rho GTPases (4, 6), a recent study
conducted on myeloid-differentiated HL-60 cells demonstrated that
dominant-negative Cdc42 disrupted the stimulation of inositol
1,4,5-trisphosphate formation mediated via the chemoattractant
receptors while inhibiting Rac2 activation (12). These findings
strongly support the notion that Rac2 and possibly Rac1 and Cdc42 are
critically involved in receptor-mediated stimulation of
PLC
2 activity.
To date, the molecular mechanism of PLC
stimulation by either
heterotrimeric G proteins or Rho GTPases is largely unknown. The lipid
nature of the substrate emphasizes the importance of understanding the
mode of PLC
2 association with the membrane and its
regulation by multiple stimulators (13). However, the knowledge and
understanding of these processes are still lacking, especially in live
cells. For example, the postulated recruitment of PLC
by
heterotrimeric G proteins to the membrane could not be supported by
experimental data (14-16). Therefore, interactions with other proteins
could determine the cellular localization of PLC
. Here, we
investigated the membrane interactions of PLC
2 and their
modulation by the constitutively active Rho GTPase Rac2(12V).
Rho GTPases have been shown to regulate a wide range of cellular
functions in their active GTP-bound state, including reorganization of
the actin cytoskeleton, gene expression and cell cycle progression (reviewed in Refs. 17-19). Rho GTPases in their inactive, GDP-bound states are present in the cytosol complexed with guanine nucleotide dissociation inhibitors (GDIs) (20, 21). Upon stimulation, Rho GTPases
are released from the GDI, translocated from the cytosol to specific
membranes (22-24) and activated (undergo GDP/GTP exchange) by guanine
nucleotide exchange factors containing a Dbl homology domain (25). The
translocation of Rho GTPases from the cytosol to membranes following
their activation offers the intriguing possibility that activated Rho
GTPases target a soluble PLC
2 to its membrane-associated substrate.
To gain more insight into the role of Rac2 in regulating the membrane
interactions and activation of PLC
2 in live cells, we
have generated green fluorescent protein (GFP)-tagged
PLC
2 (GFP-PLC
2) and
GFP-PLC
2
. The enzymatic activity of these constructs was greatly enhanced in the presence of constitutively active Rac2(V12)
in transfected cells. To study the lateral diffusion of the
GFP-PLC
2 constructs in the plasma membrane and/or their exchange between membrane-bound and cytoplasmic pools, we employed fluorescence recovery after photobleaching (FRAP). Both forms of
PLC
2 exhibited a mixture of exchange and lateral
diffusion, suggesting transient association with the plasma membrane of
live cells. Constitutively active Rac2(12V), but not wild-type (wt) Rac2 (Rac2(wt)), slowed down the fluorescence recovery times of GFP-PLC
2 and GFP-PLC
2
and increased
their translocation to the membrane. These effects, which suggest
enhanced membrane association, specifically required the putative PH
domain of PLC
2, which could not be replaced by the
equivalent region of GFP-PLC
1. Our studies reveal that
the effects of Rac2(12V) on the mode of PLC
2-membrane interactions depend on its C-terminal region, whose presence is required for the exchange to become dominant in response to activated Rac2. These observations implicate the PLC
2 C-terminal
region, known to interact with G
q and to be capable of
dimerization (26), in a new role- regulating the exchange of
PLC
2 after its recruitment to the plasma membrane by
activated Rac2.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmids containing cDNA sequences encoding
the Rho GTPases Rac2(wt), Rac2(12V), RhoA(14V), HA-Rac2(wt),
HA-Rac2(12V), and HA-RhoA(14V) (4) were produced by ligation of the
respective coding sequences without noncoding sequences into the
BamHI/EcoRI site of pcDNA3.1(+) (Invitrogen).
To add a HA epitope tag to a construct, a 5' primer containing the
sequence encoding the epitope was used. The cDNAs of
PLC
2 and the deletion mutant PLC
2
,
lacking a C-terminal region necessary for stimulation by
q subunits (Phe819-Glu1166) were
inserted in-frame with GFP into the EcoRI/SalI
site of pEGFP-N1 (Clontech) to generate plasmids
encoding GFP-PLC
2 and GFP-PLC
2
. The
cDNA of the PLC
1/PLC
2 chimera was
generated by replacing the cDNA sequence encoding the N-terminal
amino acids of PLC
2 (residues 1-138) by the
corresponding residues of PLC
1 (1-142) using the PCR
overlap extension method. The chimeric cDNA was inserted into
pEGFP-N1 as described above to generate the GFP-PLC
1/PLC
2 plasmid.
Cell Culture and Transfections--
All cell lines were
maintained in Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf serum as described (27). For FRAP and confocal microscopy
experiments, COS-7 cells grown on glass coverslips in 35-mm dishes were
transfected using the DEAE-dextran method (28) with 150 ng of plasmid
DNA encoding one of the GFP-PLC
2 derivatives together
with 850 ng of plasmid DNA encoding either untagged or HA-tagged
Rac2(wt), Rac2(12V), or RhoA(14V); cells singly transfected with a
GFP-PLC
2 derivative received instead 850 ng of empty
pcDNA3.1 vector DNA. 24-h post-transfection, they were taken for
the FRAP or confocal microscopy studies.
For studies on stimulation of GFP-PLC
2, COS-7 or HEK293
cells were seeded in 12-well plates at a density of 1 × 105 cells per well and grown overnight in DMEM with 10%
fetal calf serum. COS-7 cells were transiently transfected using
LipofectAMINE 2000 (Invitrogen), adding to each well 1 µg of DNA
mixed with 2 µl of LipofectAMINE 2000 reagent in 0.2 ml of Opti-MEM
(Invitrogen). In co-transfection experiments, 0.5 µg of cDNA
encoding GFP-PLC
2 constructs and 0.1 µg of cDNA
encoding Rac2 constructs was added. The total amount of DNA was
maintained constant by adding pcDNA3.1(+). HEK293 cells were
transiently transfected with the same amounts of DNA using CalPhos
Mammalian Transfection Kit (Clontech) according to
the manufacturer's instructions.
Antibodies and Reagents--
Mouse anti-GFP antibodies were
obtained from Roche Molecular Biochemicals, rabbit anti-Rac2 from Santa
Cruz Biotechnology, and HA.11 rabbit serum against the influenza
hemagglutinin (HA) tag (Ref. 29; anti-HA) from Covance. Alexa
594-conjuated goat anti-rabbit (G
R) IgG was from Molecular Probes,
and normal goat IgG was from Jackson ImmunoResearch.
Peroxidase-conjugated G
R and anti-mouse IgG were from Sigma. The
lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC16) was obtained from Molecular Probes and
incorporated into the plasma membrane of live cells as described (30).
Myo-[2-3H(N)]inositol (22-24 Ci/mmol) was
from PerkinElmer Life Sciences.
Fluorescence Recovery after Photobleaching (FRAP)--
FRAP
studies were conducted as described earlier (31, 32), using previously
described instrumentation (33). The experiments were performed 24-26 h
post-transfection on COS-7 cells plated on glass coverslips and
transfected with GFP-PLC
2 derivatives as described
above. All experiments were done at 22 °C, in Hank's balanced salt
solution supplemented with 20 mM HEPES, pH 7.2. The
monitoring Argon ion laser beam (488 nm, 1.2 microwatt) was focused
through the microscope (Zeiss Universal) to a Gaussian radius of
0.85 ± 0.02 µm (63× objective) or 1.36 ± 0.04 µm (40× objective). A brief pulse (6 milliwatts, 4-6 ms for the 63×
objective, and 10-20 ms for the 40× objective) bleached 50-70% of
the fluorescence in the illuminated region. The time course of
fluorescence recovery was followed by the attenuated monitoring beam.
The apparent characteristic fluorescence recovery time (
), and the
mobile fraction were derived by nonlinear regression analysis, fitting
to a lateral diffusion process with a single
value (34).
Immunofluorescence Microscopy--
Immunofluorescence confocal
microscopy was employed to detect the co-localization of
GFP-PLC
2 with HA-Rac2(12V) at the plasma membrane. COS-7
cells were co-transfected with GFP-PLC
2 together with
HA-tagged Rac2(wt), Rac2(12V), or RhoA(14V) as described under cell
culture and transfections. After 24 h, the cells were fixed with
4% paraformaldehyde in phosphate-buffered saline (45 min, 22 °C)
and permeabilized with 0.5% Triton X-100 (5 min) in the same buffer.
After blocking with normal goat IgG (200 µg/ml, 22 °C, 30 min, in
phosphate-buffered saline supplemented with 2% bovine serum albumin),
they were incubated successively in the same buffer (1 h, 22 °C,
with three extensive washes after each incubation) with the following
antibodies: (i) rabbit anti-HA (1:3500 dilution) and (ii) Alexa
594-G
R IgG (5 µg/ml). The cells were mounted with Prolong antifade
solution (Molecular Probes) and subjected to analysis by
immunofluorescence microscopy.
Determination of Inositol Phosphate Levels--
24 h
post-transfection, COS-7 or HEK293 cells were washed with PBS.
Myo-[2-3H(N)]inositol (10 µCi/ml) was added,
followed by addition of LiCl 20 min after addition of radiolabeled
inositol to a final concentration of 10 mM. Incubation was
continued for 18 h. Inositol phosphate formation was stopped and
total inositol phosphates were then separated and measured as described
(35). In vitro phospholipase C activity was determined using
phospholipid vesicles containing [3H]PtdInsP2
as described previously (5).
Subcellular Fractionation--
For subcellular fractionation,
HEK293 cells (2.7 × 106) were grown on 100-mm dishes.
They were transiently transfected using the CalPhos transfection kit
(Clontech) with vectors encoding GFP-PLC
2 derivatives (7 µg of DNA) alone or together
with plasmids encoding Rac2 derivatives (7 µg of DNA). After 24 h, the cells were scraped into 0.15 ml of hypotonic buffer (20 mM Tris/HCl, pH 7.5, 2 mM EDTA, 3 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 µM leupeptin, 1 µg/ml aprotinin, 2 µg/ml soybean trypsin inhibitor) and disrupted
by 10 passages through a 0.5 × 25 mm needle. After removal of
nuclei by centrifugation (300 × g, 10 min),
particulate (P) and soluble (S) fractions were separated by
centrifugation (12,000 × g, 15 min). Soluble and particulate fractions (80 µg of protein) were analyzed by SDS-PAGE and immunoblotting using anti-GFP or anti-Rac2 antibodies.
Immunoreactive proteins were visualized using the ECL Western blotting
detection system (Amersham Biosciences).
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RESULTS |
Constitutively Active Rac2 Stimulates GFP-PLC
2 and
GFP-PLC
2
--
To study the effect of wild-type and
constitutively active Rac2 on the activity of PLC
2 and
on its interactions with the plasma membrane in live cells, we prepared
GFP fusion proteins of full-length human PLC
2 and of the
C-terminal deletion mutant PLC
2
. To confirm that the
GFP tag did not impair their activities, both GFP-PLC
2
and GFP-PLC
2
were expressed in transiently
transfected HEK293 cells, and reconstituted in a cell-free system with
G protein 
dimers or GTP
S-activated Rac2 and phospholipid
vesicles containing [3H]-labeled PtdInsP2 as
described earlier (4, 5). The activities of both GFP constructs were
greatly stimulated by either 1 mM free Ca2+, G
protein 
dimers, or GTP
S-activated Rac2 (not shown); these responses were similar to those observed with the untagged enzymes. To
investigate whether the activity of GFP-PLC
2 is
stimulated by activated Rac2 in intact cells, the production of
inositol phosphates was measured in COS-7 cells transiently transfected with vector containing the cDNA encoding each
GFP-PLC
2 construct, either alone or together with vector
containing the cDNAs encoding Rac2(wt) or Rac2(12V). Immunochemical
analysis of whole cell lysates of the transfected cells using anti-GFP
antibodies (Fig. 1, inset) indicated that the cells expressed equal amounts of the GFP constructs (170 and 130 kDa for GFP-PLC
2 and
GFP-PLC
2
, respectively), which were absent from cells
transfected with control plasmid. Fig. 1 demonstrates that no
stimulation of inositol phosphate formation by Rac2(wt) and Rac2(12V)
was detected in cells expressing GFP alone. Little, if any, stimulation
was observed in cells singly transfected with a vector encoding
GFP-PLC
2. In contrast, co-expression of Rac2(12V), but
not of Rac2(wt), with GFP-PLC
2 caused a marked (~17-fold) stimulation of inositol phosphate formation. Similar results (not shown) were obtained when HEK293 cells were employed in
place of COS-7 cells, or when Rac2(12V) or Rac2(wt) carrying an
N-terminal HA tag replaced the untagged Rac constructs. Rac2(12V), but
not the wild-type protein also activated GFP-PLC
2
;
the only difference from PLC
2 was that the Rac2-induced
stimulation was somewhat lower (~14-fold) (Fig. 1). The ability of
constitutively active Rac to stimulate both GFP-PLC
2 and
GFP-PLC
2
is in accord with earlier in
vitro studies on the untagged forms of these proteins, although
the extent of stimulation was higher for PLC
2
than for PLC
2 in the cell-free system (36). The reasons for
this discrepancy are currently unknown, but may be related to the fact that the substrate is presented in artificial phospholipid vesicles rather than in native membrane bilayers in the cell-free system.

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Fig. 1.
Stimulation of the activity of
GFP-PLC 2 and
GFP-PLC 2 by Rac2(12V) in intact
cells. COS-7 cells were transfected with vectors encoding GFP,
GFP-PLC 2, GFP-PLC 2 , or
GFP-PLC 1/PLC 2 (0.5 µg of DNA) together
with vectors encoding Rac2(wt), Rac2(12V), or empty pcDNA3.1 vector
(0.1 µg of DNA). 24 h after transfection, the cells were
incubated for 18 h in the presence of
myo-[2-3H(N)]inositol (10 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were
determined as described under "Experimental Procedures." The values
shown correspond to the mean ± S.D. of triplicate determinations.
The inset depicts immunochemical analysis of whole cell
lysates containing GFP (lane 1), GFP-PLC 2
(lane 2), GFP-PLC 2 (lane 3), or
GFP-PLC 1/PLC 2 (lane 4).
Aliquots of the four samples were subjected to Western blotting using
anti-GFP antibodies.
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To further characterize the mechanisms of GFP-PLC
2
activation by Rac2(12V), we employed a GFP-tagged
PLC
1/PLC
2 chimera. In this chimera, the
N-terminal putative PH domain of PLC
2 (amino acids
1-138) was swapped with the equivalent region of PLC
1
(amino acids 1-142), resulting in an isozyme, which is barely
activated by Rho GTPases (36). PLC
1/PLC
2
has recently been shown to be sensitive to stimulation by 
dimers, but not by GTP
S-activated Rho GTPases, in a cell-free system
(6). Fig. 1 shows that this chimera was not activated by Rac2(12V) in
intact COS-7 cells, demonstrating a specific requirement for the
N-terminal region of PLC
2.
Activated Rac2 Increases the Translocation of
GFP-PLC
2 and GFP-PLC
2
to the Plasma
Membrane--
To investigate the mechanism by which Rac2(12V) mediates
PLC
2 stimulation, we examined the ability of activated
Rac2 to induce translocation of GFP-PLC
2 and
GFP-PLC
2
from the cytoplasm to the membrane fraction.
HEK293 cells were co-transfected with a GFP-PLC
2
construct together with either empty vector or vectors encoding
Rac2(wt) or Rac2(12V). Transfected cells were fractionated, and
aliquots of the postnuclear particulate (P) and soluble (S) fractions
were analyzed by immunoblotting using anti-GFP or anti-Rac antibodies.
Fig. 2 shows that GFP-PLC
2
and GFP-PLC
2
were soluble either with or without
co-expression of Rac2(wt). In contrast, co-expression with Rac2(12V)
resulted in translocation of considerable amounts of
GFP-PLC
2 or GFP-PLC
2
to the
particulate fraction (Fig. 2). This effect was not observed when the
GFP-PLC
1/PLC
2 chimera was co-expressed
with Rac2(12V). The above effects were obtained on cells expressing
similar levels of Rac2(wt) and Rac2(12V) (Fig. 2). Similar results (not
shown) were obtained with HA-Rac2(wt) and HA-Rac2(12V). These findings
indicate that constitutively active Rac2 effectively mediates
translocation of GFP-PLC
2 and GFP-PLC
2
to the particulate membrane fraction, and that this effect requires the
putative PH domain of PLC
2.

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Fig. 2.
Translocation of
GFP-PLC 2 to particulate fractions
of HEK293 cells by constitutively active Rac2. HEK293 cells were
co-transfected with vectors encoding GFP-PLC 2,
GFP-PLC 2 , or
GFP-PLC 1/PLC 2 (7 µg of DNA each)
together with either empty pcDNA3.1 vector (control), Rac2(wt), or
Rac2(12V) (7 µg of DNA each). After harvesting, cells were
homogenized, and aliquots (80 µg of protein) of the postnuclear
particulate (P) and soluble (S) fractions were
subjected to SDS-PAGE followed by immunoblotting. Similar results were
obtained in three independent experiments. The upper three
panels were probed with anti-GFP antibodies. The bottom
panel employed anti-Rac2 antibodies. Similar to Rac2(12V),
Rac2(wt) is mainly present in the particulate fraction, presumably due
to the inclusion of non-plasma membrane intracellular membrane
particles in this fraction, as reported earlier for overexpressed
Rac2(wt) (Ref. 24; see also Fig. 3Bi).
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To obtain further support for this notion in whole cells, we performed
confocal fluorescence microscopy analysis. Since a large fraction of
GFP-PLC
2 remains cytosolic even in the presence of
activated Rac2, it is difficult to detect its translocation to the
plasma membrane by standard fluorescence microscopy. We therefore took
advantage of the fact that activated Rac2 is largely associated with
the plasma membrane (22, 23). Thus, analysis of the co-localization of
GFP-PLC
2 with Rac2(12V) allows the identification of
membrane translocation of PLC
2, with the added advantage
that Rac2(12V) translocation is visualized simultaneously. To this end,
COS-7 cells were co-transfected with vector encoding GFP-PLC
2 together with vectors encoding either
HA-Rac2(12V) or HA-Rac2(wt). The cells were fixed and permeabilized,
and the HA-tagged Rac2 proteins were labeled with anti-HA rabbit IgG
followed by Alexa 594-G
R IgG. Dual fluorescence images (GFP, green;
Alexa 594, red) were collected, overlaid and examined for
co-localization using the co-localization function of the confocal
imaging program (Zeiss LSM 510). The results of a typical experiment
are depicted in Fig. 3. Upon
co-expression with HA-Rac2(12V), a large fraction of the
GFP-PLC
2 population became co-localized with
HA-Rac2(12V) at the rim of the cells, exhibiting typical plasma
membrane labeling (Fig. 3A, panel ii). On the
other hand, this effect was much less pronounced following
co-expression with HA-Rac2(wt) (Fig. 3B, panel
ii). The effect is mediated specifically by some Rho GTPases and
not by others; this is indicated by the failure of co-expression with
HA-RhoA(14V), judged by immunofluorescence to be expressed at levels
comparable to those of the HA-tagged Rac2 proteins, to translocate
GFP-PLC
2 to the plasma membrane (Fig. 3C, panel ii). Thus, the Rac2 effect is not mediated indirectly by raising the level of the substrate, PtdInsP2 (see
below). Taken together, these data demonstrate that activated Rac2
specifically mediates translocation of GFP-PLC
2 to the
plasma membrane in whole cells.

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Fig. 3.
Confocal fluorescence microscopy demonstrates
that GFP-PLC 2 is translocated to
the plasma membrane in the presence of Rac2(12V). COS-7 cells were
co-transfected with GFP-PLC 2 together with HA-Rac2(wt),
HA-Rac2(12V), or HA-RhoA(14V) as described under "Experimental
Procedures." After fixation with paraformaldehyde and
permeabilization, the HA-tagged proteins were labeled with rabbit
anti-HA followed by Alexa 594-G R IgG ("Experimental
Procedures"). Dual images (green fluorescence for GFP,
red for HA) were collected on the Zeiss LSM 510 confocal
microscope fitted with non-leaking green and red fluorescence filters.
The green and red images were superimposed and analyzed by the
co-localization function of the LSM 510 software. Bar, 10 µm. Left panels (Ai-Ci), superimposed green
and red images. Middle panels (Aii-Cii) depict
the cellular localization of superimposed pixels showing a high level
of both green and red fluorescence intensities (i.e.
co-localization), defined by the upper right quarter (shown
by a red-lined square) of the fluorograms (Aiii-Ciii). The
fluorograms show the red and green fluorescence intensities on a
pixel-by-pixel basis; the amount of pixels is shown by a pseudo-color
representation (on an increasing scale from blue to red). The much
higher number of pixels in the upper right quarter of
panel Aiii relative to Biii and Ciii
reflects the high level of co-localization between
GFP-PLC 2 and HA-Rac2(12V), shown in panel Aii
to occur at the rim of the cell (at the plasma
membrane).
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FRAP Studies Demonstrate Enhancement of PLC
2-Plasma
Membrane Interactions by Rac2(12V)--
To characterize the
interactions of GFP-PLC
2 derivatives with the plasma
membrane in live cells, we conducted FRAP studies on
GFP-PLC
2 and GFP-PLC
2
transiently
expressed in COS-7 cells. Because the enzyme is mostly cytosolic, we
have focused the laser beam in these measurements on flat cell regions
near the cell periphery so that the beam illuminated both top and
bottom membranes, and the cytoplasm contribution was relatively low due to the thin cell volume in such regions. The fluorescence recovery of a
purely cytoplasmic protein is expected to occur at a very fast rate,
due to relatively unrestricted diffusion in the cytosol. This rate is
determined by the characteristic fluorescence recovery time
(the
time required to attain half of the recoverable fluorescence intensity
for a Gaussian bleach profile) (31). Indeed, the fluorescence recovery
of unfused GFP (which is cytosolic) occurred at a rate faster than the
experimental time scale (Fig.
4A), resulting in curves
showing immediate recovery with extremely short
. On the other hand,
GFP-PLC
2 fluorescence recovery was significantly slower
and could thus be accurately measured under the same experimental conditions (Fig. 4B). This demonstrates that despite its
mainly cytosolic localization, GFP-PLC
2 experiences
interactions with cellular structures (most likely the plasma membrane;
see below and under "Discussion") that retard its fluorescence
recovery rate (i.e. increase the
value).

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Fig. 4.
Typical FRAP curves demonstrating that
Rac2(12V) reduces the fluorescence recovery rate of
GFP-PLC 2. FRAP experiments
were conducted 24 h after transfection on COS-7 cells transfected
with GFP (A), GFP-PLC 2 (B), or
GFP-PLC 2 together with an excess of Rac2(12V)
(C; see "Experimental Procedures"). The experiments
employed a 63× objective, resulting in a laser beam focused to a
Gaussian radius of 0.85 ± 0.02 µm. The dots
represent the fluorescence intensity; solid lines are the
best fit, derived as described under "Experimental Procedures."
A, unfused GFP exhibits free diffusion in the cytoplasm,
resulting in extremely fast fluorescence recovery. This indicates that
free diffusion in the cytoplasm occurs on a faster time scale and does
not contribute significantly to the measurements depicted in
panels B and C. B,
GFP-PLC 2 fluorescence recovery is significantly slower
than that of free GFP, enabling accurate determination of the
characteristic fluorescence recovery time ; the fluorescence
recovery is nearly complete (mobile fraction of 99%). The
curve was obtained focusing the laser beam on the plasma
membrane in flat cell regions, as done in all FRAP measurements in the
current study; when the beam was focused instead inside the cell, a
fast recovery resembling the cytoplasmic GFP (A) was
obtained. C, co-expression with Rac2(12V) increases of
GFP-PLC 2. The mobile fraction remained high (92% for
the specific curve shown; the average value of many such measurements
was around 97%).
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In view of the effects of activated Rac2 on the cellular localization
of GFP-PLC
2 (Figs. 2 and 3), we examined the effects of
co-expression of GFP-PLC
2 with Rac2(12V) on the
fluorescence recovery rate of GFP-PLC
2. If the higher
of GFP-PLC
2 relative to free cytoplasmic GFP is due
to interactions of the enzyme with the plasma membrane, it should be
further increased by Rac2(12V). As can be seen in Fig. 4 (showing
typical fluorescence recovery curves) and Fig.
5 (depicting the average data from many
such measurements), this indeed was the case. Co-expression with
Rac2(12V) dramatically increased
of GFP-PLC
2
(3.6-fold). On the other hand, co-expressed Rac2(wt) failed to induce
such an effect (Fig. 5). Qualitatively similar results were obtained
with GFP-PLC
2
, whose
value was also elevated
(2.9-fold) by co-expression with Rac2(12V) but not with Rac2(wt) (Fig.
5). The only difference was that the
values of
GFP-PLC
2
were smaller than those of GFP-PLC
2 in the absence of Rac2(12V) (1.7-fold) or in
its presence (2-fold); these shorter fluorescence recovery times are
suggestive of weaker retardation and therefore of weaker interactions
with the plasma membrane. These findings are in accord with the
contribution of C-terminally located basic residues of PLC
isozymes
to association with the membrane via loose interaction with acidic
phospholipids (16, 37). The effect of Rac2(12V) on the fluorescence
recovery rate of GFP-PLC
2 requires the putative PH
domain of PLC
2, and cannot be replaced by the equivalent
region of PLC
1, as demonstrated by the inability of
Rac2(12V) (as well as Rac2(wt)) to alter
of
GFP-PLC
1/PLC
2 (Fig.
6A). The increase in
of
GFP-PLC
2 is not a general effect that can be mediated by
any of the Rho GTPases, including those that may elevate the level of
PtdInsP2, since it was not affected significantly by
RhoA(14V) (Fig. 6B). These results are in accord with the
ability of Rac2(12V) and the inability of Rac2(wt) or RhoA(14V) to
translocate GFP-PLC
2 to the membrane and to mediate its
activation (Figs. 1-3).

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Fig. 5.
Rac2(12V) but not Rac2(wt) reduce the
fluorescence recovery rate of GFP-PLC 2 and
GFP-PLC 2 . The FRAP experiments were performed
with the 63× objective as in the legend to Fig. 4, on COS-7 cells
transfected with GFP-PLC 2 or GFP-PLC 2 ,
alone or together with either Rac2(12V) or Rac2(wt). To ensure that the
great majority of cells expressing GFP-tagged constructs co-express the
Rac2 constructs, the latter were introduced at plasmid concentrations
5.7-fold higher than that of the GFP-PLC 2 constructs.
Each bar is the mean ± S.E. of 30-60 measurements.
The mobile fractions were high throughout (93-100%). Similar results
were obtained using HA-tagged Rac2(12V) or Rac2(wt) in place of the
untagged constructs. The value of GFP-PLC 2
co-expressed with Rac2(12V) was significantly higher than of singly
expressed GFP-PLC 2 (p < 0.0001, Student's t test); so was the case for the effect of
Rac2(12V) on of GFP-PLC 2 (p < 0.0001). The effects of Rac2(wt) on the values of
GFP-PLC 2 or of GFP-PLC 2 were not
statistically significant (p > 0.05). Similar results
were obtained when HA-Rac2(12V) and HA-Rac2(wt) were used in place of
the untagged constructs.
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|

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Fig. 6.
The Rac2(12V)-mediated increase in
of GFP-PLC 2
depends on its putative PH domain and is not mediated by
RhoA(14V). FRAP studies were conducted as in the legend to Fig. 4,
using a 63× objective. Each bar is the mean ± S.E. of
30-60 measurements. The mobile fractions were 93-100% throughout.
HA-tagged constructs of Rac2(wt), Rac2(12V), or RhoA(14V) yielded
similar results. A, COS-7 cells were transfected with
GFP-PLC 1/PLC 2 alone or together with an
excess of Rac2(wt) or Rac2(12V) as in the legend to Fig. 5. Neither
Rac2(wt) nor Rac2(12V) had a significant effect on of the
PLC 2 chimera whose putative PH domain has been swapped
with that of PLC 1 (p > 0.08 in both
cases). B, COS-7 cells were transfected with
GFP-PLC 2, alone or together with an excess of RhoA(14V).
Co-expression with RhoA(14V) had no statistically significant effect on
(p > 0.06).
|
|
Beam Size Analysis Reveals Mixed Exchange and Lateral Diffusion of
GFP-PLC
2 and GFP-PLC
2
at the Plasma
Membrane--
A significant fraction of GFP-PLC
2 or
GFP-PLC
2
is cytoplasmic. Therefore, the enzymes can
interact with the membrane transiently by exchange (binding to and
dissociating from the membrane). Alternatively, they may stably
associate with the membrane, resulting in lateral diffusion and/or
gliding along the inner membrane surface. To evaluate the relative
contributions of these processes, we employed a beam size test (30,
38-40), where the area illuminated by the laser beam in the FRAP
experiment is increased, and the effect of changing the beam size on
the characteristic fluorescence recovery time
is determined. The
two modes of interaction predict highly different effects. For dynamic
exchange with a cytoplasmic pool,
reflects the chemical relaxation
time, which is independent of the beam size (30, 38-40). For lateral
diffusion,
is the characteristic diffusion time
D,
directly proportional to the illuminated area (
D =
2/4D, where
is the Gaussian laser beam
radius, and D is the lateral diffusion coefficient). The
results (Fig. 7) suggest that the fluorescence recovery of both GFP-PLC
2 and
GFP-PLC
2
occurs by a mixture of exchange and lateral
diffusion. The expected ratio between the characteristic fluorescence
recovery times with the two beam sizes employed is 2.56 (the ratio
between the areas illuminated by the two beam sizes) for a process of
pure lateral diffusion, or 1 (no dependence on beam size) for pure
exchange. As is evident from Fig. 7, the ratios obtained are
intermediate, differing significantly either from 1 or from 2.56, strongly supporting the notion that both processes contribute to the
fluorescence recovery.

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Fig. 7.
Beam size dependence of the fluorescence
recovery rates of GFP-PLC 2 and
GFP-PLC 2 indicate a mixed contribution of exchange
and lateral diffusion. The FRAP experiments were conducted as in
the legend to Fig. 4, on COS-7 cells transfected with
GFP-PLC 2 or GFP-PLC 2 . Each bar is the
mean ± S.E. of 30-60 measurements. The mobile fractions were
always high (98-99%). Two beam sizes were generated using a 63×
objective (mean ± S.E. values: Gaussian radius = 0.85 ± 0.02 µm, 2 = 0.72 ± 0.034 µm2; n = 39) or a 40× objective
( = 1.36 ± 0.04 µm, 2 = 1.85 ± 0.11 µm2; n = 39). For both
GFP-PLC 2 derivatives, the ratios between the (40×)
and (63×) values were in between the ratio expected for pure
lateral diffusion (equal to the measured ratio between the
2 values, 1.85/0.72 = 2.56 ± 0.30) and the
value of 1 (independence of the beam size) expected theoretically for
exchange. The differences between the (40×) and (63×) values
were highly significant for either GFP-PLC 2 or
GFP-PLC 2 (p < 0.0001), indicating
that the ratios of (40×)/ (63×) are significantly different from
the value of 1 expected for exchange. For either
GFP-PLC 2 or GFP-PLC 2 , the
(40×)/ (63×) ratio was also significantly different from the
ratio of (2.56 ± 0.30) measured between the two beam sizes
(p < 0.001 in both cases).
|
|
Rac2(12V) Diverts GFP-PLC
2 toward Exchange and
GFP-PLC
2
toward Lateral Diffusion--
To explore
whether constitutively active Rac2 has different effects on the two
modes of GFP-PLC
2 interaction with the membrane, we
performed the beam size test on cells co-expressing
GFP-PLC
2 and Rac2(12V). The results (Fig.
8) clearly demonstrate differential effects of activated Rac2 on the lateral diffusion and exchange of
GFP-PLC
2. In the presence of constitutively active Rac2,
the ratio between the
values of GFP-PLC
2 measured
with the two beam sizes was reduced from 2 to 1.1; the latter ratio is
not significantly different from the value expected for pure exchange (ratio of 1; Fig. 8). When two processes contribute to the fluorescence recovery, the faster will dominate, since once the fluorescence has
recovered in the bleached region, any subsequent events will involve
the replacement of one fluorescent molecule by another fluorescent
molecule. Therefore, the domination by exchange demonstrates that in
the presence of Rac2(12V) the exchange of GFP-PLC
2
between membrane-bound and cytoplasmic pools becomes significantly
faster than its lateral diffusion. However, the fact that the
values measured with both beam sizes are higher than in the absence of Rac2(12V) (Fig. 8) indicates that both processes are slowed down in the
presence of constitutively active Rac2, but the reduction in the
exchange rate is much less than in the lateral diffusion rate.

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Fig. 8.
Rac2(12V) diverts the fluorescence recovery
of GFP-PLC 2 toward exchange and of
GFP-PLC 2 toward lateral diffusion. FRAP studies
employing the beam size test were conducted as in the legend to Fig. 7,
on COS-7 cells expressing GFP-PLC 2 or
GFP-PLC 2 , alone or together with an excess of
Rac2(12V). The constitutively active Rac2 shifted the ratio of
(40×)/ (63×) obtained for GFP-PLC 2 from 2.0 to
1.1. The latter value is essentially similar to the value of 1 expected
for exchange, as indicated by the statistically insignificant
difference between the (40×) and (63×) of
GFP-PLC 2 co-expressed with Rac2(12V) (p > 0.25). An opposite effect (a shift in the ratio from 1.7 to 2.3, close to the value of 2.56 expected for pure lateral diffusion) was
observed for GFP-PLC 2 upon co-expression with
Rac2(12V). In the latter case, the difference between the (40×) and
(63×) was highly significant (p < 0.0001); on the
other hand, the difference between the ratio of (40×)/ (63×) for
GFP-PLC 2 in the presence of Rac2(12V) and the
measured ratio between the areas of the two beam sizes (2.56 ± 0.30, n = 39) was not statistically significant
(p > 0.1), suggesting that in the presence of
Rac2(12V) GFP-PLC 2 recovers essentially as expected
for lateral diffusion.
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|
Similar experiments were performed on GFP-PLC
2
. Fig.
8 shows that this mutant responded very differently to co-expression with Rac2(12V). In the presence of Rac2(12V), the ratio between the
values of GFP-PLC
2
increased from 1.7 to 2.3, opposite to the effect on GFP-PLC
2. The ratio of 2.3 is
very close to and not significantly different from the value expected
for pure lateral diffusion (2.56, the ratio between the areas
illuminated by the two beam sizes employed) (see Fig. 8). This suggests
that in the presence of Rac2(12V), the interaction of
GFP-PLC
2
with the plasma membrane is dominated by
lateral diffusion. Thus, in the presence of Rac2(12V) the lateral
diffusion of GFP-PLC
2
is significantly faster than
its exchange, although the rates of both processes are reduced as
indicated by the increase in the
values of
GFP-PLC
2
measured with both beam sizes (Fig. 8).
 |
DISCUSSION |
PLC
2 plays important roles in several signal
transduction pathways elicited by chemoattractants (41).
Chemoattractant receptors activate pertussis toxin-sensitive
heterotrimeric G proteins and Rho GTPases, eliciting a wide range of
responses in leukocytes (42-45). Because PLC
2 is a
soluble enzyme (46, 47) while its substrate is localized to the plasma
membrane, it is highly likely that activation of PLC
2
requires recruitment to the plasma membrane. Thus, the interactions of
PLC
2 with the membrane and their regulation by
heterotrimeric G protein subunits and/or Rho GTPases are highly important. However, the data on such interactions are lacking and these
processes have not been explored in live cells. In the current study,
we have employed FRAP to investigate the interactions of GFP-tagged
PLC
2 (wild-type and mutants) with the plasma membrane in
live cells and their modulation by the Rho GTPase Rac2. Our studies
demonstrate that the membrane interactions of GFP-PLC
2 and GFP-PLC
2
are characterized by a mixture of
exchange and lateral diffusion. To our knowledge, this is the first
report of such a mixed mechanism for an intracellular protein. These interactions are strongly enhanced by constitutively active Rac2(12V), and the enhancement requires the putative PH domain of
PLC
2. Although Rac2(12V) augments the membrane
interactions of both GFP-PLC
2 and
GFP-PLC
2
, it does so by altering the membrane association of the two PLC
2 derivatives in different
ways. For GFP-PLC
2, Rac2(12V) shifts the dominant
fluorescence recovery mechanism to nearly pure exchange, while for
GFP-PLC
2
lateral diffusion becomes dominant. These
findings suggest that the C-terminal region of PLC
2
(Phe819-Glu1166, which is missing in the
PLC
2
mutant) is required for exchange of
membrane-associated PLC
2 in the presence of activated
Rac2, suggesting a novel function for this region.
An important outcome of this study is the demonstration that both
stimulation and membrane translocation of GFP-PLC
2 and GFP-PLC
2
can be mediated by Rac2(12V) in intact
cells. This indicates that PLC
2 can be activated in live
cells not only by subunits of heterotrimeric G proteins, but also by
the Rho GTPase Rac2. This conclusion is supported by the recent
demonstration (12) that a dominant-inhibitory form of Cdc42 inhibits
Rac GTP-loading and chemoattractant-induced inositol
1,4,5-trisphosphate formation by the endogenous PLC
2 in
human promyelocytic HL-60 cells. Future studies should clarify whether
PLC
2 translocation and stimulation are distinct events.
The specificity of the Rac2-mediated stimulation and membrane
translocation is supported by their strict dependence on the putative
PH domain of PLC
2, as demonstrated by the lack of
response in the GFP-PLC
1/PLC
2 chimeric
construct containing the PH domain of PLC
1. This result
is in accord with the failure of PLC
1 to be activated by
Rho GTPases (36). The current results obtained in cells with the GFP
fusion proteins of PLC
2 and PLC
2
are
in agreement with in vitro studies employing the untagged proteins (36), indicating distinct structural requirements for stimulation of PLC
2 by G protein
q
subunits and Rac2. Moreover, the confocal microscopy studies (Fig. 3)
demonstrate that the translocation of GFP-PLC
2, which is
mediated only by the activated form of Rac2, is to the plasma membrane.
The failure of RhoA(14V) to translocate GFP-PLC
2 to the
plasma membrane is consistent with our previous results on the
inability of this GTPase to activate PLC
2 in
vitro (4), and suggests that this effect is Rac-specific. The fact
that RhoA(14V), which like Rac1 stimulates the activity of
phosphatidylinositol 4-phosphate 5-kinase type I (PIP5K I) (48, 49) did
not enhance the membrane localization of GFP-PLC
2, strongly suggests that the effects of Rac2 are not mediated indirectly by elevation of the level of PtdInsP2 via stimulation of
PIP5K I.
A major fraction of PLC
2 is cytoplasmic (Refs. 46 and
47; see also Figs. 2 and 3). To examine whether free diffusion in the
cytoplasm may interfere with FRAP studies on the interactions of
GFP-PLC
2 with the plasma membrane, we measured the
diffusion of free GFP, which is cytoplasmic. The fluorescence recovery
of this protein occurred on a much faster time scale than that of GFP-PLC
2 derivatives (Fig. 4); it was nearly complete
right after the bleach (Fig. 4A) and did not contribute
significantly to the FRAP measurements on GFP-PLC
2
derivatives. The slower recovery of GFP-PLC
2 and
GFP-PLC
2
suggests that they are retarded by rate-limiting interactions. This slower recovery indicates that such
interactions are manifested at least to some degree already prior to
transfection with activated Rac2. The marked increase in characteristic
fluorescence recovery times (
) of GFP-PLC
2 and
GFP-PLC
2
mediated by Rac2(12V) (Fig. 5) is in accord
with its ability to translocate these proteins to the membrane and to
stimulate their activity (Figs. 1-3), suggesting that the above interactions are with the plasma membrane. This notion is supported by
the observation that when the laser beam is focused inside the cell
rather than on the plasma membrane, the fluorescence recovery of the
GFP-PLC
2 derivatives becomes as fast as that of the
cytosolic GFP. The increase in the
values in the presence of
Rac2(12V) suggests stronger association of GFP-PLC
2 and
GFP-PLC
2
with the membrane; the enhanced association
is likely mediated via binding (direct or indirect) of the
GFP-PLC
2 derivatives to Rac2(12V), which exhibits
enhanced plasma membrane localization relative to Rac2(wt) (Fig. 3,
A and B).
As explained under Results, FRAP studies employing different laser beam
sizes can distinguish between exchange and lateral diffusion (30,
38-40). The interpretation of
depends on the process (chemical
relaxation time for exchange, characteristic diffusion time for lateral
diffusion) (38). Thus, one can compare between the
values of two
proteins only when their fluorescence recovery occurs by the same
process (similar relative contributions of exchange and diffusion for
each protein). Our results clearly demonstrate that the fluorescence
recovery of both GFP-PLC
2 and GFP-PLC
2
occurs by a mixture of exchange and lateral diffusion (Fig. 7). The
(40×)/
(63×) ratios are comparable for the two proteins (Fig.
7), demonstrating that the contribution by exchange and lateral
diffusion for both proteins is comparable. This allows to directly
compare their
values; the shorter
of GFP-PLC
2
indicates that its interactions with the plasma membrane are weaker, suggesting that the C-terminal region of PLC
2
contributes to the membrane association. This is in accord with
previous biochemical observations (16, 37). It is interesting to note
that the mixed mode of membrane interactions found in the current
studies differs from our former observations on the fluorescence
recovery of GFP-tagged Ras proteins, whose fluorescence recovery
occurred solely by lateral diffusion, reflecting stable association
with the plasma membrane (30, 40). To the best of our knowledge, this
is the first demonstration of such a mixed mechanism for fluorescence
recovery of an intracellular protein. The ability to undergo exchange
suggests that the interactions of GFP-PLC
2 and
GFP-PLC
2
with the plasma membrane are transient.
Interestingly, Rac2(12V) has diametrically opposed effects on the mode
of interaction (exchange versus lateral diffusion) of
GFP-PLC
2 and GFP-PLC
2
with the plasma
membrane. Thus, Rac2(12V) shifted the fluorescence recovery of
GFP-PLC
2 toward exchange, while that of
GFP-PLC
2
was diverted toward lateral diffusion (Fig.
8). In the latter case, we could therefore calculate the lateral
diffusion coefficient (D) for GFP-PLC
2
co-expressed with Rac2(12V). The result (D = (1.3 ± 0.2) × 10
8 cm2/s, 22 °C,
n = 46, ×63 objective) was very close to that of the lipid probe DiIC16 in the plasma membrane of the same cells
and under the same conditions (D = (1.0 ± 0.16) × 10
8 cm2/s, n = 20). This value, which is typical for the lateral diffusion of lipid
probes in cell membranes (50, 51), further reinforces the conclusion
that in the presence of Rac2(12V) the fluorescence recovery of
GFP-PLC
2
occurs mainly by lateral diffusion. This does not imply that GFP-PLC
2
does not undergo
exchange; however, its exchange rate must be significantly slower than
the lateral diffusion rate. It is important to note that in the
presence of Rac2(12V) it is not possible to compare directly the
values of GFP-PLC
2 and GFP-PLC
2
,
because their fluorescence recovery now occurs by different mechanisms
(exchange versus lateral diffusion). The domination by
lateral diffusion in the case of GFP-PLC
2
in the
presence of Rac2(12V) (Fig. 8) does not mean that this mutant exhibits
stronger interactions with the membrane as compared with
GFP-PLC
2 under the same conditions. Rather, it indicates that the interactions of these two proteins with the membrane are
different. For example, one possibility is that in the case of
GFP-PLC
2, interactions with the lipid substrate play a
more prominent role, resulting in enhanced dissociation and
reassociation (exchange) after each catalytic cycle.
The differential effects of Rac2(12V) on the membrane interactions of
GFP-PLC
2 and GFP-PLC
2
assign a novel
role to the C-terminal region missing in the GFP-PLC
2
mutant; we propose a role for this region in the Rac2-mediated
regulation of PLC
2 exchange between membrane-bound and
cytoplasmic pools. The C-terminal region of PLC
isozymes was
formerly implicated in the activation of PLC
s by G
q
(3), and was recently shown to mediate dimerization (26). This
capability may be related to the role of this region in facilitating
exchange of PLC
2, as the interactions with the membrane
may be affected by such dimerization. In conclusion, we report here
that constitutively active Rac2 translocates PLC
2 to its
lipid substrate at the plasma membrane, stimulating its enzymatic
activity in live cells; the C-terminal region of PLC
2 plays an important role in these Rac2-mediated effects.