Rac2 Regulation of Phospholipase C-beta 2 Activity and Mode of Membrane Interactions in Intact Cells*

Daria IllenbergerDagger , Claudia WalliserDagger , Joachim StrobelDagger , Orit Gutman§, Hagit Niv§, Verena GaidzikDagger , Yoel Kloog§, Peter GierschikDagger , and Yoav I. Henis§

From the Dagger  Department of Pharmacology and Toxicology, University of Ulm, 89069 Ulm, Germany and the § Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Received for publication, November 25, 2002, and in revised form, December 23, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase C-beta (PLCbeta ) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta 2 isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta 2 with the plasma membrane. For either GFP-PLCbeta 2 or GFP-PLCbeta 2Delta , a C-terminal deletion mutant lacking the region required for stimulation by Galpha q, these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta 2 and GFP-PLCbeta 2Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta 2. Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta 2, but had the opposite effect on GFP-PLCbeta 2Delta , where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta 2 C-terminal region, regulating its exchange between membrane-bound and cytosolic states.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The activity of phospholipase C-beta (PLCbeta )1 enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is stimulated with different orders of efficacy by G protein alpha q subunits and by G protein beta gamma dimers (1-3). In addition, the PLCbeta 2 isozyme is specifically activated in vitro by the Rho GTPases Rac and Cdc42, but not by RhoA (4-6). As for all PLCbeta isozymes, activation by alpha q requires the C-terminal region of PLCbeta 2, and mutants carrying deletions in this region, such as the mutant PLCbeta 2Delta that lacks the Phe819-Glu1166 segment, are resistant to stimulation by alpha q but are susceptible to activation by Rho GTPases and G protein beta gamma subunits (3, 4, 7). Recent studies (4-6) show that beta gamma dimers and Rho GTPases activate PLCbeta 2 by interacting with different regions of the effector enzyme. Thus, the PLCbeta 2 catalytic subdomains X and Y are sufficient for efficient stimulation by beta gamma , whereas the putative pleckstrin homology (PH) domain of PLCbeta 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 PLCbeta 2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLCbeta 2 and Rac1, Rac2 and Cdc42 (8-11). Moreover, in accord with our in vitro studies on PLCbeta 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 PLCbeta 2 activity.

To date, the molecular mechanism of PLCbeta 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 PLCbeta 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 PLCbeta 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 PLCbeta . Here, we investigated the membrane interactions of PLCbeta 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 PLCbeta 2 to its membrane-associated substrate.

To gain more insight into the role of Rac2 in regulating the membrane interactions and activation of PLCbeta 2 in live cells, we have generated green fluorescent protein (GFP)-tagged PLCbeta 2 (GFP-PLCbeta 2) and GFP-PLCbeta 2Delta . 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-PLCbeta 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 PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta and increased their translocation to the membrane. These effects, which suggest enhanced membrane association, specifically required the putative PH domain of PLCbeta 2, which could not be replaced by the equivalent region of GFP-PLCbeta 1. Our studies reveal that the effects of Rac2(12V) on the mode of PLCbeta 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 PLCbeta 2 C-terminal region, known to interact with Galpha q and to be capable of dimerization (26), in a new role- regulating the exchange of PLCbeta 2 after its recruitment to the plasma membrane by activated Rac2.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PLCbeta 2 and the deletion mutant PLCbeta 2Delta , lacking a C-terminal region necessary for stimulation by alpha q subunits (Phe819-Glu1166) were inserted in-frame with GFP into the EcoRI/SalI site of pEGFP-N1 (Clontech) to generate plasmids encoding GFP-PLCbeta 2 and GFP-PLCbeta 2Delta . The cDNA of the PLCbeta 1/PLCbeta 2 chimera was generated by replacing the cDNA sequence encoding the N-terminal amino acids of PLCbeta 2 (residues 1-138) by the corresponding residues of PLCbeta 1 (1-142) using the PCR overlap extension method. The chimeric cDNA was inserted into pEGFP-N1 as described above to generate the GFP-PLCbeta 1/PLCbeta 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-PLCbeta 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-PLCbeta 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-PLCbeta 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-PLCbeta 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 (Galpha R) IgG was from Molecular Probes, and normal goat IgG was from Jackson ImmunoResearch. Peroxidase-conjugated Galpha 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-PLCbeta 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 (tau ), and the mobile fraction were derived by nonlinear regression analysis, fitting to a lateral diffusion process with a single tau  value (34).

Immunofluorescence Microscopy-- Immunofluorescence confocal microscopy was employed to detect the co-localization of GFP-PLCbeta 2 with HA-Rac2(12V) at the plasma membrane. COS-7 cells were co-transfected with GFP-PLCbeta 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-Galpha 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-PLCbeta 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constitutively Active Rac2 Stimulates GFP-PLCbeta 2 and GFP-PLCbeta 2Delta -- To study the effect of wild-type and constitutively active Rac2 on the activity of PLCbeta 2 and on its interactions with the plasma membrane in live cells, we prepared GFP fusion proteins of full-length human PLCbeta 2 and of the C-terminal deletion mutant PLCbeta 2Delta . To confirm that the GFP tag did not impair their activities, both GFP-PLCbeta 2 and GFP-PLCbeta 2Delta were expressed in transiently transfected HEK293 cells, and reconstituted in a cell-free system with G protein beta gamma dimers or GTPgamma 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 beta gamma dimers, or GTPgamma S-activated Rac2 (not shown); these responses were similar to those observed with the untagged enzymes. To investigate whether the activity of GFP-PLCbeta 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-PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta , 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-PLCbeta 2. In contrast, co-expression of Rac2(12V), but not of Rac2(wt), with GFP-PLCbeta 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-PLCbeta 2Delta ; the only difference from PLCbeta 2 was that the Rac2-induced stimulation was somewhat lower (~14-fold) (Fig. 1). The ability of constitutively active Rac to stimulate both GFP-PLCbeta 2 and GFP-PLCbeta 2Delta is in accord with earlier in vitro studies on the untagged forms of these proteins, although the extent of stimulation was higher for PLCbeta 2Delta than for PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta by Rac2(12V) in intact cells. COS-7 cells were transfected with vectors encoding GFP, GFP-PLCbeta 2, GFP-PLCbeta 2Delta , or GFP-PLCbeta 1/PLCbeta 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-PLCbeta 2 (lane 2), GFP-PLCbeta 2Delta (lane 3), or GFP-PLCbeta 1/PLCbeta 2 (lane 4). Aliquots of the four samples were subjected to Western blotting using anti-GFP antibodies.

To further characterize the mechanisms of GFP-PLCbeta 2 activation by Rac2(12V), we employed a GFP-tagged PLCbeta 1/PLCbeta 2 chimera. In this chimera, the N-terminal putative PH domain of PLCbeta 2 (amino acids 1-138) was swapped with the equivalent region of PLCbeta 1 (amino acids 1-142), resulting in an isozyme, which is barely activated by Rho GTPases (36). PLCbeta 1/PLCbeta 2 has recently been shown to be sensitive to stimulation by beta gamma dimers, but not by GTPgamma 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 PLCbeta 2.

Activated Rac2 Increases the Translocation of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta to the Plasma Membrane-- To investigate the mechanism by which Rac2(12V) mediates PLCbeta 2 stimulation, we examined the ability of activated Rac2 to induce translocation of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta from the cytoplasm to the membrane fraction. HEK293 cells were co-transfected with a GFP-PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta 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-PLCbeta 2 or GFP-PLCbeta 2Delta to the particulate fraction (Fig. 2). This effect was not observed when the GFP-PLCbeta 1/PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta to the particulate membrane fraction, and that this effect requires the putative PH domain of PLCbeta 2.


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Fig. 2.   Translocation of GFP-PLCbeta 2 to particulate fractions of HEK293 cells by constitutively active Rac2. HEK293 cells were co-transfected with vectors encoding GFP-PLCbeta 2, GFP-PLCbeta 2Delta , or GFP-PLCbeta 1/PLCbeta 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).

To obtain further support for this notion in whole cells, we performed confocal fluorescence microscopy analysis. Since a large fraction of GFP-PLCbeta 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-PLCbeta 2 with Rac2(12V) allows the identification of membrane translocation of PLCbeta 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-PLCbeta 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-Galpha 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-PLCbeta 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-PLCbeta 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-PLCbeta 2 to the plasma membrane in whole cells.


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Fig. 3.   Confocal fluorescence microscopy demonstrates that GFP-PLCbeta 2 is translocated to the plasma membrane in the presence of Rac2(12V). COS-7 cells were co-transfected with GFP-PLCbeta 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-Galpha 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-PLCbeta 2 and HA-Rac2(12V), shown in panel Aii to occur at the rim of the cell (at the plasma membrane).

FRAP Studies Demonstrate Enhancement of PLCbeta 2-Plasma Membrane Interactions by Rac2(12V)-- To characterize the interactions of GFP-PLCbeta 2 derivatives with the plasma membrane in live cells, we conducted FRAP studies on GFP-PLCbeta 2 and GFP-PLCbeta 2Delta 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 tau  (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 tau . On the other hand, GFP-PLCbeta 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-PLCbeta 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 tau  value).


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Fig. 4.   Typical FRAP curves demonstrating that Rac2(12V) reduces the fluorescence recovery rate of GFP-PLCbeta 2. FRAP experiments were conducted 24 h after transfection on COS-7 cells transfected with GFP (A), GFP-PLCbeta 2 (B), or GFP-PLCbeta 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-PLCbeta 2 fluorescence recovery is significantly slower than that of free GFP, enabling accurate determination of the characteristic fluorescence recovery time tau ; 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 tau  of GFP-PLCbeta 2. The mobile fraction remained high (92% for the specific curve shown; the average value of many such measurements was around 97%).

In view of the effects of activated Rac2 on the cellular localization of GFP-PLCbeta 2 (Figs. 2 and 3), we examined the effects of co-expression of GFP-PLCbeta 2 with Rac2(12V) on the fluorescence recovery rate of GFP-PLCbeta 2. If the higher tau  of GFP-PLCbeta 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 tau  of GFP-PLCbeta 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-PLCbeta 2Delta , whose tau  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 tau  values of GFP-PLCbeta 2Delta were smaller than those of GFP-PLCbeta 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 PLCbeta 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-PLCbeta 2 requires the putative PH domain of PLCbeta 2, and cannot be replaced by the equivalent region of PLCbeta 1, as demonstrated by the inability of Rac2(12V) (as well as Rac2(wt)) to alter tau  of GFP-PLCbeta 1/PLCbeta 2 (Fig. 6A). The increase in tau  of GFP-PLCbeta 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-PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta . The FRAP experiments were performed with the 63× objective as in the legend to Fig. 4, on COS-7 cells transfected with GFP-PLCbeta 2 or GFP-PLCbeta 2Delta , 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-PLCbeta 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 tau  value of GFP-PLCbeta 2 co-expressed with Rac2(12V) was significantly higher than tau  of singly expressed GFP-PLCbeta 2 (p < 0.0001, Student's t test); so was the case for the effect of Rac2(12V) on tau  of GFP-PLCbeta 2Delta (p < 0.0001). The effects of Rac2(wt) on the tau  values of GFP-PLCbeta 2 or of GFP-PLCbeta 2Delta 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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Fig. 6.   The Rac2(12V)-mediated increase in tau  of GFP-PLCbeta 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-PLCbeta 1/PLCbeta 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 tau  of the PLCbeta 2 chimera whose putative PH domain has been swapped with that of PLCbeta 1 (p > 0.08 in both cases). B, COS-7 cells were transfected with GFP-PLCbeta 2, alone or together with an excess of RhoA(14V). Co-expression with RhoA(14V) had no statistically significant effect on tau  (p > 0.06).

Beam Size Analysis Reveals Mixed Exchange and Lateral Diffusion of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta at the Plasma Membrane-- A significant fraction of GFP-PLCbeta 2 or GFP-PLCbeta 2Delta 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 tau  is determined. The two modes of interaction predict highly different effects. For dynamic exchange with a cytoplasmic pool, tau  reflects the chemical relaxation time, which is independent of the beam size (30, 38-40). For lateral diffusion, tau  is the characteristic diffusion time tau D, directly proportional to the illuminated area (tau D = omega 2/4D, where omega  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-PLCbeta 2 and GFP-PLCbeta 2Delta 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-PLCbeta 2 and GFP-PLCbeta 2Delta 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-PLCbeta 2 or GFP-PLCbeta 2Delta . 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 omega  = 0.85 ± 0.02 µm, omega 2 = 0.72 ± 0.034 µm2; n = 39) or a 40× objective (omega  = 1.36 ± 0.04 µm, omega 2 = 1.85 ± 0.11 µm2; n = 39). For both GFP-PLCbeta 2 derivatives, the ratios between the tau (40×) and tau (63×) values were in between the ratio expected for pure lateral diffusion (equal to the measured ratio between the omega 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 tau (40×) and tau (63×) values were highly significant for either GFP-PLCbeta 2 or GFP-PLCbeta 2Delta (p < 0.0001), indicating that the ratios of tau (40×)/tau (63×) are significantly different from the value of 1 expected for exchange. For either GFP-PLCbeta 2 or GFP-PLCbeta 2Delta , the tau (40×)/tau (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-PLCbeta 2 toward Exchange and GFP-PLCbeta 2Delta toward Lateral Diffusion-- To explore whether constitutively active Rac2 has different effects on the two modes of GFP-PLCbeta 2 interaction with the membrane, we performed the beam size test on cells co-expressing GFP-PLCbeta 2 and Rac2(12V). The results (Fig. 8) clearly demonstrate differential effects of activated Rac2 on the lateral diffusion and exchange of GFP-PLCbeta 2. In the presence of constitutively active Rac2, the ratio between the tau  values of GFP-PLCbeta 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-PLCbeta 2 between membrane-bound and cytoplasmic pools becomes significantly faster than its lateral diffusion. However, the fact that the tau  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-PLCbeta 2 toward exchange and of GFP-PLCbeta 2Delta 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-PLCbeta 2 or GFP-PLCbeta 2Delta , alone or together with an excess of Rac2(12V). The constitutively active Rac2 shifted the ratio of tau (40×)/tau (63×) obtained for GFP-PLCbeta 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 tau (40×) and tau (63×) of GFP-PLCbeta 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-PLCbeta 2Delta upon co-expression with Rac2(12V). In the latter case, the difference between the tau (40×) and tau (63×) was highly significant (p < 0.0001); on the other hand, the difference between the ratio of tau (40×)/tau (63×) for GFP-PLCbeta 2Delta 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-PLCbeta 2Delta recovers essentially as expected for lateral diffusion.

Similar experiments were performed on GFP-PLCbeta 2Delta . 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 tau  values of GFP-PLCbeta 2Delta increased from 1.7 to 2.3, opposite to the effect on GFP-PLCbeta 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-PLCbeta 2Delta with the plasma membrane is dominated by lateral diffusion. Thus, in the presence of Rac2(12V) the lateral diffusion of GFP-PLCbeta 2Delta is significantly faster than its exchange, although the rates of both processes are reduced as indicated by the increase in the tau  values of GFP-PLCbeta 2Delta measured with both beam sizes (Fig. 8).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PLCbeta 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 PLCbeta 2 is a soluble enzyme (46, 47) while its substrate is localized to the plasma membrane, it is highly likely that activation of PLCbeta 2 requires recruitment to the plasma membrane. Thus, the interactions of PLCbeta 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 PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta 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 PLCbeta 2. Although Rac2(12V) augments the membrane interactions of both GFP-PLCbeta 2 and GFP-PLCbeta 2Delta , it does so by altering the membrane association of the two PLCbeta 2 derivatives in different ways. For GFP-PLCbeta 2, Rac2(12V) shifts the dominant fluorescence recovery mechanism to nearly pure exchange, while for GFP-PLCbeta 2Delta lateral diffusion becomes dominant. These findings suggest that the C-terminal region of PLCbeta 2 (Phe819-Glu1166, which is missing in the PLCbeta 2Delta mutant) is required for exchange of membrane-associated PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta can be mediated by Rac2(12V) in intact cells. This indicates that PLCbeta 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 PLCbeta 2 in human promyelocytic HL-60 cells. Future studies should clarify whether PLCbeta 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 PLCbeta 2, as demonstrated by the lack of response in the GFP-PLCbeta 1/PLCbeta 2 chimeric construct containing the PH domain of PLCbeta 1. This result is in accord with the failure of PLCbeta 1 to be activated by Rho GTPases (36). The current results obtained in cells with the GFP fusion proteins of PLCbeta 2 and PLCbeta 2Delta are in agreement with in vitro studies employing the untagged proteins (36), indicating distinct structural requirements for stimulation of PLCbeta 2 by G protein alpha q subunits and Rac2. Moreover, the confocal microscopy studies (Fig. 3) demonstrate that the translocation of GFP-PLCbeta 2, which is mediated only by the activated form of Rac2, is to the plasma membrane. The failure of RhoA(14V) to translocate GFP-PLCbeta 2 to the plasma membrane is consistent with our previous results on the inability of this GTPase to activate PLCbeta 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-PLCbeta 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 PLCbeta 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-PLCbeta 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-PLCbeta 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-PLCbeta 2 derivatives. The slower recovery of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta 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 (tau ) of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta 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-PLCbeta 2 derivatives becomes as fast as that of the cytosolic GFP. The increase in the tau  values in the presence of Rac2(12V) suggests stronger association of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta with the membrane; the enhanced association is likely mediated via binding (direct or indirect) of the GFP-PLCbeta 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 tau  depends on the process (chemical relaxation time for exchange, characteristic diffusion time for lateral diffusion) (38). Thus, one can compare between the tau  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-PLCbeta 2 and GFP-PLCbeta 2Delta occurs by a mixture of exchange and lateral diffusion (Fig. 7). The tau (40×)/tau (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 tau  values; the shorter tau  of GFP-PLCbeta 2Delta indicates that its interactions with the plasma membrane are weaker, suggesting that the C-terminal region of PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta with the plasma membrane are transient.

Interestingly, Rac2(12V) has diametrically opposed effects on the mode of interaction (exchange versus lateral diffusion) of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta with the plasma membrane. Thus, Rac2(12V) shifted the fluorescence recovery of GFP-PLCbeta 2 toward exchange, while that of GFP-PLCbeta 2Delta was diverted toward lateral diffusion (Fig. 8). In the latter case, we could therefore calculate the lateral diffusion coefficient (D) for GFP-PLCbeta 2Delta 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-PLCbeta 2Delta occurs mainly by lateral diffusion. This does not imply that GFP-PLCbeta 2Delta 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 tau  values of GFP-PLCbeta 2 and GFP-PLCbeta 2Delta , because their fluorescence recovery now occurs by different mechanisms (exchange versus lateral diffusion). The domination by lateral diffusion in the case of GFP-PLCbeta 2Delta in the presence of Rac2(12V) (Fig. 8) does not mean that this mutant exhibits stronger interactions with the membrane as compared with GFP-PLCbeta 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-PLCbeta 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-PLCbeta 2 and GFP-PLCbeta 2Delta assign a novel role to the C-terminal region missing in the GFP-PLCbeta 2Delta mutant; we propose a role for this region in the Rac2-mediated regulation of PLCbeta 2 exchange between membrane-bound and cytoplasmic pools. The C-terminal region of PLCbeta isozymes was formerly implicated in the activation of PLCbeta s by Galpha 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 PLCbeta 2, as the interactions with the membrane may be affected by such dimerization. In conclusion, we report here that constitutively active Rac2 translocates PLCbeta 2 to its lipid substrate at the plasma membrane, stimulating its enzymatic activity in live cells; the C-terminal region of PLCbeta 2 plays an important role in these Rac2-mediated effects.

    ACKNOWLEDGEMENT

We thank Dr. Alexander Barbul for his invaluable assistance with the confocal microscopy.

    FOOTNOTES

* This work was supported in part by Grant I-599-165.13/98 from the G.I.F., the German-Israeli Foundation for Scientific Research and Development (to Y. I. H. and P. G.) and by Grant SFB497 from the Deutsche Forschungsgemeinschaft (to D. I. and P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 972-3-640-9053; Fax: 972-3-640-7643; E-mail: henis@post.tau.ac.il.

Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211971200

    ABBREVIATIONS

The abbreviations used are: PLCbeta , phospholipase C-beta ; D, lateral diffusion coefficient; FRAP, fluorescence recovery after photobleaching; Galpha R, goat anti-rabbit IgG; GDI, guanine nucleotide dissociation inhibitor; GFP, green fluorescent protein; HA, influenza hemagglutinin; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; tau , apparent characteristic fluorescence recovery time; wt, wild-type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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