Rac-mediated Stimulation of Phospholipase Cγ₂ Amplifies B Cell Receptor-induced Calcium Signaling^,

Background: Phospholipase Cγ₂ (PLCγ₂) is stimulated by Rac GTPases through direct protein-protein interaction.

Results: The Rac-PLCγ₂ interaction markedly enhances B cell-receptor-mediated Ca²⁺ mobilization and nuclear translocation of the Ca²⁺-regulated transcription factor NFAT in B cells.

Conclusion: Rac-mediated stimulation of PLCγ₂ activity amplifies B cell receptor-induced Ca²⁺ signaling.

Significance: A specific Rac-resistant PLCγ₂ variant is used to determine the physiological cell signaling relevance of a functional Rac-PLCγ₂ interaction in an appropriate cellular context.

Abstract

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca²⁺. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ₂ (PLCγ₂), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ₂ to functionally reconstitute cultured PLCγ₂-deficient DT40 B cells and to examine the effects of the Rac-PLCγ₂ interaction on BCR-mediated changes of intracellular Ca²⁺ and regulation of Ca²⁺-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ₂ interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca²⁺ release from intracellular stores; (iii) Ca²⁺ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca²⁺-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ₂ activity serves to amplify B cell receptor-induced Ca²⁺ signaling.

Introduction

Inositol phospholipid-specific phospholipases C (PLC)⁴ catalyze the formation of inositol 1,4,5-trisphosphate (InsP₃) and diacylglycerol (DAG) from plasma membrane lipid substrate phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) (1). Both the rise of the former two and the decline of the latter may serve as intracellular signals to regulate a myriad of cell functions (2). In B lymphocytes, receptors for cell surface immunoglobulins such as the B cell receptors (BCR), cleavage fragments of the third complement component (CD19/CD21) (3), bacterial, viral, or autoimmunity host DNA (toll-like receptors) (4), and even certain G-protein-coupled chemokine receptors (5) mediate activation of PLCγ₂, one of the two human PLCγ isoforms. The activity of PLCγ₂ controls many B cell functions, such as protein kinase signaling, nucleocytoplasmic trafficking of transcription factors, proliferation, differentiation, cytoskeletal reorganization, cell adhesion and migration, immunological synapse formation, affinity maturation, autoimmunity, homing to and retention in tissue microenvironments, survival, and susceptibility to transformation (6, 7).

Inactivation of the PLCγ₂ gene in the mouse caused specific defects in most cell types of hematopoietic origin, except for T cells (8, 9). Mice lacking PLCγ₂ showed reduced numbers of mature conventional B cells, a block in pro-B cell differentiation, B1 B cell deficiency, absence of IgM receptor-mediated Ca²⁺ responses, and B cell-mitogen-induced cell proliferation. PLCγ₂ also plays important roles in pre-BCR-mediated early B cell development, in BAFF receptor-mediated survival, and in activation of light-chain loci for recombination as well as receptor editing of self-reactive B cells (10–12). Mutationally activated forms of PLCγ₂ have been identified in mice subjected to N-ethyl-N-nitrosourea mutagenesis (Ali5 and Ali14) and, more recently, in patients with inherited forms of autoinflammation and immunodeficiency (13–16). These defects also lead to deregulation of B cell functions.

Several of the changes described for PLCγ₂^−/− B cells, e.g. defective Ca²⁺ signaling, failure to proliferate in response to immunoglobulin receptor stimulation, impediment of B cell development, and failure to mount humoral responses to TD and TI antigens, were also observed in mice carrying deletions in all three genes encoding Vav guanine nucleotide exchange factors of Rho GTPases, Vav1, -2, and -3 (17). These results were difficult to interpret mechanistically because Vav proteins elicit both RhoGEF-dependent and -independent effects (18). However, some of the B cell defects were also observed in mice lacking either Rac2 (19) or both Rac1 and Rac2 (20), including a reduced ability of BCR or CD19 (co)ligation to increase [Ca²⁺]_i, suggesting that at least some of the B cell defects commonly observed in PLCγ₂ and Vav1/2/3-null mice were due to loss of Rac activation. At that time, the available evidence suggested that Rac GTPases might activate PLCγ₂ indirectly by enhancing the activity of phosphatidylinositol 4-phosphate 5-kinase (21), thus increasing the level of PtdInsP₂, the substrate of both PLCγ₂ and phosphoinositide 3-kinase (20). Enhanced availability of substrate to the former and enhanced formation of PtdInsP₃ by the latter were expected to activate PLCγ₂ (22).

We have previously shown that Rac GTPases, but not Cdc42 or RhoA, activate PLCγ₂, but not PLCγ₁, by direct protein-protein interaction (23). Neither enhanced formation of PtdInsP₂ nor PtdInsP₃ nor protein tyrosine phosphorylation are involved in this effect. Unlike activation of PLCβ₂, which is mediated by Rac interacting with the N-terminal PH domain of this effector, activation of PLCγ₂ involves binding of Rac to the bipartite, split PH domain (spPH) juxtaposed between the two halves, X and Y, of the PLCγ₂ catalytic domain (24). The three-dimensional structures of the heterodimeric complex between PLCγ₂ spPH and GTPγS-activated Rac2, monomeric spPH, and monomeric Rac2 liganded with either GTPγS or GDP allowed us to elucidate the conformational changes that accompany the formation of the signaling active PLCγ₂-spPH/Rac2 heterodimer (25). A residue unique for spPH of PLCγ₂, but not PLCγ₁, Phe-897, was found to be particularly important for the functional and structural PLCγ₂-spPH/Rac2 interaction. Replacement of Phe-897 to the corresponding glutamine residue of PLCγ₁, F897Q, did not affect the overall three-dimensional structure of the PLCγ₂ spPH domain, but it specifically blocked the interaction of the mutant domain with activated Rac2 (24, 25).

Rac GTPases are present at many intracellular crossroads of B cell signaling. They receive inputs from numerous cell surface receptors to regulate and integrate a host of intracellular signaling proteins, including PLCγ₂ and many proteins involved in cytoskeletal organization (26). This has made it intrinsically difficult to judge the pertinence of the PLCγ₂-Rac interaction observed in cell-free experiments and overexpression studies to cell signaling in more physiologically relevant cellular contexts. In this work, we have used DT40 B cells genetically deficient in PLCγ₂ for functional reconstitution with the Rac-resistant PLCγ₂ mutant F897Q to study the effects of a specific loss of the PLCγ₂-Rac interaction on BCR-mediated cell signaling. The results reveal that loss of this functional interaction causes a marked decrease of BCR-mediated Ca²⁺ release from intracellular stores, Ca²⁺ entry from the extracellular compartment, and nuclear translocation of the Ca²⁺-regulated transcription factor NFAT. Some of these changes have previously been observed in Rac-deficient mice (19, 20). Hence, the results may provide a mechanistic explanation for findings on the role of Rac GTPases in B cell signaling obtained in vivo that have as yet remained largely unexplained. In addition, these insights into BCR-mediated cell signaling may also apply to the mechanisms of action of other B cell receptors such as CD19/CD21, to other cells of hematopoietic origin, e.g. platelets, and to human diseases, such as certain immunodeficiencies. To our knowledge, this is the first time that a Rho-resistant but otherwise normal Rho effector was reintroduced into a genetically Rho effector-deficient background to determine the relevance of the functional Rho effector interaction in a biologically highly relevant context.

Experimental Procedures

Antibodies and Reagents

Mouse monoclonal antibody reactive against the c-Myc epitope (9B11, catalogue no. 2276) was from Cell Signaling. Mouse monoclonal antibody reactive against β-actin (AC-15, catalogue no. A3854), poly-l-lysine (catalogue no. P6282), and ionomycin (catalogue no. I-0634) was from Sigma. Anti-phosphotyrosine antibody (catalogue no. 05-321, 4G10) was purchased from Millipore. Mouse anti-chicken IgM (M-4, catalogue no. 8300-01) was obtained from SouthernBiotech. Alexa Fluor® 488 goat anti-mouse antibody (catalogue no. A-11029), fluo-4 acetoxymethyl ester (catalogue no. F-14201), Pluronic®-F127 (catalogue number P-3000MP), and thapsigargin (catalogue no. T-7459) were from Molecular Probes® (Life Technologies, Inc.). Trypsin (catalogue no. 1418475001) was from Roche Applied Sciences, and puromycin was from InvivoGen.

cDNA Cloning

Because the 5′ end of the mRNA encoding chicken PLCγ₂ was unknown at the time, 5′ rapid amplification of cDNA ends (27) was used to gather this information and produce full-length PLCγ₂ cDNAs from reverse-transcribed DT40 cell mRNA. Two presumably allelic variants were found, which are identical at the protein level to each other and to database entry XP_414166, except for a Gln to His divergence at position 865. Based on the higher frequency (7/10) of His-865 among PCR products of DT40 cell mRNA, this haplotype was used herein for further studies. A histidine is present at this position in PLCγ₂ of numerous species ranging from fish, such as coelacanth, to mammals, such as cattle or sheep. The plasmid NFAT1c-td-RFP611 encodes amino acids 1–400 of mouse NFAT1c fused to a pseudo-monomeric tandem dimer red fluorescent protein, td-RFP611 (28).

Expression Constructs and Reconstitution of PLCγ₂^−/− DT40 B Cells with Wild-type or F897Q Mutant PLCγ₂

The F897Q variant of chicken PLCγ₂ was created by site-directed mutagenesis using the primers 5′-GCAACTGATAAAGTAGAAGAACTGCAGGAATGGTACCAAAGTGTCCGTGAA-3′ (sense) and 5′-TTCACGGACACTTTGGTACCATTCCTGCAGTTCTTCTACTTTATCAGTTGC-3′ (antisense). The chicken PLCγ₂ deletion mutants PLCγ₂ΔPCI and PLCγ₂ΔPCI^F897Q lacking the phospholipase C inhibitor (PCI) peptide (amino acids 727–734) were constructed by site-directed mutagenesis using the primers 5′-GAGAAGCACCCGCTGCCTGTGACTGAGGAGC-3′ (sense) and 5′-GCTCCTCAGTCACAGGCAGCGGGTGCTTCTC-3′ (antisense). For expression in COS-7 cells, the cDNAs of C-terminally c-Myc epitope-tagged wild-type (WT) PLCγ₂, PLCγ₂^F897Q, PLCγ₂ΔPCI, and PLCγ₂ΔPCI^F897Q were ligated into the BamHI/NotI site of pcDNA3.1(+). For production of recombinant baculoviruses, the cDNAs of c-Myc epitope-tagged PLCγ₂ and PLCγ₂^F897Q were ligated into the BamHI/NotI site of pVL1393. To create stably transfected DT40 cell clones, cDNAs encoding c-Myc epitope-tagged PLCγ₂ and PLCγ₂^F897Q were ligated into the BglII/SmaI site of the expression vector pExpress, in which the expression of a cloned cDNA is controlled by the chicken β-actin promoter and an SV40 poly(A) signal (29). The pExpress cDNA expression cassette was then combined with the vector pLoxPuro by using the restriction site SpeI generating an expression vector, in which only the selectable marker is flanked by mutant loxP sites (29). For electroporation, the plasmids PLCγ₂-pLoxPuro and PLCγ₂^F897Q-pLoxPuro were linearized by the restriction enzyme NotI, precipitated, and resuspended in distilled water to a final concentration of 1 μg/μl. To obtain nontargeted integration of the constructs into the genome of DT40 B cells, 20–30 μg of linearized plasmid DNA were introduced into 20 × 10⁶ PLCγ₂^−/− DT40 cells by electroporation at 250 V and 950 microfarads resulting in a time constant of 22 ms. For selection, the cells were incubated for 11 days at 37 °C and 10% CO₂ in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated (56 °C, 30 min) fetal calf serum (FCS), 1% (v/v) heat-inactivated chicken serum (catalogue no. C5405, Sigma), 50 μm β mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin (henceforth referred to as supplements) and 0.5 μg/ml puromycin. For FRAP experiments, human PLCγ₂ fused C-terminally to enhanced green fluorescent protein (GFP) was constructed by inserting the PLCγ₂ cDNA into the KpnI site of pEGFPN3 (Clontech). The F897Q variant of human PLCγ₂-pEGFPN3 was produced by site-directed mutagenesis using the primers 5′-GACAGGGTGGAGGAGCTCCAGGAGTGGTTTCAGAGCATC-3′ (sense) and 5′-GATGCTCTGAAACCACTCCTGGAGCTCCTCCACCCTGTC-3′ (antisense).

Cell Culture and Analysis of Inositol Phosphate Formation

Chicken WT (RCB1464) and PLCγ₂-deficient (PLCγ₂^−/−; RCB1469) DT40 B cells (all from Cell Bank, RIKEN BioResource Center, Japan) were cultured at 37 °C in a humidified atmosphere of 90% air and 10% CO₂ in RPMI 1640 medium containing supplements. DT40 B cells stably reconstituted with either WT or mutant PLCγ₂ were cultured in RPMI 1640 medium containing supplements and 0.5 μg/ml puromycin. COS-7 cells were maintained at 37 °C in a humidified atmosphere of 90% air and 10% CO₂ in Dulbecco's modified Eagle's medium (Invitrogen, catalogue no. 41965-039) supplemented with 10% (v/v) FCS (Invitrogen, catalogue no. 10270-106), 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from PAA Laboratories). Transfection of COS-7 cells and analysis of inositol phosphate formation to determine PLC activity were carried out as described previously (24).

Preparation of DT40 Cell Lysates

DT40 cells (15 × 10⁶) were lysed in 240 μl of ice-cold buffer A containing 20 mm Tris/HCl, pH 7.5, 2 mm EDTA, 2 μg/ml soybean trypsin inhibitor, 3 mm benzamidine, 0.1 mm PMSF, 1 μm pepstatin, 1 μm leupeptin, and 1 μg/ml aprotinin by forcing the suspension eight times through a 0.45 × 25-mm needle attached to a disposable syringe. Nuclei and unbroken cells were removed from the cell lysate by centrifugation at 300 × g for 10 min at 4 °C. Fifty μg of cell lysate protein were subjected to SDS-PAGE, and immunoblotting was performed using antibodies reactive against the c-Myc epitope and β-actin.

Expression of Proteins in Sf9 Cells and Measurement of PLC Activity in Vitro

The production of crude preparations of isoprenylated, recombinant human Rac2 and chicken PLCγ₂ in Sf9 insect cells and the determination of PLC activity in vitro were carried out as described previously (24).

Determination of the Intracellular Calcium Concentration

To monitor the changes in [Ca²⁺]_i in individual DT40 B cells in real time, 3 × 10⁵ DT40 B cells per channel were seeded into six channel microscopy slides (uncoated ibidi® μ-slide VI^0.4, ibidi, catalogue no. 80601), which had been coated with poly-l-lysine (0.01% (w/v)), and incubated for 45 min at 37 °C and 10% CO₂ to allow for adhesion to the substrate. Next, unbound cells were flushed out with the culture medium, and the adherent cells were loaded with 2 μm fluo-4 AM, 0.02% (v/v) Pluronic® F-127, by incubation at room temperature for 30 min in RPMI 1640 medium containing supplements. Subsequently, cells were washed twice with buffer B (20 mm HEPES/NaOH, pH 7.4, 143 mm NaCl, 6 mm KCl, 1 mm MgSO₄, 5.6 mm glucose). To discriminate between mobilization of Ca²⁺ from intra- and extracellular sources, BCR stimulation was either performed in buffer B or in buffer B supplemented with 1 mm CaCl₂. The experiments were performed at room temperature.

Spinning Disc Confocal Fluorescence Microscopy

Fluorescence images were taken with the acquisition software Andor iQ 1.6 using a spinning disk confocal microscope assembled in our laboratory from individual components as described earlier (30). Briefly, its main components are a CSU10 scan head (Yokogawa, Tokyo, Japan), an inverted microscope (Axio Observer, Zeiss) with oil immersion objective (UPlanSApo 60×/1.35 NA, Olympus), environmental control system (PeCon), an image splitter unit (OptoSplit II, Cairn Research), and an EMCCD camera (DV-887, Andor). Diode-pumped solid-state lasers were employed for fluorescence excitation at 473 nm (HB-Laser LSR473-100) and 532 nm (HB-Laser LSR532-250). Time-lapse confocal fluorescence images consisting of 512 × 512 pixels were acquired in a field of 1024 × 1024 μm² with a depth resolution of ∼2 μm, time resolution of 1.5 frames/s, and exposure time for each frame of 400 ms. The 473 nm excitation laser power measured at the optical fiber output to the spinning disc module was set to ∼4 milliwatts throughout the study.

Quantitative Analysis of the Ca²⁺ Responses

The fluorescence intensity fluctuations of the Ca²⁺ -sensitive dye fluo-4 were baseline-corrected and normalized according to the maximal intensity in the presence of ionomycin to account for the differences in fluorescent dye loading between individual cells (Fig. 4A and supplemental Video S1). Then, baseline-corrected and normalized Ca²⁺ response traces were subjected to further analysis using algorithms developed in-house and implemented in MATLAB (MathWorks). A Ca²⁺ response peak was defined by a transient positive divergence from the baseline with a minimum of 10% in intensity of the maximum observed in the presence of ionomycin. The number of responding cells was calculated as the number of traces with at least one Ca²⁺ response peak and expressed as the percentage of the total number of traces in each experiment. Latency was defined as a mean time lapse between the time of ligand addition and the time corresponding to the maximum of the first Ca²⁺ response peak. Cells that did not show any detectable Ca²⁺ spike during the initial observation time for the release of Ca²⁺ from internal stores, 365.1 s, were considered to display maximal latency equal to the time of observation. The integrated intensity was calculated as the mean area under the curve of all individual traces corresponding to single cells. The peak frequency was calculated for cells showing discrete Ca²⁺ spikes with baseline resolution, i.e. after addition of anti-IgM at concentrations of 4, 40, and 400 ng/ml, and expressed in millihertz (10⁻³ Hz). The same cells were used to calculate the peak amplitude as the average intensity of the Ca²⁺ peaks in percentage of the ionomycin maximum. The integrated intensities, peak frequencies, and peak amplitudes of nonresponding cells were set to zero.

FIGURE 4.

Functional reconstitution of B cell receptor-mediated intracellular Ca²⁺ release and extracellular Ca²⁺ entry into PLCγ₂^−/− DT40 B cells. A, normalization of BCR-mediated oscillatory changes in cytosolic Ca²⁺ in PLCγ₂^−/− DT40 cells stably expressing wild-type PLCγ₂. Left panel, the mean fluorescence intensities monitored inside the three cells selected in supplemental Video S1 and marked in cyan, magenta, and blue were measured on a single-cell level using spinning disc confocal fluorescence microscopy. The right panel shows the same data after correction for basal fluorescence intensity and normalization according to the maximal intensity in the presence of ionomycin. This treatment corrects variations in fluorescent dye loading between cells. The arrowheads indicate the addition of the various extracellular reagents. B, qualitative analysis. Unmodified DT40 cells (panel a; PLCγ₂^+/+), PLCγ₂^−/− cells (panel b), PLCγ₂^−/− cells stably expressing wild-type (panel c), or F897Q mutant PLCγ₂ (panel d) (60 cells each) were treated as indicated by arrowheads in the following sequences: 40 ng/ml anti-IgM; no Ca²⁺ → 40 ng/ml anti-IgM; 1 mm Ca²⁺ → 4 μm ionomycin; 1 mm Ca²⁺ in panels a, c, and d, and 400 ng/ml anti-IgM; no Ca²⁺ → 400 ng/ml anti-IgM; 1 mm Ca²⁺ → 4 ng/ml trypsin; 1 mm Ca²⁺ → 4 μm ionomycin; 1 mm Ca²⁺ in panel b. Insets, snapshots from the corresponding time-lapse image series. C, expression of wild-type PLCγ₂ in PLCγ₂^−/− DT40 cells restores the quantitative features of B cell-receptor-mediated increases in cytosolic Ca²⁺ to the phenotype observed in wild-type DT40 cells. Top panels, unmodified DT40 cells (PLCγ₂^+/+) or PLCγ₂^−/− DT40 cells stably expressing wild-type PLCγ₂ (PLCγ₂^−/− + WT) were sequentially treated as in Fig. 4B, panel a, except that anti-IgM was used at 400 ng/ml. Bottom panels, the intensity traces obtained for both groups of cells were quantitatively analyzed as indicated for the percentage of responding cells, integrated peak intensity, peak frequency, and peak amplitude. A total number of 213 and 196 cells, respectively, was analyzed in three independent experiments (n = 3). D, PLCγ₂^−/− DT40 cells stably expressing wild-type or F897Q mutant PLCγ₂ show similar Ca²⁺ responses to thapsigargin. PLCγ₂^−/− cells stably expressing wild-type (left panel) or F897Q mutant PLCγ₂ (right panel) (78 cells each) were treated at the times indicated by the arrowheads in the following sequence: 100 nm thapsigargin; no Ca²⁺ → 100 nm thapsigargin; 1 mm Ca²⁺ → 4 μm ionomycin; 1 mm Ca²⁺.

Analysis of Nuclear NFAT1c Translocation

To study the BCR-mediated, Ca²⁺-dependent translocation of the transcription factor NFAT into the nucleus, 30 μg of cDNA encoding NFAT1c-td-RFP611 was transferred into 10⁷ PLCγ₂^−/− DT40 B cells per cuvette that had or had not been reconstituted with either WT or F897Q mutant PLCγ₂ by nucleofection (Amaxa® Nucleofector® Technology, Cell Line Nucleofector® kit T, program B-023). Forty eight h after nucleofection, 6 × 10⁵ DT40 B cells per channel were allowed to adhere to poly-l-lysine-coated ibidi® 6-channel microscopy slides. After two washing steps with buffer B, the cells were treated for 60 min with either 40 μg/ml of anti-IgM or 2 μm ionomycin in buffer B containing 1 mm Ca²⁺. The translocation of NFAT1c-td-RFP611 was analyzed at room temperature on a single cell level by spinning disc confocal microscopy over a time period of 60 min using a 532-nm excitation laser for fluorescence imaging.

Indirect Immunofluorescence Staining

For immunofluorescence staining, 3 × 10⁵ DT40 cells per channel were allowed to adhere to tissue culture-treated 6-channel microscopy slides (ibiTreat, ibidi® μ-slide VI^0.4, ibidi, catalogue no. 80606,) as described above. The cells were then fixed by incubation for 15 min in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. After washing the cells twice with PBS, the nonreacted aldehyde groups were quenched by treatment for 10 min with 50 mm NH₄Cl in PBS. The cells were then washed four times with PBS, and nonspecific protein-binding sites in the channels were blocked by incubation for 1 h with buffer C (PBS, pH 7.4, 5% (v/v) FCS, 0.02% (w/v) sodium azide, 0.01% (w/v) saponin, 0.1% (v/v) Triton X-100). The cells were then incubated for 45 min with buffer C containing the primary antibody reactive against c-Myc epitope (1:2000). After four washing steps with buffer D (PBS, pH 7.4, 0.01% (w/v) saponin, 0.1% (v/v) Triton X-100), the cells were incubated for 45 min with buffer C containing the secondary goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1:1000). After four further washing steps with buffer D, the expression of PLCγ₂ protein was analyzed on a single cell level by spinning disk confocal microscopy employing a 473-nm excitation laser for fluorescence and a halogen lamp for transmission light imaging. All steps were done at room temperature.

Analysis of PLCγ₂ Phosphorylation by Immunoprecipitation

H₂O₂ enhances tyrosine phosphorylation of PLCγ₂ mediated by the tyrosine kinases Syk and Btk (31). Aliquots (12 × 10⁶ each) of PLCγ₂- or PLCγ₂^F897Q-expressing DT40 cells were treated for 5 min with 2 mm H₂O₂ in 400 μl of PBS, pH 7.4, at 37 °C in a water bath. After addition of 4 ml of ice-cold PBS and centrifugation at 300 × g for 5 min, the pellet was resuspended in 400 μl of ice-cold lysis buffer (10 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, 0.2 mm Na₃VO₄, phosphatase inhibitor mixture 2 (1:100; v/v) (Sigma), 0.1 mm PMSF, 10 μm leupeptin, 2 μm pepstatin, 2 μg/ml soybean trypsin inhibitor, 1 μg/ml aprotinin, 3 mm benzamidine, 1 mm DTT, 16 μg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, and 16 μg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone), and the cells were lysed by incubation for 30 min at 4 °C. After centrifugation at 100,000 × g for 1 h at 4 °C, aliquots of the supernatants (1 mg of protein each) were added to anti-c-Myc antibody-protein A complexes, which had been prepared by preincubation of anti-c-Myc antibody (1:1000 (v/v) in 500 μl of PBS) with 30 μl of protein A-agarose beads (50% (v/v) slurry) overnight at 4 °C and three washes with lysis buffer. After incubation for 2 h at 4 °C, the beads were washed three times with lysis buffer and once with PBS and then subjected to SDS-PAGE and Western blotting using anti-phosphotyrosine or anti-c-Myc antibody (1:1000 (v/v) each).

FRAP Experiments

FRAP studies (32, 33) were conducted as described earlier (34). The experiments were performed 24–26 h post-transfection on COS-7 cells transfected with PLCγ₂-GFP derivatives. All experiments were conducted at 22 °C, in Hanks' balanced salt solution supplemented with 20 mm HEPES, pH 7.2. The monitoring argon ion laser beam (488 nm, 1.2 microwatts; Innova 70C, Coherent) was focused through the microscope (AxioImager.D1, Carl Zeiss MicroImaging) to a Gaussian spot with a radius ω = 0.77 ± 0.03 μm (×63/1.4 NA oil-immersion objective) or 1.17 ± 0.05 μm (×40/1.2 NA water immersion objective). Experiments were conducted with each beam size (beam size analysis is described in Refs. 35, 36). The ratio between the illuminated areas (ω²(×40)/ω²(×63)) was 2.28 ± 0.17 (n = 59). After a brief measurement at the monitoring intensity, a 5-milliwatt pulse (4–6 ms or 10–20 ms for the ×63 and ×40 objectives, respectively) bleached 50–70% of the fluorescence in the spot. Fluorescence recovery was followed by the monitoring beam. The apparent characteristic fluorescence recovery time (τ) and the mobile fraction (R_f) were derived from the FRAP curves by nonlinear regression analysis, fitting to a lateral diffusion process with a single τ value (37). The significance of differences between τ values measured with the same beam size was evaluated by Student's t test. To compare ratio measurements (τ(×40)/τ(×63) and ω²(×40)/ω²(×63)), we employed bootstrap analysis, which is preferable for comparison between ratios (38), as described by us earlier (36), using 1000 bootstrap samples.

Miscellaneous

SDS-PAGE and immunoblotting were performed according to standard protocols using antibodies reactive against the c-Myc epitope for wild-type and mutant PLCγ₂. Immunoreactive proteins were visualized using the ECL Western blotting detection system (GE Healthcare). All experiments were performed at least three times. Similar results and identical trends were obtained each time. Data from representative experiments are shown as means ± S.E. of triplicate determinations, if not stated otherwise in the figure legends. Unless stated otherwise, the significance of differences was assessed by using either the unpaired t test with two-tailed p values or repeated measures analysis of variance with Tukey's post test (Fig. 7B), both contained in GraphPad InStat®, version 3.10. Statistically significant effects are denoted by ***, p < 0.001; **, 0.001 < p < 0.01; and *, 0.01 < p < 0.05. Nonsignificant (ns) changes are denoted by ns, 0.05 < p. In Figs. 1, D and E, and 6, A–C, the data were fitted by nonlinear least squares curve fitting to three or four parameter dose-response equations using GraphPad Prism®, version 5.04. In certain cases, the global curve fitting procedure contained in Prism® was used to determine whether the best fit values of selected parameters differed between data sets. The simpler model was selected unless the extra sum of squares F-test had a p value of less than 0.05. In Fig. 6A, right panel, the data were fitted to the equation of a bell-shaped dose response curve provided by Prism®, with manual adjustments.

FIGURE 7.

BCR-induced translocation of the transcription factor NFAT1c into the nucleus is dependent on a functional interaction between PLCγ₂ and Rac. A, PLCγ₂^−/− cells or PLCγ₂^−/− cells stably expressing either wild-type or F897Q mutant PLCγ₂ were transiently transfected with a vector encoding NFAT1c-td-RFP611. Adherent cells were treated with either anti-IgM (upper panels) or ionomycin (lower panels). Snapshots were taken of the same visual fields at the beginning of the experiment in the absence of ligand (−) and at the end of each experiment in the presence of the stimulus (+).Examples of visible NFAT1c translocation events are marked by arrowheads. B, quantitative analysis of the data shown. ns, not significant.

FIGURE 1.

PLCγ₂ mutant F897Q is resistant to stimulation by activated Rac2 in intact cells and in a cell-free system. A, changes in the conformations of the PLCγ₂-interacting surface of Rac2 upon activation of Rac2 with GTPγS (gray) and of the PLCγ₂ spPH residue Phe-897 upon interaction with activated Rac2. The hydrophobic Rac2 residues that change their positions upon Rac2 activation and interact with PLCγ₂ spPH are shown in white (GDP-liganded Rac2) versus red (GTPγS-liganded Rac2). Phe-897 displays flexibility in the known structures of the free spPH domain and adopts two conformations (I and II, pale yellow) (25). This flexibility is stabilized in the complex with activated Rac2 (wheat). The side chain of the polar residue Gln in the F897Q mutant (light blue) is likely to collide with the hydrophobic side chain of Rac2 Leu-67. The structures correspond to database entries 2W2T, 2W2V, 2W2W, and 2W2X (25). The position of Gln-897 was predicted in Swiss-Model. B, COS-7 cells were cotransfected with 1 μg/well of vector encoding either WT PLCγ₂ (WT) or its F897Q mutant (F897Q) together with either empty vector (Mock) or with 10 ng/well of vector encoding Rac2 (Rac2) or its constitutively active mutant Rac2^G12V (Rac2^G12V). Inositol phosphate formation was measured as described under “Experimental Procedures” (upper panel). Cells from one well each were analyzed by SDS-PAGE and immunoblotting (lower panel). C, left panel, soluble fractions of Sf9 cells infected with baculoviruses encoding β-galactosidase (Control), WT PLCγ₂ (WT), or its Phe-897 mutant (F897Q) were incubated with phospholipid vesicles containing [³H]PtdInsP₂. Right panel, soluble fractions of baculovirus-infected Sf9 cells were adjusted to contain similar basal PLC activity according to the left panel. Aliquots (10 μl) of these samples were reconstituted with aliquots of detergent extracts prepared from membranes of Sf9 cells infected with baculoviruses encoding either β-galactosidase or Rac2 and incubated in the presence of 100 μm GDP or GTPγS with phospholipid vesicles containing [³H]PtdInsP₂. Inset, aliquots (10 μl) of the samples were subjected to SDS-PAGE and immunoblotting. Lane 1, control; lane 2, WT, lane 3, F897Q. D, wild-type and F897Q mutant PLCγ₂ are similarly sensitive to activation by Ca²⁺. Aliquots of the soluble fractions of baculovirus-infected Sf9 cells were incubated at increasing concentrations of free Ca²⁺ with phospholipid vesicles containing [³H]PtdInsP₂. For both wild-type and F897Q mutant PLCγ₂, half-maximal and maximal stimulatory effects were observed at ∼140 nm and 10 μm free Ca²⁺. E, PLCγ₂ variants ΔPCI^WT and ΔPCI^F897Q lacking the PCI peptide have similar constitutive activities but show a striking difference in their sensitivity to Rac2. Tyrosine phosphorylation-induced activation of PLCγ is mediated by removal of intramolecular autoinhibition caused by an octapeptide (⁷²⁶YRKMRLRY in human PLCγ₂; PCI) within the C-terminal Src homology domain contained in the Src homology domain tandem between the two catalytic subdomains X and Y. Deletion of the PCI peptide causes activation of PLCγ₂ (88). Upper left panel, COS-7 cells were transfected with increasing amounts of vector encoding ΔPCI^WT or ΔPCI^F897Q or with a single amount (1000 ng) of vector encoding either wild-type PLCγ₂ or its F897Q mutant. Upper right panel, COS-7 cells were cotransfected with vectors encoding either of the ΔPCI variants together with empty vector (Mock) or with 50 ng/well of vector encoding Rac2 or Rac2^G12V. Lower panel, expression of wild-type and mutant PLCγ₂ isozymes in upper left panel. Cells from one well each were analyzed by SDS-PAGE and immunoblotting. F, wild-type and the F897Q mutant PLCγ₂ do not differ in their ability to be tyrosine-phosphorylated by treatment of DT40 cells with H₂O₂. PLCγ₂ phosphorylation in PLCγ₂^−/− DT40 cells stably transfected with wild-type or the F897Q mutant PLCγ₂ was analyzed in the absence (−) or presence (+) of H₂O₂. PLCγ₂ was immunoprecipitated from cell extracts using anti-c-Myc antibody, and Western blotting was performed with either anti-phosphotyrosine (pY) antibody (top panel) or anti-c-Myc antibody (bottom panel).

FIGURE 6.

A, functional interaction of PLCγ₂ with activated Rac enhances the proportion of cells responding to BCR ligation with an increase in [Ca²⁺]_i. PLCγ₂^−/− stably expressing either wild-type or F897Q mutant PLCγ₂ were treated in the absence (left panel) or presence (right panel) of 1 mm extracellular Ca²⁺ with medium supplemented with increasing concentrations of anti-IgM. The data correspond to the means ± S.E. of three independent measurements for each ligand concentration, each performed on 60–80 cells. The best fit values obtained for the EC₅₀ values in A and the corresponding half-maximal proportional Ca²⁺ responses as well as the EC₅₀ value estimated in the right panel are marked by dashed lines. B and C, functional interaction of PLCγ₂ with activated Rac enhances the BCR-mediated increase in integrated Ca²⁺ fluorescence activity in the absence and in the presence of extracellular Ca²⁺. PLCγ₂^−/− stably reconstituted with either WT (left panels) or F897Q mutant of PLCγ₂ (right panels) were treated in the absence (B) or presence (C) of 1 mm extracellular Ca²⁺ with increasing concentrations of anti-IgM. Data obtained for single cells are shown. Nonresponding cells are shown individually below the abscissae. The mean values were fit by nonlinear least squares curve fitting. The standard errors of the best fit values of the maximal intensities are indicated by shading. B, 202, 173, 195, 174, and 185 (WT) and 121, 186, 201, 192, and 196 (F897Q) single cells were analyzed with increasing concentrations of anti-IgM. C, these cell numbers were 201, 174, 195, 173, and 185 (WT) and 121, 187, 200, 191, and 196 (F897Q). D, latency (time between anti-IgM addition and first Ca²⁺ peak) was plotted as a function of ligand concentration in the absence of extracellular Ca²⁺. Cells that showed no Ca²⁺ response during the observation time of 365.1 s were considered to have maximal latency equal to the time of observation. Data are shown as mean ± S.E. of 121–202 values obtained by analysis of all individual traces (corresponding to single cells) from three independent experiments in each group for each ligand concentration. E, peak frequency (left panel) and peak amplitude (right panel) calculated for the cells treated with anti-IgM in concentrations of 4, 40, and 400 ng/ml in the absence of extracellular Ca²⁺.

Results

Point Mutation F897Q Renders the DT40 Cell PLCγ₂ Orthologue Specifically Resistant to Stimulation by Activated Rac2

Mutational and structural analyses have shown that the stimulation of human PLCγ₂ by activated Rac GTPases is due to an interaction of the Rac switch I and II regions with specific residues in the C-terminal half of the PLCγ₂ spPH domain (24, 25). Fig. 1A illustrates the importance of the PLCγ₂ residue Phe-897, contained within the C-terminal α helix of spPH, in this respect. Phe-897 forms the core of a hydrophobic pocket on PLCγ₂ spPH, which interacts with several hydrophobic residues of the Rac2 switch I and II regions, such as Val-36 (I), Phe-37 (I), Trp-56 (II), Leu-67 (II), and Leu-70 (II). Rac2-PLCγ₂ complex formation is thought to have two consequences as follows: (i) conversion of GTP-bound Rac2 from conformational state 1 to state 2. These states were initially described for H-Ras as having low versus high affinity, respectively, toward Ras effectors (39). (ii) Stabilization of the Phe-897 side chain was in one of the two conformational states observed in the crystal structures of free spPH (25). Replacement of Phe-897 in human PLCγ₂ spPH by the corresponding human PLCγ₁ residue glutamine is expected to reduce the hydrophobic momentum of the Rac2 binding pocket on PLCγ₂ spPH and, possibly, interfere with the reorientation of the Leu-67 side chain upon complex formation (Fig. 1A). Consistent with this view, the F897Q substitution blocked activation of human PLCγ₂ by constitutively active Rac2 and abolished binding of GTPγS-activated Rac2 to PLCγ₂ spPH, while leaving the overall fold of PLCγ₂ spPH unaffected (24, 25).

To minimize the differences between the cellular system to be functionally reconstituted and analyzed here and unmodified DT40 B cells, a cDNA encoding chicken PLCγ₂ was produced using mRNA from DT40 cells as a template. The encoded protein shares 83% identical residues with human PLCγ₂. Within the C-terminal helices of the two spPH domains, 14 of 17 residues, including Phe-897, are identical, and three are conserved. The experiments shown in Fig. 1, B and C, examined the consequences of the F897Q mutation on the ability of the DT40 PLCγ₂ orthologue (henceforth referred to as PLCγ₂) to interact with activated Rac2. Fig. 1B shows that constitutively active Rac2^G12V, but not WT Rac2, caused a marked (∼19-fold) stimulation of PLCγ₂ activity in COS-7 cells, but it was ineffective in control cells lacking PLCγ₂ and in cells expressing the F897Q mutant. These observations were confirmed in a cell-free system (Fig. 1C). The F897Q mutation did not affect activation of PLCγ₂ by calcium ions or by loss of autoinhibition (Fig. 1, D and E) and had no influence on the extent of PLCγ₂ protein tyrosine phosphorylation in response to H₂O₂ (Fig. 1F).

To measure the effect of the F897Q mutation on the interactions of PLCγ₂ with the plasma membrane in intact cells, FRAP experiments were performed on GFP-tagged WT and the F897Q mutant PLCγ₂. PLCγ₂-GFP fluorescence recovery occurred with a fluorescence recovery time (τ) of 0.079 s (Fig. 2A, left panel). Coexpression of constitutively active Rac2^G12V enhanced the interaction of the enzyme with the plasma membrane, resulting in an ∼1.5-fold increase in τ of PLCγ₂-GFP to 0.115 s (Fig. 2A, right panel). Interestingly, FRAP beam size analysis experiments showed that the WT enzyme and the variant F897Q did not differ in their modes of membrane interaction, as the fluorescence recovery of both PLCγ₂-GFP and PLCγ₂^F897Q-GFP occurred by a mixture of binding to and dissociation from the membrane, referred to as exchange, and of stable association with the membrane, resulting in lateral diffusion. In the presence of Rac2^G12V, the fluorescence recovery of WT PLCγ₂-GFP was shifted toward lateral diffusion, as the ratio between the fluorescence recovery times with the two beam sizes (×40 and ×63 objectives) was 2.3 (Fig. 2, B and C). This ratio is indistinguishable from that expected for recovery by pure lateral diffusion (2.28 ± 0.17, the ratio between the areas illuminated by the two beam sizes employed). Consequently, in the presence of Rac2^G12V, the interaction of PLCγ₂-GFP with the plasma membrane is dominated by lateral diffusion, suggesting that the exchange rate becomes much slower than the lateral diffusion rate (i.e. a shift to stable membrane interactions). In marked contrast, Rac2^G12V had no effect at all on the mode of membrane association of the mutant PLCγ₂^F897Q-GFP, which correlates well with our observations that Rac2^G12V cannot interact with and fails to stimulate PLCγ₂^F897Q. We conclude that PLCγ₂ is not compromised in its overall activity and in its mode of membrane interaction by the F897Q mutation, in contrast to its specific loss of regulation by activated Rac.

FIGURE 2.

FRAP beam size analysis demonstrates that wild-type and F897Q mutant PLCγ₂ display similar membrane interactions, but only that of wild-type PLCγ₂ is augmented by Rac2^G12V. A, COS-7 cells were cotransfected with a vector encoding GFP-tagged wild-type PLCγ₂ (WT) together with either empty vector (left panel) or vector encoding Rac2^G12V (WT + Rac2^G12V; right panel). The typical FRAP curves shown were obtained using a ×63 objective. The solid lines represent the best fit obtained by nonlinear regression analysis. The best fit τ values are depicted in each panel. The mobile fractions (R_f) were above 0.93 in all cases and therefore are not shown. B and C, FRAP beam size analysis. Transfection was as in A, except that in some studies the GFP-tagged mutant (F897Q) replaced its wild-type counterpart (WT). Bars represent the means ± S.E. of 30–60 measurements. The studies employed ×40 and ×63 objectives, yielding a beam size ratio of 2.28 ± 0.17 (n = 59). Thus, this τ(×40)/τ(×63) ratio is expected for FRAP by lateral diffusion (C, upper dashed line). A τ ratio of 1 (C, lower dotted line) indicates recovery by exchange (35). In the absence of Rac2^G12V, both the τ values (comparing values measured with the same beam size (B) and the τ(×40)/τ(×63) ratios (C) of WT and F897Q mutant PLCγ₂ were similar (p > 0.4 in all cases; Student's t test), suggesting similar lateral diffusion in and exchange rate from the plasma membrane. Although coexpression of Rac2^G12V had no significant effect on either the τ values or τ ratio of the F897Q mutant (p > 0.4), it significantly enhanced the membrane association of wild-type PLCγ₂, reflected in a slower τ(×40) (**, p < 0.002) and in a significant increase of its τ ratio (*, p < 0.02; bootstrap analysis) from an intermediate value (characterizing recovery by a mixture of lateral diffusion and exchange) to 2.3, similar to the value expected for pure lateral diffusion.

Functional Reconstitution of Wild-type and F897Q Mutant PLCγ₂ into PLCγ₂^−/− DT40 B Cells

Next, genetically PLCγ₂-deficient DT40 cells were stably reconstituted with either isogenic WT PLCγ₂ or the PLCγ₂^F897Q mutant such that the resultant cell clones were indistinguishable in terms of enzyme expression and subcellular distribution (Fig. 3). Spinning disc confocal fluorescence microscopy was then used to determine the regulatory influence of Rac on the kinetic parameters of BCR-mediated changes in [Ca²⁺]_i at the level of individual DT40 B cells. Cells were loaded with the fluorescent Ca²⁺ indicator fluo-4, and authentic PLCγ₂^+/+ DT40 cells were compared with PLCγ₂^−/− cells, before and after stable reconstitution of the latter with either WT or F897Q mutant PLCγ₂, for their ability to respond to BCR ligation with increases in [Ca²⁺]_i. In accordance with earlier studies (40), cells were first treated with 40 ng/ml anti-IgM in the absence of extracellular Ca²⁺ to measure the BCR-mediated Ca²⁺ release from intracellular stores. After some time, the effect of the same concentration of anti-IgM was determined in the presence of 1 mm extracellular Ca²⁺ to also allow for the entry of extracellular Ca²⁺ into the cells. Finally, the Ca²⁺ ionophore ionomycin was added to normalize the fluorescence intensities obtained for the individual cells (Fig. 4A). The latter values were subsequently used to normalize the individual intensity time traces. Sixty cells from each cell type were analyzed individually in each experiment (Fig. 4B). Each of the curves shown represents the fluorescence intensity time trace of a single cell in the observation area. Fig. 4B, upper left panel, shows that addition of anti-IgM to WT DT40 caused oscillations of [Ca²⁺]_i in most but not all cells. In responding cells, the oscillations commenced after a lag time of ≥∼2 min. Although some cells displayed a less oscillatory and more monophasic increase in [Ca²⁺]_i upon addition of extracellular Ca²⁺ accompanied by a loss of spiking, most responding cells showed a relatively homogeneous oscillatory behavior in the absence and presence of extracellular Ca²⁺. In PLCγ₂^−/− cells, by contrast, none of the cells responded to addition of anti-IgM (400 ng/ml), regardless of whether Ca²⁺ was absent from or present in the incubation medium (Fig. 4B, upper right panel). Addition of 4 ng/ml trypsin to these cells in the presence of extracellular Ca²⁺ caused the appearance of a single [Ca²⁺]_i peak, presumably by activation of G-protein-coupled PAR2 receptors endogenously present in DT40 cells, followed by activation of endogenous PLCβ (41). In this case, almost all cells responded, and the lag time was much shorter (∼10 s). Fig. 4B, lower panels, shows that although both PLCγ₂ and PLCγ₂^F897Q were able to reconstitute oscillatory Ca²⁺ responses to PLCγ₂^−/− DT40 cells following BCR ligation, substantial quantitative differences were apparent already at the level of visual assessment of the two sets of time traces. Specifically, the proportion of cells responding to anti-IgM as well as both the peak amplitudes and the frequencies of the spikes were considerably lower, and the mean latencies of the overall responses were distinctly longer for cells expressing PLCγ₂^F897Q rather than WT PLCγ₂. A more detailed, quantitative characterization of this difference will be presented below in Figs. 5 and 6. A quantitative comparison of the [Ca²⁺]_i transients observed in PLCγ₂^+/+ DT40 cells versus PLCγ₂^−/− cells reconstituted with WT PLCγ₂ in response to 400 ng/ml anti-IgM is shown in Fig. 4C (upper panels). There were no significant differences between the two cell types in the proportions of responding cells, integrated peak intensities, peak frequencies, and amplitudes (Fig. 4C, lower panels). Thus, the PLCγ₂-deficient cells reconstituted with WT PLCγ₂ bear a close resemblance to their native counterparts. Fig. 4D shows that PLCγ₂^−/− cells reconstituted with WT versus F897Q mutant PLCγ₂ do not appreciably differ in [Ca²⁺]_i responses triggered by addition of thapsigargin, an inhibitor of the sarco-/endoplasmic reticulum Ca²⁺-ATPase (40, 41). Thus, the F897Q mutation and, by extension, interaction of PLCγ₂ with activated Rac have no effect on BCR-independent [Ca²⁺]_i mobilization in DT40 B cells.

FIGURE 3.

Stable reconstitution of PLCγ₂^−/− DT40 B cells with wild-type versus F897Q mutant PLCγ₂. PLCγ₂-deficient DT40 cells (89) were stably reconstituted with either isogenic wild-type or F897Q mutant PLCγ₂. A, aliquots of the lysates (50 μg of protein) of native DT40 cells (PLCγ₂^+/+), PLCγ₂-deficient DT40 cells (PLCγ₂^−/−), and from three independent clones PLCγ₂^−/− DT40 cells stably expressing similar quantities of either wild-type (A–C) or F897Q mutant PLCγ₂ (a–c) were subjected to SDS-PAGE and immunoblotting (upper panel). The same membrane was subsequently probed with an anti-β-actin antibody to control for equal loading of samples (lower panels). All six clones were used for experimentation in this study, with no differences detected between clones A–C and a–c, respectively. B, cells from the clones A and c were analyzed by indirect fluorescence staining (left panels). Right panels, corresponding phase contrast images. C, mean fluorescence intensities of the three images each, as shown in B, were corrected for background staining. Similar results were obtained for other pairs of clones.

FIGURE 5.

Rac-insensitive mutant PLCγ₂^F897Q is quantitatively impaired in its ability to restore B cell receptor-mediated Ca²⁺ flux in PLCγ₂^−/− DT40 cells. PLCγ₂^−/− cells stably expressing wild-type (left column) or F897Q mutant PLCγ₂ protein (right column) were treated with increasing concentrations of anti-IgM. The treatment was performed as indicated by the arrowheads in the following sequence: anti-IgM; no Ca²⁺ → anti-IgM; 1 mm Ca²⁺ → 4 μm ionomycin; 1 mm Ca²⁺. Forty nine to 60 cells were analyzed in each single experiment.

Concentration Dependence on Anti-IgM of [Ca²⁺]_i Response Impairment in DT40 B Cells Expressing Wild-type Versus Rac-insensitive F897Q Mutant PLCγ₂

To determine the influence of the Rac-PLCγ₂ interaction on [Ca²⁺]_i changes in DT40 B cells in response to increasing extents of BCR ligation, PLCγ₂^−/− DT40 cells stably reconstituted with either WT or F897Q mutant PLCγ₂ were treated with increasing concentrations of anti-IgM in the absence of extracellular Ca²⁺, followed by the same concentration of anti-IgM in the presence of 1 mm extracellular Ca²⁺. Cells were treated with 4 μm ionomycin at the end of each experiment for baseline correction and normalization of the [Ca²⁺]_i responses in single cells. Fig. 5, left panels, shows that oscillatory [Ca²⁺]_i responses developed in cells reconstituted with WT PLCγ₂ at anti-IgM concentrations between 4 and 400 ng/ml. At 40–400 ng/ml anti-IgM, the responses were similar in both their frequencies and intensities, in the absence or presence of extracellular Ca²⁺. At 4 μg/ml anti-IgM (Fig. 5, next-to-lowest left panel), individual Ca²⁺ oscillations appeared to coalesce into single major asymmetric peaks in many traces. These peaks reached their maximum intensities within seconds after anti-IgM addition in the absence of extracellular Ca²⁺ to gradually decline to base levels over the next 6 min. Addition of extracellular Ca²⁺ to anti-IgM at this time point led to a second, more symmetric [Ca²⁺]_i wave, reaching a maximum after about 1.5 min and declining with similar kinetics thereafter. At still higher concentrations of anti-IgM, 40 μg/ml, the pattern was qualitatively similar but somewhat reduced in quantitative terms, both in the absence and presence of extracellular Ca²⁺. In cells expressing the Rac-insensitive mutant F897Q of PLCγ₂ (Fig. 5, right panels), there was a striking loss of the [Ca²⁺]_i responses, in particular at low concentrations of anti-IgM, e.g. 40 ng/ml. In each case, this effect was clearly evident both with and without extracellular Ca²⁺. At high (4 and 40 μg/ml) concentrations of anti-IgM, there also was a loss of coalescence of individual oscillations in responding cells and a decrease in the overall duration of the [Ca²⁺]_i response to BCR ligation, in particular in the absence of extracellular Ca²⁺.

The results of further quantitative analyses of the differences between the [Ca²⁺]_i responses of cells expressing WT versus F897Q PLCγ₂ are shown in Fig. 6. Thus, in the absence of extracellular Ca²⁺, there was a marked reduction in the anti-IgM sensitivity of the latter cells by an order of magnitude, but no change in the maximal extent, when the proportion of responding cells was used as a response parameter (Fig. 6A, left panel). In the presence of extracellular Ca²⁺, the major difference was a distinct loss of the proportion of responding cells, which was similar (∼55%) at low and intermediate anti-IgM concentrations, 4–400 ng/ml, and even more striking (∼80%) at high anti-IgM concentrations (Fig. 6A, right panel). There was no apparent change in the sensitivity of the cells to anti-IgM. Integration of the fluorescence intensities in single cells over time and evaluation of their concentration dependence on anti-IgM at the level of the means showed a reduction of the maximal mean intensity in cells expressing the F897Q mutant by 64% in the absence of extracellular Ca²⁺ (Fig. 6B). In its presence, this loss amounted to 82% (Fig. 6C).

Fig. 6D shows that resistance of PLCγ₂ to regulation by Rac also resulted in a longer latency of the [Ca²⁺]_i response to addition of anti-IgM. This became evident at low anti-IgM concentrations (40 ng/ml) and more prominent at all higher concentrations. [Ca²⁺]_i peak frequencies and amplitudes were only analyzed for the three lowest ligand concentrations, 4–400 ng/ml, and in the absence of extracellular Ca²⁺, mostly because of peak coalescence at higher anti-IgM concentrations. By assigning a peak frequency of zero to nonresponding cells, we determined a reduction of this parameter by ∼86 and ∼55% in cells expressing the F897Q mutant rather than WT PLCγ₂ (Fig. 6E, left panel). Likewise, decreases of peak amplitudes amounting to ∼84 and ∼58% were observed for the cells harboring the mutant at 40 and 400 ng/ml anti-IgM, respectively (Fig. 6E, right panel). Collectively, these results indicate that the regulation of PLCγ₂ by Rac is not an absolute requirement for BCR-mediated Ca²⁺ release at most BCR ligand concentrations tested herein, both in the absence and presence of extracellular Ca²⁺. However, although the qualitative patterns of [Ca²⁺]_i responses to BCR ligation are similar in the absence and presence of PLCγ₂ regulation by Rac, there are striking quantitative differences. These are readily evident at the level of the proportion of responding cells and their sensitivity to anti-IgM, the latency of the response after addition of anti-IgM, as well as both the peak frequency and amplitude of the oscillatory [Ca²⁺]_i responses.

Dependence of BCR-mediated Nuclear Translocation of the Transcription Factor NFAT1c on Functional Rac-PLCγ₂ Interaction

Within minutes after BCR-mediated B cell activation, several transcription factors, including the Ca²⁺-dependent transcription factor NFAT1c, are translocated into the nucleus to induce transcription of regulatory genes involved in B cell-fate decisions (6). To determine the role of a functional Rac-PLCγ₂ interaction in this response, a C-terminally truncated NFAT1c construct consisting of the N-terminal transactivation and the calcineurin-binding domain (amino acids 1–400) fused to the red fluorescent protein td-RFP611 was introduced into PLCγ₂^−/− DT40 B cells and PLCγ₂^−/− DT40 B cells stably expressing either WT or F897Q mutant PLCγ₂, and its nuclear translocation following BCR ligation was analyzed by spinning disk fluorescence microscopy (Fig. 7). Treatment of cells with ionomycin, resulting in PLCγ₂-independent nuclear translocation of NFAT1c-td-RFP611 in almost all transfected cells, was used as a positive control (Fig. 7, A, lower panels, and B, right panel). In PLCγ₂-deficient cells, there was little, if any, nuclear translocation of the fluorescent reporter protein in response to anti-IgM (Fig. 7A, upper panels), consistent with the concept that this event is absolutely dependent on PLCγ₂-induced Ca²⁺ mobilization. In cells expressing WT PLCγ₂, NFAT1c-td-RFP611 translocated into the nuclei in about 80% of the cells (Fig. 7B, left panel). Importantly, only 16% of the cells expressing the Rac-insensitive PLCγ₂ mutant F897Q displayed nuclear NFAT1c-td-RFP611, such that uncoupling of PLCγ₂ from Rac caused an 80% decrease of a cellular response directly related to altered gene transcription and, by extension, cell fate decisions in B cells.

Discussion

This study shows that specific interference with a functional Rac-PLCγ₂ interaction in intact DT40 B cells precipitates several clear alterations in BCR-mediated Ca²⁺ signaling. First, the sensitivity of Ca²⁺ release from internal stores to the BCR ligand anti-IgM is reduced by an order of magnitude. Second, the extent of the Ca²⁺ signal in the presence of extracellular Ca²⁺, also involving entry of extracellular Ca²⁺ into the cells, is greatly diminished. Third, Ca²⁺-regulated, NFAT-mediated transcriptional regulation is largely decreased.

Several lines of evidence have already suggested that PLCγ₂ and Rac are both activated by BCR ligation, although a direct interaction of the two signaling molecules has received relatively little attention (6, 26, 42, 43). Thus, several elements of the canonical B cell receptor signaling cascade, e.g. Syk (44), Btk (45), and BLNK (46, 47), are known to physically interact with and activate the Rac activator Vav. BCR cross-linking caused activation of both Rac1 and Rac2 within minutes (48). Rac cooperated with PLCγ₂ in BCR-mediated activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, SRF, and NFAT (49, 50). Dominant negative Rac1 suppressed these cooperative relationships. These results were interpreted to suggest that the signaling pathways controlling the activities of Rac and PLCγ₂ are activated by BCR ligation in parallel to converge at points distal to enhanced formation of InsP₃ and DAG. Btk, BLNK, Vav, and PLCγ₂ form highly coordinated microsignalosomes in a process dependent on Syk and Lyn (51). Formation of these complexes is important for amplification of signaling and, hence, for appropriate B cell activation, in particular at low antigen concentrations. The results presented here strongly suggest that signal amplification is caused within or in the vicinity of these sites by convergence of signals emanating from activated BCR through a direct interaction of activated Rac with PLCγ₂.

Calcium oscillations arise from a complex interplay between PLC-mediated generation of InsP₃ and DAG, InsP₃ receptor-mediated release of intracellular Ca²⁺, and entry of extracellular Ca²⁺ mediated by channels that are either store-operated, activated by DAG, or subject to other regulatory controls (52). These incremental changes may be rapidly overturned by InsP₃ metabolism and Ca²⁺ efflux from the cytosol (6, 53). All three InsP₃ receptors are present in DT40 B cells and display differential sensitivities to InsP₃ in these cells (54). Expression of only one of the three in InsP₃ receptor-deficient cells resulted in very distinct oscillatory responses to similar degrees of BCR activation. This finding suggests that InsP₃ receptors are a major regulatory site of Ca²⁺ oscillations in these cells. In addition, feedback mechanisms may exist in B cells giving rise to spatiotemporal dynamics of the levels of InsP₃ (55) and, by extension, of DAG. For example, the transient receptor potential cation channel type 3-mediated increases in [Ca²⁺]_i were shown in B cells to promote translocation of PLCγ₂ to the plasma membrane and further activation of the enzyme, by a process involving physical interaction with TRPC3 cation channels (56). Interestingly, some aspects of the latter interaction appeared to be independent of the phospholipase C activity of PLCγ₂ (40, 56). Selective and direct inhibition of TRPC3 channels by pyrazole-3 eliminated the Ca²⁺ influx-dependent PLCγ₂ plasma membrane translocation and the late oscillatory phase of the BCR-induced Ca²⁺ response (57). The same TRPC3 inhibitor also suppressed Rac1 activation without affecting total Rac1 protein abundance in cardiomyocytes (58), whereas activated Rac1 enhanced the rapid vesicular translocation and membrane insertion of another transient receptor potential cation channel, TRPC5 (59). Thus, the interaction of PLCγ₂ with Rac may influence BCR-mediated Ca²⁺ oscillations in several ways.

Previous studies have shown that the interaction of PLCγ₂ with Rac2^G12V markedly enhances its enzymatic activity in vitro and in intact cells and coincides with a translocation of PLCγ₂ from the soluble to the particulate fraction of intact cells (23). Here, we demonstrate that binding of activated Rac to WT PLCγ₂ also changes the mode of plasma membrane association of the latter from a mixture of exchange and lateral diffusion to almost pure lateral diffusion (Fig. 2). This suggests that Rac-PLCγ₂ interaction reduces the exchange rate between membrane-associated and cytoplasmic WT PLCγ₂ such that it becomes negligible relative to its lateral diffusion rate, demonstrating a stronger membrane association. Hence, in intact cells, Rac-activated WT PLCγ₂ mainly travels along the plasma membrane, allowing for a spatio-temporal pattern of encounter with both its lipid substrate and its protein interaction partners, e.g. TRPC3, that is different from that of its unliganded or Rac-resistant counterparts.

The dependence of the Ca²⁺ oscillations in cells expressing WT PLCγ₂ on low to intermediate concentrations of anti-IgM (≤4 μg/ml) suggests that signal transduction occurs mostly through frequency modulation, although there is also a clear increase in the amplitude with increasing BCR ligation. It is also evident that, in this concentration range of anti-IgM, the spiking behavior of the cells is largely independent of the presence of extracellular Ca²⁺. The relative independence of oscillation frequency on external Ca²⁺ suggests that DT40 B cells are highly efficient at recycling their internal Ca²⁺ (53). In aggregate, these findings indicate that a minimal model based on reversible desensitization of InsP₃ receptors may suffice to explain the oscillatory pattern observed at low to intermediate anti-IgM concentrations (53, 60). According to the minimal model, [Ca²⁺]_i oscillations are driven by a coupled process of Ca²⁺-induced activation and obligatory intrinsic inactivation of the Ca²⁺-sensitized state of InsP₃ receptors. Ca²⁺ spikes are initiated by the Ca²⁺-mediated conversion of low affinity InsP₃ receptors from their low to the high affinity type (60). In this paradigm, the decrease in latency and the increase in oscillation frequency observed in Fig. 6, D and E, would be due to the decrease in the time required to generate a sufficient Ca²⁺ trigger signal to initiate the first or next Ca²⁺ release spike, respectively. Enhanced and accelerated translocation of PLCγ₂ to the plasma membrane containing its phospholipid substrate by activated Rac would allow for a decrease in the time necessary for reaching this threshold level, such that considerably lower levels of activated BCR are required to elicit a given [Ca²⁺]_i response. Of note, enhanced association of PLCγ₂ with the plasma membrane following interaction with activated Rac is indeed observed in the FRAP beam size analysis experiments (Fig. 2), and the absence of this effect on the PLCγ₂ mutant defective in Rac interactions (F897Q) is in accord with the much lower quantitative effect of anti-IgM in cells expressing this mutant on the Ca²⁺ oscillations (Fig. 6E). In addition to temporal changes of PLCγ₂ activation by Rac, spatial changes may come into play. Given that the Ca²⁺ conductances are similar for the three InsP₃-R subtypes (61), the increase in amplitude observed with increasing anti-IgM concentrations may be due to expression of the receptors at different cellular levels in DT40 B cells, with higher abundance of subtypes with lower InsP₃ sensitivity (InsP₃-R1 (4.7 μm); InsP₃-R2 (0.35 μm); InsP₃-R3 (18.6 μm); EC₅₀ values for InsP₃-mediated Ca²⁺ release in permeabilized cells expressing a single InsP₃-R subtype in parentheses (54)).

When extracellular Ca²⁺ is provided to cells incubated without Ca²⁺ for several minutes at high concentrations of anti-IgM, the main effect of a loss of Rac regulation by PLCγ₂ is a marked reduction in the number of responding cells, which cannot be recuperated by increasing the concentration of anti-IgM. Several scenarios may explain this observation: (i) the interaction of PLCγ₂ with activated Rac could strengthen the productive interaction of PLCγ₂ with plasma membrane channels such as TRPC3 allowing entry of extracellular Ca²⁺, either by altered temporal or spatial interaction of PLCγ₂ with the membrane (cf. Fig. 2). If TRPC3 channels were involved in Rac activation in B cells, as they appear to be in cardiomyocytes (see above), this would further reinforce this process. (ii) Cells could be desensitized to BCR-mediated Ca²⁺ signaling with continued anti-IgM exposure. They can become less sensitive to this course of action if the Rac-PLCγ₂ interaction is intact. Both homologous and heterologous desensitization of B cell membrane-immunoglobulin-mediated Ca²⁺ mobilization have long been known to occur within minutes of anti-immunoglobulin exposure (62). Interestingly, anti-IgM-treated B cells are hyper-responsive to AlF₄⁻ and mastoparan (63). Although the two reagents are known as activators of heterotrimeric G proteins, they also appear to activate Rho GTPases, including Rac (64, 65). If this was to occur in B cells, the results presented here and by Cambier et al. (63) may indicate that anti-IgM-mediated B cell activation affects a function(s) of the macromolecular complex involving PLCγ₂, without impinging upon the activation of the enzyme by Rac, and that the complex is protected, at least in part, when the Rac-PLCγ₂ interaction is intact. The markedly diminished effect of high concentrations of anti-IgM on [Ca²⁺]_i observed in cells lacking the functional Rac-PLCγ₂ interaction does not appear to require exposure of B cells to anti-IgM in the absence of extracellular Ca²⁺, because the inhibitory effect was also evident at the level of nuclear NFAT1c-td-RFP611 translocation, which was assayed in the continued presence of extracellular Ca²⁺ (Fig. 7).

Under these conditions, the insensitivity of PLCγ₂ to activated Rac led to a marked reduction in BCR-mediated nuclear translocation of NFAT1c-td-RFP611 (Fig. 7), strongly suggesting that the changes observed at the level of [Ca²⁺]_i are effectively transduced to the transcriptional level. In B lymphocytes, the activity of the Ca²⁺-regulated transcriptional regulators c-Jun N-terminal kinase (JNK), NFκB, and NFAT are differentially regulated by the amplitude and duration of oscillatory Ca²⁺ signals (66). NF-κB and JNK are selectively activated by a large transient [Ca²⁺]_i rise, whereas NFAT is activated by a low, sustained Ca²⁺ plateau. These results and findings obtained in T lymphocytes (67) and basophilic leukemia cells (68) are consistent with the notion that nuclear translocation of NFAT functions as a working memory of Ca²⁺ signals by decoding Ca²⁺ oscillations (69). In general, this process appears to be more cost-effective than translation of continuous Ca²⁺ signals. In this study, nuclear translocation and, hence, activation of NFAT1c-td-RFP611 was studied at 40 μg/ml anti-IgM, allowing for similar numbers of responders (∼92%) when cells expressing either WT or F897Q mutant PLCγ₂ were analyzed for BCR-mediated Ca²⁺ release from internal stores (Fig. 6A), with an overall reduction of the integrated fluorescence intensity in the latter cells by 64% (Fig. 6B). In the presence of extracellular Ca²⁺, both the proportion of responding cells and the mean integrated single cell fluorescence intensity were reduced by more than 80% (Fig. 6, A and C). Because NFAT activation is highly dependent on both InsP₃-induced intracellular Ca²⁺ release and store-operated Ca²⁺ entry mediated by CRAC channels (50, 70), it is likely that reductions of both responses participate in reducing nuclear NFAT translocation. Furthermore, analysis of the Ca²⁺ oscillatory behavior in the absence of extracellular Ca²⁺ at 40 μg/ml anti-IgM revealed that the majority of responses (>75%) consisted of more than one Ca²⁺ spike in cells expressing WT PLCγ₂, whereas this parameter was reduced to 35% in cells expressing the F897Q mutant, showing only a single Ca²⁺ spike in most cases (cf. Fig. 5, lower panels). Thus, reduced [Ca²⁺]_i spiking from intracellular sources may also contribute to the substantial decrease in nuclear NFAT translocation caused by resistance of PLCγ₂ to activation by Rac.

Because Rac GTPases are activated in B cells by quite a number of cell surface receptors in addition to the BCR, the signaling pathway convergence shown in this work is very well suited to detect coincident extracellular signals and to ensure signaling reliability by correctly interpreting the emergence of signals according to the particular cellular context and the specific (patho)physiological circumstances (71). Thus, agonist activation of receptors for integrins, chemokines, and pathogen-derived ligands has also been shown to activate Rac, which provides the GTPase with the potential to act as a central B cell signaling hub. For integrins, it is interesting to note that coligation of the β₂α_L integrin LFA-1 and BCR has been shown to cause an ∼10-fold reduction in the amount of BCR ligand required for B cells to make a tight contact with target membranes containing the receptor ligands (72). Using a similar experimental design, Henderson et al. (73) showed that Rac2 plays an important role in outside-in signaling from LFA-1 that leads to firm adhesion of murine B cells to ICAM-1. Interestingly, the Rac2 deficiency could be bypassed by treatment of B cells with phorbol ester and ionomycin, thus mimicking enhanced activation of PLCγ₂. Although the molecular mechanisms of Rac activation by LFA-1 in B cells are unknown, they may be similar to those encountered in T cells (74–76), although a non-Vav RacGEF(s) may be involved in this case (73). Hence, simultaneous activation of both BCR and LFA-1, e.g. at the immunological synapse, may give rise to locally enhanced Rac activation, allowing for an increased potency of BCR ligand to mediate integrin activation, at least in part by enhanced activity of PLCγ₂ (73, 77).

A positive interaction between Rac and PLCγ₂ may also be (patho)physiologically relevant in platelets, where PLCγ₂ is activated downstream of other immunoreceptor tyrosine-based activation motif-coupled receptors, such as the major platelet collagen receptor glycoprotein VI or CLEC-2 (78), and by integrin α₂β₁ (79). Inactivation of the Rac1 gene in the mouse caused defective thrombus formation on collagen under flow conditions (78), and pharmacological inhibition of Rac in human platelets led to reduced PLCγ₂ activity and impaired α_IIbβ₃ fibrinogen receptor stimulation (79). Intriguingly, the functional consequences of Rac1 deficiency were particularly striking at low and intermediate concentrations of GPVI or CLEC-2 receptor agonists (78), suggesting that Rac may enhance the sensitivity of PLCγ₂ stimulation to extracellular ligands in platelets as well. Hence, pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

The regulation of B cells by BCR ligation is intertwined with their regulation by certain chemokines, such as the CXCR4 and CXCR5 agonists CXCL12 and CXCL13, respectively (5). Activated CXCR4 and CXCR5 appear to be coupled to both Btk and PLCγ₂ and, through a yet unidentified RacGEF (possibly DOCK2), to activation of Rac. Recently, Rac2 has been shown to be critical for LFA-1-mediated adhesion of mouse B cells in response to CXCL12 or CXCL13 (73). It remained unclear whether Rac2 causes enhanced adhesion directly by augmenting inside-out activation of integrins or indirectly by fostering receptor-mediated activation of PLCγ₂. Recently, pathogen-derived signals have been suggested to activate Rac in murine B cells via Toll-like receptor TLR4 and MyD88 (80). Activated TLR4 activation also enhanced CXCR5-mediated Rac activation. Of note, TLR4 was found in other leukocytes to be coupled to activation of PLCγ₂ (81, 82), raising the possibility that Rac is of regulatory importance in that respect as well.

The enhanced BCR ligand sensitivity observed for cells expressing WT PLCγ₂ versus its F897Q mutant is reminiscent of the reduced threshold for antigen receptor stimulation observed in human peripheral blood B lymphocytes upon coligation of CD19 with BCR (3). In mouse B cells, the costimulatory effect of CD19 on BCR-mediated increases in [Ca²⁺]_i was clearly reduced in B cells from rac2^−/− mice (19). Previously, these effects were mainly ascribed to the known interactions of CD19 with the RhoGEF Vav and to the positive interaction of Vav, presumably via activated Rac, with phosphatidylinositol 4-phosphate 5-kinase and/or PI3K, followed by indirect activation of PLCγ₂ (6, 42). Our current results strongly suggest that Rac enhances BCR-mediated Ca²⁺ signaling in B cells by direct interaction with and activation of PLCγ₂.

Several families have been described with members affected by homozygous mutations in the CD19 gene causing undetectable or substantially decreased levels of CD19 in B cells. In these patients, suffering from an antibody-deficiency syndrome, there were marked alterations in Ca²⁺ mobilization in B cells following anti-IgM treatment (83). Similar defects of Ca²⁺ mobilization were observed in patients with defective CD81 and CD21, which function in a complex with CD225 and CD19 and cooperate with BCR to mediate antigen recognition (84, 85). Thus, the stimulatory interaction of PLCγ₂ with Rac may not only play a pivotal role in determining the sensitivity of the BCR to stimulation by antigen, but may also contribute to BCR coreceptor signaling and to functional alterations of the latter in human disease such as certain forms of monogenetic common variable immunodeficiency (86). The very recent observation of a RAC2 loss-of-function mutation in two siblings with characteristics of a common variable immunodeficiency is consistent with this view (87).