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Originally published In Press as doi:10.1074/jbc.M313098200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 13, 12009-12019, March 26, 2004
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The Rap GTPases Regulate Integrin-mediated Adhesion, Cell Spreading, Actin Polymerization, and Pyk2 Tyrosine Phosphorylation in B Lymphocytes*

Sarah J. McLeod{ddagger}§, Andrew J. Shum{ddagger}, Rosaline L. Lee{ddagger}, Fumio Takei¶, and Michael R. Gold{ddagger}||

From the {ddagger}Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada

Received for publication, December 1, 2003 , and in revised form, December 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365–1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756–41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and {alpha}4 integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-mediated adhesion plays an important role in B lymphocyte maturation, trafficking, and activation. The major integrins on B cells are lymphocyte function-associated antigen-1 (LFA-1 1; {alpha}L{beta}2 integrin), very late antigen-4 (VLA-4; {alpha}4{beta}1 integrin), and {alpha}4{beta}7 integrin. LFA-1 binds to intercellular adhesion molecule-1 (ICAM-1; CD54) (1). VLA-4 and {alpha}4{beta}7 integrin bind to the extracellular matrix (ECM) component fibronectin and to vascular cell adhesion molecule-1 (VCAM-1; CD106) (1, 2). During B cell development, VLA-4 on B cell progenitors binds to VCAM-1 on bone marrow stromal cells. This interaction plays an essential role in B cell development by retaining B cell precursors in the bone marrow, where they can receive survival signals and differentiation signals (3–5). Once a B cell exits the bone marrow, it must migrate into and within lymphoid organs to encounter antigens. The entry of B lymphocytes into the lymph nodes, Peyer's patches, bone marrow, and white pulp cords of the spleen involves adhesion mediated by the LFA-1, VLA-4, and {alpha}4{beta}7 integrins (5–8). These integrins allow B cells to bind to endothelial cells that line the blood vessels and then transmigrate across the endothelial cell layer into the lymphoid tissue. Once a B cell has bound antigen within the lymphoid organ, it must receive T cell help to become activated and to differentiate into an antibody-producing plasma cell. The cognate interaction between T helper cells and B cells is stabilized by multiple receptor-ligand interactions, including the binding of LFA-1 to ICAM-1 (9). Finally, germinal center B cells use both LFA-1 and VLA-4 to bind to follicular dendritic cells and receive survival signals (10, 11).

Integrins on the surface of resting lymphocytes normally have low affinity and low avidity for their ligands. However, external stimuli such as chemokines and antigens generate intracellular signals that rapidly activate integrins, converting them to a clustered high affinity/high avidity state that can bind adhesion molecules on other cells or ECM components (1, 12). The nature of this "inside-out" signaling that promotes integrin activation is not completely understood. Many signaling pathways contribute to integrin activation, including the pathways that involve phosphatidylinositol 3-kinase, protein kinase C, Fyb/ADAP (adhesion and degranulation adaptor protein), and the Rho GTPase (12–15).

Once activated, integrins can bind their ligands and initiate intracellular signaling pathways that regulate many aspects of cell behavior, including cell survival, proliferation, and differentiation and reorganization of the actin cytoskeleton (16). The reorganization of a cell's actin cytoskeleton and the resulting morphological changes are important for many cellular processes, including cell-cell adhesion, cell motility, lymphocyte extravasation, and cell migration on an ECM substrate or across cell surfaces (17). Cell-cell and cell-substrate interactions are often accompanied by cell spreading, a flattening of the cell that increases the surface area of the contact region with other cells or the ECM, thereby allowing stronger adhesion.

The Rap GTPases appear to play a key role in cell adhesion and post-adhesion events. In mammalian cells, there are four 23-kDa Rap proteins, Rap1A, Rap1B, Rap2A, and Rap2B, each encoded by a separate gene. The Rap1A and Rap1B proteins are 97% identical and assumed to be functionally equivalent. The same is true for Rap2A and Rap2B, which have 60% overall identity to the Rap1 proteins. Although the effector-binding regions of Rap1 and Rap2 are identical at eight of nine residues, it is not clear whether Rap1 and Rap2 have identical, overlapping, or distinct functions. Until recently, little was known about the function of the Rap proteins in mammalian cells. However, a number of reports have now shown that expressing constitutively active Rap1 promotes integrin activation in T cells and myeloid cells, whereas blocking the activation of endogenous Rap GTPases prevents receptor-induced integrin activation (13, 18–26). Moreover, in Dictyostelium discoideum and Drosophila melanogaster, orthologs of the mammalian Rap1 GTPase regulate cell morphology, actin polymerization, and cell migration (27–29).

In B cells, integrin-dependent adhesion, actin polymerization, and cell spreading can be induced by antigens and anti-Ig antibodies that cluster the B cell antigen receptor (BCR), by chemokines such as stromal cell-derived factor-1 (SDF-1), and by phorbol esters (30–36). We have previously shown that these stimuli activate the Rap1 and Rap2 GTPases in B cells (37, 38). In this study, we investigated whether Rap activation mediates the effects of anti-Ig antibodies, SDF-1, and phorbol esters on integrin-dependent adhesion, cell spreading, and actin polymerization in B cells, processes that are important for chemokine-induced migration and for chemokine- or antigen-induced B cell adhesion. We show that activation of endogenous Rap in B cells is important for these stimuli to induce LFA-1- and {alpha}4 integrin-dependent adhesion. Moreover, by expressing a constitutively active form of Rap2, we show for the first time that Rap2 can promote integrin-dependent adhesion. We also show that Rap activation in B cells is important for cell spreading, actin polymerization, and the phosphorylation of Pyk2, a tyrosine kinase involved in regulating cell morphology as well as chemokine-induced B cell migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—The A20 and WEHI-231 murine B cell lines were obtained from American Type Culture Collection (Manassas, VA). The M2-10B4 murine bone marrow stromal cell line (39, 40) was a gift from Dr. C. J. Eaves (British Columbia Cancer Agency). All cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 50 µM 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, 15 units/ml penicillin, and 50 µg/ml streptomycin (complete medium).

Expression of RapGAPII and Rap2V12 in B Cell Lines—cDNAs encoding FLAG-tagged RapGAPII or Rap2V12 in the pMSCVpuro vector (Clontech) were gifts from Dr. Michiyuki Matsuda (Osaka University, Osaka, Japan). A20 cell clones stably expressing RapGAPII, Rap2V12, or the empty pMSCVpuro vector were generated by electroporation (41). All experiments were performed using RapGAPII-expressing A20 clone 16, and the results were confirmed using clone 3. The Rap2V12-expressing A20 cells were an oligoclonal bulk population that was not subjected to single cell cloning. Bulk populations of WEHI-231 cells stably expressing RapGAPII, Rap2V12, or the empty pMSCVpuro vector were generated by retrovirus-mediated gene transfer (42), followed by selection with 0.25 µg/ml puromycin. The expression of FLAG-RapGAPII or FLAG-Rap2V12 was detected by immunoblotting with anti-FLAG monoclonal antibody M2 (Sigma).

Rap Activation Assays—Cells were resuspended at 2 x 107/ml in 0.5 ml of modified HEPES-buffered saline (43) and stimulated with goat anti-mouse Ig antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), recombinant human SDF-1 (R&D Systems, Minneapolis, MN), or phorbol 12,13-dibutyrate (PdBu; Sigma). The cells were solubilized by adding an equal volume of Rap lysis buffer (50 mM Tris-HCl (pH 7.5), 1% Igepal (ICN, Costa Mesa, CA), 200 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM Na3VO4) to the cell suspension and then removing insoluble material by centrifugation. Rap activation assays were performed as described previously (37, 38). Briefly, a glutathione S-transferase (GST) fusion protein containing the Rap1/2-binding domain of the RalGDS protein (GST-RalGDS) was used to selectively precipitate the active GTP-bound forms of Rap1 and Rap2, which were then detected by sequential immunoblotting with antibodies to Rap2 (Transduction Laboratories, Lexington, KY) and Rap1 (Santa Cruz Biotechnology, Santa Cruz, CA).

Flow Cytometry—To analyze the cell-surface expression of the LFA-1 and {alpha}4 integrins, cells were stained with 30 µg/ml rat anti-mouse LFA-1 monoclonal antibody TIB-213 (FD441.8; American Type Culture Collection) (44) or rat anti-mouse {alpha}4 integrin monoclonal antibody PS/2 (a gift from Dr. B. Chan, Robarts Research Institute, London, Ontario, Canada) (3), followed by fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc.).

Soluble ICAM-1 and VCAM-1—The soluble extracellular domain of ICAM-1 was produced in NS-1 cells and purified from culture supernatants by affinity chromatography as described previously (45). The VCAM-1-Fc fusion protein was produced in COS cells and purified from the culture supernatant by protein A affinity chromatography. The plasmid encoding the VCAM-1-Fc fusion protein was constructed as follows. cDNA encoding domains 1–3 of murine VCAM-1 was generated by reverse transcription-PCR using RNA from the murine endothelial cell line ENDO-D1. After sequencing, the PCR product was subcloned into the Fc fusion vector pIG, which carries the genomic fragment encoding the hinge and constant regions of human IgG1 (46).

Adhesion Assays Using Immobilized ICAM-1 or VCAM-1—Adhesion assays were performed using a modification of the method described by Welder et al. (45). Nunc Maxisorp 96-well plates were coated at room temperature for 60–90 min with either soluble ICAM-1 or VCAM-1-Fc (30 µg/ml) diluted in 0.1 M carbonate buffer (pH 9.6). The wells were washed three times with phosphate-buffered saline and blocked with 0.5 mg/ml bovine serum albumin in phosphate-buffered saline for 30 min at room temperature. A20 cells were resuspended at 1.25 x 106/ml in Hanks' buffered saline solution with 2% fetal calf serum (binding buffer) and stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma) or anti-IgG antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 25 min at 37 °C. The cells were then pelleted, resuspended at 1.25 x 106/ml in binding buffer containing 10 µg/ml 5-chloromethylfluorescein diacetate (Molecular Probes, Inc., Eugene, OR), and incubated for 30 min at 37 °C. When the cells were stimulated with SDF-1, they were stained with 5-chloromethylfluorescein diacetate prior to being stimulated with SDF-1 for 20 min. After being labeled and stimulated, the cells were pelleted and resuspended at the same concentration in binding buffer, except for cells to be treated with neutralizing antibody to either LFA-1 or {alpha}4 integrins. These cells were resuspended at 1.25 x 107/ml in 50 µl of binding buffer containing 50 µg/ml anti-LFA-1 monoclonal antibody TIB-213 or anti-{alpha}4 integrin monoclonal antibody PS/2, incubated for 5 min at room temperature, and diluted to 1.25 x 106/ml in binding buffer. For each sample, 1.25 x 105 cells in 0.1 ml of binding buffer was added to triplicate wells of the soluble ICAM-1- or VCAM-1-Fc-coated 96-well plate. The plates were incubated at 37 °C for 25 min, and the total fluorescent signal (excitation at 485 nm and emission at 530 nm) from each well was measured using a Bio-Tek FL600 microplate fluorescence reader (Bio-Tek Instruments Inc., Winooski, VT). The wells were then washed manually five to six times with 37 °C binding buffer to remove non-adhering cells, and the fluorescent signal from each well was measured again. Data are represented as the percent of cells that remained adhered after washing. This was calculated by dividing the post-wash fluorescence (remaining adhered cells) by the pre-wash fluorescence (total input cells) for each well.

Cell-Cell Adhesion Assays—To assess the homotypic aggregation of WEHI-231 cells, 2 x 106 cells were resuspended in 2 ml of complete medium and added to each well of a 6-well plate. The cells were incubated with 2 nM PdBu in the presence or absence of 30 µg/ml anti-LFA-1 monoclonal antibody TIB-213 for 16 h at 37 °C and then photographed. The number of single cells and aggregates, as well as the number of cells per aggregate, was determined for several random fields. To assess the adhesion of A20 cells to the M2-10B4 bone marrow stromal cell line, A20 cells (2 x 106 in 1 ml of complete medium) were plated on a 75% confluent monolayer of M2-10B4 cells in a 6-well plate. Where indicated, 30 µg/ml anti-{alpha}4 integrin monoclonal antibody PS/2 or 10 µg/ml rabbit anti-CXCR4 antibody (Santa Cruz Biotechnology) was added to the medium. The A20 cells were allowed to adhere for 4–5 h before washing the wells twice with complete medium and then photographing the remaining cells. The number of A20 cells adhering to the M2-10B4 cells was determined by counting random fields of cells.

Cell Spreading Assays—A20 cells (7.5 x 105 cells in 1.5 ml of complete medium) were added to each well of a 6-well tissue culture plate and cultured with or without 10 µg/ml goat anti-mouse IgG antibodies. The cells were allowed to adhere and spread overnight at 37 °C and then were observed by light microscopy. For each well, three different observers each determined the number of spread cells in three random fields containing ~300 cells each. In addition, the cells were photographed using a camera system attached to an Olympus IX70 microscope. Cells that had a flattened morphology or were phase-dark were scored as spread.

Actin Polymerization Assays—The amount of polymerized F-actin was analyzed on a single cell basis by flow cytometry essentially as described by Bleul et al. (47). Single cell suspensions of A20 cells (4 x 105 cells in 0.4 ml of modified HEPES-buffered saline) were added to polypropylene microcentrifuge tubes and stimulated with 15 nM PMA for various times at 37 °C. Reactions were stopped by adding 100 µl of phosphate-buffered saline containing 9% formaldehyde, 2 µg/ml FITC-phalloidin (Sigma), and 0.25 mg/ml lysophosphatidylcholine (Sigma). After 10 min at 37 °C, the mean fluorescence intensity was determined using a FACScan flow cytometer and taken as a measure of the amount of polymerized F-actin. Where indicated, the cells were incubated with rat anti-mouse LFA-1 monoclonal antibody TIB-213 (30 µg/ml final concentration) for 30 min at 37 °C before being stimulated with PMA.

Pyk2 Tyrosine Phosphorylation—Where indicated, 6-well tissue culture plates were coated with a collagen/fibronectin ECM (48). The collagen/fibronectin ECM was prepared by adding 1.5 ml of a 2% gelatin solution (type B from bovine skin, Sigma) to each well of the plate and then incubating the plate overnight at 37 °C. The next morning, the gelatin was aspirated; 1.5 ml of fetal calf serum was added to each well; and after incubating the plate at 37 °C for 1 h, the wells were washed with phosphate-buffered saline. Vector control and RapGAPII-expressing A20 cells (5 x 106 cells in 1 ml of modified HEPES-buffered saline) were added to wells that had been either left untreated or coated with the collagen/fibronectin ECM. After 20 min at 37 °C, the cells were stimulated with goat anti-mouse IgG antibodies or SDF-1. Reactions were terminated by adding 0.25 ml of cold 5x lysis buffer (50 mM Tris-HCl (pH 7.2), 5% Triton X-100, 140 mM KCl, 10 mM EDTA, 2.5 mM Na3VO4, 1 mM Na2MoO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin). The cell extracts were placed on ice for 10 min and then centrifuged for 15 min in the cold to remove insoluble material. The cell extracts were mixed with 1 µg of goat anti-Pyk2 antibody (sc-1514, Santa Cruz Biotechnology) for 1 h at 4 °C and then transferred to tubes containing 10 µl of protein G-Sepharose (Sigma) for 1 h. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose. The filters were probed with anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Inc., Charlottesville, VA) or with antibody that recognizes Pyk2 phosphorylated at either Tyr579 or Tyr580 (BioSource International, Camarillo, CA). The filters were then reprobed with goat anti-mouse Pyk2 antibody, followed by horseradish peroxidase-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rap Activation Regulates Integrin-mediated B Cell Adhesion to ICAM-1 and VCAM-1—In B cells, integrin activation and the subsequent integrin-mediated adhesion mediate many important processes and can be stimulated by phorbol esters, antigens, anti-Ig antibodies, and chemokines such as SDF-1. To investigate the role of Rap activation in cell adhesion, cell spreading, and actin polymerization in B cells, we blocked the activation of endogenous Rap1 and Rap2 by expressing a Rap-specific GTPase-activating protein, RapGAPII, in A20 murine B lymphoma cells (41). RapGAPII promotes the conversion of active GTP-bound Rap1 and Rap2 to the inactive GDP-bound forms while having no effect on the activation of other GTPases, including Ha-Ras (49), R-Ras (49), RhoA (50), and Rac1 (38). Using a Rap activation assay in which a GST-RalGDS fusion protein is used to selectively precipitate the active GTP-bound forms of Rap1 and Rap2, we have shown that RapGAPII expression significantly inhibited the ability of phorbol esters (Fig. 1A), anti-Ig antibodies (41), and SDF-1 (38)2 to activate Rap1 and Rap2 in A20 cells and in other B cell lines. Rap1 activation was completely blocked by RapGAPII expression, whereas Rap2 activation was decreased by at least 80%, as determined by densitometry. RapGAPII expression can therefore be used as a loss-of-function approach for investigating the function of Rap activation in B cells.



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  		FIG. 1.
RapGAPII expression blocks the activation of endogenous Rap1 and Rap2 and inhibits B cell adhesion to ICAM-1 and VCAM-1. A, vector control and RapGAPII-expressing A20 cells were stimulated with 100 nM PdBu for the indicated times. Rap activation assays were performed using a GST-RalGDS fusion protein to selectively precipitate the active GTP-bound forms of Rap1 and Rap2, which were detected by immunoblotting with specific antibodies. Molecular mass markers (in kilodaltons) are shown to the left. Similar results were obtained in multiple independent experiments using this clone of RapGAPII-expressing A20 cells as well as another clone. A similar inhibition of Rap1 and Rap2 activation was observed when the cells were stimulated with a different phorbol ester (PMA) instead of PdBu. B–F, vector control and RapGAPII-expressing A20 cells were stimulated with 15 nM PMA (B and E), 10 µg/ml anti-IgG (C and F), or 100 ng/ml SDF-1 (12.5 nM) (D) before being assayed for their ability to bind immobilized ICAM-1 (B–D) or immobilized VCAM-1 (E and F). The data are expressed as the percent of cells that bound firmly and represent the means ± S.D. for triplicate wells in the same experiment. For each panel, similar results were obtained in at least three independent experiments. The use of neutralizing antibody to LFA-1 (B–D) or {alpha}4 integrins (E and F) showed that the adhesion to ICAM-1 and VCAM-1 was mediated by LFA-1 and {alpha}4 integrins, respectively. As a specificity control, we showed that anti-{alpha}4 integrin antibody did not block LFA-1-dependent adhesion to ICAM-1. G, vector control (upper panels) and RapGAPII-expressing (lower panels) A20 cells were analyzed for LFA-1 and {alpha}4 integrin expression by flow cytometry. The thin lines represent unstained cells, whereas the thick lines represent cells stained with rat anti-LFA-1 monoclonal antibody TIB-213 or rat anti-{alpha}4 integrin monoclonal antibody PS/2, followed by FITC-conjugated mouse anti-rat IgG antibodies.

 

To assess whether Rap activation is involved in B cell adhesion mediated by LFA-1 or {alpha}4 integrins, we compared the ability of vector control and RapGAPII-expressing A20 cells to bind to recombinant ICAM-1 or VCAM-1 that had been adsorbed onto microtiter wells. We found that blocking the activation of endogenous Rap1 and Rap2 via the expression of RapGAPII significantly inhibited the ability of A20 cells to bind to ICAM-1 and VCAM-1. Phorbol esters, anti-IgG antibodies, and SDF-1 increased the ability of the vector control A20 cells to bind to ICAM-1 (Fig. 1, B–D). This adhesion was mediated by LFA-1 and could be blocked by neutralizing antibody to LFA-1. The PMA-, anti-IgG-, and SDF-1-induced binding of the Rap-GAPII-expressing cells to ICAM-1 was reduced by 70–80% compared with the vector control cells (Fig. 1, B–D). Similarly, phorbol esters and anti-IgG antibodies increased the ability of vector control A20 cells to bind to VCAM-1 via {alpha}4 integrins, and this response was significantly reduced in the RapGAPII-expressing cells (Fig. 1, E and F). Other experiments indicated that Rap activation was also important for SDF-1 to induce {alpha}4 integrin-dependent adhesion (see Fig. 4). Thus, the activation of Rap1 and/or Rap2 is important for both LFA-1- and {alpha}4 integrin-mediated B cell adhesion. Because RapGAPII expression did not decrease the cell-surface expression of LFA-1 or {alpha}4 integrins (Fig. 1G), these results indicate that Rap activation is important for extracellular stimuli to convert LFA-1 and {alpha}4 integrins to their active states that have high avidity/affinity for their ligands.



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  		FIG. 4.
RapGAPII expression inhibits the adhesion of A20 cells to the M2-10B4 bone marrow stromal cell line. A, vector control (upper left panel) and RapGAPII-expressing (lower panel) A20 cells were added to a monolayer of M2-10B4 cells and incubated for 4–5 h at 37 °C. After washing away unbound cells, the fibroblast-like M2-10B4 cells and any bright, round A20 cells that adhered were photographed. Scale bars = 200 µm. The number of vector control and RapGAPII-expressing cells adhering to M2-10B4 cells was determined by counting three random fields of cells (upper right panel). The data represent the mean number of adherent A20 cells ± S.E. for three independent experiments. B, neutralizing antibody to CXCR4 or {alpha}4 integrins inhibited the binding of vector control A20 cells to the M2-10B4 stromal cell line. Vector control A20 cells were plated on monolayers of M2-10B4 stromal cells in the presence or absence of antibody to CXCR4 or {alpha}4 integrins. The number of A20 cells adhering to M2-10B4 cells was determined by counting three random fields of cells. As a comparison, the effects of RapGAPII expression are also shown. The data are expressed as the percent of vector control cells that adhered in the absence of neutralizing antibody and represent the means ± S.E. for three independent experiments.

 
To support the idea that Rap activation plays a role in integrin-mediated B cell adhesion, we used a complementary gain-of-function approach in which we expressed a constitutively active mutant form of Rap2, Rap2V12, in A20 cells. Rap2 activation assays on the transfected A20 cells confirmed that the FLAG-tagged Rap2V12 protein was constitutively active and that the total amount of activated Rap2 in these cells after stimulation with either phorbol esters (Fig. 2A) or anti-IgG antibodies (41) was significantly greater than in vector control cells. When we examined integrin-mediated adhesion, we found that Rap2V12 expression enhanced both phorbol ester- and anti-IgG-induced adhesion to ICAM-1 and VCAM-1 (Fig. 2, C–F). This was most evident when the cells were stimulated with suboptimal concentrations of PMA or anti-IgG antibodies. Because Rap2V12 expression did not increase the cell-surface expression of LFA-1 or {alpha}4 integrins (Fig. 2B), these results support the idea that Rap-GTP and, in particular, Rap2-GTP can promote integrin activation.



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  		FIG. 2.
Rap2V12 expression increases the amount of activated Rap2 in A20 cells and increases B cell adhesion to ICAM-1 and VCAM-1. A, vector control and Rap2V12-expressing A20 cells were stimulated with 100 nM PdBu for the indicated times, and Rap2 activation was analyzed as described in the legend to Fig. 1. Molecular mass markers (in kilodaltons) are shown to the left. Similar results were obtained in two independent experiments. B, vector control (upper panels) and Rap2V12-expressing (lower panels) A20 cells were analyzed for LFA-1 and {alpha}4 integrin expression by flow cytometry. The thin lines represent unstained cells, whereas the thick lines represent cells stained with rat anti-LFA-1 monoclonal antibody TIB-213 or rat anti-{alpha}4 integrin monoclonal antibody PS/2, followed by FITC-conjugated mouse anti-rat IgG antibodies. C–F, vector control and Rap2V12-expressing A20 cells were stimulated with the indicated concentrations of PMA or anti-IgG antibodies before being assayed for their ability to bind immobilized ICAM-1 (C and D) or immobilized VCAM-1 (E and F). The data are expressed as the percent of cells that bound firmly and represent the means ± S.D. for triplicate wells in the same experiment. For each panel, similar results were obtained in at least three independent experiments. For all conditions shown, the difference between the values for the vector control and Rap2V12-expressing cells was statistically significant (p < 0.05), as determined by Student's t test. The use of neutralizing antibody to LFA-1 (C and D) or {alpha}4 integrins (E and F) showed that the adhesion to ICAM-1 and VCAM-1 was mediated by LFA-1 and {alpha}4 integrins, respectively.

 
Rap Activation Regulates Cell-Cell Adhesion in B Cells— Next, we investigated whether Rap activation in B cells is important for cell-cell interactions that depend on LFA-1 or {alpha}4 integrins. As a simple model of cell-cell adhesion, we made use of the observation that treating B cells with phorbol esters results in homotypic aggregation and the formation of large clusters of cells (51). This response could be readily observed using the WEHI-231 murine B cell line (Fig. 3C). To examine the role of Rap activation in the homotypic adhesion of WEHI-231 cells, we expressed RapGAPII in these cells and showed that it completely blocked phorbol ester-induced Rap1 activation and inhibited Rap2 activation by >80% at all time points, as determined by densitometry (Fig. 3A). When vector control WEHI-231 cells were treated overnight with the phorbol ester PdBu, the cells aggregated into large clusters, with very few single cells remaining (Fig. 3C). This homotypic adhesion was mediated by LFA-1 because the addition of anti-LFA-1 antibody completely blocked the clustering (Fig. 3C). When the RapGAPII-expressing cells were treated with PdBu, they formed far fewer clusters; the clusters that did form contained fewer cells; and there were more single cells (Fig. 3C). In two independent experiments, counting at least 500 cells in random fields showed that only 8.4 ± 1.0% of the vector control cells remained as single cells after the overnight PdBu treatment, whereas 55.8 ± 5.0% of the RapGAPII-expressing cells remained as single cells that did not aggregate. RapGAPII expression had no effect on the cell-surface expression of LFA-1 (Fig. 3B), indicating that Rap activation regulates the ability of LFA-1 to bind to ICAM-1. Thus, activation of the Rap GTPases is important not only for integrin-mediated adhesion of B cells to purified adhesion molecules, but also for LFA-1-mediated cell-cell adhesion in B cells.



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  		FIG. 3.
RapGAPII expression inhibits phorbol ester-induced homotypic adhesion in WEHI-231 cells. A, vector control and RapGAPII-expressing WEHI-231 cells were stimulated with 100 nM PdBu for the indicated times. Rap activation was analyzed as described in the legend to Fig. 1. Molecular mass markers (in kilodaltons) are shown to the left. Similar results were obtained in two independent experiments. B, vector control (upper panel) and RapGAPII-expressing (lower panel) WEHI-231 cells were analyzed for LFA-1 expression by flow cytometry. The thin lines represent unstained cells, whereas the thick lines represent cells stained with rat anti-LFA-1 monoclonal antibody TIB-213, followed by FITC-conjugated mouse anti-rat IgG antibodies. C, vector control and RapGAPII-expressing WEHI-231 cells were cultured for 16 h with medium alone, with medium containing 2 nM PdBu, with or medium containing 2 nM PdBu plus 30 µg/ml anti-LFA-1 before being photographed. Scale bars = 50 µm. Similar results were obtained in three independent experiments.

 
We also examined the role of Rap activation in a more physiological model of B cell adhesion, the binding of B lineage cells to bone marrow stromal cells. SDF-1-mediated adhesion to bone marrow stromal cells plays a critical role in B cell development. This interaction depends on the SDF-1-induced activation of VLA-4, which allows B cells to bind to VCAM-1 on the stromal cells (52). We compared the ability of vector control and RapGAPII-expressing A20 cells to adhere to the M2-10B4 bone marrow stromal cell line, which secretes SDF-1 (40). When vector control A20 cells were added to a monolayer of M2-10B4 cells and cultured for 4–5 h, the A20 cells adhered strongly to the M2-10B4 cells (Fig. 4A). The binding of the A20 cells to the M2-10B4 cells was dependent on CXCR4 signaling as well as {alpha}4 integrin-mediated interactions because both anti-CXCR4 antibody and {alpha}4 integrin-specific blocking antibody reduced the adhesion of the A20 cells to the M2-10B4 cells (Fig. 4B). In contrast to the vector control A20 cells, when the RapGAPII-expressing A20 cells were added to a monolayer of M2-10B4 cells, far fewer cells attached and remained adhered after washing the wells (Fig. 4A). Examining random fields of cells from three independent experiments showed that blocking Rap activation via the expression of RapGAPII caused a 70% reduction in the number of cells that bound to the M2-10B4 cells (Fig. 4, A and B), an effect similar to that caused by blocking CXCR4 signaling or blocking the binding of {alpha}4 integrins to their ligands (Fig. 4B). Thus, Rap activation is important for B cells to undergo cell-cell interactions and contributes to the ability of B lineage cells to bind to bone marrow stromal cells.

Rap Activation Regulates Cell Spreading—Cell-cell and cell-substrate interactions mediated by integrins are often accompanied by cell spreading, a flattening of the cell that increases the surface area of the contact region with other cells or the ECM, thereby allowing stronger adhesion. Having shown that integrin-mediated adhesion in B cells is regulated by Rap GTPases, we investigated whether Rap activation is also important for B cell spreading. We compared the anti-IgG-induced spreading of vector control A20 cells with that of RapGAPII-expressing A20 cells. Unstimulated vector control A20 cells grown overnight in the presence of fetal calf serum on tissue culture-treated plastic dishes displayed a mixture of morphologies, with ~75% of the cells exhibiting a round, detached morphology and ~25% of the cells having an adherent, spread morphology, which corresponded to the cells appearing darker by phase-contrast microscopy and having several membrane projections (Fig. 5, A and B). When the vector control A20 cells were cultured overnight with anti-IgG antibodies, the number of spread cells rose from 25 to ~75%, and both the number of projections per cell and the length of these projections increased following stimulation with anti-IgG antibodies (Fig. 5, A–C). In contrast to the vector control cells, unstimulated RapGAPII-expressing A20 cells rarely showed a spread phenotype in culture and tended to be uniformly round cells that did not adhere to tissue culture plastic (Fig. 5, A and B). Even after stimulation with anti-IgG antibodies, only ~5% of the RapGAPII-expressing A20 cells appeared spread (Fig. 5, A and B). The significantly decreased anti-IgG-induced spreading of the RapGAPII-expressing A20 cells could be due to their inability to adhere to the tissue culture dish, which may be coated with serum-derived ECM components. Interestingly, the small number of RapGAPII-expressing cells that did adhere and spread after anti-IgG stimulation failed to show the dramatic cell projections that were seen in the anti-IgG antibody-treated vector control A20 cells (Fig. 5A, lower right panel). This suggests that Rap activation regulates the extension of membrane processes in addition to regulating cell adhesion. Similar to what we observed for anti-IgG antibody stimulation, phorbol ester-induced cell spreading was also inhibited when Rap activation was blocked by the expression of RapGAPII.2 Thus, in B cells, both anti-IgG- and phorbol ester-induced cell spreading, as well as the extension of membrane projections, is strongly dependent on Rap activation.



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  		FIG. 5.
RapGAPII expression inhibits cell spreading in A20 cells. A, vector control (upper panels) and RapGAPII-expressing (lower panels) A20 cells were grown overnight in tissue culture wells in the presence or absence of 10 µg/ml anti-IgG antibodies and then photographed. Scale bars = 50 µm. B, to quantitate cell spreading, three independent fields of ~300 cells were counted, and cell bodies that were phase-dark or had a flattened morphology were scored as spread. The data are expressed as the percent of cells that were spread and represent the means ± S.E. for three independent experiments. C, a region of the upper right panel in A was enlarged to show anti-IgG antibody-treated vector control cells with eyelash-like cell projections. Scale bar = 50 µm. Similar results were obtained using another RapGAPII-expressing A20 clone.

 
Rap Activation Regulates Actin Polymerization—Both cell spreading and the extension of membrane projections require reorganization of the actin cytoskeleton as well as new actin polymerization. Because blocking Rap activation prevented cell spreading and dramatically suppressed the extension of membrane projections, we hypothesized that the Rap GTPases regulate actin polymerization in B cells. To test this, we stimulated vector control and RapGAPII-expressing A20 cells with the phorbol ester PMA, a strong stimulus that mimics the diacylglycerol produced in response to signaling by the BCR or CXCR4. The cells were then permeabilized and stained with FITC-phalloidin, a specific probe for F-actin (Fig. 6). The amount of polymerized F-actin in the cells was determined by flow cytometry. Stimulating the vector control A20 cells with PMA caused a rapid increase in polymerized F-actin that could be detected within 30 s (Fig. 6A). The response peaked at 5 min and was sustained for at least 45 min. Although the PMA-induced increase in the total amount of polymerized F-actin in the cells ranged from 15 to 40%, the responses were highly reproducible. This appears to be an integrin-independent response because blocking antibody to LFA-1 did not inhibit this phorbol ester-induced actin polymerization (Fig. 6B). The binding of {alpha}4 integrins to ECM components is unlikely to occur in this assay because the cells are stimulated in serum-free medium while in suspension in polypropylene tubes.



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  		FIG. 6.
RapGAPII expression inhibits phorbol ester-induced actin polymerization. A, vector control and RapGAPII-expressing A20 cells were stimulated with 15 nM PMA for the indicated times, fixed, and stained with FITC-phalloidin. The mean fluorescence intensity (MFI) was determined by flow cytometry and taken as a measurement of polymerized F-actin. For both the vector control and RapGAPII-expressing cells, the data are expressed as the percent of F-actin present in the corresponding unstimulated cells, which is indicated as 100%. Note that the amount of polymerized actin in unstimulated RapGAPII-expressing A20 cells was 20.0 ± 4.9% lower than that in unstimulated vector control cells. Each data point represents the mean ± S.E. for six independent experiments. The differences between the PMA-induced increases in F-actin levels in the vector control cells versus the RapGAPII-expressing cells were statistically significant, as determined by Student's t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, vector control A20 cells were incubated with or without neutralizing antibody to LFA-1 and then stimulated with 15 nM PMA for the indicated times before quantitating the amount of polymerized F-actin as described for A. Each data point represents the mean ± S.D. for two independent experiments.

 
The amount of polymerized F-actin in unstimulated RapGAPII-expressing A20 cells was 20.0 ± 4.9% lower (mean ± S.E.; p = 0.026 for six independent experiments, as determined by Student's t test) than that in unstimulated vector control A20 cells. Moreover, the PMA-induced increase in polymerized F-actin levels was significantly smaller at all time points in the RapGAPII-expressing cells compared with the vector control cells (Fig. 6A). The onset of the increase in F-actin levels also appeared to be slightly delayed in the Rap-GAPII-expressing cells (Fig. 6A). Thus, Rap activation regulates the basal levels of polymerized F-actin in A20 cells and also contributes to the rapid integrin-independent burst of actin polymerization that can be induced by phorbol esters.

Rap Activation Regulates Tyrosine Phosphorylation of the Pyk2 Tyrosine Kinase—In B cells, integrin engagement, as well as antigen receptor and chemokine receptor engagement, has been shown to result in the tyrosine phosphorylation and activation of the Pyk2 tyrosine kinase (53, 54). Pyk2 is related to the focal adhesion kinase (FAK), and both of these kinases have been implicated in mediating integrin-triggered cell adhesion, spreading, and motility (55, 56). Pyk2 also plays an essential role in chemokine-induced B cell migration, as B cells from Pyk2-deficient mice are unable to migrate toward SDF-1 and several other chemokines (57). Because Rap activation in B cells is required for integrin activation (Figs. 1 and 2), cell spreading (Fig. 5), and SDF-1-induced migration (38), we hypothesized that Pyk2 might be regulated by Rap.

Tyrosine phosphorylation of Pyk2 regulates Pyk2 kinase activity, the SH2 (Src homology 2) domain-dependent binding of Src family kinases to Pyk2, and the ability of Pyk2 to regulate cell morphology (55, 58, 59). In A20 cells, we found that both anti-Ig antibodies and SDF-1 induced Pyk2 tyrosine phosphorylation and that this was significantly enhanced by integrin engagement (Fig. 7A). When vector control A20 cells were stimulated in polypropylene tubes3 or in untreated tissue culture wells (Fig. 7A), anti-IgG antibodies caused a modest increase in Pyk2 tyrosine phosphorylation, and SDF-1 caused very weak tyrosine phosphorylation. However, if the A20 cells were plated on wells coated with a collagen/fibronectin ECM that can engage {alpha}4 integrins, the anti-IgG- and SDF-1-induced tyrosine phosphorylation of Pyk2 was significantly enhanced (Fig. 7A). Because Rap activation regulates the ability of integrins to bind their ligands and because integrin engagement contributes to BCR- and SDF-1-induced Pyk2 phosphorylation, we asked whether Pyk2 phosphorylation is regulated by Rap activation. Fig. 7B shows that preventing Rap activation via the expression of RapGAPII substantially reduced both anti-IgG- and SDF-1-induced Pyk2 tyrosine phosphorylation. Moreover, using phosphorylation state-specific antibodies, we found that RapGAPII expression inhibited the phosphorylation of Pyk2 at Tyr579 and Tyr580 (Fig. 7C). Because the phosphorylation of FAK at the analogous residues (Tyr576 and Tyr577) is required for FAK-dependent cell spreading (60), the Rap-dependent phosphorylation of Pyk2 at these sites may be critical for Pyk2 function in B cells.



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  		FIG. 7.
Anti-IgG- and SDF-1-induced tyrosine phosphorylation of Pyk2 depends on integrin engagement and Rap activation. A, vector control A20 cells were cultured for 30 min in either bare tissue culture wells or wells coated with a collagen/fibronectin ECM before being stimulated with 20 µg/ml anti-IgG antibody ({alpha}IgG) or 100 ng/ml SDF-1 (12.5 nM) for the indicated times. Anti-Pyk2 immunoprecipitates (ippt) were probed with anti-phosphotyrosine monoclonal antibody 4G10 (Anti-P-Tyr; upper panels) and then reprobed with anti-Pyk2 antibody (lower panels). The molecular mass of Pyk2 is ~110 kDa. Similar results were obtained in three independent experiments. B, vector control and RapGAPII-expressing A20 cells were cultured for 30 min in wells coated with collagen/fibronectin ECM and then stimulated with either 20 µg/ml anti-IgG antibody or 100 ng/ml SDF-1 (12.5 nM) for the indicated times. Tyrosine phosphorylation of Pyk2 was analyzed as described for A. Similar results were obtained in five independent experiments. The band indicated by the asterisk is a nonspecific band caused by the anti-IgG antibodies used to stimulate the cells. C, vector control and RapGAPII-expressing A20 cells were plated on collagen/fibronectin ECM and stimulated as described for B. Anti-Pyk2 immunoprecipitates were probed with antibody that specifically recognizes Pyk2 phosphorylated at Tyr579 (Y579; first row) or Tyr580 (Y580; third row). These blots were then reprobed with anti-Pyk2 antibody (second and fourth rows, respectively). Similar results were obtained in two independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that activation of the Rap GTPases in B lymphocytes is important for integrin-mediated adhesion as well as cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation. Using RapGAPII expression to suppress the activation of Rap1 and Rap2, we showed that Rap activation is required for anti-Ig antibodies, SDF-1, and phorbol esters to effectively induce LFA-1- and {alpha}4 integrin-dependent adhesion of B cells to purified adhesion molecules, to other B cells, and to bone marrow stromal cells. Using a complementary gain-of-function approach, we showed that the constitutively active Rap2V12 enhanced both LFA-1- and {alpha}4 integrin-dependent B cell adhesion. Although Rap1 has been implicated in integrin-mediated adhesion (13, 18–26), this is the first report that Rap2 can promote integrin activation. In addition to regulating integrin activation, we also found that Rap activation plays a critical role in BCR-induced morphological changes that include cell spreading and the extension of membrane processes. We also demonstrated a role for Rap activation in promoting actin polymerization as well as BCR- and CXCR4-induced tyrosine phosphorylation of Pyk2. Actin polymerization is required for changes in cell morphology and for the extension of membrane processes (17). Pyk2 has been linked to changes in cell morphology and is required for B cells to migrate toward a variety of chemokines (56, 57). Together with our previous finding that Rap activation is important for SDF-1-induced B cell migration (38), the data reported here indicate that Rap activation controls a number of cellular processes that regulate B cell adhesion, morphology, and migration.

Although Rap activation has been shown to link receptor signaling to integrin-dependent adhesion in T cells, myeloid cells, and Ba/F3 cells overexpressing LFA-1 (13, 19, 21–26), this is the first report that Rap activation is important for integrin-mediated adhesion in B cells. We have shown that Rap couples both tyrosine kinase-linked receptors such as the BCR and G protein-coupled receptors such as CXCR4 to cellular machinery that can promote integrin activation and cell adhesion. Although the process by which Rap-GTP promotes integrin activation is not well understood, Katagiri et al. (61) recently identified an adaptor protein called RAPL that binds Rap1-GTP as well as LFA-1. Rap1-GTP·RAPL complexes direct the membrane localization of LFA-1 and promote the clustering of LFA-1, which would increase its avidity for ICAM-1. RAPL overexpression also increases the affinity of LFA-1 for ICAM-1. It is not known if RAPL or an analogous protein mediates the Rap-dependent activation of {alpha}4 integrins.

Although previous studies have shown that expressing constitutively active Rap1V12 results in the activation of LFA-1 and {alpha}4 integrins (13, 19, 23–25), this is the first report showing that activated Rap2 can also promote the activation of these integrins. We found that expressing constitutively active Rap2V12 was sufficient to increase the binding of unstimulated A20 cells to ICAM-1 and VCAM-1 (Fig. 2). In addition, Rap2V12 expression enhanced the ability of low concentrations of anti-IgG antibodies and PMA to induce LFA-1- and {alpha}4 integrin-dependent adhesion. At higher concentrations of these stimuli, the activation of endogenous Rap proteins may be sufficient to induce maximal adhesion. Nevertheless, our data show that Rap2-GTP can promote integrin activation, at least in the A20 B cell line. In contrast to our findings, Shimonaka et al. (25) found that constitutively active Rap1V12 enhances LFA-1 activation in Ba/F3 cells, but that Rap2V12 does not. This could reflect cell type-specific functions of Rap1 versus Rap2, differences in the level of Rap2V12 expression achieved by our group versus Shimonaka et al., or the possibility that Rap2V12 is a weaker inducer of integrin activation than Rap1V12. It is not known whether Rap2-GTP can also bind RAPL and, if so, whether it does so with the same efficiency as Rap1-GTP. We have not been able to directly compare the ability of Rap1V12 and Rap2V12 to induce integrin activation in B cells because we have been unable to express constitutively active Rap1V12 in B cell lines. Nevertheless, our data establish that Rap2-GTP is able to promote integrin activation.

Although we have shown that Rap2V12 can promote integrin activation in B cell lines, it is not clear whether BCR- and CXCR4-induced integrin activation in B cells is normally mediated by Rap1, Rap2, or both of these GTPases. Our finding that RapGAPII expression inhibits integrin-mediated B cell adhesion does not distinguish between a role for endogenous Rap1 versus Rap2 because RapGAPII inhibits the activation of both Rap1 and Rap2. Preliminary experiments support the idea that Rap2 contributes to LFA-1 activation in B cells. When we expressed another Rap-specific GTPase-activating protein, SPA-1, in A20 cells, Rap1 activation was completely inhibited, whereas Rap2 activation was virtually unaffected.2 Under these conditions, in which Rap2 was activated in the absence of Rap1 activation, phorbol ester-induced adhesion to LFA-1 occurred normally.2 Although this indicates that the activation of endogenous Rap2 is sufficient to induce LFA-1 activation in B cells, it does not preclude a role for Rap1 in this process. Currently, we do not have a way to selectively inhibit the activation of Rap2A and Rap2B without also inhibiting the activation of Rap1A and Rap1B. Dominant-negative forms of Rap1 and Rap2 would inhibit the activation of both Rap1 and Rap2 because they act by sequestering upstream activators and because Rap1 and Rap2 share the same upstream activators.

In addition to showing that Rap activation regulates integrin-mediated adhesion in B cells, we also showed that cell spreading is a Rap-dependent process. Culturing A20 B lymphoma cells with anti-Ig antibodies resulted in dramatic morphological changes characterized by cell spreading and the extension of membrane processes. Both of these morphological changes were blocked when Rap activation was suppressed via the expression of RapGAPII. Consistent with the idea that Rap activation is important for the anti-IgG-induced extension of membrane processes in B cells, the membrane processes seen in the anti-IgG-treated A20 cells (Fig. 5C) resemble the "eyelash" type of membrane extensions observed by Heo and Meyer (62) when they overexpressed activated Rap2B in NIH 3T3 cells.

Cell spreading and the extension of membrane processes play an essential role in cell migration. During chemokine-induced lymphocyte migration, the cells assume a polarized morphology and then spread and extend membrane processes in the direction of movement as they move along ECM components in response to a gradient of chemoattractant. Integrin-mediated adhesion at the leading edge of the migrating cell provides the traction necessary for forward movement (63). Such directional movement is essential for B cells to enter lymphoid organs, to encounter antigens, and to receive T cell help, the key events required for a B cell to become activated and to differentiate into an antibody-producing plasma cell. In previous work, we have shown that Rap activation is essential for B cells to migrate toward the chemokine SDF-1 (38). Our findings in this study that Rap activation regulates integrin-mediated B cell adhesion as well as cell spreading and the extension of membrane processes provide possible mechanisms by which Rap activation promotes cell migration.

Both cell spreading and the extension of membrane processes are dependent on reorganization of the actin cytoskeleton as well as the formation of new actin polymers that support membrane processes and protrusions (17). In B cells, stimulation with phorbol esters has been shown to rapidly induce actin polymerization, as judged by an increase in the total amount of polymerized F-actin in the cell (31). This actin polymerization response can apparently occur in an integrin-independent manner because it occurs in cells that are in suspension (Fig. 6) and is not blocked by antibodies that prevent the binding of integrins to their ligands (Fig. 6B). We found that this rapid phorbol ester-induced actin polymerization response in B cells is partially dependent on Rap activation. In the RapGAPII-expressing A20 cells, the PMA-induced increase in polymerized F-actin was reduced and delayed compared with the response seen in the vector control cells. In addition, the amount of polymerized F-actin in unstimulated cells was significantly lower (20 ± 5%) in the RapGAPII-expressing A20 cells than in the vector control cells. Thus, Rap regulates both basal and signaling-induced actin polymerization. Although overexpressing Rap has been shown to increase the amount of F-actin in Dictyostelium cells (27), this is the first report that Rap activation regulates the amount of F-actin in mammalian cells. Thus, Rap appears to be an evolutionarily conserved regulator of the actin cytoskeleton.

Rap-GTP may regulate the actin cytoskeleton in B cells via both integrin-dependent and integrin-independent mechanisms. The contribution of Rap activation to the rapid phorbol ester-induced increase in polymerized F-actin shown in Fig. 6 likely reflects a direct, integrin-independent effect of Rap-GTP on actin polymerization because these experiments were done with cells in suspension, conditions under which integrin engagement is likely to be minimal. In contrast, the Rap-dependent spreading of anti-IgG-treated A20 cells (Fig. 5) could involve integrin signaling because these responses occurred over an 18-h time period and because the cells were cultured in the presence of serum, which contains fibronectin and other ECM components that can adhere to tissue culture plates. In fibroblasts, integrin signaling can lead to activation of Rac1 and Cdc42, GTPases that promote actin polymerization and reorganization (16).

In this study, we also show for the first time that the Pyk2 tyrosine kinase is a downstream target of Rap. Pyk2 is an important regulator of cell morphology and cytoskeletal organization as well as a key mediator of integrin signaling. Pyk2 is required for cell spreading and adhesion-dependent cytoskeletal reorganization in osteoclasts and macrophages (59, 64) and is also essential for B cells to migrate toward the chemokines SDF-1, BLC (CXCL13), and SLC (CCL12) (57). Although the mechanism by which Pyk2 regulates cell morphology and migration is not understood, Pyk2 has been reported to associate with a number of proteins that have been implicated in these processes, including FAK, paxillin, Vav, and phosphatidylinositol 3-kinase (55, 56, 65–68). We found that both anti-IgG- and SDF-1-induced tyrosine phosphorylation of Pyk2, specifically phosphorylation at Tyr579 and Tyr580, was dependent on Rap activation, as it was substantially reduced in RapGAPII-expressing cells. Tyrosine phosphorylation of Pyk2 at these sites correlates with increased kinase activity (58). Moreover, phosphorylation of FAK at sites that are equivalent to Tyr579 and Tyr580 of Pyk2 is critical for FAK-mediated cell spreading and migration (60). Thus, Rap-dependent phosphorylation of Pyk2 at these sites in B cells could be important for regulating cell morphology and migration in B cells.

We are currently investigating the mechanism by which Rap controls the phosphorylation of Pyk2 at Tyr579 and Tyr580. In fibroblasts, Src family kinases are thought to phosphorylate these sites in Pyk2 (59). It is unlikely that Rap regulates the activation of Src kinases because blocking Rap activation did not impair overall BCR-induced protein tyrosine phosphorylation.2 However, Rap-dependent integrin activation could regulate the co-localization of Pyk2 with Src kinases. Pyk2 has been shown to co-localize with Src kinases at sites of integrin activation in fibroblasts and T cells (69, 70).

In summary, we have shown that activation of the Rap GTPases promotes integrin-mediated adhesion, cell spreading, actin polymerization, and the phosphorylation of Pyk2 in B cells. By controlling B cell adhesion, morphology, and migration, Rap activation may play an important role in multiple steps in B cell development and activation.


   FOOTNOTES
 
* This work was supported by a grant from the Cancer Research Society of Canada (to M. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of graduate fellowships from the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research. Back

|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-4070; Fax: 604-822-6041; E-mail: mgold{at}interchange.ubc.ca.

1 The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1; VLA-4, very late antigen-4; ICAM-1, intercellular adhesion molecule-1; ECM, extracellular matrix; VCAM-1, vascular cell adhesion molecule-1; BCR, B cell antigen receptor; SDF-1, stromal cell-derived factor-1; RapGAPII, Rap-specific GTPase-activating protein II; PdBu, phorbol 12,13-dibutyrate; GST, glutathione S-transferase; RalGDS, Ral guanine nucleotide dissociation stimulator; FITC, fluorescein isothiocyanate; PMA, phorbol 12-myristate 13-acetate; FAK, focal adhesion kinase. Back

2 S. J. McLeod and M. R. Gold, unpublished data. Back

3 R. L. Lee and M. R. Gold, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank May Dang-Lawson for technical assistance, Cal Roskelley for critically reading the manuscript, and Hanne Ostergaard and Michiyuki Matsuda for key reagents.



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