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J. Biol. Chem., Vol. 276, Issue 44, 40518-40527, November 2, 2001

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Rho and Rho-associated Kinase Modulate the Tyrosine Kinase PYK2 in T-cells through Regulation of the Activity of the Integrin LFA-1^*

José Luis Rodríguez-Fernández^§¶, Lorena Sánchez-Martín^¶, Mercedes Rey^**, Miguel Vicente-Manzanares^**, Shuh Narumiya, Joaquín Teixidó^§§, Francisco Sánchez-Madrid^**, and Carlos Cabañas^¶¶

From the  Instituto de Farmacología y Toxicología CSIC, Facultad de Medicina, Universidad Complutense, 28040 Madrid, ^** Servicio de Inmunología, Hospital de la Princesa, 28006 Madrid, Spain, the  Department of Pharmacology, Kyoto University, Faculty of Medicine, Kyoto 606, Japan, and ^§§ Departamento de Inmunología, Centro de Investigaciones Biológicas CSIC, Velázquez 144, 28006 Madrid, Spain

Received for publication, April 2, 2001, and in revised form, July 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-L-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Lymphocyte function-associated antigen-1 (LFA-1)¹ (CD11a/CD18) is a member of the ₂ family of integrin receptors that is expressed on the membrane of lymphocytes. This molecule mediates important adhesive phenomena through interaction with its ligands intercellular adhesion molecules (ICAM-1, -2, or -3) (1-3). The  and  subunits of LFA-1 are both composed of a large extracellular amino-terminal domain, involved in the recognition of ligands, a transmembrane segment, and a small cytoplasmic carboxyl-terminal region that can potentially bind cytoskeletal and signaling components (3). Resting leukocytes that circulate in the bloodstream are mainly rounded-shaped cells that express an inactive form of LFA-1, which is functionally unable to bind its ligands. However, stimulation of different leukocyte receptors, including the TcR-CD3 complex and cytokine receptors, results in the generation of intracellular signals that lead to activation of LFA-1 and thereby the acquisition of the ability to bind its ligands (3). As to the mechanism of activation of LFA-1, a variety of experimental results indicates that under physiological conditions the ligand binding activity of LFA-1 correlates mostly with the organization of this integrin into clusters on the membrane of T-cells and not with conformational changes of individual integrin molecules, which are characterized by increased affinity for ligands (4, 5).

It has been proposed that the LFA-1 receptor that is expressed on the surface of resting T-cells is maintained in an inactive form due to the interactions of its cytoplasmic region with the network of actin fibers assembled beneath the plasma membrane (4, 5). According to this model, molecules that regulate the organization of this membrane-anchored actin cytoskeleton are potentially able to regulate the activity of LFA-1 because they could release the constraints on this integrin and allow the formation of clusters of LFA-1 with increased binding avidity for ligands (4, 5). In this regard, the small GTPase Rho, a well known regulator of the actin cytoskeleton (6, 7), may potentially perform this function as an indirect modulator of LFA-1. Rho GTPase molecules cycle between an inactive GDP-bound form and an active GTP-bound state. The cycling between these two forms is regulated in vivo by guanidine nucleotide exchange factors and GTPase-activating proteins (6, 7). Two different approaches have been used to inhibit the function of Rho. The first is the use of the C3-exoenzyme from Clostridium botulinum, an agent that ADP-ribosylates and specifically inactivates this small GTPase (8). The second approach is based on the use of N19Rho, a dominant-negative form of this small GTPase (9). Through the use of both approaches, it has been shown that Rho participates in a number of leukocyte functions, including the regulation of T-cell activation and pseudopodial extension (10, 11), B-cell homotypic aggregation (12), chemokine-induced adhesion (13), neutrophil emigration, up-regulated adhesion (13-15), NK cell mobility and cytosolic activity (16, 17), and in the maintenance of lymphocyte and monocyte shape (11, 18, 19).

Some of the downstream effects of the GTP-bound Rho are exerted through the serine/threonine kinase Rho-associated kinase (Rho kinase/ROK/ROCK, hereafter referred as ROCK) (20). ROCK phosphorylates and inhibits the enzyme myosin light chain phosphatase and also directly phosphorylates the myosin light chain (MLC), which altogether results in an increase in phosphorylated MLC and formation of actin-myosin contractile fibers (6, 7, 20). Another target of ROCK is LIM kinase (LIMK), which phosphorylates and inhibits the protein cofilin, leading to the stabilization of F-actin (20, 21). The study of the cell functions controlled by ROCK has been significantly advanced with the use of Y-27632, a specific competitive inhibitor of this kinase (22-24). Studies with this inhibitor have shown that ROCK, acting on the targets mentioned above, is able to regulate the dynamics of the actin cytoskeleton (20).

Upon activation of LFA-1, this integrin binds ICAM-1, which subsequently leads to the polarization of T-cells (25). In vivo, such polarization is observed following the interaction of the lymphocytes with antigen-presenting cells or during chemokine-directed transendothelial migration, processes where LFA-1/ICAM-1 interactions play a key role (26). Following ligand engagement, LFA-1 induces a variety of intracellular signals that are thought to be involved in the process of polarization induced by this integrin. Intracellular signals induced from LFA-1 include phospholipid hydrolysis, mobilization of Ca²⁺ from intracellular stores, activation of different isoenzymes of protein kinase C, phosphorylation of phospholipase C1 and the linker protein p130^CAS, and activation of the non-receptor tyrosine kinases FAK and PYK2 (25, 27-29). Proline-rich tyrosine kinase 2 (PYK2), also called RAFTK (for related adhesion focal tyrosine kinase), CAK (for Cell adhesion kinase ), CADTK (for calcium-dependent tyrosine kinase), or FAK2, is a tyrosine kinase expressed in cells from neural, epithelial, and hematopoietic origin (30-34). PYK2 does not harbor SH2 or SH3 domains but contains several binding sites for different SH2/SH3-containing signaling proteins. Following activation/autophosphorylation of this enzyme, PYK2 may bind cytoskeletal proteins (like paxillin), linkers (like p130^Cas), or other tyrosine kinases (including Src family members), which may potentially impinge PYK2 in a variety of signal transduction pathways (35, 36).

In this study we show that inhibition of the small GTPase Rho, or its downstream effector ROCK, leads on T-cells to clustering and activation of the LFA-1 integrin and the enhancement of the tyrosine kinase activity of PYK2. Such activation of PYK2 requires engagement of LFA-1, strongly suggesting that Rho and ROCK can regulate the tyrosine kinase PYK2 through modulation of LFA-1 activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Human T-lymphoblasts were prepared from peripheral blood mononuclear cells by treatment with 0.5% phytohemagglutinin for 48 h (37-39). Cells were washed and cultured in RPMI 1640 (BioWhittaker) containing 10% FCS (Flow Laboratories) and interleukin-2 (20 units/ml). T-lymphoblasts cultured for 10-12 days were typically used in all experiments. The T-cell line HSB-2 was cultured in RPMI 1640 containing 10% FCS.

Antibodies and Reagents-- ICAM-1-Fc chimeric protein consisting of the five domains of ICAM-1 fused to the Fc fragment of human IgG1 and the C. botulinum C3-exoenzyme were prepared as described previously (40, 41). KIM-127 and Lia 3/2.1 mAbs have been described elsewhere (42-44). The anti-LFA-1 mAb BCA-1 was from R & D Systems. The 38 and the 24 mAbs (45, 46) were a generous gift of Dr. N. Hogg (Imperial Cancer Research Fund, London) and the NKI-L16 mAb (47) was a gift of Dr. C. G. Figdor (University Medical Center, Nijmegen, The Netherlands). Anti-PYK2 and anti-RhoA antibodies were from Santa Cruz Biotechnology Inc. [-³²P]ATP (4000 Ci/mmol) and ³²P-NAD (200 Ci/mmol) were from American Radiolabeled Chemicals, Inc. Phytohemagglutinin, FITC-phalloidin, TRITC-phalloidin, protein G-Sepharose, BSA, poly-L-lysine, cytochalasin D, anti--tubulin, anti-myosin light chain (clone MY-21), and anti-total actin (AC-40) mAbs, the FITC, TRITC, and peroxidase-conjugated secondary antibodies were all purchased from Sigma. The rabbit polyclonal sera against phosphorylated myosin light chain (P-MLC20) was obtained from Dr. F. Matsumura (48). ECL reagents were from Amersham Pharmacia Biotech. Interleukin-2 was from Eurocetus. Y-27632 was a generous gift of Yoshitomi Pharmaceutical Industries. All other reagents used were of the purest grade available.

Cell Attachment Assays and Confocal Microscopy-- Cell adhesion assays, either on ICAM-1 (5 µg/ml) or on PLL (20 µg/ml)-coated dishes, were performed as described (25). Immunofluorescence staining of -tubulin and actin was performed on round glass coverslips and have been described previously (25). Immunofluorescence analysis of the organization of LFA-1 on the membrane was performed as described (49). Briefly, control and pretreated cells were washed three times in RPMI 1640 before plating them on poly-L-lysine-coated coverslips (150 × 10³ cells/coverslip), in the presence of appropriate stimuli and/or anti-LFA-1 mAbs at 10 µg/ml. Unbound cells were removed by gently washing them in warm RPMI 1640. To prevent antibody-induced clustering, cells were fixed with 1% formaldehyde in PBS for 10 min at room temperature before performing a second incubation for 25 min at 4 °C with 10 µg/ml of either FITC-conjugated sheep anti-mouse IgG or with TRITC-conjugated sheep anti-mouse IgG (for immunofluorescence analysis of the GFP-transfected cells). Before mounting the samples for fluorescence microscopy, they were washed again with PBS and distilled water. Confocal microscopy was performed using a MRC-1000 Confocal Laser Scanning System (Bio-Rad) connected to a Nikon Diaphot 200 inverted microscope. Images of 20 serial vertical cellular sections were acquired every 0.4 µm with the Bio-Rad COMOS graphical user interface and software.

Flow Cytometry Analysis of LFA-1 and Assessment of ICAM-1Fc Binding-- Flow cytometry analysis of cells stained with anti-LFA-1 antibodies (mAbs 24 and 38) was performed as described previously (46, 50). Briefly, untreated controls or T-cells pretreated with the C3-exoenzyme or Y-27632 inhibitors were washed twice in Hepes/NaCl buffer (20 mM Hepes, 150 mM NaCl, 2 mg/ml D-glucose, pH 7.4) containing 2 mM MgCl₂ and 0.5 mM CaCl₂. The control cells that were stimulated with MnCl₂ were washed in Hepes/NaCl buffer containing 0.5 mM EGTA to chelate residual calcium ions before they were incubated for 1 h in Hepes/NaCl buffer that contained 200 µM MnCl₂. After the treatments, the T-cells (5 × 10⁵ cells) were added to a 96-well round-bottom plate, where they were incubated with 10 µg/ml of the 24 or the 38 mAbs for 15 min at 37 °C in 100 µl of PBS. After this first incubation, T-cells were washed 3 times with PBS and incubated for an additional period of 30 min at 4 °C with FITC-conjugated sheep anti-mouse IgG secondary antibody in PBS. Finally, after 3 washes with PBS, cells were fixed in 2% formaldehyde in PBS, and their fluorescence was measured using a FACScan® flow cytometer (Becton Dickinson). Flow cytometry and measurement of the binding of soluble recombinant ICAM-Fc to cells was performed as described (38, 51), maintaining in all steps the cation concentrations as described above in the flow cytometry analysis of LFA-1. Soluble ICAM-1 Fc binding was detected using FITC-conjugated goat anti-human IgG secondary antibody (Fc-specific) (38, 51).

Expression Constructs and Transient Transfection Assays-- The pEGFP-C1 (green fluorescent protein (GFP)) expression vector was obtained from CLONTECH (Palo Alto, CA), and the pEGFP-C1-N19Rho, pEGFP-C1-V14Rho, and pEGFP-C1-N17Rac expression plasmids have been described previously (11). For the transient transfection experiments, HSB-2 cells (2 × 10⁷) were washed twice with cold Hanks' balanced salt solution and resuspended in 500 µl of cold Opti-MEM (Life Technologies, Inc.) before being placed in 0.4-cm electroporation cuvette (Bio-Rad). DNA (15 µg) was added, and transfections were conducted at 1200 microfarads/280 V using a Gene Pulser II electroporation system and the Capacitance Extender Plus module (Bio-Rad). After the electroporation burst, the cells were transferred to plastic dishes and cultured in RPMI 1640 containing 10% FCS and antibiotics (Flow Laboratories). 24 h after electroporation the transfection efficiency was estimated by flow cytometry, and cells with high level of GFP expression were selected by sorting on a FACS Vantage cell sorter (Becton Dickinson, Oxford, UK).

PYK2 Immunoprecipitations-- Untreated or inhibitor-pretreated T-lymphoblasts and untransfected or transfected HSB-2 cells were washed twice with RPMI, plated on dishes coated with BSA, PLL, or ICAM-1 (2.5 × 10⁶ cells/35 mm dish), as specified in the legends of the figures, and kept for 15 min on ice. The dishes were then transferred to a 37 °C incubator for an additional 40 (T-lymphoblasts) or 90 min (HSB-2 cells). Parallel dishes of T-lymphoblasts or HSB-2 cells, which were used as positive controls, were stimulated also with 10 µg/ml mAb KIM-127 for the same period. After these incubation periods on the dishes, all plated cells (both attached and in suspension) were solubilized in 1 ml of ice-cold lysis buffer (10 mM Tris/HCl, pH 7.65, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 2 mM sodium orthovanadate, 1% Triton X-100, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation at 14,000 rpm for 10 min and the pellets discarded. After centrifugation, supernatants were transferred to new tubes, and PYK2 was immunoprecipitated at 4 °C overnight with protein G-agarose-conjugated goat polyclonal anti-PYK2 antibody (C-19). Immunoprecipitates were washed three times with lysis buffer and either used for in vitro kinase reactions (see below) or extracted in 2× SDS-PAGE sample buffer (200 mM Tris/HCl, pH 6.8, 0.1 mM sodium orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol) by boiling 5 min, fractionated by one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE), and further analyzed as described under "Results" and the figure legends.

In Vitro Kinase Assays-- PYK2 kinase assays were performed as described (25, 52). Briefly, PYK2 immunoprecipitates were washed and pelleted (2,500 rpm for 10 min at 4 °C) three times in lysis buffer and twice with kinase buffer (20 mM Hepes, 3 mM MnCl₂, pH 7.35). Pellets were dissolved in 40 µl of kinase buffer, and reactions were started by adding 10 µCi of [-³²P]ATP. The reactions were carried out at 30 °C for 15 min and stopped on ice by adding 10 mM EDTA. After the kinase reactions, pellets were washed in lysis buffer containing 10 mM EDTA, extracted for 5 min at 95 °C in 2× SDS-PAGE sample buffer, and analyzed by SDS-PAGE. After fixing and drying the gels, autoradiography was performed at 80 °C. Autoradiographs were analyzed using an AGFA (Mortsel, Belgium) Studio Scan IIsi scanner, and bands were quantified using the Bio-Rad Molecular Analyst software.

Immunoblotting-- Proteins separated by SDS-PAGE were transferred to nitrocellulose using a Bio-Rad SD transblot system and blocked for 2 h using 3% non-fat dry milk in PBS, pH 7.4, and incubated for 1 h at 22 °C with the anti-PYK2 antibody (C-19), the anti-P-MLC20 anti-phospho-myosin light chain (P-MLC20), or the anti-MLC (clone MY-21), anti-actin (AC-40), or anti-RhoA antibodies, in all cases diluted 1:500 in PBS containing 3% non-fat dry milk. After incubating the membranes with horseradish peroxidase-conjugated secondary antibodies, immunoreactive bands were visualized using ECL reagents.

Treatment of the T-cells with the C3-exoenzyme and ADP-ribosylation Assay of Rho-- To inhibit Rho, lymphocytes were incubated for 16 h in 10% FCS in RPMI plus 50 µg/ml C3-exoenzyme (dissolved in 20 mM Hepes, 150 mM NaCl, pH 7.5). This long treatment was required to allow efficient entry of C3-exoenzyme into the cells (41, 53). We measured the degree of ADP-ribosylation of Rho after the C3 treatment as described (54). Briefly, equal numbers (2.5 × 10⁶) of control and C3-treated cells were washed with cold PBS, harvested in 400 µl of cold buffer (2 mM MgCl₂, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin in 50 mM Hepes, pH 7.4), and sonicated. Aliquots (100 µg) of these lysates were incubated with ³²P-NAD and 25 ng of C3-exoenzyme at 37 °C for 30 min. 2× SDS-PAGE sample buffer was added, and the samples were boiled for 5 min and run on SDS-PAGE. Gels were fixed, dried, and subjected to autoradiography at 80 °C. Ribosylated Rho is clearly identified in the autoradiographs as an apparent 21-23-kDa band (41).

Treatment with Y-27632 and Assessment of Myosin Light Chain Phosphorylation-- To inhibit ROCK and consequently the phosphorylation of the MLC, T-lymphocytes were incubated with 20 µM Y-27632 (stock solution in water) for 1 h (22-24). To measure the phosphorylation of the MLC, control and cells treated with Y-27632 were pelleted, rinsed in PBS, and precipitated with ice-cold 10% trichloroacetic acid in acetone containing 2 mM dithiothreitol. Pellets were rinsed twice with ice-cold acetone containing 10 mM dithiothreitol, dried, and solubilized in 2× SDS-PAGE sample buffer. Samples were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blotting either with the MY-21 mAbs or with the P-MLC20 anti-phospho-MLC antibody (48).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Inactivation of Rho and ROCK Reorganizes the F-actin Cytoskeleton of T-cells-- To study the involvement of the small GTPase Rho and its downstream effector ROCK in the process of LFA-1-dependent cellular adhesion and polarization, T-cells were pretreated either with the C3-exoenzyme (Rho inhibitor) or with Y-27632 (ROCK inhibitor) as specified under "Materials and Methods." We assayed first the effectiveness of these inhibitors by analyzing in T-cells maintained in suspension their effects on the ADP-ribosylation of Rho and the phosphorylation of MLC, respectively. As shown in Fig. 1A, after the 16-h pretreatment of the cells with the C3-exoenzyme, Rho molecules were very efficiently ADP-ribosylated as a further short in vitro treatment of cell extracts (30 min) with C3-exoenzyme in the presence of ³²P-NAD rendered very low incorporation of radioactivity (90% inhibition). As shown in Fig. 1B, Y-27632 treatment resulted in an 84% inhibition in the phosphorylation levels of the MLC, demonstrating the effectiveness of this inhibitor.

View larger version (73K): [in this window] [in a new window]   Fig. 1.   A, ADP-ribosylation of Rho induced by C3-exoenzyme treatment of T-cells. Upper box, cells were untreated (CONTROL) or pretreated with the C3-exoenzyme (50 µg/ml) for 16 h (C3). After the toxin pretreatment the cells were lysed, and the lysate proteins (100 µg) were subjected to an in vitro 32P-ADP-ribosylation assay as described under "Materials and Methods." As C3-exoenzyme is highly specific for Rho, 32P labeling of this protein is clearly identified in the autoradiographs. The lack of incorporation of radioactivity of Rho in vitro in the extracts demonstrates the efficiency of the pretreatment of the T-cells with the C3-exoenzyme. ADP-ribosylated Rho (ADPr-Rho) is indicated. Lower box, anti-RhoA immunoblot analysis of 100 µg of total protein, as a gel loading control. Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as percentage of in vitro inhibition of Rho ribosylation related to controls. B, inhibition of the phosphorylation of myosin light chain following treatment with Y-27632. Untreated (CONTROL) or Y-27632-pretreated (20 µM, 1 h) lymphocytes were extracted with trichloroacetic acid in acetone, and the proteins precipitated were split into two halves and dissolved in 2× SDS-PAGE sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Each nitrocellulose membrane was then subjected to Western blotting with the phosphorylated myosin light chain (P-MLC20) (upper box) and anti-total MLC (MY-21) (lower box) antibodies, respectively. The location of MLC and its phosphorylated form (MLC-P) is indicated. Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as percentage of inhibition of MLC phosphorylation related to controls. C, effect of C3-exoenzyme, Y-27632, and cytochalasin D on the F-actin staining of T-cell plated on PLL-coated dishes. T-cells were incubated untreated (CONTROL) or treated with 50 µg/ml of C3-exoenzyme for 16 h or with 20 µM Y-27632 for 1 h or with 0.1 µg/ml of cytochalasin D for 2 h. The cells were plated for 1 h on PLL-coated dishes and then fixed, permeabilized, and stained with TRITC-phalloidin to visualize F-actin. D, transfection of the HSB-2 T-lymphoblastoid cells with the GFP-N19Rho cDNA construct affects the F-actin organization of the HSB-2 cells. Transfections were performed as described under "Materials and Methods." Mock-transfected HSB-2 cells (MOCK), or cells transfected with GFP-vector, GFP-N19Rho, or GFP-V14Rho, were washed in RPMI and plated onto PLL-coated dishes for 60 min. The cells were then fixed, permeabilized, and F-actin stained with TRITC-phalloidin as specified under "Materials and Methods." GFP protein staining was analyzed using the FITC fluorescence channel.

The C3-exoenzyme and Y-27632 inhibitors were next used to analyze the involvement of Rho and ROCK in the organization of the filamentous actin (F-actin) cytoskeleton of T-cells. Control and T-cells pretreated with either of these agents or with a low concentration of cytochalasin D (0.1 µg/ml), an agent which at this concentration partially depolymerizes F-actin, were plated on PLL-coated dishes, and F-actin was stained with TRITC-phalloidin. In these experiments, cells were plated on PLL, not on the LFA-1 ligand ICAM-1, to study directly the effect of the inhibitors on the organization of F-actin without the possible influence of LFA-1/ICAM-1 interactions on F-actin. Compared with control cells, where F-actin was organized in a thick rim around the cell, in T-cells pretreated with the inhibitors the thickness of this ring was reduced, and patches of actin become evident beneath the lymphocyte membrane (Fig. 1C). The C3 and Y-27632 inhibitors affected only the organization of the F-actin fraction since both SDS-PAGE and Western blot analyses of control and inhibitor-treated cells demonstrated that the total actin levels of the cells were not altered (not shown). Furthermore, the effects of the inhibitors on the cytoskeleton were selective, as the -tubulin cytoskeletal network was not altered as detected by immunofluorescence staining (not shown). The results obtained with the C3-exoenzyme in primary T-lymphoblasts were confirmed by transfecting the T-lymphoblastoid cell line HSB-2 with a cDNA construct that expresses GFP-N19Rho, a dominant-negative inhibitor of Rho tagged with GFP (9, 11). As controls we used HSB-2 cells that were transfected with the GFP vector or with GFP-V14Rho, which encodes a constitutively active form of Rho. HSB-2 cells from the transfected population with high and homogeneous levels of GFP were sorted by FACS. These cells were subsequently plated on PLL-coated glass slides, and the F-actin organization of the transfected cells was analyzed by immunofluorescence using TRITC-phalloidin. In agreement with the experiments performed with the C3-exoenzyme, in the GFP-N19Rho transfected HSB-2 cells the thickness of the F-actin cytoskeleton was reduced, and actin was observed aggregated below the membrane (Fig. 1D). In contrast, HSB-2 cells transfected with the GFP vector or with GFP-V14Rho displayed a thick ring of F-actin that was not reorganized (Fig. 1D). The results show that the inhibition of Rho or ROCK leads to the disorganization and aggregation of the F-actin cytoskeleton in T-cells.

Inhibition of Rho or ROCK Induces an Increase in Adhesion and Morphological Changes in T-cells Plated on ICAM-1-- To analyze the role of Rho and ROCK in the control of LFA-1 function, untreated controls and T-cells pretreated with C3-exoenzyme or the Y-27632 inhibitor were plated on ICAM-1-coated dishes, and their degree of adhesion and changes in morphology were examined. As shown in Fig. 2, a low percentage of unstimulated T-cells attached to the dishes precoated with the ligand ICAM-1. However, either direct induction of activation of LFA-1 with the activating mAb KIM-127, which was used as a control stimulus to induce activation of LFA-1, or pretreatment of cells with C3-exoenzyme or Y-27632 inhibitors led to an important increase in the percentage of cells adhering to ICAM-1 (Fig. 2). Furthermore, this augmented adhesion was accompanied by profound changes in the morphology of the lymphoblasts, from a round and moderately spread morphology to an elongated and highly spread phenotype where the cells display a cell body and a long cellular projection (Fig. 2). These changes induced by the treatment of the inhibitors when the T-cells were plated on ICAM-1-coated dishes were mediated by the interaction of LFA-1 with its ligand ICAM-1 as T-cells pretreated with these inhibitors and plated onto poly-L-lysine-coated dishes attached and spread but did not exhibit any change in adhesion or morphology (Fig. 2). Moreover, when cells pretreated with the inhibitors were plated onto ICAM-1-coated dishes in the presence of different anti-LFA-1-blocking antibodies (Lia 3/2.1, 38, or BCA-1 mAbs), both the adhesion and the morphological changes were completely blocked (not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Effect of the treatment with C3-exoenzyme and Y-27632 on the morphology and LFA-1-dependent adhesion of T-cells. Lymphocytes were plated either on ICAM-1 or PLL-coated dishes. Cells untreated (CONTROL) or pretreated with the C3-exoenzyme (16 h, 50 µg/ml) or Y-27632 (20 µM, 1 h) were subsequently left unstimulated or were stimulated with the LFA-1-stimulatory mAb KIM-127 (10 µg/ml) and plated on the dishes for 1 h. The cells were fixed and stained with 0.5% of crystal violet in 20% methanol before photography. A representative experiment is shown. Bar diagrams on the right side of the figure represent quantitations of static cell adhesion assays on ICAM-1 or PLL, performed as specified under "Materials and Methods." The values represent the mean ± S.E. (n = 4) percentage of adherent cells.

The changes in morphology observed following the treatment with the C3-exoenzyme in primary T-lymphoblasts were confirmed transfecting HSB-2 T-cells with the dominant-negative inhibitor of Rho (GFP-N19Rho). HSB-2 cells were transfected with the GFP vector, with GFP-N19Rho, or with the constitutively active form of Rho (GFP-V14Rho). The cells from the transfected population with high and homogeneous levels of GFP were then sorted by FACS. These cells were plated on ICAM-1-coated glass slides, and immunofluorescence phalloidin staining was performed. As shown in Fig. 3, HSB-2 cells transfected with GFP-N19Rho displayed important changes in morphology. The morphology acquired by these cells is very similar to that exhibited by mock-transfected HSB-2 cells, where LFA-1 had been directly activated with mAb KIM-127, and also similar to the phenotype displayed by T-lymphoblasts pretreated with C3-exoenzyme or with Y-27632 (Fig. 2). In contrast, HSB-2 cells transfected with the GFP vector or with GFP-V14Rho did not exhibit any change in their morphology (Fig. 3). Taken together, the results demonstrate that inhibition of Rho or its downstream effector ROCK leads to an increase in the LFA-1-mediated adhesion to ICAM-1 and to polarization of the cells.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Transfection of the HSB-2 T-lymphoblastoid cells with the GFP-N19Rho cDNA construct induces polarization of the cells. Transfections were performed as described under "Materials and Methods." Mock-transfected HSB-2 cells either unstimulated (MOCK) or stimulated with the LFA-1-stimulatory mAb KIM-127 (10 µg/ml) (MOCK + KIM-127) or cells transfected with the GFP vector, GFP-N19Rho, or GFP-V14Rho cDNA constructs were washed in RPMI and plated onto ICAM-1-coated dishes for 90 min. The cells were then fixed, permeabilized, and F-actin stained with TRITC-phalloidin as specified under "Materials and Methods." GFP protein staining was analyzed using the FITC channel. Insets, show Nomarski images of individual cells.

Inhibition of Rho or ROCK Induces an Increase in the Avidity of LFA-1-- We analyzed in more detail how the inhibition of Rho or ROCK regulates the observed LFA-1-dependent increase in the adhesion of T-lymphocytes to ICAM-1. Such increase in adhesion could be potentially due either to an enhancement in the expression of LFA-1 and/or to changes in the affinity and/or the avidity of this integrin. FACS analysis using the mAb 38, an antibody that recognizes the  subunit of LFA-1, showed that treatment with C3-exoenzyme (Rho inhibitor) or Y-27632 (ROCK inhibitor) did not affect the level of expression of the integrin (not shown). To study the effects of the inhibitors on the affinity of LFA-1, we used the mAb 24, which recognizes an epitope expressed on the  subunit of LFA-1 only upon conformational changes that parallel a high ligand-binding affinity state of this integrin (45, 46). T-cells treated with Mn²⁺, a divalent cation that induces strong expression of the epitope recognized by the mAb 24, were used as a positive control for expression of high affinity LFA-1. MnCl₂ was used to activate LFA-1 instead of KIM-127 to rule out any interference with the secondary antibody in the FACS and immunofluorescence analyses. These experiments showed that pretreatment of the cells with the C3-exoenzyme or the Y-27632 inhibitor did not affect significantly the expression of the epitope 24, indicating that the affinity of LFA-1 was not altered (Fig. 4A). Consistent results were obtained by studying directly the ability of soluble ICAM-1 to interact with LFA-1 integrin by FACS (Fig. 4B). In these experiments, we observed that, in contrast to what was observed with the divalent cation Mn²⁺, the C3 and Y-27632 inhibitors did not increase the amount of soluble ICAM-1 binding to LFA-1 (Fig. 4B).

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Treatment with C3-exoenzyme or Y-27632 does not change the affinity and induce clustering of LFA-1. A, flow cytometry analysis of the expression of the epitope recognized by mAb 24. Untreated control cells (CONTROL), cells pretreated either with the C3-exoenzyme (C3) or the Y27632 inhibitor, and untreated cells stimulated with PDBu (75 nM) or MnCl2 (200 µM) were analyzed by FACS as described under "Materials and Methods." Results are expressed as mean fluorescence intensity. The values represent mean ± S.E. (n = 4) of mean fluorescence intensity. B, soluble ICAM-1 Fc was incubated with untreated cells (CONTROL), with cells pretreated with the C3-exoenzyme (C3), with cells pretreated the Y-27632 inhibitor, or with untreated cells that were stimulated with PDBu (75 nM) or MnCl2 (200 µM). Bound soluble ICAM-1 was detected with FITC-conjugated goat anti-human (Fc-specific) antibody and analyzed by flow cytometry. Results are expressed as mean fluorescence intensity. The values represent mean ± S.E. (n = 4) of mean fluorescence intensity. C, T-cells were left untreated (CONTROL), pretreated with the C3-exoenzyme (50 µg/ml) for 16 h (C3-EXOENZYME), or pretreated with Y-27632 (20 µM, 1 h), before plating them on PLL-coated dishes. Some untreated control cells were also stimulated for 1 h with PDBu (75 nM) or MnCl2 (200 µM). LFA-1 was detected with 10 µg/ml of the 38, 24, or NKI-L16 mAbs and analyzed by confocal microscopy as specified under "Materials and Methods." A representative experiment is shown.

We also examined the effects of the C3-exoenzyme on the distribution of LFA-1 on the T-lymphoblast membrane by confocal microscopy. For this purpose, we plated the lymphocytes on PLL-coated dishes, instead of ICAM-1, to prevent any effect of this ligand on the organization of LFA-1. The T-cells were stained in parallel with anti-LFA-1 mAb 38, which recognizes the L subunit of LFA-1 molecules, with mAb 24, which recognizes the high affinity form of LFA-1, or with mAb NKI-L16, which recognizes an epitope that is expressed only when LFA-1 is clustered on the membrane of the T-cells (47). The cells pretreated with C3-exoenzyme were compared with cells stimulated with PDBu, a phorbol ester that induces clustering of LFA-1 on the membrane, or with Mn²⁺, a divalent ion that increases the affinity of LFA-1 for its ligand. As shown in Fig. 4C, unstimulated cells, LFA-1 is expressed evenly around the membrane, with few areas of higher or concentrated fluorescence. In contrast, similarly to cells treated with PDBu, many clusters of high intensity LFA-1 fluorescence were detected by the antibodies on the T-cells that were pretreated with the C3-exoenzyme. Interestingly, clusters of LFA-1 were also observed in cells stimulated with Mn²⁺, as shown by the 38 and NKI-L16 staining, implying that this divalent ion induces not only changes in affinity but also clustering of LFA-1 on the membrane. The staining with mAb 24 in the immunofluorescence experiments confirmed the results obtained with the 24 mAb by FACS analysis (Fig. 4A), since, as observed in Fig. 4C, in the cells pretreated with C3-exoenzyme there is no increased expression of the epitope recognized by the 24 mAb. When we examined the effects of the Y-27632 inhibitor on LFA-1 molecules on the membrane of the T-cells by confocal microscopy using the 24, 38, and NKI-L16 mAbs, we observed that they were similar to those obtained upon treatment of the cells with the C3-exoenzyme (Fig. 4C). Taken together, our results show that inhibition of Rho or ROCK causes an increase in LFA-1 avidity without a major change in the affinity of this integrin.

Transfection of HSB-2 T-cells with Constitutively Active V14Rho Blocks the Changes in Organization of LFA-1-- We analyzed whether transfection of the HSB-2 T-cells with the constitutively active form of Rho GFP-V14Rho results in inhibition of the aggregation of LFA-1 molecules on the membrane of these cells. HSB-2 cells that had been transfected with the GFP vector or with GFP-V14Rho were either left unstimulated or were stimulated with 200 µM MnCl₂, an agent that we observed previously that induces the clustering of LFA-1 on the T-cell membrane (Fig. 4C). We used MnCl₂ to induce aggregation of LFA-1 instead of mAb KIM-127 to avoid the interference of this antibody in the immunofluorescence staining. The cells were plated on PLL-coated glass slides, and LFA-1 was stained with the anti-LFA-1 mAb 38 and then examined by confocal microscopy to study the effects of GFP-V14Rho expression on the distribution of LFA-1 on the membrane. As shown in Fig. 5, in contrast to mock-transfected or HSB-2 T-cells transfected with the GFP vector, which show an important aggregation of LFA-1 upon stimulation with MnCl₂, LFA-1 was not clustered on the surface of the GFP-V14Rho-expressing cells following stimulation with MnCl₂. These results indicate that the stabilization of cortical F-actin induced by V14-Rho blocks the stimulus-induced clustering of LFA-1 on the membrane of T-cells.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Transfection with the constitutively active GFP-V14Rho cDNA construct blocks the Mn2+-induced clustering of LFA-1 on the membrane of the HSB-2 T-cells. Transfections were performed as described under "Materials and Methods." Mock-transfected HSB-2 cells (MOCK) or HSB-2 cells transfected with the GFP vector or with the constitutively active GFP-V14Rho were either left unstimulated () or stimulated with 200 µM MnCl2 to activate LFA-1 (MnCl2). The cells were then washed in RPMI and plated onto PLL-coated dishes for 60 min. After this time, the cells were stained with the anti-LFA-1 mAb 38 (using anti-mouse IgG TRITC-conjugated as a secondary antibody) to analyze the distribution of LFA-1 as described under "Materials and Methods." GFP protein staining was analyzed using the FITC channel.

Cortical F-actin Under the Control of Rho Regulates the Activity of Tyrosine Kinase PYK2-- We have shown previously (25) that in T-cells plated onto ICAM-1 the activation of integrin LFA-1 leads to enhanced activity of the tyrosine kinase PYK2. As inhibition of Rho or ROCK with C3 and Y-27632, respectively, also induces the activation of LFA-1 and increases T-cell adhesion to ICAM-1, we analyzed whether these inhibitors could also cause an increase in the activity of PYK2. T-lymphocytes that have been pretreated either with the C3-exoenzyme or with Y-27632 and untreated control cells were plated and allowed to adhered to ICAM-1-coated dishes for 40 min. KIM-127 mAb was used as a positive control for stimulation of LFA-1-dependent adhesion. All plated T-cells (both adherent and in suspension) were lysed; PYK2 was immunoprecipitated, and its activity was analyzed by in vitro kinase assays. The results of these experiments demonstrate that pretreatment either with C3-exoenzyme or with Y-27632 leads to an activation of PYK2, which is similar to that observed upon direct stimulation of LFA-1 with mAb KIM-127 (Fig. 6, A and B). Consistently, transfection of HSB-2 T-cells with dominant-negative GFP-N19Rho also resulted in increased activity of PYK2, which was not observed when these cells were transfected with control GFP vector (Fig. 6C). Furthermore, when we transfected HSB-2 cells with constitutively active GFP-V14Rho, which inhibits the reorganization of LFA-1 on the cell membrane (Fig. 5), we did not observe any activation of PYK2, even after stimulation with the activating LFA-1 mAb KIM-127, and even more, the basal levels of PYK2 activity were clearly reduced (Fig. 6D).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   PYK-2 kinase activity is stimulated following inhibition of Rho or ROCK and blocked by overexpression of constitutively active GFP-V14Rho. A and B, T-cells, untreated or pretreated with C3-exoenzyme (C3) or Y-27632 (see "Materials and Methods"), were washed in RPMI medium and then plated on ICAM-1-coated dishes in the absence () or presence (+) of 10 µg/ml of mAb KIM-127 for 40 min. All plated cells (attached and in suspension) were then lysed, and the lysates were incubated with C-19 anti-PYK2 antibody to immunoprecipitate PYK2 and kinase reactions performed as described under "Materials and Methods" (IP: PYK2; IVK: PYK2). In experiments performed in parallel, lysates treated as before were also precipitated with C-19 antibody and analyzed by SDS-PAGE, followed by transfer of proteins to nitrocellulose membranes and Western blotting with anti-PYK2 antibody (IP: PYK2; WB: PYK2). Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the unstimulated and untreated controls. C, transfections of HSB-2 lymphoblastoid cells were performed as described under "Materials and Methods." Mock-transfected cells (transfection, ) were either left unstimulated () or stimulated (+) with KIM-127 (10 µg/ml), and cells were transfected with the GFP vector (vector) or GFP-N19Rho (N19Rho). Transfected cells were washed in RPMI and plated on ICAM-1-coated dishes for 90 min. All plated cells (attached and in suspension) were then lysed and kinase reactions performed as in A (IP: PYK2; IVK: PYK2). In parallel experiments Western blots were also performed (IP: PYK2; WB: PYK2). Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the untransfected and unstimulated controls. D, transfections of HSB-2 lymphoblastoid cells were performed as described under "Materials and Methods." Mock-transfected cells (transfection, ) and cells transfected with GFP-V14Rho (V14Rho) were either left unstimulated () or stimulated (+) with KIM-127 (10 µg/ml). Transfected cells were washed in RPMI and plated on ICAM-1-coated dishes for 90 min, lysed, and kinase reactions performed as as in C (IP: PYK2; IVK: PYK2). Western blots were also performed in parallel experiments (IP: PYK2; WB: PYK2). Results were quantified, normalized for differences in loading, and expressed as fold activation compared with the untransfected and unstimulated controls. A representative experiment is shown.

We analyzed further the role of F-actin cytoskeleton on the activation of PYK2 by examining the effect of transfecting HSB-2 cells with GFP-N17Rac, a dominant-negative form of the small GTPase Rac that also affects F-actin organization. We transfected HSB-2 cells with GFP-N17Rac (9, 11), with GFP vector, or with GFP-N19Rho (which was used as a positive control for changes in F-actin and LFA-1 distribution) and selected HSB-2 cells with high and homogeneous levels of GFP by FACS sorting. These cells were subsequently plated on PLL-coated glass slides, and the F-actin organization of the transfected cells was analyzed by immunofluorescence using TRITC-phalloidin. As shown in Fig. 7A, compared with the GFP vector-transfected cells, in the GFP-N17Rac-transfected HSB-2 cells the thickness of the F-actin below the cell membrane was reduced, and clusters of actin were also observed. We analyzed also the organization of LFA-1 on the membrane of the GFP-N17Rac transfectants, and as shown in Fig. 7B, transfection with N17Rac also resulted in clustering of LFA-1. Finally, we examined the effect of transfection with GFP-N17Rac on the activity of the tyrosine kinase PYK2. As shown in Fig. 7C, overexpression of N17Rac resulted in an increase in PYK2 activity, which was not observed when these cells were transfected with control GFP vector.

View larger version (61K): [in this window] [in a new window]   Fig. 7.   Transfection of the HSB-2 T-cells with the GFP-N17Rac cDNA construct alters the organization of F-actin, the clustering of LFA-1, and induces activation of PYK2. Transfections were performed as described under "Materials and Methods." Mock-transfected HSB-2 cells (MOCK) or cells transfected with GFP vector or GFP-N17Rac (N17Rac) or with GFP-N19Rho (N19Rho) used as a control were washed in RPMI and plated onto PLL-coated dishes for 60 min. A, the cells were fixed, permeabilized, and F-actin-stained with TRITC-phalloidin as specified under "Materials and Methods." GFP protein staining was analyzed using the FITC channel. B, the cells were then washed in RPMI and plated onto PLL-coated dishes for 60 min. After this time the cells were stained with the anti-LFA-1 mAb 38 (using anti-mouse IgG TRITC-conjugated as a secondary antibody) to analyze the distribution of LFA-1 (see "Materials and Methods"). GFP protein staining was analyzed using the FITC channel. A representative experiment is shown. C, mock-transfected cells (transfection, ), either left unstimulated () or stimulated (+) with KIM-127 (10 µg/ml), and cells transfected with the GFP vector or GFP-N17Rac (N17Rac) were washed in RPMI and plated on ICAM-1-coated dishes for 90 min. Upper box, all plated cells (attached and in suspension) were lysed, PYK2 immunoprecipitated, and kinase reactions performed (IP: PYK2; IVK: PYK2). Lower box, in parallel experiments Western blots to show equal loading were also performed (IP: PYK2; WB: PYK2). Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the untransfected and unstimulated controls. A representative experiment is shown.

As treatment with the C3 and Y-27632 inhibitors, or transfection with the dominant-negative (N19Rho) or constitutive active (V14Rho) forms of Rho, and the dominant-negative form of Rac (N17Rac) are affecting the F-actin cytoskeleton of lymphocytes, these results suggest that reorganization of F-actin is probably necessary for the activation of PYK2 and that the stabilization of subcortical actin blocks the activation of this kinase.

Finally, to analyze directly the role of F-actin on the activation of PYK2, we pretreated the cells with a low concentration of cytochalasin D (0.1 µg/ml) to partially depolymerize F-actin. Such treatment with cytochalasin D induced an increase in the adhesion (4, 5) and also changes in the morphology of the T-cells plated on ICAM-1-coated dishes, which acquire a polarized phenotype (Fig. 8A). In experiments performed in parallel, we analyzed the activity of the kinase PYK2 in lysates obtained from these cytochalasin D-treated cells. As shown in Fig. 8B, in contrast to the effects of the treatment with a high dose of cytochalasin D (5 µg/ml) which, as we have shown before, blocks the activation of PYK2 (25), a low concentration of cytochalasin D (0.1 µg/ml) induced activation of PYK2. Taken together, our experiments demonstrate that agents that partially disorganize the cortical F-actin cytoskeleton of T-cells, such as inhibition of Rho or ROCK or treatment with a low concentration of cytochalasin D, cause the activation of PYK2 and strongly suggest that F-actin mediates the effects of Rho and ROCK on the LFA-1-dependent activation of PYK2.

View larger version (50K): [in this window] [in a new window]   Fig. 8.   Effect of the treatment with a low concentration of cytochalasin D on the LFA-1-dependent adhesion of T-lymphocytes to ICAM-1 and on the activity of PYK2. A, untreated T-cells (), untreated T-cells stimulated with mAb KIM-127 (KIM-127), or cells that had been pretreated with 0.1 µg/ml cytochalasin D (Cyt D) for 2 h were plated for 60 min on ICAM-1-coated plastic wells. Adherent cells were then fixed and stained with 0.5% crystal violet in 20% methanol before photography. A representative experiment is shown. B, upper box, untreated T-cells () or cells pretreated with 0.1 or 5 µg/ml cytochalasin D (Cyt D) were plated for 40 min on ICAM-1-coated dishes. Untreated cells were also plated for 40 min on BSA-coated dishes. All plated cells (attached and in suspension) were then lysed, incubated with C-19 anti-PYK2 antibody to immunoprecipitate PYK2, and subjected to kinase reactions as described under "Materials and Methods" (IP: PYK2; IVK: PYK2). Lower box, in parallel experiments loaded levels of total PYK2 were determined by immunoprecipitation (IP) with the C-19 anti-PYK2 antibody and Western blot analysis (WB) with C-19 anti-PYK2 antibody. Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the untreated controls.

The Effects of Rho or ROCK on the Activity of the Tyrosine Kinase PYK2 Require Engagement of LFA-1-- We analyzed next whether the effects of Y-27632 on the tyrosine kinase activity of PYK2 required ligand engagement of LFA-1 and adhesion of the lymphocytes onto the ligand ICAM-1, or whether this inhibitor was able to cause a direct activation of PYK2. For this purpose, T-lymphoblasts pretreated with the Y-27632 were plated on ICAM-1-coated dishes in the presence or absence of the Lia 3/2.1 or 38 mAbs, two antibodies that block completely the interaction between LFA-1 and ICAM-1, and then the kinase activity of PYK2 was analyzed. The results of these experiments (Fig. 9A) show that treatment of the cells with the LFA-1-blocking mAbs inhibited the increase in PYK2 activity in response to the pretreatment with Y-27632, indicating that engagement of LFA-1 was required for the observed activation of PYK2 when ROCK was inhibited (Fig. 9A). Control experiments performed with an antibody of the same isotype showed that the effects of the blocking antibodies were specifically caused by affecting the interaction between LFA-1 with ICAM-1 (not shown). Furthermore, no increase in activity of PYK2 was observed in T-cells pretreated with C3-exoenzyme or Y-27632 and plated onto PLL-coated dishes instead of ICAM-1 (Fig. 9B). Taken together, the previous experiments demonstrate that the effects of Rho and ROCK on the activity of PYK2 require engagement of LFA-1, strongly suggesting that Rho can regulate PYK2 through modulation of the activity of LFA-1.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   C3-exoenzyme or Y-27632-induced stimulation of PYK2 kinase activity requires engagement of LFA-1. A, upper box, T-lymphocytes were preincubated for 1 h in RPMI medium without () or with Y-27632 (+), subsequently washed in RPMI, plated onto ICAM-1-coated plastic dishes, and allowed to adhere for 40 min in the absence (CONTROL) or presence of the anti-LFA-1 blocking mAbs Lia 3/2 or 38 (both at 10 µg/ml). All plated cells (attached and in suspension) were then lysed and processed for in vitro kinase reactions (IVK) as described under "Materials and Methods." Lower box, equal loading of PYK2 from extracts was confirmed by Western blotting with the anti-PYK2 antibody C-19. Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the untreated controls. B, upper box, T-lymphocytes, untreated or treated with Y-27632 or C-3 exoenzyme (C3), were washed in RPMI, plated on PLL-coated plastic dishes, and allowed to adhere for 40 min. All plated cells (attached and in suspension) were then lysed, the extracts incubated with C-19 antibody to immunoprecipitate PYK2, and in vitro kinase reactions (IVK) performed as described under "Materials and Methods." Lower box, equal loading of PYK2 from extracts was confirmed by Western blotting with the anti-PYK2 antibody C-19. Results were quantified by densitometric analysis, normalized for differences in loading, and expressed as fold activation compared with the untreated controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results presented herein demonstrate that inhibition of the small GTPase Rho or its downstream effector ROCK in T-cells induces clustering and activation of the integrin LFA-1, changes in adhesion and cell morphology, and activation of the tyrosine kinase PYK2. Importantly, the stimulatory effects of the Rho and ROCK inhibitors on the kinase activity of PYK2 are not direct and require the engagement of the integrin LFA-1. Furthermore, our experiments indicate that the effects of Rho and ROCK on LFA-1 are mediated by the F-actin cytoskeleton. The model depicted in Fig. 10 integrates the new findings presented in this paper with previous results (4, 5, 25, 55-58) on the mechanism of activation of the integrin LFA-1 in T-lymphocytes. According to this model: (a) LFA-1 is associated with the F-actin cytoskeleton through a variety of actin-associated proteins (55); (b) organized cortical F-actin acts as a restraint that maintains LFA-1 in an inactive state (4, 5, 55-57); (c) the small GTPase Rho and its downstream effector ROCK, by controlling the organization of the cortical F-actin cytoskeleton, regulates the function of LFA-1 through changes in avidity; and (d) activated LFA-1 confers T-cells the ability to attach to ICAM-1-coated substrates, leading to the polarization of the T-cells and the activation of the tyrosine kinase PYK2 (25). It should be emphasized that one of the important characteristics of this model is that the activation of PYK2, elicited by the treatment of the cells with the C3 and Y-27632 inhibitors, is not direct and requires the engagement of LFA-1, as such activation does not take place when anti-LFA-1-blocking antibodies were used to inhibit the interaction of LFA-1 and ICAM-1. Thus, our results are suggesting a novel mechanism by which the integrin LFA-1 mediates the effects of Rho on the activity of the tyrosine kinase PYK2.

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Model depicting how Rho and ROCK regulates the clustering and activation of LFA-1 on the membrane of the T-cells and the activation of PYK2. Inhibition of Rho (with the C3-exoenzyme or dominant-negative N19Rho) or ROCK (with Y-27632) leads to F-actin disorganization, and this allows the activation of LFA-1 through and increase in the clustering of LFA-1 on the membrane of the cells with no major changes in the affinity of this integrin. The engagement of immobilized ligand ICAM-1 by the activated LFA-1 molecules leads to increase in cell adhesion and polarization of the lymphocytes and to the activation of the tyrosine kinase PYK2. Expression of V14Rho has the opposite effect, i.e. stabilization of F-actin, inhibition of clustering, and avidity activation of LFA-1 on the T-cell membrane and also blockade of morphological changes and activation of PYK2 (see text for details).

According to the model presented in Fig. 10, disruption of the interactions between LFA-1 and the F-actin cytoskeletal network below the plasma membrane releases LFA-1 molecules and leads to the formation of LFA-1 clusters with increased ligand binding avidity (4, 5). This is consistent with reports showing that treatment of T-cells with calcium ionophores (49), which are known to activate proteases that disrupt these interactions between LFA-1 and cortical F-actin, or with low concentrations of the F-actin-disrupting agent cytochalasin D (4, 5, 55-57) induce an increase in the aggregation of LFA-1 molecules and the concomitant LFA-1-dependent adhesion to ICAM-1, similarly to the effects that we observe by inhibiting Rho or ROCK. Interestingly, despite the important enhancement of adhesion to ICAM-1, we did not detect any increase in the affinity of LFA-1 after treatment with the C3 and Y-27632 inhibitors. These results are in agreement with other studies that suggest the activation of LFA-1 in T-cells occurs mainly through increases in clustering, rather than through enhancement of the ligand affinity of this integrin (4, 5, 58). Accordingly, the low basal affinity of the LFA-1 molecules would be sufficient to support strong cellular adhesion when these receptors become organized into clusters on the membrane of the T-cells (58).

According to the data presented herein and our previous results (25), the engagement of activated LFA-1 by ICAM-1 leads to the polarization of T-lymphocytes and the activation of the tyrosine kinase PYK2. The molecular mechanism whereby LFA-1 regulates the polarization of the T-cells and the activation of PYK2 remains to be elucidated. With regard to the activation of PYK2, at present the mechanism of activation of this tyrosine kinase is largely unknown (35, 36). However, as it is well established that aggregation is important for the integrin-dependent activation of a variety of cytoplasmic tyrosine kinases, the clustering of LFA-1 that we observe upon the inhibition of Rho or ROCK could also be important for the activation of PYK2 (59). The polarization of T-cells that takes place upon adhesion to ICAM-1 may also play an important role in the activation of PYK2, as we have shown previously (25) that interference with changes in morphology of T-lymphocytes results in abrogation the LFA-1-mediated activation of this tyrosine kinase.

Our results suggest that Rho/ROCK have to be included in a group of signaling molecules that may regulate the activity of LFA-1 mainly through induction of integrin clustering and changes in avidity (5). In this regard, our results also imply that stimuli that affect molecules that regulate the level of active Rho (GTP-Rho), either through activation of Rho-GAPs or inhibition of Rho-GEFs, may affect the integrity of the T-cell actin cytoskeleton and modulate indirectly the avidity and activity of LFA-1, which upon ligand engagement leads to the activation of downstream signals controlling the phenotypic changes in the cells. In this regard, it has been shown for NIH 3T3 fibroblasts, which are cells normally embedded in a fibronectin matrix, that stimuli that inactivate Rho, such as exposure to the extracellular matrix protein tenascin, induce polarization of the cells that resemble the effects we observe with T-cells (60).

Inactivation of the Rho/ROCK axis seems to also have different effects depending on the cell context and type analyzed. Thus, it has been reported that the treatment of resting T-cells with C3-exoenzyme induces polarization of T-cells plated onto fibronectin ligands, although in that report it did not observe any effect on the adhesion of the cells (10). Also, C3-exoenzyme blocked the LFA-1-dependent aggregation of a B-cell line when these cells were treated with phorbol ester to induce such aggregation (12). Furthermore, in fibroblasts it has been shown that inactivation of Rho leads to the disruption of the clusters of integrin observed at the focal contacts of these cells (61). Finally, it should be pointed out that although our findings and the model presented in Fig. 10 are related mainly to LFA-1, we do not exclude that the activity of other integrin receptors on leukocytes, which could be also associated and controlled by the cortical actin cytoskeletal network below the plasma membrane (62), could also be regulated by the small GTPase Rho and its downstream effector ROCK.

In conclusion, we show that inhibition of the small GTPase Rho or its downstream effector ROCK in T-lymphocytes leads to the activation of the LFA-1 integrin through receptor clustering and avidity changes and also to the enhancement of the tyrosine kinase activity of PYK2. The activation of PYK2 requires engagement of LFA-1, strongly suggesting that Rho and ROCK can regulate the tyrosine kinase PYK2 through modulation of the activity of LFA-1.

	ACKNOWLEDGEMENTS

Y-27632 was a generous gift (to J. T.) from Yoshitomi Pharmaceutical Industries, Ltd., Japan. We acknowledge Dr. Carl Figdor for the supply of NKI-L16 mAb; Dr. Nancy Hogg for the 24 and 38 mAbs; Dr. Birgit Leitinger for the protocol to measure by FACS the affinity of LFA-1 for ICAM-1; Dr. Mariano Vitón and Dr. Pedro Lastres for their advice with the FACS; and Marigel Ollacarizqueta for assistance with the confocal microscopy.

	FOOTNOTES

^* This work was supported in part by Grants SAF 98/0080 from Comisión Interministerial de Ciencia y Tecnología, 08.1/0015/1997 and 08.3/0010.2/1999 from "Comunidad de Madrid" (to C. C.), Grants SAF99-0034-C02-01 and 2FD97-0680-C02-02 from the Ministerio de Educación y Cultura, by QLRT-1999-01036 from the European Community (to F. S.-M.), and Grant SAF 99/0057 from CICYT (to J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a "Contrato de Reincorporación" associated with Grants DGICYT PB94-0231 and CICYT SAF98/0080 from the "Ministerio Español de Educación y Cultura." To whom correspondence regarding PYK2 should be addressed. Present address: Laboratorio de Immunooncología, Hospital Gregorio Maranón, Doctor Esquerdo 46, 28007 Madrid, Spain.

^¶ Both authors contributed equally to this work.

Recipient of a fellowship "Incorporación de Técnicos a Equipos de Investigación" from "Comunidad de Madrid."

^¶¶ To whom correspondence should be addressed: Instituto de Farmacología y Toxicología CSIC, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. Tel.: 34 91 3941444; Fax: 34 91 3941470; E-mail: cacabagu@eucmax.sim.ucm.es.

Published, JBC Papers in Press, August 6, 2001, DOI 10.1074/jbc.M102896200

	ABBREVIATIONS

The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1; ROCK, Rho-associated kinase; ICAM-1, intercellular adhesion molecule-1; PLL, poly-L-lysine; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; MLC, myosin light chain; PDBu, phorbol 12,13-dibutyrate; FCS, fetal calf serum; TRITC, tetramethylrhodamine B isothiocyanate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES