Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Fernández, J. L.
Right arrow Articles by Cabañas, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Fernández, J. L.
Right arrow Articles by Cabañas, C.
(Journal of Leukocyte Biology. 2002;71:520-530.)
© 2002 by Society for Leukocyte Biology

LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca2+-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells

José Luis Rodríguez-Fernández*, Lorena Sánchez-Martín*, Cristina Alvarez de Frutos*, David Sancho{dagger}, Martyn Robinson{ddagger}, Francisco Sánchez-Madrid{dagger} and Carlos Cabañas*

* Instituto de Farmacología y Toxicología (Centro Mixto CSIC-UCM), Facultad de Medicina, Universidad Complutense, Madrid, Spain;
{dagger} Servicio de Inmunología, Hospital de la Princesa, Madrid, Spain; and
{ddagger} Celltech Ltd., Slough, United Kingdom

Correspondence: Dr. Carlos Cabañas, Instituto de Farmacología y Toxicología (CSIC-UCM), Facultad de Medicina UCM, Pabellón III, 28040 Madrid, Spain. E-mail: cacabagu{at}med.ucm.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Lymphocyte function-associated antigen (LFA-1) is a member of the ß2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca2+ mobilizing agents A23187, thapsigargin, and ionomycin induce an increase in adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) and activation and redistribution of the proline-rich tyrosine kinase-2 (PYK2) to the microtubule-organizing center (MTOC) in T-lymphoblasts. These effects are similar to those observed upon direct induction of activation of LFA-1 with the stimulatory mAb KIM-127. Most importantly, Ca2+ mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca2+-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca2+ chelator EGTA, which depletes the intracellular Ca2+, inhibited the effects of mAb KIM-127 on cell morphology and PYK2 activation. This inhibition with EGTA was not reversed by cross-linking integrin LFA-1 with specific antibodies, indicating that Ca2+ exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca2+ chelators affect the organization of the microtubular cytoskeleton and the localization of PYK2 to the MTOC area, suggesting that these agents could inhibit the activation of PYK2 by interfering with the microtubular network of T cells. Taken together, our results demonstrate for the first time an important role for the integrin LFA-1 and the microtubular cytoskeleton in the Ca2+-mediated activation of PYK2 in T-lymphocytes.

Key Words: adhesion molecules • T-lymphocytes • protein kinases • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18 or {alpha}Lß2) is a member of the ß2 family of integrins, which is expressed selectively on leukocytes. In these cells, LFA-1 mediates adhesive phenomena through interaction with its ligands, intercellular adhesion molecule-1 (ICAM-1; CD54), ICAM-2 (CD102), or ICAM-3 (CD50). The modulation of the ability of LFA-1 to interact with its ligands allows leukocytes to perform important functions in processes such as migration into tissues at sites of inflammation or infection [1 2 3 ]. Resting leukocytes, which circulate in the bloodstream, express a form of LFA-1 that is "inactive," i.e., unable to interact with its ligands. However, upon stimulation of different cell-surface receptors, including receptors for chemokines and the T-cell receptor (TcR)-CD3 complex, a variety of intracellular signals are induced that lead to the "activation" of the LFA-1 integrin and to the acquisition of the ability of this integrin to bind ligands [3 ]. One of the stimuli that leads to the activation of LFA-1 is an increase in the intracellular Ca2+ levels, which may elicit this effect by altering the LFA-1 avidity/affinity for its ligand ICAM-1 [4 , 5 ].

Although in vivo LFA-1 is generally activated by stimulation of different surface receptors, in vitro, this integrin can be activated artificially from without the cells with divalent cations or with specific "activating" or "stimulatory" monoclonal antibodies (mAb) [1 2 3 ]. This type of antibody binds to the {alpha} or the ß subunits of LFA-1, altering the conformation of this integrin to a state of increased affinity for its ligands. Activating antibodies include NKI-L16, which is specific for the {alpha} subunit, and KIM-127, which is directed to the ß2 subunit [5 6 7 ]. Upon activation and engagement of its ligands, LFA-1 integrin induces the generation of intracellular signals in T-lymphocytes, including mobilization of Ca2+ from intracellular stores and activation of tyrosine kinases [3 , 8 9 10 11 12 ].

Proline-rich tyrosine kinase-2 (PYK2), also called focal adhesion kinase (FAK2), related adhesion focal tyrosine kinase (RAFTK), and Ca2+-dependent tyrosine kinase (CADTK), belongs to a family of nonreceptor tyrosine kinases whose canonical member is FAK [13 14 15 16 17 18 ]. In contrast to FAK, which is expressed ubiquitously, PYK2 is restricted to cells of hematopoietic, neural, and epithelial origin [13 14 15 16 17 18 ]. PYK2 shares significant homology with FAK (60% identity in the central catalytic domain and 40% identity in C and N termini) and like FAK, does not contain SH2 or SH3 domains but contains several sites for binding SH2/SH3 containing signaling proteins [18 ]. It is believed that the interactions of PYK2 with intracellular signaling molecules confer this kinase the ability to function in a variety of cellular transduction pathways [18 ]. In this regard, PYK2 has been implicated in the regulation of ion channels [15 ], c-Jun N-terminal kinase (JNK) [19 20 21 22 23 24 25 ], p44/42 mitogen-activated protein kinase (p44/42 MAPK) [15 , 26 , 27 ], p38 MAPK [28 ], and p70 S6 kinase (p70S6K) [29 ].

PYK2 can be regulated by members of the integrin family of adhesion receptors. In this regard, PYK2 displays ß1 integrin-dependent phosphorylation in natural killer cells, B lymphocytes, megakaryocytes, transfected COS cells, and ß2- and ß3-dependent tyrosine phosphorylation in T-lymphocytes [12 , 27 , 30 31 32 ]. In the case of ß1 and ß3 integrins, tyrosine phosphorylation of PYK2 has been stimulated experimentally by direct cross-linking of these integrins with specific antibodies [27 , 30 , 31 ]. It is well-established that PYK2 can also be stimulated by receptors and stimuli that lead to the elevation of intracellular Ca2+ in PC12, epithelial, B cells, megakaryocytes, and platelets [14 , 18 , 19 , 33 , 34 ]. However, in spite of the recognized importance of Ca2+, it is not clear how this cation induces the activation of PYK2. Because this tyrosine kinase is not directly activated by Ca2+ or calmodulin in vitro, it has been suggested that PYK2 should be regulated indirectly by a Ca2+-dependent cellular event, although the nature of such an event has not been elucidated [15 ]. In this paper, we have investigated the role of Ca2+ in the signaling from LFA-1 and specifically, in the LFA-1-mediated activation of the tyrosine kinase PYK2. Our results demonstrate for the first time an important role for the LFA-1 integrin and the T-cell microtubular cytoskeleton in the Ca2+-mediated activation of PYK2 in T-lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Cells and culture conditions
Human T-lymphoblasts were prepared from peripheral blood mononuclear cells as described [35 , 36 ]. T-lymphoblasts cultured for 10–16 days were typically used in all experiments.

Antibodies and reagents
A dimeric form of an ICAM-1-Fc chimeric protein consisting of the five domains of ICAM-1, fused to the Fc fragment of human immunoglobulin G (IgG)1, was prepared as described previously [37 ]. The LFA-1-stimulatory mAb KIM-127 is specific for the ß2 integrin subunit [6 , 7 ]. The anti-LFA-1 {alpha} subunit (CD11) 38 and 24 [38 , 39 ] mAb were a generous gift of Dr. N. Hogg [Imperial Cancer Research Fund (ICRF), London]. The anti-CD31 mAb TP1/15, which was used as a control, the anti-LFA-1 ß subunit (CD18) Lia 3/2 mAb, and the anti-LFA-1 {alpha} subunit TP1/40 mAb have been described previously [40 , 41 ]. The PYK2 antipeptide polyclonal antibody C-19 was from Santa Cruz Biotechnology (Santa Cruz, CA). Thapsigargin, ionomycin, A23187, ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), phytohemagglutinin (PHA), bovine serum albumin (BSA), colchicine, poly-L-lysine (PLL), L-{alpha}-lysophosphatidylcholine, the anti-{alpha}-tubulin mAb, and the fluorescein isothiocyanate (FITC)- and TRITC-conjugated secondary antibodies were all purchased from Sigma Chemical Co. (St. Louis, MO). FITC-phalloidin and fluorocrome Fluo-3/AM were from Molecular Probes (Eugene, OR). Protein G-agarose was from Boehringer Mannheim (Lewes, UK). Enhanced chemiluminescence (ECL) reagents were from Amersham (Little Chalfont, UK). Interleukin (IL)-2 was from Eurocetus (Amsterdam, The Netherlands). All other reagents used were of the purest grade available.

Cell-attachment assays and LFA-1 integrin cross-linking experiments
Cell-adhesion assays were performed as described elsewhere [12 , 35 , 39 , 42 ]. To induce LFA-1 cross-linking in cells under attachment conditions, the mAb used (at 10 µg/ml) were immobilized for 2 h at 37°C on plastic plates that have been precoated overnight with 10 µg/ml rabbit anti-mouse antibody; then the dishes were blocked with 1% BSA in phosphate-buffered saline (PBS) for 1 h and finally, washed extensively with RPMI before adding cells. To induce cross-linking of LFA-1 on cells in suspension, T-lymphocytes were incubated in RPMI at 4°C with the suitable mAb (at 10 µg/ml), followed by a 20-min incubation at 37°C in the presence of rabbit anti-mouse antibody (25 µg/ml).

Flow cytometric analysis of LFA-1 and the content of F-actin
Flow cytometry analysis of cells stained with anti-LFA-1 antibodies was performed as described previously [43 , 44 ]. Unless otherwise specified, cells were washed and incubated in RPMI medium containing the appropriate concentrations of different stimuli prior to their addition to the wells of a 96 round-bottom well plate. Approximately 5 x 105 cells were incubated in each well with 2.5 µg of the 24 or 38 mAb for 15 min at 37°C in 100 µl PBS. After this first incubation, cells were washed three 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 three washes with PBS, cells were fixed in 2% formaldehyde in PBS, and their fluorescence was measured using a FACScan® flow cytometer (Becton Dickinson, San Jose, CA). The content of F-actin was performed as described [45 ]. Briefly, cells were permeabilized, fixed, and stained in a single step by the addition of a solution containing 0.25 mg/ml L-{alpha}-lysophosphatidyl-choline, 4% formaldehyde, and 2 U/ml FITC-phalloidin. Cells were incubated at room temperature for 10 min, washed twice with PBS, and analyzed on the flow cytometer as above.

Measurement of intracellular-free calcium
Changes in the levels of intracellular-free calcium were measured by flow cytometry with the calcium-sensitive fluorochrome Fluo-3/AM as described previously [46 ]. Briefly, human T-lymphoblasts (2x106 Fluo-3-loaded cells) were analyzed in each assay. After the establishment of the basal level of Ca2+ during 60 s, the cells were stimulated with the calcium mobilizers in a final volume of 0.5 ml RPMI. Cells were acquired during the assay using the FACScan® cytometer and analyzed using the CellQuest software (Becton Dickinson).

Digital confocal microscopy
For immunofluorescence analysis, round-glass coverslips (13 mm {phi}) were precoated with recombinant ICAM-1-Fc protein and blocked with 1% BSA as indicated above. T-lymphoblasts were washed twice in HEPES/NaCl buffer (20 mM HEPES, 150 mM NaCl, 2 mg/ml glucose, pH 7.4) and allowed to adhere to the ICAM-1-Fc-coated coverslips for 60 min at 37°C. After two washes in PBS, adherent cells were fixed and permeabilized at room temperature with 1.85% formaldehyde and 0.1% Triton X-100 in PBS. Cells were then incubated for 60 min with a goat anti-PYK2 polyclonal antibody. Then, cells were washed extensively in PBS/0.5% BSA and incubated for 60 min with the corresponding anti-goat FITC-conjugated secondary antibody. In some experiments, the samples were washed again with PBS and double-stained with the anti-{alpha}-tubulin mAb for 60 min, washed in PBS/0.5% BSA, followed by an incubation for 60 min with an anti-mouse TRITC-conjugated secondary antibody, or stained with TRITC-conjugated phalloidin. Before mounting the samples for fluorescence microscopy, they were washed again with PBS and distilled water. Confocal microscopy was performed using an MRC-1000 confocal laser-scanning system (Bio-Rad, Watford, UK) connected to a Nikon Diaphot 200 inverted microscope. Images of 20 serial vertical-cellular sections were acquired every 0.5 µm with the Bio-Rad COMOS graphical user-interface and software.

{alpha}-Tubulin fractionation
Cells (5x105 cells per treatment) were preincubated with RPMI alone, RPMI containing 3 mM EGTA, or RPMI containing 10 µM colchicine, then washed in PBS, and permeabilized (8 min) in warm PBS containing 0.1% Triton X-100 [47 ]. After this period, the Triton X-100 soluble fraction was removed, and the Triton X-100 insoluble fraction was dissolved in PBS buffer. Equal volumes of the two fractions were dissolved in 2 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample, boiled, and run in SDS-PAGE electrophoresis buffer (see below) [12 ]. After SDS-PAGE, proteins were stained with Coomassie brilliant blue or transferred to nitrocellulose membranes to perform Western blotting of {alpha}-tubulin.

Immunoprecipitation and Western blotting
Immunoprecipitations were performed as described [12 ]. Briefly, T-lymphoblasts (2.5x106 cells, unless otherwise stated) were washed twice with RPMI, plated on dishes coated with BSA or ICAM-1, and after 15 min on ice, stimulated with 10 µg/ml mAb KIM-127 for 35 min. The stimulation was terminated by solubilizing the cells in 1 ml ice-cold lysis buffer [10 mM Tris/HCl, pH 7.65, 5 mM ethylenediaminetetraacetate (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 were discarded. After centrifugation, supernatants were transferred to fresh tubes, and proteins were immunoprecipitated at 4°C overnight with protein G-agarose-linked goat polyclonal anti-PYK2 antibody (C-19). Immunoprecipitates were washed three times with lysis buffer and used for in vitro kinase reactions (see below) or extracted in 2 x 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 for 5 min, fractionated by one-dimensional SDS-PAGE, and further analyzed as described in Results and in the figure legends. SDS-PAGE proteins were transferred to nitrocellulose, blocked using 3% nonfat dried milk in PBS, pH 7.2, and incubated for 2 h at 22°C with either the anti-PYK2 polyclonal antibody or the anti-{alpha}-tubulin mAb, both diluted 1:500 in PBS containing 3% nonfat dried milk. After incubating membranes with horseradish peroxidase (HRP)-conjugated secondary antibodies, immunoreactive bands were visualized using ECL reagents.

In vitro kinase reactions
Reactions were performed as described [12 , 48 ]. Briefly, immunoprecipitates were washed and pelleted (2500 rpm 10 min in the cold) 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 kinase buffer, and reactions were started by adding 10 µCi [{gamma}-32P]adenosine 5'-triphosphate (ATP). The reactions were carried out at 30°C for 15 min and were stopped on ice by adding 10 mM EDTA. After the in vitro kinase reactions, the pellets were washed in lysis buffer containing 10 mM EDTA, extracted for 5 min at 95°C in 2 x SDS-PAGE sample buffer, and fractionated by SDS-PAGE. After fixing and drying the gels, autoradiography was performed at -80°C. Autoradiograms were analyzed using an AGFA StudioScan-II-si scanner, and bands were quantified using the Bio-Rad molecular analyst software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Ca2+ mobilization induces morphological changes and activation of LFA-1 integrin in T-lymphoblasts
To study the effects of intracellular Ca2+ in adhesion and morphology of T-lymphocytes, cells were stimulated with thapsigargin or the Ca2+ ionophores A23187 and ionomycin to induce an increase in the cytosolic Ca2+ concentration. In the absence of stimulation, most T cells grow in suspension, remain as individual cells, and display a rounded appearance [12 ]. Treatment with Ca2+ mobilizers induced an important and rapid increase in the intracellular Ca2 levels in these cells (Fig. 1 A ), which was followed by an enhancement in the LFA-1-dependent lymphoblast adhesion to ICAM-1 (Fig. 1B) and significant changes in lymphoblast morphology (Fig. 1C) . Adhering lymphoblasts changed their shape from a round and moderately spread morphology to an elongated and highly spread phenotype, characterized by the presence of a cell body and a long cellular projection (Fig. 1C , right panel). These changes induced by Ca2+ mobilizers were similar to those observed upon direct induction of activation of LFA-1 molecules on T cells using the activating mAb KIM-127 (Fig. 1B and 1C) and were not induced when the cells were plated on PLL instead of ICAM-1 (Fig. 1C , left panel), demonstrating the requirement for the integrin LFA-1.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 1. Ca2+ mobilizers induce an increase in the intracellular Ca2+ levels, enhancement of adhesion to ICAM-1, morphological remodeling, and activation of LFA-1. (A) T cells were loaded with Fluo3, and Ca2+ levels were measured as indicated in Materials and Methods. Prior to the addition (arrow) of the corresponding stimuli (ionomycin, 7 µM; thapsigargin, 5 µM; and A23187, 4 µM), basal Ca2+ levels were recorded for 60 s. Addition of a vehicle solution did not exert any effect on Ca2+ levels (not shown). Data are represented as density-plot profiles. (B) T-lymphoblasts were washed in RPMI medium and then plated on ICAM-1-Fc-coated plastic dishes and allowed to adhere for 60 min in the absence (NO STIMULUS) or the presence of different LFA-1 stimulating agents: 10 µg/ml mAb KIM-127, 4 µM A23187, 7 µM ionomycin, or 5 µM thapsigargin. Cell-adhesion assays were carried out as specified in Materials and Methods. The values represent the mean ± SE (n=4) percentage of adhesion with respect to the maximum adhesion obtained with the KIM-127 mAb. (C) T-lymphoblasts plated on ICAM-Fc or on PLL-coated dishes were left unstimulated or stimulated with the KIM-127 mAb or different Ca2+-mobilizing agents as in (B). The cells were fixed and stained with 0.5% crystal violet in 20% methanol before photography. A representative experiment is shown. (D) T-lymphoblasts were washed in RPMI medium and then incubated with RPMI alone (NO STIMULUS), RPMI plus 200 µM Mn2+, 4 µM A23187, 7 µM ionomycin, or 5 µM thapsigargin for 40 min. Then the expression of the LFA-1 "activation-reporter" epitope detected by mAb 24 was measured by flow cytometry as described in Materials and Methods. Bars represent percentage of positive cells for the activation epitope 24, and the values above the bars represent mean fluorescence intensity. Note that the experiments were performed in RPMI medium that contains Ca+2 ions, which exerts inhibitory effects upon LFA-1 function [43 ]. A representative experiment out of three is shown.

 
Because Ca2+ mobilization affects adhesion onto ICAM-1, we analyzed the effects of Ca2+ on the activity of LFA-1. T cells were stimulated with Ca2+ mobilizers, and then the expression of the LFA-1 "activation-reporter" epitope detected by mAb 24 [38 , 39 ] was analyzed by flow cytometry. As shown in Figure 1D , all Ca2+ mobilizers induced expression of the epitope 24 on T cells, although to a lower extent than the expression of 24 induced by Mn2+, a potent activator that increases the ligand affinity of LFA-1 [42 , 43 ]. Treatment with Ca2+ mobilizers did not have any effect on the total expression of LFA-1 as demonstrated with the anti-{alpha}L LFA-1 38 mAb [38 ] by fluorescein-activated cell sorter (FACS) analysis (not shown). Taken together, these results show that Ca2+ mobilization results in increased LFA-1-dependent adhesion, remodeling of lymphoblast morphology, and increased ligand-binding affinity of LFA-1.

Ca2+ mobilizers induce microtubular remodeling and relocalization of the tyrosine kinase PYK2 in lymphoblasts
Because we have demonstrated previously that the tyrosine kinase PYK2 localizes to the microtubule-organizing center (MTOC) following activation of LFA-1 [12 ], we studied whether Ca2+ mobilizers could also induce changes in the microtubular cytoskeleton and in the localization of PYK2. Lymphoblasts plated on ICAM-1, unstimulated or treated with the Ca2+ mobilizer A23187 or with Mn2+ as a control, were stained by double-labeling immunofluorescence with an anti-{alpha}-tubulin mAb and with an anti-PYK2 antibody. The {alpha}-tubulin staining displayed by the cells stimulated with Ca2+ mobilizers was similar to the staining shown by T cells when LFA-1 was stimulated directly from outside with Mn2+ (Fig. 2 A ), with the characteristic microtubular network stemming from the MTOC, which is typically located between the cell body and the long cytoplasmic projection [12 ]. In the cells stimulated with Ca2+ mobilizers, such as ionomycin, thapsigargin (not shown), or A23187 (Fig. 2A) , PYK2 distributed mainly to the MTOC area, as demonstrated by the costaining with the anti-{alpha}-tubulin antibody (Fig. 2A) . When the staining of {alpha}-tubulin and PYK2 was performed in cells plated on dishes covered with PLL instead of ICAM-1 (Fig. 2B) , it was observed that although PYK2 also localized in these cells to the MTOC area, the staining in this region is more diffuse, and neither Mn2+ nor A23187 induces relocation of PYK2 (Fig. 2B) .



View larger version (91K):
[in this window]
[in a new window]
  		Figure 2. Organization of {alpha}-tubulin and location of PYK2 to the MTOC area in the Ca2+-stimulated cells. Lymphoblasts were allowed to adhere onto ICAM-1-Fc (A) or PLL-coated dishes (B) for 90 min in HEPES/NaCl buffer containing 1 mM Mg2+ and 1 mM Ca2+ (unstimulated), in HEPES/NaCl buffer containing 200 µM Mn2+ [stimulated (Mn2+)], or in RPMI containing 4 µM A23187 [stimulated (A23187)]. The cells were fixed, permeabilized, and doubled-stained with the C-19 anti-PYK2 antibody and with an anti-{alpha}-tubulin mAb ({alpha}-TUBULIN). Immunofluorescence was analyzed by confocal microscopy as described in Materials and Methods. Arrowheads indicate the position of the MTOC.

 
Ca2+-induced activation of PYK2 in lymphoblasts requires engagement of LFA-1
We wanted to confirm that Ca2+ mobilizers induce an increase in the kinase activity of PYK2 in T cells, similarly to what has been described in other cellular systems [14 , 18 , 19 , 33 , 34 ]. For this purpose, T cells plated in ICAM-1-coated dishes were stimulated with different ionophores and then lysed and processed for in vitro kinase analysis of PYK2. As shown in Figure 3 , Ca2+ mobilization induced activation of PYK2 when T cells were plated in ICAM-1-coated dishes. However, it is most interesting that when the same experiments were performed with T cells maintained in suspension on BSA (not shown) or plated in ICAM-1-coated dishes in the presence of the mAb 38 or Lia 3/2, two anti-LFA-1-blocking antibodies that prevent the interaction between ICAM-1 and LFA-1 [38 , 40 ], the activation of PYK2 was blocked (Fig. 3) . These results demonstrate clearly that Ca2+ mobilization alone is not sufficient for induction of activation of PYK2 in T-lymphoblasts and that engagement of LFA-1 is required for this process.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Ca2+-induced stimulation of PYK2 kinase activity requires engagement of LFA-1. T-lymphoblasts plated on ICAM-1-Fc-coated plastic dishes and stimulated with 4 µM A23187, 5 µM thapsigargin (TG), 7 µM ionomycin, or 10 µg/ml mAb KIM-127 were allowed to adhere for 40 min in the absence (-) or presence (+) of the anti-LFA-1-blocking mAb 38 (CD11a) or Lia 3/2 (CD18). The cells were then lysed, the extracts were incubated with the C-19 antibody to immunoprecipitate PYK2, and the in vitro kinase reactions (IVK) were performed as described in Materials and Methods. PYK2 levels were determined by immunoprecipitation (IP) with the C-19 anti-PYK2 antibody and Western blot analysis (WB) with the C-19 anti-PYK2 antibody. The results shown are representative of three independent experiments.

 
Ca2+ mobilization is necessary for LFA-1-mediated morphological remodeling and activation of PYK2
Because T-lymphoblasts stimulated with Ca2+ mobilizers undergo important morphological changes when they are plated on ICAM-1-coated dishes, we examined further the Ca2+ dependence of these morphological changes by incubating the cells with EGTA. This nonpermeable chelator blocks Ca2+ influx through the plasma membrane and depletes the intracellular stores of this cation. EGTA pretreatment affects only marginally the degree of adhesion of the cells to ICAM-1; however, as shown in Figure 4 A , cells pretreated with EGTA, plated on dishes coated with ICAM-1, and stimulated with KIM-127 mAb displayed a round-shape morphology compared with non-EGTA-pretreated controls (Fig. 4A) .



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. Ca2+ mobilization is necessary for LFA-1-mediated morphological remodeling and activation of PYK2. (A) T-lymphoblasts were washed in RPMI medium and then left untreated (CONTROL) or pretreated with 3 mM EGTA for 30 min (EGTA). The cells were then allowed to attach for 60 min on ICAM-Fc-coated dishes in the absence (ICAM-1) or presence of 10 µg/ml mAb KIM-127 (ICAM-1+KIM-127). Attached cells were fixed and stained with 0.5% crystal violet in 20% methanol prior to photography. A representative experiment out of four is shown. (B) T-lymphoblasts were pretreated as in (A) in the absence (CONTROL) or presence of EGTA (EGTA) and then allowed to attach on ICAM-Fc-coated dishes in the absence (-) or presence (+) of 10 µg/ml mAb KIM-127. Cells were then lysed, and half of the lysates was incubated with C-19 antibody to immunoprecipitate PYK2 and kinase reactions performed as described in Materials and Methods (IP:PYK2; IVK:PYK2). The other half of the lysates was also immunoprecipitated with C-19 antibody and analyzed by SDS-PAGE, followed by transfer of proteins to nitrocellulose membranes and Western blotting with an anti-PYK2 antibody (IP:PYK2; WB:PYK2). The results shown are representative of three independent experiments.

 
Previously, we have shown that preventing the changes in the morphology of lymphoblasts, which follow induction of LFA-1 activation, results in a blockade of the increase in the activity of PYK2 [12 ]. Because EGTA pretreatment of T cells was blocking the morphological changes induced after activation of LFA-1, we analyzed the effect of this agent on the activation of PYK2. Figure 4B shows that when compared with untreated controls cells, pretreatment of the lymphoblasts with EGTA resulted in a complete inhibition of the LFA-1-dependent activation of PYK2. Taken together, these results show that Ca2+ mobilization is an event necessary for the morphological changes induced by LFA-1 and for the activation of PYK2.

Cross-linking LFA-1 with specific antibodies does not overcome the inhibition of T-cell morphological changes and PYK2 activity by Ca2+ chelator agents
Next, we tried to dissect the role played by the morphological changes and by LFA-1 in the observed effects after Ca2+ mobilization. To analyze further the role of LFA-1, we used several anti-LFA-1 antibodies to induce specific clustering of this integrin, because clustering of surface receptors is sufficient in many instances to induce intracellular signaling, including specific activation of tyrosine kinases [49 ]. Control cells and T-lymphocytes, which were pretreated with the Ca2+ chelator EGTA, were subsequently plated on dishes coated with the anti-LFA-1 mAb 38 (not shown), TP1/40, or 24 or with the anti-CD31 mAb TP1/15, which was used as a control (Fig. 5 A ). All of the anti-LFA-1 antibodies used induced attachment and spreading of the T cells to a different extent (Fig. 5A) . The same results were observed when the cells were plated onto the F(ab)'2 fragments of these antibodies (not shown). Furthermore, as observed in Figure 5A , in contrast with the control cells, which displayed a polarized morphology, when the cells were pretreated with a combination of EGTA and BAPTA-AM (an agent that chelates intracellular calcium; not shown) or EGTA alone, they maintained a rounded shape even after induction of cross-linking with the anti-LFA-1 TP1/40 or 24 mAb. These results suggest that the signals induced upon clustering of LFA-1 were not sufficient to induce morphological changes in T-lymphocytes in the absence of Ca2+ mobilization.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Cross-linking of LFA-1 with specific antibodies does not overcome the inhibition of T-cell morphological changes and PYK2 activity caused by Ca2+ chelator agents. (A) T-lymphoblasts were washed in RPMI medium and then left untreated (CONTROL) or pretreated with 3 mM EGTA for 30 min (EGTA). The cells were then allowed to attach for 60 min on ICAM-Fc-coated dishes in the absence (ICAM-1) or presence of 10 µg/ml mAb KIM-127 (ICAM-1+KIM-127) or on dishes precoated with the anti-LFA-1 mAb TP1/40 and 24 or the anti-CD31 mAb TP1/15, which was used as a control [IgG (CONTROL)]. Attached cells were fixed and stained with 0.5% crystal violet in 20% methanol prior to photography. A representative experiment out of four is shown. (B) T-lymphoblasts were pretreated as in (A) in the absence (CONTROL) or presence of 50 µM BAPTA-AM plus 3 mM EGTA (BAPTA/EGTA). The cells were then allowed to attach on ICAM-Fc-coated dishes precoated with rabbit anti-mouse IgG ({alpha}-MIgG) and anti-LFA-1 TP/1 40 (TP1/40) or 24 mAb for 10 min. Cells were then lysed, half of the lysates was incubated with C-19 antibody to immunoprecipitate PYK2, and kinase reactions were performed as described in Materials and Methods. The results shown are representative of three independent experiments.

 
In parallel experiments, the LFA-1 integrin molecules on control T cells or cells pretreated with a combination of EGTA and BAPTA were induced to cluster with the TP1/40 or the 24 mAb immobilized on plastic. PYK2 was immunoprecipitated from lysates obtained from these cells and the activity of this kinase assayed. As shown in Figure 5B , PYK2 activation was inhibited in the cells that were pretreated with BAPTA/EGTA. Similar results were obtained when LFA-1 aggregation was induced in cells maintained in suspension (not shown). Taken together, these results indicate that in the presence of Ca2+ chelators, LFA-1 clustering is not sufficient to induce changes in T-cell morphology or the activation of PYK2, implying that the inhibitory effects of the chelators are exerted downstream of this integrin.

EGTA pretreatment affects the microtubular organization and the localization of PYK2 to the MTOC
Because Ca2+ mobilizers induce remodeling of the cytoskeleton (Fig. 2) , and EGTA was interfering with the morphological changes induced by LFA-1 (Figs. 4A and 5A) , we analyzed whether this agent could be affecting the organization of the actin or the microtubular networks of T cells. Immunofluorescence staining showed that in the polarized controls and the rounded EGTA-pretreated cells, actin is present mostly in the cell periphery, as a prominent subplasmalemmal network, with no apparent differences in the organization or thickness of this structure between the control and EGTA-pretreated cells (Fig. 6 A ). Accordingly, FACS analysis and Coomasie-blue staining of the cytoskeletal actin fractions after SDS-PAGE showed that there were no differences in the amount of F-actin in the EGTA-treated cells compared with the control cells (not shown). In contrast, the immunofluorescence staining of the {alpha}-tubulin in stimulated controls and cells pretreated with EGTA showed that the microtubular network of the latter was less organized compared with the control cells (Fig. 6B) . We confirmed the effects of EGTA on the tubulin organization of the T cells by Western blot analysis. The comparison of the levels of unpolymerized {alpha}-tubulin of control cells maintained in RPMI medium, cells pretreated with 3 mM EGTA in RPMI, and cells pretreated with the microtubular depolymerizing agent colchicine confirmed that EGTA pretreatment induces depolymerization of {alpha}-tubulin (Fig. 6C) . Furthermore, in addition to these effects in the organization of {alpha}-tubulin, we also observed by immunofluorescence that the staining of PYK2 at the MTOC region of stimulated cells is reduced in the EGTA-pretreated cells (Fig. 6A and 6B) . Because we have demonstrated previously that disruption of the microtubular cytoskeleton inhibits the localization to the MTOC and activation of PYK2 [12 ], the results described above suggest that the effects of EGTA on the microtubular organization may contribute to explain the inhibitory effects of this chelator on the activity of PYK2.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 6. Effects of EGTA pretreatment on the actin and microtubular networks and on PYK2 localization to the MTOC region. T-lymphoblasts were washed in RPMI medium and then left untreated (CONTROL) or pretreated with 3 mM EGTA for 60 min (EGTA). The cells were then transferred to HEPES/NaCl medium and allowed to attach on ICAM-Fc-coated dishes in the presence of 1 mM Mg2+ and 1 mM Ca2+ (UNSTIMULATED) or in the presence of 200 µM Mn2+ (STIMULATED). (A) The cells were fixed, permeabilized, and doubled-stained with the anti-PYK2 antibody (PYK2) and phalloidin (ACTIN) as specified in Materials and Methods. Arrowheads indicate the position of PYK2. The results shown are representative of three independent experiments. (B) The cells were fixed, permeabilized, and doubled-stained with the anti-PYK2 antibody (PYK2) and an anti-{alpha}-tubulin antibody ({alpha}-TUBULIN) as specified in Materials and Methods. Arrowheads indicate the position of the MTOC. (C) T-lymphoblasts were washed in RPMI medium and then incubated in the absence (-) or the presence (+) of 3 mM EGTA or 20 µM colchicine. Detergent-soluble (SOLUBLE) and detergent-insoluble (POLYMERIZED) {alpha}-tubulin was fractionated and analyzed by Western blotting with anti-{alpha}-tubulin antibodies as described in Materials and Methods. The position of {alpha}-tubulin ({alpha}-TUB) is indicated with an arrow. Note that exposure times of soluble and polymerized {alpha}-tubulin blots are different. The results shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 REFERENCES
 
In this study, we have analyzed the role of Ca2+ in the signaling induced from the integrin LFA-1 and in the regulation of the activity of the tyrosine kinase PYK2 in T cells. We have identified the integrin LFA-1 and the microtubular cytoskeleton as two distinct targets for Ca2+ in these cells that are involved in the modulation of the activity of the tyrosine kinase PYK2.

To analyze the role of Ca2+ mobilization in the signaling induced by LFA-1 in T cells, we increased the intracellular Ca2+ concentration using three different Ca2+ mobilizers, ionomycin, thapsigargin, and A23187. These agents increased T-cell adhesion to ICAM-1, induced changes in cell morphology and polarization and reorganization of the microtubular cytoskeleton, and also led to redistribution to the MTOC and activation of the tyrosine kinase PYK2. All these effects closely resembled those observed when LFA-1 was activated from outside the cells with the activating mAb KIM-127, suggesting that Ca2+ mediates some of the LFA-1-dependent changes [12 ]. Importantly, the changes induced by intracellular Ca2+ were not observed when PLL was used as a substrate instead of ligand ICAM-1, clearly indicating the specific integrin dependence. It is most interesting that we demonstrate the novel finding that activation of the tyrosine kinase PYK2 induced by these Ca2+ mobilizers was prevented by an antibody blockade of LFA-1/ICAM-1 interaction, demonstrating the requirement for integrin engagement in the Ca2+-induced activation of PYK2. To our knowledge, this is the first study demonstrating an important role for an integrin in the Ca2+-dependent activation of PYK2. The use of adherent cells in which integrins are engaged constitutively has probably hampered the identification of such a role for integrins in previous studies. However, in line with our results, it has been shown that PYK2 in monocyte cells could not be activated by agents that induced an increase in intracellular Ca2+ unless the cells are attached to a substrate, suggesting that cell adhesion was necessary for the Ca2+-dependent activation of PYK2 in these cells [50 ].

When Ca2+ chelators (such as EGTA) were used to deplete the intracellular Ca2+ stores, the morphological changes induced in T-lymphoblasts with the activating mAb KIM-127 were inhibited. Furthermore, the signaling from LFA-1 was also altered following pretreatment with EGTA, because activation of PYK2 upon induction of clustering of this integrin with different antibodies, a method that induces activation of tyrosine kinases FAK and PYK2 in other cell settings [27 , 30 , 31 , 49 ], was completely inhibited. Because induction of aggregation of LFA-1 was not sufficient to overcome the inhibitory effects of chelators on cell morphology and PYK2 activity, the results suggested that the Ca2+ chelators were exerting their effects downstream of LFA-1. To examine this possibility, we analyzed the cytoskeleton of the cells. Immunofluorescence, Western blot, and FACS analyses showed that no important changes in the F-actin content take place in the cells pretreated with EGTA compared with control cells. In contrast, our analysis indicated that EGTA pretreatment affected mainly the microtubular system of the lymphocytes, as revealed by an important increase in the unpolymerized {alpha}-tubulin fraction of the microtubules. Therefore, these results point out that preventing the increase in intracellular Ca2+ has important effects on the tubulin network of T cells. Because we have shown that interference with the microtubular organization of the T-lymphocytes prevents the redistribution to the MTOC and inhibits the activation of PYK2 (ref. [12 ]; and unpublished results), and we observed that the localization of PYK2 to the MTOC was altered when the cells were pretreated with EGTA, our data suggest that the inhibitory effects of Ca2+ chelators on PYK2 activity involve the microtubular network in T cells. The results showing that Ca2+ chelators affect the microtubular network organization were unexpected and suggest that the function of some molecular components, which are involved in the control of microtubule dynamics in T cells, is regulated by intracellular Ca2+. In this regard, potential molecular candidates to play such a role include several microtubule associated proteins (MAPs), calcineurin, calmodulin, calreticulin, calpain, and classical protein kinase C (PKC; ref. [51 ] and references therein). It is interesting that it has been shown recently that a classical PKC isoform [PKC ß(I)] is crucially involved in LFA-1-mediated regulation of the polarization of T cells [52 ]. Because phorbol esters, which are well-known activators of classical PKCs, also stimulate PYK2, and PKC ß(I) colocalizes with this tyrosine kinase at the MTOC [12 , 52 , 53 ], it is possible that Ca2+ mobilization could activate PKC ß(I), and this may lead to T-cell polarization and PYK2 activation. In this regard, an interesting aspect of this present study and from previous work in our laboratory is the correlation between the redistribution of PYK2 to the MTOC and activation of this kinase [12 ]. The MTOC is emerging as a structure, where different regulatory molecules localize in T-lymphocytes and other leukocytes [12 , 46 , 52 53 54 ].

Our results are particularly relevant with respect to the role of Ca2+ in the regulation of the activity of PYK2. Since it was first described, this tyrosine kinase has been known to display a strong requirement for Ca2+ for its activity in vivo, as demonstrated by the stimulatory and inhibitory effects that Ca2+ mobilizers and chelators, respectively, exert on this enzyme [14 , 18 , 19 , 33 , 34 ]. Because PYK2 is not activated directly by Ca2+ or calmodulin in vitro [15 ], this suggests that PYK2 is regulated by a Ca2+-dependent event in intact cells. Our results show for the first time that LFA-1-mediated cell adhesion and the microtubular network are relevant components involved in the Ca2+-dependent regulation of the activity of PYK2 in T-lymphocytes.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
REFERENCES
 
A recent paper shows that the activity of the ß2 integrin may be modulated by the tubulin cytoskeleton in lymphocytes: Zhou, X., Li, J., Kucik, D. F. (2001) The microtubule cytoskeleton participates in control of ß2 integrin avidity. J. Biol. Chem., 276, 44762–44769.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from CICYT SAF 98/0080 (to C. C.) and a grant from Comunidad de Madrid (to C. C.). J. L. R-F. was supported by a "Contrato de Reincorporación," associated to grants DGICYT PB94-0231 and CICYT SAF98/0080, awarded by the "Ministerio Español de Educación y Cultura." The salary of C. A. d. F. was part of grant SAF 98/0080 from CICYT, and L. S-M. is the recipient of a fellowship "Incorporación de Técnicos a Equipos de Investigación" from "Comunidad de Madrid." We are grateful to Mariano Vitón (Hospital La Princesa) for his assistance with the flow cytometer, to Marigel Ollacarizqueta for her assistance with confocal microscopy, and to Nancy Hogg (ICRF, UK) and Joaquín Teixidó (CIB-CSIC, Madrid, Spain) for their generous sharing of reagents and for reviewing the manuscript.


   FOOTNOTES
 
Correspondence concerning PYK2 and current address of José Luis Rodríguez-Fernández: Laboratorio de Inmuno-Oncología, Hospital Gregorio Marañón, 28007 Madrid, Spain. E-mail: rodrifer39@yahoo.com

Received September 3, 2001; revised October 4, 2001; accepted October 29, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 

  1. Harris, E. S., McIntyre, T. M., Prescott, S. M., Zimmerman, G. A. (2000) The leukocyte integrins J. Biol. Chem. 275,23409-23412[Free Full Text]
  2. Gahmberg, C. G. (1997) Leukocyte adhesion: CD11/CD18 integrins and intercellular adhesion molecules Curr. Opin. Cell Biol. 9,643-650[Medline]
  3. van Kooyk, Y., Figdor, C. G. (1997) Signaling and adhesive properties of the integrin leucocyte function-associated antigen 1 (LFA-1) Biochem. Soc. Trans. 25,515-520[Medline]
  4. Stewart, M. P., McDowall, A., Hogg, N. (1998) LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain J. Cell Biol. 140,669-707
  5. van Kooyk, Y., Weder, P., Hogervorst, F., Verhoeven, A. J., van Seventer, G., te Veld, A. A., Borst, J., Keizer, C. G., Figdor, C. G. (1991) Activation of LFA-1 through a Ca2+-dependent epitope stimulates lymphocyte adhesion J. Cell Biol. 112,345-354[Abstract/Free Full Text]
  6. Ortlepp, S., Stephens, P. E., Hogg, N., Figdor, C. C., Robinson, M. K. (1995) Antibodies that activate ß2 integrins can generate different ligand binding states Eur. J. Immunol. 25,637-643[Medline]
  7. Stephens, P., Romer, J. T., Shock, A., Ortlepp, S., Figdor, C., Robinson, M. K. (1995) KIM127, an antibody that promotes adhesion, maps to a region of CD18 that includes cysteine-rich repeats Cell Adhes. Commun. 3,375-384[Medline]
  8. Sjaastad, M. D., Nelson, W. J. (1997) Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium BioEssays 19,47-55[Medline]
  9. Kanner, S. B., Grosmaire, L. S., Ledbetter, J. A., Damle, N. K. (1993) ß2-Integrin LFA-1 signaling through phospholipase C-{gamma} 1 activation Proc. Natl. Acad. Sci. USA 90,7099-7103[Abstract/Free Full Text]
  10. Arroyo, A. G., Campanero, M. R., Sánchez-Mateos, P., Zapata, J. M., Ursa, M. A., del Pozo, M. A., Sánchez-Madrid, F. (1994) Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase J. Cell Biol. 126,1277-1286[Abstract/Free Full Text]
  11. Lub, M., van Kooyk, Y., Figdor, C. G. (1995) Ins and outs of LFA-1 Immunol. Today 16,479-489[Medline]
  12. Rodríguez-Fernández, J. L., Gómez, M., Luque, A., Hogg, N., Sánchez-Madrid, F., Cabañas, C. (1999) The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes Mol. Biol. Cell 10,1891-1907[Abstract/Free Full Text]
  13. Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S., Pasztor, L. M., White, R. A., Groopman, J. E., Avraham, H. (1995) Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain J. Biol. Chem. 270,27742-27751[Abstract/Free Full Text]
  14. Earp, H. S., Huckle, W. R., Dawson, T. L., Li, X., Graves, L. M., Dy, R. (1995) Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells. Separation of the major calcium-regulated tyrosine kinase from p125FAK J. Biol. Chem. 270,28440-28447[Abstract/Free Full Text]
  15. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., Schlessinger, J. (1995) Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions Nature 376,737-745[Medline]
  16. Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., Sasaki, T. (1995) Cloning and characterization of cell adhesion kinase ß, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily J. Biol. Chem. 270,21206-21219[Abstract/Free Full Text]
  17. Herzog, H., Nicholl, J., Hort, Y. J., Sutherland, G. R., Shine, J. (1996) Molecular cloning and assignment of FAK2, a novel human focal adhesion kinase, to 8p11.2-p22 by nonisotopic in situ hybridization Genomics 32,484-486[Medline]
  18. Avraham, H., Park, S. Y., Schinkmann, K., Avraham, S. (2000) RAFTK/Pyk2-mediated cellular signaling Cell. Signal. 12,123-133[Medline]
  19. Yu, H., Li, X., Marchetto, G. S., Dy, R., Hunter, D., Calvo, B., Dawson, T. L., Wilm, M., Anderegg, R. J., Graves, L. M., Earp, H. S. (1996) Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation J. Biol. Chem. 271,29993-29998[Abstract/Free Full Text]
  20. Liu, Z., Ganju, R. K., Wang, J., Schweitzer, K., Weksler, S., Avraham, S., Groopman, J. E. (1997) Characterization of signal transduction pathways in human bone marrow endothelial cells Blood 90,2253-2259[Abstract/Free Full Text]
  21. Ganju, R. K., Dutt, P., Wu, L., Newman, H., Avraham, H., Avraham, S., Groopman, J. E. (1998) ß-Chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK Blood 91,791-797[Abstract/Free Full Text]
  22. Liu, Z. Y., Ganju, R. K., Wang, J. F., Ona, M. A., Hatch, W. C., Zheng, T., Avraham, S., Gill, P., Groopman, J. E. (1997) Cytokine signaling through the novel tyrosine kinase RAFTK in Kaposi’s sarcoma cells J. Clin. Investig. 99,1798-1804[Medline]
  23. Zohn, I., Yu, H., Li, X., Cox, A. D., Earp, H. S. (1995) Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase Mol. Cell. Biol. 15,6160-6168[Abstract]
  24. Tokiwa, G., Dikic, I., Lev, S., Schlessinger, J. (1996) Activation of Pyk2 by stress signals and coupling with JNK signaling pathway Science 273,792-794[Abstract]
  25. Li, X., Yu, H., Graves, L. M., Earp, H. S. (1997) Protein kinase C and protein kinase A inhibit calcium-dependent but not stress-dependent c-Jun N-terminal kinase activation in rat liver epithelial cells J. Biol. Chem. 272,14996-15002[Abstract/Free Full Text]
  26. Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., Schlessinger, J. (1996) A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation Nature 383,547-550[Medline]
  27. Mainiero, F., Gismondi, A., Soriani, A., Cippitelli, M., Palmieri, G., Jacobelli, J., Piccoli, M., Frati, L., Santoni, A. (1998) Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon gamma production in human natural killer cells J. Exp. Med. 188,1267-1275[Abstract/Free Full Text]
  28. Pandey, P., Avraham, S., Kumar, S., Nakazawa, A., Place, A., Ghanem, L., Rana, A., Kumar, V., Majumder, P. K., Avraham, H., Davis, R. J., Kharbanda, S. (1999) Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism J. Biol. Chem. 274,10140-10144[Abstract/Free Full Text]
  29. Graves, L. M., He, Y., Lambert, J., Hunter, D., Li, X., Earp, H. S. (1997) An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells J. Biol. Chem. 272,1920-1928[Abstract/Free Full Text]
  30. Astier, A., Avraham, H., Manié, S. N., Groopman, J., Canty, T., Avraham, S., Freedman, A. S. (1997) The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after ß1-integrin stimulation in B cells and binds to p130cas J. Biol. Chem. 272,228-232[Abstract/Free Full Text]
  31. Ma, E. A., Lou, O., Berg, N. N., Ostergaard, H. L. (1997) Cytotoxic T lymphocytes express a ß3 integrin which can induce the phosphorylation of focal adhesion kinase and the related PYK-2 Eur. J. Immunol. 27,329-335[Medline]
  32. Li, J., Avraham, H., Rogers, R. A., Raja, S., Avraham, S. (1996) Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes Blood 88,417-428[Abstract/Free Full Text]
  33. Hiregowdara, D., Avraham, H., Fy, Y., London, R., Avraham, S. (1997) Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin J. Biol. Chem. 272,10804-10810[Abstract/Free Full Text]
  34. Raja, S., Avraham, S., Avraham, H. (1997) Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism J. Biol. Chem. 272,10941-10947[Abstract/Free Full Text]
  35. Luque, A., Gómez, M., Puzon, W., Takada, Y., Sánchez-Madrid, F., Cabañas, C. (1996) Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common ß1 chain J. Biol. Chem. 271,11067-11075[Abstract/Free Full Text]
  36. Cantrell, D. A., Smith, K. A. (1984) Interleukin 2 T cell system. A new model for cell growth Science 224,1312-1315[Abstract/Free Full Text]
  37. Berendt, A. R., McDowall, A., Craig, A. G., Bates, P. A., Sternberg, M. J. E., Marsh, K., Newbold, C. I., Hogg, N. (1992) The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site Cell 68,71-81[Medline]
  38. Dransfield, I., Hogg, N. (1989) Regulated expression of Mg2+ binding epitope on leukocyte integrin alpha subunits EMBO J 8,3759-3765[Medline]
  39. Dransfield, I., Cabañas, C., Barrett, J., Hogg, N. (1992) Interaction of leukocyte integrins with ligand is necessary but not sufficient for function J. Cell Biol. 116,1527-1535[Abstract/Free Full Text]
  40. Campanero, M. R., del Pozo, M. A., Arroyo, A. G., Sánchez-Mateos, P., Hernández-Caselles, T., Craig, A., Pulido, R., Sánchez-Madrid, F. (1993) ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway J. Cell Biol. 123,1007-1016[Abstract/Free Full Text]
  41. Sancho, D., Santis, A. G., Alonso-Lebrero, J. L., Viedma, F., Tejedor, R., Sánchez-Madrid, F. (2000) Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors J. Immunol. 165,3868-3875[Abstract/Free Full Text]
  42. Stewart, M. P., Cabañas, C., Hogg, N. (1996) T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1 J. Immunol. 156,1810-1817[Abstract]
  43. Dransfield, I., Cabañas, C., Craig, A., Hogg, N. (1992) Divalent cation regulation of the function of the leukocyte integrin LFA-1 J. Cell Biol. 116,219-226[Abstract/Free Full Text]
  44. Cabañas, C., Hogg, N. (1993) Ligand intercellular adhesion molecule 1 has a necessary role in activation of integrin lymphocyte function-associated molecule 1 Proc. Natl. Acad. Sci. USA 90,5838-5842[Abstract/Free Full Text]
  45. Weber, C., Katayama, J., Springer, T. A. (1996) Differential regulation of ß1 and ß2 integrin avidity by chemoattractants in eosinophils Proc. Natl. Acad. Sci. USA 93,10939-10944[Abstract/Free Full Text]
  46. Sancho, D., Nieto, M., Llano, M., Rodríguez-Fernández, J. L., Tejedor, R., Avraham, S., Cabañas, C., López-Botet, M., Sánchez-Madrid, F. (2000) The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing J. Cell Biol. 149,1249-1262[Abstract/Free Full Text]
  47. Rodríguez Fernández, J. L., Geiger, B., Salomon, D., Ben-Ze’ev, A. (1993) Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells J. Cell Biol. 122,1285-1294[Abstract/Free Full Text]
  48. Rodríguez-Fernández, J. L., Rozengurt, E. (1998) Bombesin, vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine induce focal adhesion kinase activation in intact Swiss 3T3 cells J. Biol. Chem. 273,19321-19328[Abstract/Free Full Text]
  49. Rodríguez-Fernández, J. L. (1999) Why do so many stimuli induce tyrosine phosphorylation of FAK? BioEssays 21,1069-1075[Medline]
  50. Li, X., Hunter, D., Morris, J., Haskill, J. S., Earp, H. S. (1998) A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal J. Biol. Chem. 273,9361-9364[Abstract/Free Full Text]
  51. Berridge, M. J., Lipp, P., Bootman, M. D. (2000) The versatility and universality of calcium signals Nat. Rev. (Mol. Cell. Biol.) 1,11-21[Medline]
  52. Volkov, Y., Long, A., McGrath, S., Eidhin, D. N., Kelleher, D. (2001) Crucial importance of PKC-ß(I) in LFA-1-mediated locomotion of activated T cells Nat. Immunol. 2,508-514[Medline]
  53. Volkov, Y., Long, A., Kelleher, D. (1998) Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes ß(I) and {delta} J. Immunol. 161,6487-6495[Abstract/Free Full Text]
  54. Herreros, L., Rodríguez-Fernández, J. L., Brown, M. C., Alonso-Lebrero, J. L., Cabañas, C., Sánchez-Madrid, F., Longo, N., Turner, C. E., Sánchez-Mateos, P. (2000) Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton J. Biol. Chem. 275,26436-26440[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Sanchez-Martin, N. Sanchez-Sanchez, M. D. Gutierrez-Lopez, A. I. Rojo, M. Vicente-Manzanares, M. J. Perez-Alvarez, P. Sanchez-Mateos, X. R. Bustelo, A. Cuadrado, F. Sanchez-Madrid, et al.
Signaling through the Leukocyte Integrin LFA-1 in T Cells Induces a Transient Activation of Rac-1 That Is Regulated by Vav and PI3K/Akt-1
J. Biol. Chem., April 16, 2004; 279(16): 16194 - 16205.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Fernández, J. L.
Right arrow Articles by Cabañas, C.
Right arrow Search for Related Content
PubMed