* Instituto de Farmacología y Toxicología (Centro Mixto CSIC-UCM), Facultad de Medicina, Universidad Complutense, Madrid, Spain;
Servicio de Inmunología, Hospital de la Princesa, Madrid, Spain; and
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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Key Words: adhesion molecules • T-lymphocytes • protein kinases • signal transduction
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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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 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 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.
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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 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 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--lysophosphatidylcholine, the anti--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--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
) 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--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.
-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 -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--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 [
-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.
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 View larger version (48K): [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.
|
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--tubulin mAb and
with an anti-PYK2 antibody. The -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--tubulin antibody (Fig. 2A)
. When the staining of -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 a new window] |
Figure 2. Organization of -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--tubulin mAb (-TUBULIN). Immunofluorescence was analyzed by
confocal microscopy as described in Materials and Methods. Arrowheads
indicate the position of the MTOC.
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 View larger version (28K): [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.
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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.
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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 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 (-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.
|
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 -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
-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 -tubulin (Fig. 6C)
.
Furthermore, in addition to these effects in the organization of
-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 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--tubulin antibody (-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)
-tubulin was fractionated and analyzed by Western blotting with
anti--tubulin antibodies as described in Materials and Methods. The
position of -tubulin (-TUB) is indicated with an arrow. Note that
exposure times of soluble and polymerized -tubulin blots are
different. The results shown are representative of three independent
experiments.
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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 -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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Received September 3, 2001; revised October 4, 2001; accepted October 29, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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1 activation Proc. Natl. Acad. Sci. USA 90,7099-7103
J. Immunol. 161,6487-6495This article has been cited by other articles:
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  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]
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