(Received for publication, October 25, 1995; and in revised form, December 21, 1995)
From the
The B-lymphoblastoid cell line JY undergoes homotypic
aggregation in a lymphocyte function-associated antigen-1
(LFA-1)-mediated, intracellular adhesion molecule-1 (ICAM-1)-dependent
manner when stimulated with phorbol 12-myristate 13-acetate or
anti-LFA-1 antibodies. Under conditions that lead to cell aggregation,
we observed rapid tyrosine phosphorylation of
p130, a protein previously identified to be
phosphorylated on tyrosine in both v-src- and
v-crk-transformed cells. Phosphorylation of
p130
was dependent on binding of LFA-1 to its
ligand, ICAM-1, as demonstrated by the use of anti-ICAM-1 antibodies.
Several observations suggest that this event may be an important step
in the signaling pathway initiated by LFA-1. p130
phosphorylation was rapidly reversible upon disengagement of
the LFA-1
ICAM-1 complex and required cell adhesion since binding
of phorbol 12-myristate 13-acetate-stimulated JY cells to purified
ICAM-1 or cross-linking of either LFA-1 or ICAM-1 was not sufficient to
induce phosphorylation of p130
. The
integrin-stimulated phosphorylation of p130
created binding sites that were recognized in vitro by the SH2 domain of c-CrkII, a key adaptor protein involved in
cell differentiation and transformation. Moreover, we also showed that
the LFA-1-stimulated tyrosine phosphorylation of p130
induces the formation of a
p130
CrkII and
p130
CrkL complex in intact cells. This
observation suggests that adhesion mediated by the interaction of LFA-1
and ICAM-1 initiates a signaling cascade that involves the activation
of protein tyrosine kinases and leads to the regulation of
protein-protein interaction via SH2 domains, a key process shared with
growth factor signaling pathways.
Integrins form a family of structurally related proteins that
play a role in mediating cell-cell and cell-extracellular matrix
interactions(1, 2) . A subset of this family, the
integrins, plays a central role in the adhesion
events of the immune and inflammatory
responses(1, 3, 4) . The leukocyte integrins
share the common
subunit (CD18) noncovalently
associated with one of three
subunits,
(CD11a),
(CD11b), and
(CD11c) to form LFA-1, (
)Mac-1,
and p150,95, respectively(5, 6) . LFA-1 is the
receptor for three ligands that are members of the Ig superfamily,
ICAM-1, ICAM-2, and
ICAM-3(7, 8, 9, 10) . Ligand binding
is divalent cation dependent, requires energy, but is not affected by
disruption of the actin cytoskeleton by cytochalasin
B(7, 11, 12) . In resting leukocytes, LFA-1
mediates little binding activity; however, activation of an array of
signal transduction pathways by cell surface receptors including CD2,
the T-cell receptor complex, Ig, and major histocompatibility complex
class II as well as through activation of protein kinase C by phorbol
esters, results in enhanced avidity of LFA-1 for its ligands (13) . Activation occurs through increased affinity of LFA-1
for at least one of its ligands, ICAM-1(14) . It is a transient
and regulated process that is associated with enhanced ligand binding,
adhesion, and subsequent de-adhesion(15) .
The signal
transduction pathways initiated by the activation of integrins have
recently begun to emerge(16, 17) . The integrin family mediates the adhesive interaction of cells with
extracellular matrix components such as fibronectin, vitronectin, and
collagen that are associated with cytoskeletal rearrangement, changes
in the cytoplasmic pH, intracellular Ca
levels,
protein tyrosine phosphorylation, and gene
expression(16, 17) . These events are mediated by a
synergistic mechanism that involves receptor occupancy and
aggregation(18) . Binding of integrins to extracellular matrix
induces phosphorylation of p125
, tensin, paxillin, Syk,
and, more recently, cortactin and p130
(19, 20, 21, 22, 23, 24, 25, 26) .
The phosphorylation of p125
leads to the association with src-family tyrosine kinases through specific SH2 domain
interactions(16) . This association results in the
phosphorylation of p125
at additional sites creating
binding sites for the adaptor protein, GRB2, thus forming a
multiprotein complex that may lead to the activation of the
mitogen-activated kinase
cascade(16, 17, 27) . These observations
indicate that the integrin-ligand interaction initiates a signaling
cascade that shares elements utilized by growth factor receptors and
that modulates protein-protein complex formation through the
interaction of SH2 domains and tyrosine-phosphorylated proteins (28, 29, 30) .
The signaling pathways that
are activated upon engagement of the integrin family
members are less well understood. Protein phosphorylation occurs upon
integrin activation. These include
p58
, paxillin, and a set of yet uncharacterized
proteins p70, p115, and p140(31, 32, 33) .
Although the
integrins share a common
subunit, the biological response may be a function of the
associated
subunit, as demonstrated for the phosphorylation of
paxillin observed in tumor necrosis factor-treated neutrophils that is
dependent upon Mac-1 activation(34) . Studies carried out with
lymphocytes demonstrated that LFA-1 provides co-stimulatory signals for
T-cell activation(35) ; engagement of LFA-1 with antibody or
purified ICAM-1 results in inositol phospholipid hydrolysis and
increased intracellular Ca
(35) . Accordingly,
it has been reported that phospholipase C
is phosphorylated on
tyrosine residues upon cross-linking of LFA-1 by antibody(36) .
It has also been reported that activation of LFA-1 in B cells (JK32.1)
and T-cells (JM) results in enhanced tyrosine phosphorylation of a set
of yet unknown proteins(37, 38) . In this report, we
demonstrate that activation of LFA-1, by either stimulation of inside
out signaling pathways or through a
subunit-specific
activating antibody, results in enhanced tyrosine phosphorylation of
p130
and subsequent association with crkII. Phosphorylation is reversible, dependent upon the
interaction of LFA-1 with its ligands and requires cellular adhesion.
The specific antibody CBR LFA-1/2 was prepared as described
previously(39) . The anti-p130
, FAK, CrkII, and
paxillin mAb were purchased from Transduction Laboratories, Lexington,
KY. The antiphosphotyrosine antibody 4G10 was purchased from Upstate
Biotechnologies, Inc., Lake Placid, New York. The anti-ICAM-1 antibody
RR 1/1 was a gift of R. Rothlein, Boerhinger Ingleheim, Ridgefield, CN.
The polyclonal antibodies against p120
and Crk II, and
Protein A/G-agarose were purchased from Santa Cruz Biotechnology, Santa
Cruz, CA. CBR-IC 1/4 was obtained from the Fifth International Workshop
and Conference on Human Leukocyte Differentiation
Antigens(40) . Leupeptin, aprotinin, benzamidine, Triton X-100,
and phorbol 12-myristate 13-acetate were obtained from Calbiochem, La
Jolla, CA.
Where indicated, cells were incubated with 2 µM cytochalasin B for 5, 10, and 30 min as described previously (41) prior to stimulation. Incubation with the signal transduction pathway inhibitors was performed for 30 min with staurosporine (5 µg/ml) and genistein (50 µg/ml), at room temperature.
Figure 1:
Stimulation of tyrosine phosphorylation
of p130 in JY cells by PMA and the
activating antibody CBR LFA-1/2. The B-lymphoblastoid cell line
JY was incubated in the absence of stimuli (lanes 1, 4, 7, and 10) with PMA (50 ng/ml; lanes
2, 5, 8, and 11) or CBR LFA-1/2 (10
µg/ml; lanes 3, 6, 9, and 12)
for 10 min at 37 °C. Samples in lanes 1, 4, 7, and 10 were incubated with the blocking antibody
RR 1/1 to reduce background aggregation. Lysates were prepared as
described under ``Experimental Procedures.''
Immmunoprecipitation of lysates was performed with
anti-p120
, anti-p130
,
anti-paxillin, and anti-p125
antibodies (all Ab at 1
µg/ml). The immunoprecipitated proteins were subjected to
SDS-polyacrylamide gel electrophoresis (8%) and transferred to
nitrocellulose as described. The immunoblot was probed with
antiphosphotyrosine 4G10 as described under ``Experimental
Procedures.'' The molecular mass markers are indicated along the left border.
Figure 3:
Reversibility of p130phosphorylation. Cells were incubated in the absence (lane
1) or the presence (lanes 2-5) of PMA (50 ng/ml)
for 2 (lane 2) and 10 min (lane 3). After 10 min at
37 °C, samples presented in lanes 4 and 5 were
treated with EDTA (10 mM) for 5 min. The sample in lane 5 was washed 3 times in L15 medium to remove EDTA, and cells were
then restimulated with PMA. Lysates were immunoprecipitated with the
anti-p130
antibody and immunoblotted with the
antiphosphotyrosine antibody 4G10 as described under
``Experimental Procedures.''
Figure 2:
Effect of the ICAM-1 blocking antibody RR
1/1 on p130 phosphorylation. JY cells were
treated with or without CBR LFA-1/2 (10 µg/ml) in the presence (lanes 1 and 2) or absence (lane 3) of RR
1/1 for 10 min at 37 °C. Lysates were prepared and
immunoprecipitated with mAb directed against p130
(2 µg/ml). After SDS-PAGE and transfer to
nitrocellulose, the immunoblot was probed with the antiphosphotyrosine
antibody 4G10 (A) or, after stripping the immunoblot, the
anti-p130
antibody (2 µg/ml) (B),
and detected as described under ``Experimental
Procedures.''
Figure 4:
Phosphorylation of p130 in JY cells bound to purified ICAM-1 (A) or upon
cross-linking of ICAM-1 (B). A, JY cells were treated
with the anti-ICAM-1 antibody RR 1/1 (lanes 2, 3, 6, and 7). After washing unbound antibody (lanes
2 and 6), cells were treated with (lanes 1, 2, 3, 5, 6, and 7) or
without PMA (lane 4) and allowed to aggregate in solution (lanes 4-7) or to bind to immobilized ICAM-1 (lanes
1-3). Lysates were prepared from cells collected under both
conditions and immunoprecipitated with the anti-p130
antibody. After SDS-PAGE and transfer to nitrocellulose, the
immunoblot was probed with the antiphosphotyrosine antibody 4G10 as
described under ``Experimental Procedures.'' B,
cells were incubated with the anti-ICAM-1 antibody and cross-linked (lanes 1 and 2) as described under
``Experimental Procedures,'' with PMA 50 ng/ml (lanes 1 and 3) or RR 1/1 (lane 4). After lysis, the
samples were analyzed, subjected to SDS-PAGE, and, after transfer to
nitrocellulose, probed with the antiphosphotyrosine antibody 4G10 as
described under ``Experimental
Procedures.''
Cytoskeletal
integrity plays a critical role in the modulation of protein kinase
activity through integrins(19, 21) .
In order to determine its role in modulation of p130
phosphorylation by LFA-1, we examined the effect of cytochalasin
on activation of p130
phosphorylation. At time points as
long as 30 min of cytochalasin treatment, there was no change in the
phosphorylation of p130
in response to phorbol esters.
When the PMA stimulation was carried out in the presence of the protein
kinase C inhibitor, staurosporine, or the protein tyrosine kinase
inhibitor, genistein, the tyrosine phosphorylation of p130
was blocked. This result supports the conclusion that PMA induces
p130
phosphorylation via a tyrosine kinase cascade
initiated by the activated LFA-1 molecule.
We probed whether ICAM-1 may be
playing a role in signaling p130 phosphorylation by
examining whether antibody-mediated cross-linking of ICAM-1 alone was
able to stimulate phosphorylation of p130
. Cells were
incubated with the non-blocking, non-activating, anti-ICAM-1 antibody,
CBR IC-1/4. Upon cross-linking of the bound antibodies, we could not
detect p130
phosphorylation nor did we observe cell
aggregation (Fig. 4B). PMA-stimulated phosphorylation
of p130
as well as cell aggregation was demonstrated in
the presence of these antibodies, indicating that the cells remained
functional. We also saw no enhancement of p130
phosphorylation in response to antibody mediated cross-linking of
LFA-1. These results collectively support the conclusion that cell-cell
adhesion is required to stimulate phosphorylation of
p130
.
Figure 5:
Formation of a
p130CrkII complex. In panels A and B, JY cell lysates (10
cells) were
prepared from cells treated with the anti-ICAM-1 antibody
RR1/1(-) or PMA (+) and immunoprecipitated with antibodies
directed against crkII and crkL (2 µg/ml). After
SDS-PAGE and transfer to nitrocellulose, the immunoblots were probed
with a mAb against phosphotyrosine (4G10) (panel A) or
p130
(panel B). In panels C and D, JY cell lysates (10
cells) were
prepared from cells treated with RR1/1(-) or PMA (+).
Lysates were precipitated with the GST alone, GST fused with the SH2
domain of crkII (GST SH2-crkII) and, in panel
C, an antibody directed against p130
(
-cas). Complexes were analyzed after SDS-PAGE
and transfer to nitrocellulose with the antiphosphotyrosine antibody
4G10 (panel C) or an antibody directed against
p130
(panel
D).
Adhesion of lymphocytes that is mediated by the interaction
of LFA-1 with its ligands is a regulated process(15) . LFA-1,
like other integrin family members, undergoes activation that is
initiated by an array of stimuli such as T-cell receptor cross-linking,
chemokines, and phorbol esters(13) . This inside-out signaling
process renders the receptor competent to engage its cognate ligand. It
has become evident that binding of the integrins to their ligands is
associated with a signaling complex that transduces, from outside to
inside, signals that result in cellular changes that ultimately
regulate the adhesive properties of the cell. Although protein kinase
activities are required for LFA-1 activation, the exact nature and
regulation of the kinases involved are unknown(42) . Unlike the
family of integrins, LFA-1-mediated adhesion does not
activate or stimulate the phosphorylation of the p125
kinase (Fig. 1) or other members of the c-src gene family(38, 48) . However, here we
demonstrate that adhesion mediated through LFA-1
ICAM-1 binding
leads to tyrosine phosphorylation of p130
. The
phosphorylation of p130
has recently been reported to
occur in fibroblasts in a
integrin-dependent
manner(19, 21) . p130
was initially
described as a tyrosine-phosphorylated protein in both v-src- (49, 50) and v-crk- (51, 52) transformed cells. The phosphorylated protein
forms a complex with both v-Src and v-Crk in a tyrosine-dependent
manner, presumably via the SH2 domain present in these adapter proteins
and suggests a role in a signaling
cascade(43, 46, 47, 52) .
We
found that tyrosine phosphorylation of p130 occurs in a
ligand-dependent manner in that either antibodies that disrupt the
ICAM-1
LFA-1 interaction (Fig. 2) or chelation of cations
that are required for LFA-1
ICAM-1 results in loss of p130
phosphorylation (Fig. 3). Phosphorylation of p130
was accomplished by stimulation of both inside out signaling
pathways by phorbol esters as well as through direct activation of
LFA-1 by an antibody directed against the
subunit ( Fig. 2and Fig. 3). Moreover, we have been able to
demonstrate that p130
phosphorylation is a reversible
process that requires the direct engagement of LFA-1 with ICAM-1 (Fig. 3). Since we were able to block the PMA-induced tyrosine
phosphorylation of p130
with genistein, it is likely that
LFA-1 mediated, ICAM-1 dependent phosphorylation of p130
is due to activation of tyrosine kinase activity. In contrast to
phosphorylation mediated through
integrins, LFA-1
stimulated phosphorylation of p130
occurs by 2 min and
was not affected by treatment of the JY cells with cytochalasin. This
parallels the observation that cytochalasin did not block the homotypic
aggregation of JY cells(41) .
We further probed whether the
biochemical changes induced by the engagement of LFA-1 with ICAM-1 in
JY cells could be initiated by signals generated either by the LFA-1 or
ICAM-1 moiety. We attempted to define whether p130 phosphorylation required output signals from both the LFA-1 and
ICAM-1 molecules by studying its phosphorylation under conditions that
eliminated the contribution of the cell-bound ICAM-1 to the aggregation
process. If we assume that under our experimental conditions the
anti-ICAM-1 antibody remained bound to the cellular ICAM-1, the result
depicted in Fig. 4suggests that activation of the tyrosine
kinases involved in p130
phosphorylation requires the
membrane-bound form of ICAM-1. We further probed this through studies
to examine the effects of antibody cross-linking of either ICAM-1 or
LFA-1. Under these conditions, we were not able to mimic p130
phosphorylation. These observations support the conclusion that
p130
undergoes tyrosine phosphorylation in an cell-cell
adhesion-dependent manner and parallels the observed dependence on
platelet adhesion for p125
phosphorylation(24) .
This may be due to the fact that the LFA-1
ICAM-1 interaction may
be through dimerized ICAM-1 (53) and we were not able to
sufficiently mimic this in vitro or that additional steps that
are critically dependent upon LFA-1
ICAM-1 binding and cell-cell
adhesion are called into play in inducing phosphorylation of
p130
.
The function of tyrosine-phosphorylated
p130 is unknown. The unique structure of this protein
suggests that it may participate in signal transduction by mediating
protein-protein interactions via SH2 and SH3 domains. The primary
structure of p130
reveals the presence of nine sites with
the YXXP motif that can be recognized by the Crk-SH2
domain(46, 54) . Indeed, here we have shown that the
endogenous CrkII protein associates with the integrin-stimulated,
tyrosine-phosphorylated p130
. Moreover, we have shown
that the LFA-1-mediated phosphorylation of p130
creates
binding sites for the isolated SH2 domain of CrkII (Fig. 5).
The exact role that the p130CrkII complex may
play in signaling by LFA-1 is unknown; however, with the observation
that LFA-1-stimulated phosphorylation of p130
is
reversible upon disruption of the LFA-1/ICAM-1 interaction (Fig. 3), it may play a significant role in the overall
signaling by LFA-1. Crk has been shown to interact with two GTP
exchange proteins, Sos and C3G, through its SH3
domain(55, 56, 57) . Thus, a possible role
for the p130
CrkII/C3G multiprotein complex in the
integrin-mediated tyrosine phosphorylation would be regulating the
activity of the Ras family of GTPases. Indeed, LFA-1-mediated cell
aggregation requires the activity of the small GTP-binding protein
RhoA, a known modulator of the actin cytoskeleton(41) .
The
tyrosine kinase(s) that is activated by the LFA-1ICAM-1 complex
and that leads to p130
phosphorylation is unknown. c-Abl
and the Src-family of kinases have been suggested as potential kinases
that mediate
-dependent p130
phosphorylation(19, 58) . However, we have not
detected phosphorylated p130
in BCR-Abl-transformed
cells(59) . In addition, activation of the c-Src family of
kinases by LFA-1 has not been detected(48) . The results
presented in this paper indicate that it will be possible to use the
enhanced phosphorylation of p130
as a scaffolding to
elucidate the signal transduction cascade initiated by the
LFA-1
ICAM-1 interaction and correlate the findings with the
observed changes in cellular adhesion, cell spreading, and subsequent
de-adhesion.