©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Adhesion through the Interaction of Lymphocyte Function-associated Antigen-1 with Intracellular Adhesion Molecule-1 Induces Tyrosine Phosphorylation of p130 and Its Association with c-CrkII (*)

(Received for publication, October 25, 1995; and in revised form, December 21, 1995)

Lilli Petruzzelli (1) Mimi Takami (1) Roman Herrera (2)(§)

From the  (1)Department of Internal Medicine, University of Michigan and the Department of Veteran's Affairs Medical Center, Ann Arbor, Michigan 48109-0668 and the (2)Department of Signal Transduction Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 p130was 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-1bulletICAM-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 p130bulletCrkII and p130bulletCrkL 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.


INTRODUCTION

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 beta(2) integrins, plays a central role in the adhesion events of the immune and inflammatory responses(1, 3, 4) . The leukocyte integrins share the common beta(2) subunit (CD18) noncovalently associated with one of three alpha subunits, alpha (CD11a), alpha (CD11b), and alpha (CD11c) to form LFA-1, (^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 beta(1) 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 beta(2) integrin family members are less well understood. Protein phosphorylation occurs upon beta(2) integrin activation. These include p58, paxillin, and a set of yet uncharacterized proteins p70, p115, and p140(31, 32, 33) . Although the beta(2) integrins share a common beta(2) subunit, the biological response may be a function of the associated alpha 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 beta(2) 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.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

The B-lymphoblastoid cell line JY was maintained in RPMI 1640 media (Life Technologies, Inc.) containing 5% heat-inactivated fetal calf serum (Sigma). 16 h prior to experiments, cells were washed in RPMI 1640 and resuspended in RPMI 1640 containing 0.2% fetal calf serum.

The beta(2) 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.

Activation of Cells

JY cells were collected by centrifugation at 400 times g for 5 min at 21 °C. Cells were resuspended in Leibovitz's L15 media (Life Technologies, Inc.) at 21 °C at a concentration of 4-8 times 10^6 cells/ml unless otherwise indicated. 1 ml of cells was used per assay point, and assays were performed in 1.5-ml microcentrifuge tubes (unless otherwise indicated). Background aggregation was blocked by preincubation of the control specimen with the anti-ICAM-1 antibody RR 1/1 for 10 min at 21 °C. Cells were stimulated by either the addition of PMA at 50 ng/ml or the beta(2) activating antibody, CBR LFA-1/2, at 10 µg/ml. The cells were warmed to 37 °C and rotated for 10 min (unless otherwise indicated in the figure legend). After 10 min, aggregation was confirmed by light microscopy. The cells were centrifuged for 2 min in a microcentrifuge at 2000 rpm, the supernatant was decanted, and the pellet was resuspended in 1 ml of Lysis buffer (Hepes, pH 7.4 (50 mM), NaCl (0.15 M), EGTA (1 mM), sodium pyrophosphate (10 mM) sodium fluoride (100 mM), sodium vanadate (3 mM), Triton X-100 (1%), glycerol (10%), EDTA 1 mM, benzamidine (1 mM), leupeptin (1 µg/ml), and aprotinin (0.5 µg/ml)). After 10 min on ice, samples were centrifuged at 13,000 rpm at 4 °C for 15 min, and the supernatant was collected.

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.

Cell Binding to Immobilized ICAM-1

ICAM-1 was purified and immobilized on 24-well polystyrene plates (Falcon 3847; Becton Dickinson, Lincoln Park, NJ) as described previously(42) . Cells were incubated in the presence or absence of the anti-ICAM-1 antibody RR 1/1 for 10 min at room temperature. Unbound antibody was removed by washing 3 times in L15. The washed cells (250 µl at 1 times 10^7 cells/ml) were then added to wells containing immobilized ICAM-1 and centrifuged at 200 times g for 2 min (four wells were used for each time point). The base of the plate was submerged in a 37 °C water bath for 10 min with the indicated stimulus, after which unbound cells were collected from the supernatant of each well. Bound cells were removed by incubation with lysis buffer and were added to the corresponding unbound cells. Cells were processed as describe above.

Immunoprecipitation

Solubilized cell extracts were incubated with antibody as indicated in the figure legends for 3 h at 4 °C. The immune complexes were collected by incubation with Protein G/A plus agarose and analyzed by immunoblotting following SDS-PAGE. Individual proteins were detected with the antibodies specified in the figure legends, and phosphotyrosine-containing proteins were detected with the antiphosphotyrosine antibody 4G10 (1 µg/ml final). Bound antibodies were detected with horseradish peroxidase-linked secondary antibody followed by enhanced chemiluminescence (ECL) according to manufacturer's directions (Amersham Corp.). Immunoblots were probed with the anti-p130 antibody to confirm that an equal amount of protein was loaded in each sample.

In Vitro Binding to GST-SH2 Domain of Crk 11

The glutathione S-transferase (GST) fusion protein containing the SH2 domain of CrkII was the generous gift of Drs. Birge and Hanafusa(43) . Production and purification of both GST or GST-SH2 fusion proteins was carried out as described previously(28) . The binding assay was carried out by incubating the GST fusion proteins bound to glutathione-agarose with the cell extracts prepared as described above. After 1 h at 4 °C, the beads were washed with the same buffer and the bound proteins were analyzed as described for the immunoprecipitates.

Cross-linking of ICAM-1 and LFA-1

Cells (2 times 10^7) were incubated with the indicated LFA-1 or ICAM-1 antibodies at 10 µg/ml for 10 min at room temperature in L15 medium. Unbound antibody was removed by washing twice in L15. Bound antibody was cross-linked by the addition of rabbit anti-mouse IgG (20 µg/ml) for 10 min at 37 °C. Lysates were prepared as described above, and the antibody complexes were removed by adsorption to Protein A-Sepharose for 30 min at 4 °C. p130 was precipitated from the lysates as described above.


RESULTS

Activation of LFA-1 Induces Tyrosine Phosphorylation of p130

In order to probe the signaling events that are triggered upon binding of LFA-1 to ligands, we examined whether a protein tyrosine kinase cascade was initiated from this interaction. In a preliminary experiment, using the c-src SH2 domain as a probe, we identified a protein, p110, that was phosphorylated upon homotypic aggregation of the B-lymphoblastoid cell line JY (data not shown). We then tested whether this protein was one of the previously described proteins shown to play a role in the signal transduction cascade initiated by both growth factors and beta(1) integrin family members such as p120, p125, p130, or paxillin. Serum-starved JY cells were induced to aggregate by stimulation with either phorbol esters or by the activating anti-beta(2) antibody, CBR LFA-1/2, that bypasses intracellular signaling pathways(39) . Stimulated and unstimulated cell extracts were immunoprecipitated with mAb to p130, p120, p125, or paxillin (M(r) 70,000), and the complexes were analyzed by immunoblot with an antiphosphotyrosine antibody (Fig. 1). Under the conditions where homotypic aggregation was confirmed to have taken place, we observed tyrosine phosphorylation of p130 but not of c-Cbl, FAK, or paxillin. The nature of the tyrosine-phosphorylated band (M(r) 160,000) seen in the anti-Cbl immunoprecipitate remains unknown. A nonspecific band (M(r) 100,000) is present in the immunoprecipitates of the three monoclonal antibodies. This result demonstrates that cell aggregation induces tyrosine phosphorylation of p130 in lymphocyte cell lines. We detected p130 phosphorylation at a maximal level as early as 2 min after stimulation with PMA ( Fig. 1and see Fig. 3) or CBR LFA-1/2 (data not shown).


Figure 1: Stimulation of tyrosine phosphorylation of p130 in JY cells by PMA and the beta(2) 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.''



LFA-1/ICAM-1 Interaction Is Required for p130 Phosphorylation

The leukocyte integrin LFA-1 was previously shown to be the critical component in mediating phorbol ester-stimulated homotypic aggregation of the B-cell line JY(44). We used two tools to probe the role of LFA-1 in mediating phosphorylation of p130: 1) the beta(2)-activating antibody CBR LFA-1/2 that stimulates binding of LFA-1 bearing cells to ICAM-1 in the absence of intracellular signaling pathways (39) and 2) the anti-ICAM-1 blocking antibody, RR 1/1. Cells that were stimulated with CBR LFA-1/2 and allowed to aggregate exhibited enhanced phosphorylation of p130 (Fig. 2). We carried out the aggregation assay in the presence of RR 1/1, which prevents the LFA-1-mediated, ICAM-1-dependent homotypic aggregation of these cells(45) . As seen in Fig. 2A, the tyrosine phosphorylation of p130 required the engagement of this receptor with its ligand, since it could not be detected when the aggregation was blocked by RR1/1. Reprobing of the blot with anti-p130 showed that an equal amount of p130 was present in each lane (Fig. 2B). Similarly, PMA treatment of JY cells led to tyrosine phosphorylation of p130 that was blocked by the anti-ICAM-1 antibody (Fig. 4A).


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 beta(1) 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.

Phosphorylation of p130 Is Reversible upon Cell Disaggregation

Divalent cations play a critical role in adhesion of LFA-1 to its ligands, and, in the absence of the divalent cation magnesium, regulated adhesion through LFA-1 does not occur(7) . We utilized this observation to probe whether phorbol ester-induced phosphorylation of p130 was reversible. If cells are allowed to aggregate and are then incubated with an EDTA-containing buffer, there is a rapid disaggregation of the cells. Concomitant with this process, we observed a loss of tyrosine-phosphorylated p130 (Fig. 3). If the cells were then washed in L15 medium that contains adequate divalent cations to mediate adhesion through LFA-1 and then stimulated with phorbol esters, aggregation again takes place and coincides with the reappearance of p130 phosphorylation on tyrosine residues (Fig. 3). This demonstrates that EDTA treatment did not damage the cells and supports the conclusion that p130 undergoes tyrosine phosphorylation in a regulated manner that is dependent on the ligand-receptor interaction.

Requirement for Cell-Cell Adhesion in Mediating the LFA-1/ICAM-1-dependent p130 Phosphorylation

The interaction of LFA-1 with its ligands is required to induce p130 phosphorylation in JY cells as well as the T-cell lines SKW3 and Jurkat (data not shown). We utilized purified ligand to determine whether membrane-bound ICAM-1 was required to initiate phosphorylation of p130. After incubation of JY cells in the presence or absence of the anti-ICAM-1 antibody RR1/1 to prevent cell-cell adhesion followed by removal of unbound antibody (Fig. 4A, lanes 2 and 6), we compared phosphorylation of p130 in cells that underwent homotypic aggregation in response to PMA to those cells that were allowed to adhere to purified ICAM-1 (Fig. 4A). Adhesion to ICAM-1 was confirmed by determining the number of unbound cells after PMA stimulation and was greater than 90%. Under conditions where cells were adherent to ICAM-1 but still able to form cell-cell contact (Fig. 4A, lane 1) p130 phosphorylation was observed. This is in contrast to conditions where LFA-1 was activated but cell-cell contact was blocked by RR1/1 (Fig. 4A, lane 2); markedly reduced phosphorylation of p130 was observed despite binding to ICAM-1 immobilized on the plate. The level of phosphorylation was comparable with that seen with cells aggregated in solution under the same conditions (Fig. 4A, lane 6). Thus, cell-cell adhesion appears to be a critical component in mediating LFA-1bulletICAM-1-dependent phosphorylation of p130. We have also examined phosphorylation under conditions where cells were adherent to purified LFA-1, and again we did not see stimulation of p130 phosphorylation.

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.

LFA-1-mediated Phosphorylation of p130 Induces the Formation of a p130bulletCrkII Complex

Previously it was reported that in v-crk transformed cells, p130 was phosphorylated on tyrosine residues and associated with v-Crk through the SH2 domain of Crk(46, 47) . We tested the hypothesis that the LFA-1 mediated phosphorylation of p130 on tyrosine leads to the formation of a p130bulletc-CrkII complex. Cell lysates prepared from basal or PMA activated cells were incubated with antibodies directed against CrkII and CrkL (Fig. 5, A and B), and the complexes were analyzed by immunoblotting with antiphosphotyrosine (Fig. 5A) or anti-p130 antibodies (Fig. 5B). As is seen in Fig. 5A, both the anti-CrkII and anti-crkL antibodies immunoprecipitated a phosphoprotein of M(r) 130,000 only from the extracts prepared from PMA-stimulated cells. Furthermore, this protein was recognized on immunoblot specifically by p130 antibody (Fig. 5B). We did not detect p130 in CrkII or CrkL immunoprecipitates from PMA-treated cells incubated with RR 1/1. An additional band appears in the negative control on the phosphotyrosine immunoblot but not in the anti-Cas blot, suggesting that this phosphotyrosine-containing protein is not related to p130. In order to further address the specificity of the interaction, we examined whether the isolated crkII SH2 domain formed a complex with phosphorylated p130in vitro. The GST-SH2 domain of Crk recognized a phosphoprotein of M(r) 130,000 (Fig. 5C, lane 4) that was also seen in the anti-cas immunoblot (Fig. 5D, lane 4) in extracts prepared from cells stimulated with PMA but not in unstimulated cells. This result indicates that LFA-1-mediated phosphorylation of p130 generates a recognition motif that is the binding site for the SH2 domain of CrkII.


Figure 5: Formation of a p130bulletCrkII complex. In panels A and B, JY cell lysates (10^8 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 (10^7 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 (alpha-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).




DISCUSSION

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 beta(1) 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-1bulletICAM-1 binding leads to tyrosine phosphorylation of p130. The phosphorylation of p130 has recently been reported to occur in fibroblasts in a beta(1) 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-1bulletLFA-1 interaction (Fig. 2) or chelation of cations that are required for LFA-1bulletICAM-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 beta 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 beta(1) 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-1bulletICAM-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-1bulletICAM-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 p130bulletCrkII 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 p130bulletCrkII/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-1bulletICAM-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 beta(1)-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-1bulletICAM-1 interaction and correlate the findings with the observed changes in cellular adhesion, cell spreading, and subsequent de-adhesion.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48105. Tel.: 313-998-5963; Fax: 313-996-5668; Herrerr{at}aa.wl.com.

(^1)
The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1 (CD11a, CD18); ICAM, intracellular adhesion molecule; PMA, phorbol 12-myristate 13-acetate; SH2 and SH3, src homology 2 and 3, respectively; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We thank Jennifer Osterwisch for expert technical assistance and Drs. D. Lockwood for encouragement in this project, Vered Ribon for helpful discussions, and A. Saltiel, G. Nunez and R. Todd for critical review of this manuscript. We also thank J. Brown for preparation of the manuscript.


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