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

Abstract

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the ␤2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the ␤2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2. INTRODUCTIONIntegrins are heterodimeric cell surface proteins that function in cell adhesion, cytoskeleton anchorage, and the transduction of cellular stimuli into cytoplasmic signals (Clark and Brugge, 1995;Schwartz et al., 1995).The ␤2-integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is selectively expressed on leukocytes and mediates important adhesive phenomena of these cells through interaction with its ligands intercellular adhesion molecule 1 (ICAM-1; CD54), ICAM-2 (CD102), and ICAM-3 (CD50). The resting leukocytes, which circulate in the bloodstream, express an inactive form of LFA-1 that is functionally unable to mediate interactions with ICAMs. Induction § Corresponding author. E-mail address: cacabagu@eucmax. sim.ucm.es.© 1999 by The American Society for Cell Biology 1891 of leukocyte activation through different cell surface receptors, including the T cell receptor-CD3 complex and receptors for cytokines, results in the generation of intracellular signals that lead to activation of LFA-1 molecules. This "inside-out" mechanism of LFA-1 activation brings about a change in the conformation of the extracellular region of the integrin, which is functionally reflected by the ability of the cells to adhere to LFA-1 ligands. Activation of LFA-1 molecules can also be induced directly from outside the cells by altering the extracellular divalent cation conditions, i.e. by addition of micromolar conc...

SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility

“…6). Rap1 and VAV-1 have also been implicated in outside-in signaling (24,46). Cytoskeleton protein talin binds LFA-1 and controls LFA-1 activation (47), and our preliminary data also showed that M12 reduced the association of LFA-1-talin in resting states.…”

“…In addition to mediating ICAM-1 adhesion, it can generate outside-in signals that costimulate T cells (25,41,42). The nature of the outside-in pathway has been unclear, but may involve PYK-2 (proline-rich tyrosine kinase 2) and FAK (24,25). ADAP and its binding to SLP-76 can regulate TcR mediated inside-out signaling for integrin activation (9,10,14).…”

“…One possibility is that M12 might bind and occupy SKAP1 to block endogenous ADAP binding to SKAP1. Alternatively, previous work has implicated kinases PYK-2 and FAK in LFA-1 outside-in signaling using affinity activating antibodies (24,25). Our previous work showed that FYN phosphorylates ADAP (12, 13).…”

“…In addition to inside-out signaling, LFA-1 generates so-called ''outside-in'' signals that costimulate cell function (4,24,25). ␣4␤1 integrin VLA-4 binding to its ligands fibronectin or VCAM-1 (vascular cell adhesion molecule-1) enhances T cell proliferation via FAK (focal adhesion kinase) (26) and the stabilization of microclusters (27).…”

LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse

“…These results raise the possibility that LFA-1 may transduce specific costimulatory signals that can impact both on T cell activation and differentiation. Cross-linking of LFA-1 has been shown to activate phospholipase C (27)(28)(29), PI3K and JNK (30), Pyk2 (31), DNAM (CD226) (32), cytohesion and ERK (33,34), and JAB1 (34,35). But whether these signals are induced during T cell Ag recognition in the context of APC and how these signals might be integrated with TCR-derived signaling are not yet clear.…”