Lymphocyte Function-associated Antigen-1-mediated T Cell Adhesion Is Impaired by Low Molecular Weight Phosphotyrosine Phosphatase-dependent Inhibition of FAK Activity

Paola Chiarugi,Lucia Magnelli,Giovanni Raugei,Giacomo Cozzi,Giampietro Ramponi,Elisa Giannoni,Maria Letizia Taddei,Tania Fiaschi,Francesca Buricchi

doi:10.1074/jbc.m302686200

Abstract

Protein tyrosine phosphorylation is one of the earliest signaling events detected in response to lymphocyte function-associated antigen-1 (LFA-1) engagement during lymphocyte adhesion. In particular, the focal adhesion kinase p125FAK, involved in the modulation and rearrangement of the actin cytoskeleton, seems to be a crucial mediator of LFA-1 signaling. Herein, we investigate the role of a FAK tyrosine phosphatase, namely low molecular weight phosphotyrosine phosphatase (LMW-PTP), in the modulation of LFA-1-mediated T cell adhesion. Overexpression of LMW-PTP in Jurkat cells revealed an impairment of LFA-1-dependent cell-cell adhesion upon T cell receptor (TCR) stimulation. Moreover, in these conditions LMW-PTP causes FAK dephosphorylation, thus preventing the activation of FAK downstream pathways. Our results also demonstrated that, upon antigen stimulation, LMW-PTP-dependent FAK inhibition is associated to a strong reduction of LFA-1 and TCR co-clustering toward a single region of T cell surface, thus causing an impairment of receptor activity by preventing changes in their avidity state. Because co-localization of both LFA-1 and TCR is an essential event during encounters of T cells with antigen-presenting cells and immunological synapse (IS) formation, we suggest an intriguing role of LMW-PTP in IS establishment and stabilization through the negative control of FAK activity and, in turn, of cell surface receptor redistribution.

Highlights

Lymphocytes have a dual function that requires them to circulate in a nonadhesive form through blood and lymph and become adherent when they have to interact with endothelial cells for transmigration into sites of inflammation or infection, to stabilize cell-cell contact with antigen-presenting cells (APCs)1 for the establishment of a proper immune response, or to act as effector cells to lyse their targets [1]
Because co-localization of both lymphocyte function-associated antigen-1 (LFA-1) and T cell receptor (TCR) is an essential event during encounters of T cells with antigen-presenting cells and immunological synapse (IS) formation, we suggest an intriguing role of low molecular weight phosphotyrosine phosphatase (LMW-PTP) in IS establishment and stabilization through the negative control of FAK activity and, in turn, of cell surface receptor redistribution
Because tyrosine phosphorylation is a critical event in integrin-mediated signal transduction, we investigated the role of a phosphotyrosine phosphatase, namely low molecular weight phosphotyrosine phosphatase (LMW-PTP) in the modulation of T cell adhesion

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Unless specified, all reagents were obtained from Sigma. The stimulatory anti-CD3 monoclonal antibodies (mAbs) were purified from OKT3 hybridoma culture medium. Cells were stimulated for 20 min at 4 °C with 10 ␮g/ml anti-CD3 mAbs, followed by a 1-h incubation at 37 °C in the presence of rabbit anti-mouse Abs (25 ␮g/ml). To measure cell-cell adhesion, probe Jurkat cells resuspended in complete medium were stimulated with anti-CD3 mAbs as previously described, added to HUVEC monolayers (2.5 ϫ 105 cells in 500 ␮l/well), and incubated with target cells. Immunofluorescence Microscopy Analysis—Stable transfected Jurkat cells were washed once in RPMI, resuspended in fresh medium at a concentration of 5 ϫ 105 cells/ml, and allowed to adhere to 20 ␮g/ml poly-L-lysine-coated coverslips. Superimposition analyses were performed with Confocal Assistant version 4.02

RESULTS

DISCUSSION

Methods