Adhesion to fibronectin promotes the activation of the p125(FAK)/Zap-70complex in human T cells

Abstract

The beta1 integrins are a family of heterodimeric adhesion receptors involved in cell-to-cell contacts and cell-to-extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, alpha4beta1 and alpha5beta1, can function as costimulatory molecules in T-cell receptor (TCR)-dependent T-cell activation. In the current study the Jurkat T-cell line was used as a model system to investigate the TCR-independent role of cell adhesion to fibronectin in the activation of Zap-70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, was induced. Moreover, clustering of fibronectin receptors led to the detection of a p125FAK/Zap-70 complex. Finally, while the complex between fak-B, another protein kinase localized to focal adhesion sites, and Zap-70 was detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap-70 complex appeared specifically induced following fibronectin-mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the beta1 integrin family mediate signalling pathways in T cells.

Figure 1

Cell adhesion to fibronectin. Jurkat cells were added to plastic‐immobilized BSA (10 µg/ml) (a) or FN (10 µg/ml) in the absence (b) or in the presence (c) of the anti β1 functional mAb 4B4. Adherent cells were analysed with an inverted microscope and micrographs were made using Nomarski optics.

Figure 2

Induction of p125^FAK tyrosine phosphorylation following adhesion to fibronectin Phosphorylation of p125^FAK was examined in Jurkat cells adherent to plastic‐immobilized fibronectin (10 µg/ml) for 10 or 30 min or incubated onto BSA‐treated dishes for 30 min. Aliquots of cell lysate containing equivalent amounts of proteins as determined by the Lowry assay were used for immunoprecipitation. p125^FAK was incubated with specific antibodies and immunocomplexes were attached to protein A; then, immunoprecipitated complexes were fractionated on a 10% polyacrylamide gel under reducing conditions and immunoblotted with an anti‐phosphotyrosine mAb.

Figure 3

Coprecipitation of the p125^FAK/Zap‐70 complex following adhesion to FN. (a) Association of FAK‐related members with Zap‐70 was examined in Jurkat cells plated for 30 min onto BSA‐ or FN‐coated (10 µg/ml) dishes. Cell lysates were incubated with the anti‐p125^FAK antibodies and immunocomplexes were attached to protein A–Sepharose, fractionated on a 10% polyacrylamide gel, transferred to nitrocellulose and immunoblotted with an anti Zap‐70 antibody. (b) Association of p125^FAK with Zap‐70 was examined in Jurkat cells plated for 30 min onto BSA‐ (10 µg/ml) and for 30 or 45 min onto FN‐coated (10 µg/ml) dishes. Cell lysates were incubated with anti‐p125^FAK specific antibodies and immunocomplexes were processed as in (a). The samples contained equal protein amounts by protein assay and equal quantities of p125^FAK by Western blotting.