Steel factor enhances integrin-mediated tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) and paxillin

Abstract

Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp25FAK)) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin βl and combination of anti-α4 with antiα5 antibodies, indicating α4β1 and α5βl integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10-7 mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF1 cells to fibronetin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125(FAK) and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125(FAK) and paxillinin a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125(FAK). Wortmannin, at 10-7 mol/L, completely abolished SLF-induced enhancement of pp125(FAK) tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125(FAK) tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-α4 antibody also inhibited SLF-induced enhancement of pp125(FAK) tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125(FAK) through activation of integrin ('inside-out' signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.