STEEL FACTOR ENHANCES INTEGRIN-MEDIATED TYROSINE PHOSPHORYLATION OF FOCAL ADHESION KINASE (PP125(FAK)) AND PAXILLIN

Integrin-mediated interaction of hematopoietic progenitor cells with b one marrow stromal extracellular matrix components is important in hem atopoiesis. Focal adhesion kinase (pp125(FAK)) plays a central role in signal transduction through integrin receptors, We studied matrix-int egrin interaction and subsequent signaling in human growth factor-depe ndent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibrone ctin-coated wells was blocked by antiintegrin beta 1 and combination o f anti-alpha 4 with anti-alpha 5 antibodies, indicating alpha 4 beta 1 and alpha 5 beta 1 integrin mediated adherence. Steel factor (SLF) in creased TF-1 adhesion to fibronectin dose-dependently and 10(-7) mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and i mmunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphor ylation of several proteins identified as pp125(FAK) and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosph orylation of pp125(FAK) and paxillin in a dose-dependent manner, Treat ment with SLF without plating on fibronectin did not induce tyrosine p hosphorylation of pp125(FAK). Wortmannin, at 10(-7) mol/L, completely abolished SLF-induced enhancement of pp125(FAK) tyrosine phosphorylati on, while c-kit autophosphorylation was not affected, This suggests th at increase of pp125(FAK) tyrosine phosphorylation was mediated throug h a wortmannin sensitive pathway, rather than by direct action on c-ki t tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-alpha 4 antibody also inhibited SLF-induced enhancement of p p125(FAK) tyrosine phosphorylation without detachment of TF-1 cells, T hese data suggest that SLF enhances integrin-fibronectin-dependent tyr osine phosphorylation of pp125(FAK) through activation of integrin ('' inside-out'' signaling) and following integrin occupancy. This establi shes a novel linkage between c-kit/SLF pathway and integrin fibronecti n signaling. (C) 1997 by The American Society of Hematology.