Steel factor enhances integrin-mediated tyrosine phosphorylation of focal adhesion kinase (pp125FAK) and paxillin.
Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) 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 beta 1 and combination of anti-alpha 4 with anti-alpha 5 antibodies, indicating alpha 4 beta 1 and alpha 5 beta 1 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 TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10(-7) mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK 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-alpha 4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK 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.
Takahira
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Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Gotoh
A
A
Ritchie
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A
Broxmeyer
H E
HE
D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D019089:Stem Cell Factor / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D016023:Integrins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D019089:Stem Cell Factor; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human