Abstract

We have used mutant macrophages which are deficient in expression of Src-family kinases to define an integrin signaling pathway that is required for macrophage adhesion and migration. Following ligation of surface integrins by fibronectin, the p120^c-cbl (Cbl) protein rapidly becomes tyrosine phosphorylated and associated with the Src-family kinases Fgr and Lyn. In hck^-/-fgr^-/-lyn^-/- triple mutant cells, which are defective in spreading on fibronectin-coated surfaces in vitro and show impaired migration in vivo, Cbl tyrosine phosphorylation is blocked, Cbl protein levels are low, adhesion-dependent translocation of Cbl to the membrane is impaired and Cbl-associated, membrane-localized phosphatidylinositol 3 (PI-3)-kinase activity is dramatically reduced. In contrast, adhesiondependent activation of total cellular PI-3 kinase activity is normal in mutant cells, demonstrating that it is the membrane-associated fraction of PI-3 kinase which is most critical in regulating actin cytoskeletal rearrangements that lead to cell spreading. Treatment of wild-type cells with the Src-family-specific inhibitor PP1, Cbl antisense oligonucleotides or pharmacological inhibitors of PI-3 kinase blocks cell spreading on fibronectin surfaces. These data provide a molecular description for the role of Src-family kinases Hck, Fgr and Lyn in ₁-integrin signal transduction in macrophages.