Hck enhances the adherence of lipopolysaccharide-stimulated macrophages via Cbl and phosphatidylinositol 3-kinase

Src family tyrosine kinases have previously been proposed to mediate some o f the biological effects of lipopolysaccharide on macrophages, Accordingly, we have sought to identify substrates of Src family kinases in lipopolysac charide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells wi th lipopolysaccharide was found to induce gradual and persistent tyrosine p hosphorylation of Cbl in an Src family kinase-dependent manner. Immunopreci pitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cell s, while expression of an activated form of Hck in Bac1.2F5 cells induces t yrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimula tion. The Src homology 3 domain of Hck can directly bind Cbl, and this inte raction is important for phosphorylation of Chi. Association of the p85 sub unit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following l ipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression e xperiments indicate that phosphorylation of Cbl by Hck can facilitate the a ssociation of p85 with Cbl, Lipopolysaccharide treatment also stimulates th e partial translocation of lick to the cytoskeleton of Bac1.2F5 cells. Nota bly, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. T hus, we postulate that Hck enhances the adherence of lipopolysaccharide-sti mulated macrophages, at least in part, via Cbl and PI 3-kinase.