Integrin alpha v beta 5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin.

The macrophage colony-stimulating factor (M-CSF) is able to induce the expression of the alpha v beta 5 integrin receptor on the surface of cultured human macrophages (De Nichilo, M. O., and Burns, G. F. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2517-2521). In the present study, we establish that the adhesion of M-CSF-treated macrophages to vitronectin is mediated by the integrin alpha v beta 5, and show by indirect immunofluorescence analysis that alpha v beta 5 and the cytoskeletal protein paxillin localize to focal contacts upon adhesion to vitronectin. Immunoprecipitation and Western blot analysis revealed that M-CSF-treated macrophages do not express focal adhesion kinase (FAK), thereby providing direct evidence for integrin-dependent localization of paxillin to focal contacts in the absence of FAK expression. Investigation of paxillin phosphorylation by two-dimensional phosphoamino acid analysis indicates that paxillin is 99% phosphorylated on serine residue(s) in response to vitronectin adhesion, and only 1% phosphorylated on tyrosine. Stimulation of protein kinase C (PKC) activity with the phorbol ester phorbol 12-myristate 13-acetate enhances paxillin phosphorylation, while two selective inhibitors of PKC, GF109203X and chelerythrine chloride, effectively block the phosphorylation of paxillin induced in response to vitronectin adhesion. Taken together, these data demonstrate that in M-CSF-treated macrophages adherent to vitronectin, paxillin localizes to focal contacts in the absence of FAK expression and is predominantly phosphorylated on serine residue(s) in a PKC-dependent manner.