Integrin -dependent Serine Phosphorylation of Paxillin in Cultured Human Macrophages Adherent to Vitronectin

doi:10.1074/jbc.271.18.11016

Integrin -dependent Serine Phosphorylation of Paxillin in Cultured Human Macrophages Adherent to Vitronectin (*)

From the Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892

^§ To whom correspondence should be addressed:
Laboratory of Developmental Biology, National Institute of Dental Research, Bldg. 30, Rm. 404, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370.
Tel.: 301-402-1558; Fax: 301-402-0897; mnichilo{at}yoda.nidr.nih.gov.

Abstract

The macrophage colony-stimulating factor (M-CSF) is able to induce the expression of the αβ 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 αβ, and show by indirect immunofluorescence analysis that αβ 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ECM

extracellular matrix

M-CSF

macrophage colony-stimulating factor

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

PY

phosphotyrosine

FAK

focal adhesion kinase

PBS

phosphate-buffered saline

BSA

bovine serum albumin.
- Received December 13, 1995.