Phosphorylation of focal adhesion kinase (pp125(FAK)) is increased in human keratinocytes induced to migrate by extracellular matrices

During the healing process of skin wounds, human keratinocytes migrate acro ss a provisional matrix of the wound bed. The mechanisms by which keratinoc ytes migrate on connective tissue are not known. In this study, we examined the role of focal adhesion kinase (FAK), an 125 kDa protein that co-locali zes with focal adhesions in cells plated on extracellular matrix. We induce d human keratinocytes into various states of migration by plating them on e xtracellular matrices that minimally, moderately, or strongly induce cellul ar migration, and then examined the expression of FAK at the protein level and its degree of tyrosine phosphorylation using Western immunoblotting and immunoprecipitation. In highly migratory human keratinocytes, we found tha t three proteins were predominantly tyrosine phosphorylated, one of them be ing FAK. Tyrosine phosphorylation of FAK tightly correlated with the level of cellular motility but not cell attachment to the matrix. Time course exp eriments demonstrated that in highly motile keratinocytes, tyrosine phospho rylation of FAK peaked at 12 h, the time when maximal migration on the matr ix ensues. In contrast to FAK, the beta1 integrin subunit of human keratino cytes that configures with the alpha2, alpha3, and alpha5 integrin subunits to form integrin receptors for matrix, did not display tyrosine phosphoryl ation linked to motility. Using anti-sense oligonucleotides to FAK, we demo nstrate that FAK is required for human keratinocyte migration, but not for focal adhesion formation. (C) 2001 Wiley-Liss, Inc.