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 across a provisional matrix of the wound bed. The mechanisms by which keratinocytes 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-localizes with focal adhesions in cells plated on extracellular matrix. We induced human keratinocytes into various states of migration by plating them on extracellular matrices that minimally, moderately, or strongly induce cellular 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 that three proteins were predominantly tyrosine phosphorylated, one of them being FAK. Tyrosine phosphorylation of FAK tightly correlated with the level of cellular motility but not cell attachment to the matrix. Time course experiments demonstrated that in highly motile keratinocytes, tyrosine phosphorylation of FAK peaked at 12 h, the time when maximal migration on the matrix ensues. In contrast to FAK, the beta1 integrin subunit of human keratinocytes that configures with the alpha2, alpha3, and alpha5 integrin subunits to form integrin receptors for matrix, did not display tyrosine phosphorylation linked to motility. Using anti-sense oligonucleotides to FAK, we demonstrate that FAK is required for human keratinocyte migration, but not for focal adhesion formation.
Yurko
M A
MA
Department of Dermatology, Northwestern University Medical School, Chicago, Illinois, USA.
O'Toole
E A
EA
Woodley
D T
DT
D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000737:chemistry / Q000502:physiology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000201:enzymology / Q000502:physiology*; D007797:Laminin / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007797:Laminin; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human