Differential regulation of focal adhesion kinase and paxillin phosphorylation by the small GTP-binding protein Rho in human corneal epithelial cells.
OBJECTIVE
Lysophosphatidic acid (LPA), which activates the small guanosine triphosphate (GTP)-binding protein Rho, was previously shown to promote migration of the rabbit corneal epithelium in culture. The signaling pathway responsible for this effect of LPA was examined in this study with a human corneal epithelial (HCE) cell line.


METHODS
The activation of Rho was detected with a pull-down assay. Tyrosine phosphorylation of paxillin and of focal adhesion kinase (FAK) were examined both by immunofluorescence staining and by immunoprecipitation and immunoblot analyses. Expression of integrins alpha5 and beta1 was evaluated by immunoblot analysis.


RESULTS
Incubation of cells with LPA (10 micro M) for 2 min resulted in marked activation of Rho, and this effect was blocked by pretreatment with the Rho inhibitor exoenzyme C3 (2 micro g/ml) for 24 h. Tyrosine phosphorylation of both paxillin and FAK was detected in HCE cells under basal conditions by immunofluorescence staining, immunoprecipitation, and immunoblot analyses. LPA induced a concentration- and time-dependent increase in the tyrosine phosphorylation of paxillin, which was maximal at a concentration of 10 micro M and a time of 2 min. Exoenzyme C3 inhibited LPA-induced paxillin phosphorylation. Neither LPA nor exoenzyme C3 affected tyrosine phosphorylation of FAK or expression of integrins alpha5 and beta1.


CONCLUSIONS
LPA induces Rho activation and the consequent tyrosine phosphorylation of paxillin in HCE cells, and these effects likely contribute to the promotion of corneal epithelial migration by this agent.
Saito
Jun
J
Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube, Japan.
Morishige
Naoyuki
N
Chikama
Tai-Ichiro
T
Gu
Jianguo
J
Sekiguchi
Kiyotoshi
K
Nishida
Teruo
T
D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D015151:Immunoblotting; D039482:Integrin alpha5 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013194:Staining and Labeling; D014443:Tyrosine / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000502:physiology*
D003598:Cytoskeletal Proteins; D039482:Integrin alpha5; D019012:Integrin beta1; D008246:Lysophospholipids; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D020741:rho GTP-Binding Proteins; C032881:lysophosphatidic acid