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Q. Pan, C. Gao, P. Zelenka; Cdk5 Phosphorylated at Tyrosine-15 Co-Localizes With Early Focal Adhesions in Cultured Human Corneal Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3370.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that Cdk5, a member of the cyclin-dependent kinase family of proline-directed protein kinases, regulates adhesion and migration of corneal epithelial cells. These findings suggest that Cdk5 may affect the function of focal adhesions, the macromolecular assemblies that mediate cell to matrix adhesion. As a first test of this hypothesis, this study determines the subcellular localization of the activated, tyrosine phosphorylated form, Cdk5(pY15), with respect to known focal adhesion proteins.
Human corneal limbal epithelial (HCLE) cells were cultured with keratinocyte serum free medium. At 80~90% confluence, cells were replated onto fibronectin coated dishes or slides. After 30min or 36 hr, cells were fixed and immunostained with phosphospecific antibody to Cdk5 (pY15) in conjunction with antibodies directed to focal adhesion kinase (FAK), paxillin, or vinculin. Immunofluorescence was observed using secondary antibodies labeled with fluorescein (488nm) and rhodamine (568nm).
Immunofluorescence of spreading cells 30min after plating showed Cdk5(pY15) co-localized with both FAK and paxillin in focal adhesions. Co-localization with paxillin was strongest at the distal tips of focal adhesions and decreased proximally. Cdk5(pY15) staining in focal adhesions was primarily distal to vinculin, with occasional, small regions of co-localization. Cdk5(pY15) staining was not seen in mature focal adhesions in the cell interior, which were positive for paxillin and vinculin. In resting cells (36hr after plating), Cdk5(pY15) was diffusely localized in the cytoplasm.
Cdk5(pY15) is transiently associated with focal adhesions in the early stages of formation, but is lost as focal adhesions undergo maturation. These findings suggest Cdk5 may exert its effects on corneal epithelial cell adhesion by affecting focal adhesion assembly.
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