Cdk5 Is Essential for Recruitment of Active Src to Focal Adhesions

Abstract

Purpose: : Cdk5 regulates cell adhesion and migration in the corneal epithelium. The present study explores the possible involvement of Cdk5 in focal adhesion assembly in spreading human corneal limbal epithelial (HCLE) cells.

Methods: : HCLE cells were plated on fibronectin and examined after 60min. Endogenous Cdk5 was suppressed by stable or transient transfection with siCdk5. Cdk5 kinase activity and Y15 phosphorylation were inhibited by dominant negative Cdk5(K33T) or Cdk5(Y15F); to inhibit Cdk5 kinase activity without inhibiting Y15 phosphorylation, we used dominant negative Cdk5(D144N) or the pharmacological inhibitor, olomoucine. Phosphorylated and unphosphorylated focal adhesion proteins were detected by immunofluorescence microscopy or by immunoblotting with specific antibodies. Binding of cSrc to FAK was determined by immunoprecipitation and immunoblotting. Subcellular fractions were isolated by differential detergent solubility.

Results: : Cdk5, Cdk5 (pY15), and p35 co-localized with vinculin and other focal adhesion (FA) proteins at FAs. Suppressing Cdk5 expression with siRNA reduced formation of vinculin-containing FAs (75.1%, p<0.05). siCdk5 also reduced Src(Y416) phosphorylation, prevented binding of Src to FAK, and blocked Src-dependent phosphorylation of FAK (Y576) and paxillin(Y118), indicating that active Src failed to join focal adhesions. In addition, siCdk5 decreased Src levels in a detergent-insoluble cytoskeletal fraction (39 %, p< 0.05), indicating that focal adhesions fail to link to the cytoskeleton in the absence of Cdk5. Although siCdk5 reduced focal adhesion formation, it did not block FAK(Y397) autophosphorylation or affect adhesion to fibronectin at 4^oC, demonstrating that integrin engagement was not affected. Like siCdk5, dominant negative Cdk5 constructs Cdk5 (K33T) and Cdk5 (Y15F) reduced focal adhesion formation. In contrast, focal adhesions were not blocked by Cdk5 (D144N) or olomoucine, indicating that Cdk5 enzymatic activity is not involved.

Conclusions: : Expression of Y15 phosphorylated Cdk5 protein is essential for efficient recruitment of active Src to nascent focal adhesion sites in spreading corneal epithelial cells.

Keywords: cell adhesions/cell junctions • cornea: epithelium