Abstract
Abstract: :
Purpose: Cdk5 forms a complex with Src in both lens epithelial cells and lens fibers, which may play a role in Cdk5–dependent regulation of cell–matrix and cell–cell adhesion. This study investigates the nature of the Cdk5–Src complex and determines the effect of Cdk5 activity and phosphorylation status on complex formation. Methods: A glutathione S–transferase Cdk5 fusion construct (GST–Cdk5) was incubated with in vitro translated, radiolabeled Src. Complexes were isolated by affinity chromatography on glutathione conjugated beads (GST pull–down). Known phosphorylation sites of Cdk5 were altered by site–directed mutagenesis. Cos7 cells were transiently transfected with wild type and mutated Src and Cdk5, or green fluorescent protein (GFP) fusions of these proteins. Proteins were immunoprecipitated using anti–GFP antibody and immunoblotted with antibodies to Cdk5, Src, and Cdk5(pY15). Results: GST pull–down experiments showed that in vitro translated, radiolabeled Src bound toGST–Cdk5, but not to GST alone, suggesting a direct interaction between these proteins. In transfected Cos7 cells, GFP–Cdk5 co–immunoprecipitated with Src. Site specific mutation of Cdk5 at known phosphorylation sites, (Y15F and S159A), did not prevent association with Src, indicating that prior phosphorylation of Cdk5 is not required for association with Src. Cdk5 mutations that abolish kinase activity also maintained the ability to complex with Src. In addition, loss of Cdk5 kinase activity did not prevent phosphorylation of Cdk5 at Y15, a site associated with increased Cdk5 activity. Conclusions: Src is able to bind directly to Cdk5. Since the interaction was not abolished by the Y15F mutation, interaction between pY15 of Cdk5 and the SH2 domain of Src is unlikely to form the basis for the interaction. The previously reported phosphorylation of Src(S75) by Cdk5 also has no effect on complex formation, since Cdk5 kinase activity was not required for complex formation. Future studies will use these mutations to explore the functional consequences of Cdk5–Src complex formation.
Keywords: cell adhesions/cell junctions • phosphorylation