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D.R. Ledee, R.N. Fariss, P.S. Zelenka; The Cdk5 Activating Protein, p39, Interacts With Nonmuscle Myosin Light Chain . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1893.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To explore Cdk5–dependent regulation of epithelial cell adhesion and migration by identifying lens proteins that interact with Cdk5, p35, and p39. Methods: A yeast two–hybrid E18 rat lens cDNA library was screened using Cdk5, p35, and p39 as baits. Clones that grew without histidine and expressed beta–galactosidase were sequenced. Interactions were confirmed by glutathione–S–transferase affinity chromatography (GST–pull down assay). Co–transfections with green fluorescent protein (GFP)–myosin light chain (MLC) and hemagglutinin (HA)–tagged p39, p35, or Cdk5 were performed. Cell extracts were immunoprecipitated with anti–GFP antibody and immunoblotted with anti–HA antibody. Subcellular localization was determined by confocal fluorescence microscopy in N/N1003a lens epithelial cells. Results: Sequencing a p39–interacting clone identified MLC. In pull–down assays,GST–MLC interacted with radiolabeled p39, but weakly or not at all with p35 or Cdk5. GST–pull–down assays with p39 deletion products mapped the MLC binding site to the p39 N–terminus. Cdk5 and p39 both co–immunoprecipitated with MLC when co–transfected in Cos1 cells. Confocal fluorescence microscopy showed co–localization of p39 and MLC along cytoplasmic filaments and along cell boundaries. Conclusions: The Cdk5 activating protein, p39, interacts specifically with nonmuscle MLC. Cdk5 does not directly interact with MLC but may form an intracellular complex via p39. This interaction may target Cdk5/p39 kinase activity to cytoskeletal sites involved in adhesion and migration.
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