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B. K. Tripathi, P. S. Zelenka; The Cdk5 Activating Protein P39 Directly Links Muskelin to Myosin and Stress Fibers. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1218.
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We have recently shown that opposite ends of p39, a cyclin dependent kinase 5 (Cdk5) activating protein, bind myosin essential light chain (MLC) and the LisH domain protein, muskelin, both of which are known to affect cytoskeletal organization. Here we seek to determine whether p39 specifically links muskelin to myosin and the significance of these interactions to cytoskeletal organization in lens.
Human lens epithelial cells were allowed to spread on fibronectin for 2 hr. Immunoprecipitation and immunoblotting with specific antibodies were used to confirm the interaction of endogenous proteins in lens epithelial cell lines and whole rat lens extracts. Cdk5 activity was inhibited by a pharmacological inhibitor (olomoucine,15 µM) and by dominant negative GFP-Cdk5(D144N). Cdk5 and p39 expression were suppressed by specific siRNAs. Lens epithelial cells were transfected with fluorescence-tagged fusion constructs, and subcellular localization of proteins was determined by confocal fluorescence microscopy. RT-PCR primers were designed to detect alternatively spliced isoforms of myosin heavy chain (MHC), MHC II-B and MHC II-B1 (10 amino acid insert).
The alternatively spliced isoform, MHC II-B1, a known substrate of Cdk5, was expressed in lens epithelia and fibers. Immunoprecipitation and immunoblotting of lens epithelial cell lines and rat lens extracts confirmed that endogenous MHC II-B and myosin regulatory light chain (MRLC) formed a complex containing MLC, p39, and muskelin. Suppression of p39 by siRNA significantly reduced co-immunoprecipitation of muskelin with MLC, MRLC, and MHC II-B. Fluorescence-tagged p39 and MLC fusion proteins colocalized along stress fibers and cortical actin filaments. Immunofluorescence microscopy demonstrated that muskelin colocalized with microtubules, MRLC, and contracting stress fibers during cell migration.
p39 is essential for formation of a protein complex connecting muskelin to myosin. This interaction thus targets Cdk5/p39 kinase activity to cytoskeletal substrates such as MHC II-B1 during cell adhesion and migration, and may serve to link microtubules to stress fibers.
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