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P. S. Zelenka, D. R. Ledee; Muskelin Interacts With Melanin-Concentrating Hormone Receptor 1 Interacting Zinc-Finger Protein, MIZIP. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2012.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the role of muskelin in epithelial cell adhesion and migration by identifying lens proteins with which it interacts.
A yeast two-hybrid E18 rat lens cDNA library was screened using the complete cDNA of muskelin as bait. Clones that grew without histidine and expressed beta-galactosidase were sequenced. Interactions were confirmed by glutathione-S-transferase affinity chromatography (GST-pull down assay) and co-immunoprecipitation. Subcellular localization was determined by confocal fluorescence microscopy in N/N1003a lens epithelial cells transiently transfected with GFP tagged MIZIP and Myc tagged Muskelin.
Sequencing of muskelin-interacting clones identified MIZIP. In pull-down assays, GST-muskelin interacted with radiolabeled MIZIP. GST-pull-down assays with muskelin deletion products mapped the MIZIP binding site to the 5th kelch repeat. Co-immunoprecipition assays of transiently transfected Cos 1 cells with tagged MIZIP and muskelin, as well as co- immunoprecipition of endogenous protein from mouse lens confirmed interaction. Confocal fluorescence microscopy of GFP tagged MIZIP and Myc tagged Muskelin transiently transfected into N/N1003a cells showed co-localization of muskelin and MIZIP along cytoplasmic filaments and perinuclear region.
Previously, our lab showed muskelin interaction with the CDK5 activating protein, p39, and proposed a role for muskelin as a scaffolding protein tethering multiple proteins to cytoskeletal structures. In this study muskelin interacts with MIZIP, a protein shown to interact α- and ß-tubulin, further supporting a role for muskelin in the structural mechanisms of cells.
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