PURPOSE: To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein. METHODS: cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy. RESULTS: Rat Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, alpha-actinin-1, alpha-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. alpha-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with alpha-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and alpha-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and alpha-actinin-1 were preferentially located along the basal aspect. CONCLUSIONS: These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.
PURPOSE: To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein. METHODS: cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy. RESULTS:RatPdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, alpha-actinin-1, alpha-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. alpha-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with alpha-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and alpha-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and alpha-actinin-1 were preferentially located along the basal aspect. CONCLUSIONS: These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.
Authors: Jonathan M Elkins; Carina Gileadi; Leela Shrestha; Claire Phillips; Jing Wang; João R C Muniz; Declan A Doyle Journal: Protein Sci Date: 2010-04 Impact factor: 6.725
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