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AD Marmorstein, PJ McLaughlin, J Stanton, L Yan, JW Crabb, LY Marmorstein; Bestrophin Interacts with the b-catalytic Subunit of Protein Phosphatase 2A . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2902.
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Purpose: Bestrophin is encoded by the vitelliform macular dystrophy 2 (VMD2) gene, which is mutated in Best macular dystrophy (BMD), a dominantly inherited macular degenerative disease. Bestrophin is a 68 kDa basolateral plasma membrane protein of unknown function and is expressed only in retinal pigment epithelial (RPE) cells. The purpose of this study is to elucidate the function of bestrophin and to identify proteins with which bestrophin interacts. Methods: A bestrophin complex was immunoaffinity-purified from porcine RPE lysates. Proteins in the complex were identified by MALDI-TOF mass spectrometry. Results were verified by reciprocal immunoprecipitation. Phosphorylation status of bestrophin was determined by labeling of the protein with [32P]. Results: A major band at 68 kDa was identified as bestrophin, and a second band of ∼36 kDa was determined to be the b-catalytic subunit of the serine/threonine protein phosphatase 2A (PP2Ac). This finding was confirmed by reciprocal immunoprecipitation with antibodies against PP2Ac using both human and porcine RPE lysates. The carboxy-terminal cytoplasmic domain of bestrophin appears to be sufficient for the interaction with PP2Ac as demonstrated by a pull-down assay using a fusion of this domain with glutathione-S-transferase (GST). We also found that bestrophin is phosphorylated when expressed ectopically in RPE-J cells, and that this phosphorylation is partially sensitive to okadaic acid, an inhibitor of PP2A. Conclusion: Bestrophin interacts with PP2Ac. These data suggest that bestrophin participates in a signal transduction pathway and may be involved in transducing the signal that generates the light peak of the EOG.
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