title: Bestrophin interacts physically and functionally with protein phosphatase 2A.
abstract: Bestrophin is a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial cells (RPE). It is encoded by the VMD2 gene, which is mutated in Best macular dystrophy, a disease characterized by a depressed light peak in the electrooculogram. Recently it was proposed that bestrophin is a chloride channel responsible for generating the light peak. To investigate its function further, we immunoaffinity purified a bestrophin complex from RPE lysates and identified bestrophin and the beta-catalytic subunit of protein phosphatase 2A (PP2A) as members of the complex by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Protein-protein interaction between bestrophin and PP2Ac and the structural subunit of PP2A, PR65, was confirmed by reciprocal immunoprecipitation. The C-terminal cytoplasmic domain of bestrophin was sufficient for the interaction with PP2A as demonstrated by a pulldown assay using a fusion of this domain with glutathione S-transferase. Bestrophin was phosphorylated when expressed in RPE-J cells and this phosphorylation was sensitive to okadaic acid. Purified PP2A effectively dephosphorylated bestrophin in vitro. These data suggest that bestrophin is in the signal transduction pathway that modulates the light peak of the electrooculogram, that it is regulated by phosphorylation, and that phosphorylation of bestrophin is in turn regulated by PP2A.
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Lihua Y
authors.initials: LY
authors.lastname: Marmorstein
authors.forename: Precious J
authors.initials: PJ
authors.lastname: McLaughlin
authors.forename: J Brett
authors.initials: JB
authors.lastname: Stanton
authors.forename: Lin
authors.initials: L
authors.lastname: Yan
authors.forename: John W
authors.initials: JW
authors.lastname: Crabb
authors.forename: Alan D
authors.initials: AD
authors.lastname: Marmorstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000075002:Bestrophins; D002478:Cells, Cultured; D018118:Chloride Channels; D003600:Cytosol / Q000737:chemistry; D004585:Electrooculography; D005136:Eye Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008027:Light; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D015398:Signal Transduction; D013552:Swine
chemical_list: C113133:BEST1 protein, human; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2