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U. Wolfrum, P. Trojan, S. Rausch, D. Wünschig, A. Gießl, A. Pulvermüller; Light–Dependent Phosphorylation of Centrins Regulates Binding to Transducin in Mammalian Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1091.
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Centrins are members of the parvalbumin superfamily of Ca2+–binding EF–hand proteins. In ciliated cells, like vertebrate photoreceptors, 4 centrins are differentially localized in the transition zone of cilia – the photoreceptor connecting cilium. Our previous studies revealed Ca2+–dependent binding of centrins to the ß–subunit of the visual G–protein transducin. There is evidence that the centrin/G–protein complex assembly regulates the light driven, adaptive translocation of transducin through the photoreceptor cilium. Here we analyzed whether or not the functions of centrins are further regulated via phosphorylation in mammalian photoreceptors.
In in vitro and ex vivo phosphorylation assays were applied bacterial expressed centrins and centrins in explanted rat retinas; immunocytochemistry; immunoelectron microscopy; recombinant protein expression; protein–protein interaction assays including co–immunoprecipitation, Blot overlay, GST–pull down, and kinetic light scattering assays. MALDI–MS analysis of protein phosphorylation.
Centrins were differentially phosphorylated in a light–dependent manner in mammalian photoreceptors. Immunocytochemistry confirmed co–localization of centrins with transducin and with the protein kinase CK2 in the connecting cilium. Specific CK2 inhibition dramatically reduced the phosphorylation of centrins. Sequence analysis of centrins identified the target sequences for CK2–mediated phosphorylation. Specific CK2 phosphorylation sites in centrin molecules were pinpointed using deletion constructs and via MALDI–MS. Decreased binding affinities of phosphorylated centrins to transducin were observed in kinetic light–scattering assays.
To our knowledge centrins are the first cytoskeletal elements which are light–dependently phosphorylated. The binding of centrins to transducin is antagonistically modulated by the free Ca2+–concentration and by light–dependent centrin phosphorylation. These molecular modifications of centrins contribute to the regulation light–driven movements of transducin between the inner and outer segment compartment of the photoreceptor cell.
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