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R.H. Lee, P.–S. Ng, K.F. Faull; In vivo phosphorylation at the serine–8 residue affects the conformation and the phosphorylation characterstics of phosducin. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1279.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Serine residue 8 of phosducin (pdc) is conserved in nearly all species and is phosphorylated (pS8) in vivo. To determine if pS8 regulates pdc actions, the characteristics of in vivo phosphorylated pdc–pS8 was investigated. Methods: Specific antibody for pS8 was used to monitor native pdc–pS8 during native gel electrophoresis, ultracentrifugation and HPLC in order to examine its conformation. The state of pdc–pS8 phosphorylation in rod outer segments was determined by 2D gel electrophoresis and LC–ESIMS was used to detect a candidate pdc–pS8 binding protein. Results: Pdc–pS8, partially purified from bovine retinal extract, exists in complex with ßγ–transducin. During native gel electrophoresis, the pdc–pS8 complex migrates faster than both the pdc and pdc–pS73 complexes. Unlike both the pdc and the pdc–pS73 complexes which sediment as a symmetric 80–kDa molecule, the pdc–pS8 complex shows an asymmetric sedimentation profile, suggesting the existence of a population of higher molecular weight complex. LC–ESIMS detected in the highly purified pdc–pS8 complex an 11,714.8 Da and trypsin–sensitive molecule that is absent in the parallel pdc and pdc–pS73 samples. The asymmetric sedimentation profile and the presence of the 11–kDa molecule were abolished by pre–treatment of the pdc–pS8 sample with alkaline phosphatase, suggesting phosphorylation–dependent binding. The isoelectric points of the pdc–pS8 population in dark–adapted and okadaic acid–treated rod outer segments indicate the stoichiometry of phosphorylation as being 1 to 3, in contrast to the pdc–pS73 population as being 3 to 6. The pdc–pS8 population is largely free of pS73 and vice versa, suggesting the propensity for excluding simultaneous phosphorylation at these two sites. Conclusions: Phosphorylation at S8 confers new conformation to pdc and possibly new recognition site for binding to an 11–kDa protein. It also reduces the propensity of pdc to undergo further phosphorylation at S73 and hyper–phosphorylation at other part of the pdc molecule. Emerging evidence indicates that pdc is a multifunctional signaling protein. Our results indicate phosphorylation at S8 contributes to the regulation of pdc via multisite phosphorylation.
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