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R.H. Lee, M. Johnson, P. Ng; 2D Gel Analysis of Phosducin in Rod Outer Segments: The Effect of Light . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1528.
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Purpose: To characterize the effect of light on the state of phosducin (pdc) phosphorylation. Methods: Intact rod outer segments (IROS) were subjected to 2D gel electrophoresis followed by Sypro Ruby staining and Western blot analysis with anti-pdc-pan, anti-pS73, and anti-pS8 to identify all pdc, the pdc subpopulations with phosphorylation at Serine73 (pS73), and Serine8 (pS8), respectively. The migration patterns of IROS proteins and pdc at different states of phosphorylation were analyzed by Phoretix Evolution software for spot matching, spot densities, pI and molecular weight (m.wt.) determination. Results: 2D gel analysis of purified bovine retinal pdc revealed an array of 4 33-kDa spots at pH 5.07, 5.01, 4.95 and 4.88, respectively. Stoichiometric phosphorylation by PKA decreased the pI of each spot by an average of 0.085-pH unit. IROS that was pre-treated with alkaline phosphatase also revealed 4 pdc spots in the same location. In the dark-adapted IROS, about 12 to 16 pdc spots were detected including 2 new arrays of increasingly acidic spots with increasingly higher apparent m.wt.'s. The most acidic pdc spot was detected at pH 4.62 and 35.5 kDa. Anti-pS73 revealed a very different pattern, with most of the pdc-pS73 spots clustering between pH 4.8 to 4.6 and 34 to 35-kDa. Anti-pS8 detected an array of 5-6 spots in the 34-kDa region and between pH 4.95 to 4.70. Light abolished all 35-kDa pdc spots, decreased the densities of some 34-kDa spots, but increased, most notably, the density of the pH 5.09 spot by about 2 to 5-fold. Light also abolishes most, if not all, of the pdc-pS73 spots. Light greatly decreased the density of the most acidic pdc-pS8 spot but the total intensity of all pdc-pS8 spots was not significantly changed. Conclusions: IROS contains 4 pdc isoforms, of which the pI = 5.09 form is a major target for phosphorylation. In the dark, as much as 40% of total pdc is phosphorylated to varying extents: from 1 to 5 Serine in a pdc molecule may be phosphorylated. The predicted overlap among different phosphorylated pdc isoforms prohibits definitive spot assignment. Still, the light-induced pattern changes in the total and the selective pdc populations revealed a consistent scenario in which the steady state levels of all phosphorylation sites, except pS8, are decreased by light, with the pS73 site being most rapidly dephosphorylated. The significance of the subpopulations-specific migration patterns will be discussed.
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