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RH Lee, P-S Ng, H Yu; Multisite And Hierarchical Phosphorylation In Light Regulation Of Phosducin Phosphorylation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1390.
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Purpose: To investigate the mechanism for light regulation of phosducin (pdc) phosphorylation. Methods: Intact bovine retinal rod outer segments (IROS) were incubated under varied illumination conditions, with or without regulators for protein kinases and/or protein phosphatases. The steady state levels and the signal patterns of total pdc and the Serine73-phosphorylated pdc (pdc-pS73) after 1 and 2D gel electrophoreses were monitored by Western blot analyses with anti-pdc-pan and anti-pdc-pS73, respectively. Results: The immunochemical signals for total pdc and pdc-pS73 in dark-adapted IROS were detected as multiple bands/spots. Semi-quantitative Western blot showed that pdc-pS73 constitutes 15-35% of total pdc. At 0ºC, the pdc-pS73 spots were localized mostly to 33-34 kDa and pH 4.7-4.9, a region for mono- and diphosphorylated pdc. At 30ºC these spots showed time-dependent migration to 34.5-35 kDa and pH 4.5-4.7, a region for pdc with 2 to 4 phosphates. Exposure to light at 30ºC triggered time-dependent decreases in pdc-pS73 with concomitant loss of the total pdc signals in the di- and multiphosphorylated region. In contrast, light at 0ºC had no effect on the pdc-pS73 signals. Dark incubation at 30ºC with chelerythrine HCl, a natural benzophenanthridine alkaloid, blocked the temperature-dependent migration as well as the light-induced abolishment of the pdc-pS73 spots. Conclusion: Pdc in dark-adapted IROS exists in a variety of phosphorylated states. The pdc-pS73 population of pdc undergoes additional phosphorylation at other sites in a time- and temperature-dependent manner, resulting in the accumulation of multiphosphorylated pdc that is particularly susceptible to light-induced dephosphorylation of pS73 and some other phosphorylation sites. Conditions that block the subsequent phosphorylation of pdc-pS73 also block the light-induced dephosphorylation. Thus light regulation of pdc involves multisite phosphorylation at S73 and subsequently at one or more other sites. Multiphosphorylated pdc binds beta, gamma-transducin with markedly reduced affinity, suggesting that the dissociated pdc is the target for light-induced dephosphorylation.
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