Abstract
Purpose: :
To investigate the regulation of the photoreceptor–specific GTPase Accelerating Protein (GAP) RGS9–1 by PKCα. Normal light response kinetics require both RGS9–1 and its membrane anchor R9AP, whose interactions have been previously shown to be modified by a light–inhibited phosphorylation on Ser475 of RGS9–1 catalyzed by one or more isozymes of protein kinase C (PKC).
Methods: :
PKCα–/– and wild–type mice were compared. The presence of PKCα in mouse rod outer segments was examined by western blot of purified rod outer segments. The localization of PKCα to rod outer segments was confirmed by analysis of fractions collected from two successive sucrose gradients and iso–osmotic OptiPrep gradient purification. The amount of PKCα in purified intact rod outer segments was determined by quantitative western blot of rod outer segments and retina lysates. In vitro phosphorylation of RGS9–1 in rod outer segments by endogenous PKCα was detected by kinase assays. In vivo light–regulated phosphorylation of RGS9–1 at Ser475 was assessed by immunoprecipitation of RGS9–1 from mice retinas and followed by western blot using Ser475–phosphate–specific antibody.
Results: :
PKCα immuno–reactivity is present in wild–type mouse rod outer segments but missing in those of PKCα –/– mice. It consistently co–purifies with rod outer segments. Approximately 20% of the total retinal PKCα pool is in mouse rod outer segments. PKCα knockouts do not exhibit the light–sensitive phosphorylation of RGS9–1 on PKC target site Ser475 that is observed in wild–type mice and show slower RGS9–1 Ser475 phosphorylation in vitro.
Conclusions: :
PKCα immuno–reactivity in photoreceptor cells is genuine and not due to cross–activity and contamination. PKCα is essential for light–sensitive RGS9–1 Ser475 phosphorylation. These results indicate that rod outer segments contain modest levels of PKCα, and this enzyme is responsible for regulation of the R9AP–RGS9–1 membrane complex by Ser475 phosphorylation, thus likely regulating the rate–limiting reaction in vision.
Keywords: photoreceptors • phosphorylation • transgenics/knock-outs