Abstract
Abstract: :
Purpose: To investigate the mechanism for light–triggered dephosphorylation of phosducin (pdc) in rod outer segments. Methods: The state of phosducin phosphorylation at the serine 73 (pS73) in intact rod outer segments (IROS) was determined in light or darkness with or without the presence of varying doses of inhibitors which exhibit different relative potencies against different types of serine/threonine protein phosphatases. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase(s) involved in pdc dephosphorylation. The relative contribution of protein kinase and different phosphatases was further evaluated by examining pS73 in homogenized IROS. Results: The steady state levels of pS73 in dark–adapted IROS were increased by incubation at 30C for 20 min with okadaic acid (OKA) or calyculin–A (Caly–A) to a maximal level of 126% of the dark control. Both inhibitors exhibit similar IC50 at around 21 nM. The dark pS73 levels in the presence of saturating OKA (200 nM) were unchanged by homogenization and dilution of ATP and second messengers, indicating pS73 in dark IROS is dephosphorylated only by protein phosphatase 2A (PP2A). In the light, pS73 decreased to 30% of the dark control. OKA and Caly–A partially blocked the loss with similar IC50 at around 24 nM. In the presence of saturating OKA, the light pS73 levels were about 65% of the dark control. Under the same condition, light decreased pS73 in IROS homogenate to 15% of that in the dark homogenate. Cyclosporin A and Deltamethrin, alone or with OKA, had no effect on pS73 levels in light or in darkness. These results revealed that in the light pdc is dephosphorylated by an OKA–resistant protein phosphatase and by PP2A. Conclusions: The dephosphorylation reaction plays a major role in regulating the state of pdc phosphorylation in IROS. Pdc is an endogenous substrate for PP2A and an OKA–resistant phosphatase. In the dark, the OKA–resistant phosphatase is inactive and the pS73 levels are affected by the activities of PP2A. In the light, the OKA–resistant phosphatase is activated. The resulting accelerated dephosphorylation and the continuing action of PP2A contribute to the light–induced decrease in pS73 levels. Two OKA–resistant phosphatases, PP2C and PPEF, have been detected in ROS. Considering that this phosphatase is activated by light in IROS homogenate without the participation of a diffusible second messenger, we suggest PP2C is the light–activated phosphatase.
Keywords: phosphorylation • signal transduction • photoreceptors