The Roles of PP2A and PP2C in Dephosphorylating Phosducin in Rod Outer Segments

Abstract

Purpose: : Phosducin (pdc) in intact rod outer segment (IROS) is dephosphorylated by protein phosphatase 2A (PP2A) and 2C (PP2C). Light–activation of PP2C contributes to the accelerated loss of phosphorylated pdc in the light. This study investigates the roles of PP2A and PP2C.

Methods: : The activities and subcellular distribution of PP2A and PP2C in dark–adapted IROS were determined by in vitro phosphatase assay using ³²P–labeled pdc (phosphorylated at serine 73) and phospho–casein and by immunoblotting with antisera for phosphatases. In situ, the effects of light and/or phosphatase inhibitors on the subcellular distribution and the levels of endogenously phosphorylated pdc were monitored by a phospho–specific anti–pS73 antiserum.

Results: : IROS homogenate dephosphorylates phospho–casein, a common reference substrate for PP2A and PP2C, in Mg²⁺–independent/okadaic acid (OKA)–sensitive and Mg²⁺–dependent/OKA–resistant manner at the rate of 855.7 (PP2A) and 370.5 (PP2C) pmole/min/mg Rh, respectively. Since purified PP2A shows 3–fold higher casein activity than purified PP2C, this indicates that PP2A and PP2C are present in IROS at comparable levels. Pdc phosphorylated by PKA (at S73) is dephosphorylated in vitro by IROS PP2C and PP2A at 30% and 12%, respectively, of the rates for phospho– casein. Isotonic extraction of dark–adapted IROS removed 65% of IROS PP2C activity and anti–PP2C immunoreactivity into the soluble fraction; after sequential extraction about 25% remained membrane–bound. Parallel fractionation showed similar distribution of the endogenously phosphorylated pdc, with the membrane fraction particularly enriched with highly phosphorylated pdc. The levels of soluble, but not membrane–bound, phosphorylated pdc was significantly increased by pre–treatment with OKA. Light exposure in the presence of OKA abolished the membrane–bound phosphorylated pdc, with no effect on the soluble population.

Conclusions: : PP2A and PP2C are present at similar levels and largely in the cytosol of the IROS preparations, so is the endogenously phosphorylated pdc. Yet, in situ, only PP2A, not PP2C, dephosphorylates the soluble pdc; the highly phosphorylated pdc on the ROS membrane shows remarkable resistance to PP2A and is rapidly dephosphorylated when the membrane–associated PP2C is activated by light. Protein–protein interaction and enzyme recruitment are well–recognized mechanisms for regulating the specificity of protein phosphatases. We suggest that the selective actions of PP2A and PP2C on the soluble and membrane–associated pdc are similarly regulated.

Keywords: phosphorylation • photoreceptors • signal transduction