Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Izabela Sokal,Guang Hu,Yan Liang,Muling Mao,Theodore G Wensel,Krzysztof Palczewski

doi:10.1074/jbc.m211782200

Abstract

Inactivation of the visual G-protein transducin by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP) RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light- and Ca(2+)-sensitive phosphorylation on Ser(475). Because there are much higher RGS9-1 levels in cones than in rods, we investigated whether Ser(475) is phosphorylated in rods using Coneless mice and found that both the phosphorylation and its regulation by light occur in rods. Therefore, we used rod outer segments as the starting material for the purification of RGS9-1 kinase activity. Two major peaks of activity corresponded to protein kinase C (PKC) isozymes, PKCalpha and PKCtheta. A synthetic peptide corresponding to the Ser(475) RGS9-1 sequence and RGS9-1 were substrates for recombinant PKCalpha and PKCtheta. This phosphorylation was removed efficiently by protein phosphatase 2A, an endogenous phosphatase in rod outer segments, but not by PP1 or PP2B. Phosphorylation of RGS9-1 by PKC had little effect on its activity in solution but significantly decreased its affinity for its membrane anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKCtheta immunostaining was at higher levels in cone outer segments than in rod outer segments, as was found for the components of the RGS9-1 GAP complex. Thus, PKC-mediated phosphorylation of RGS9-1 represents a potential mechanism for feedback control of the kinetics of photoresponse recovery in both rods and cones, with this mechanism probably especially important in cones.

Highlights

Transducin (Gt),1 the visual G-protein, is inactivated when its bound GTP is hydrolyzed to GDP, a free phosphate dissoci
We report here that two protein kinase C (PKC) isozymes from ROS, PKC␣ and PKC␪, co-purify with RGS9-1 kinase activity, that both phosphorylate RGS9-1 at Ser475, and that protein phosphatase 2A is responsible for the RGS9-1 dephosphorylation at this site
Phosphorylation Occurs in Rods—RGS9-1 is highly expressed in cone but is present in rod photoreceptors cells (Fig. 1A) [16]

Summary

Introduction

Transducin (Gt), the visual G-protein, is inactivated when its bound GTP is hydrolyzed to GDP, a free phosphate dissoci-. Phosphorylated RGS9-1 is present exclusively in the cholesterolrich detergent-insoluble membrane domains (Rafts), whereas the unphosphorylated form can be readily extracted with mild detergent [19]. These results demonstrate differences in the subcellular localization between phosphorylated RGS9-1 and its unphosphorylated form. CAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 phospholabeling by more than 90%, whereas dibutyryl-cAMP stimulated it 3-fold [20]. These studies suggested PKA as the major kinase responsible for RGS9-1 phosphorylation in ROS. In ROS, RGS9-1 phosphorylation required the presence of free Ca2ϩ ions and was inhibited by light [20]

Methods

Results

Conclusion