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Subunit of Cgmp Phosphodiesterase in Vertebrate Rod Photoreceptors (Signal Transduction/Transducin/Okadaic Acid) Fumio HAYASHI*T, GRACE Y

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Subunit of Cgmp Phosphodiesterase in Vertebrate Rod Photoreceptors (Signal Transduction/Transducin/Okadaic Acid) Fumio HAYASHI*T, GRACE Y Proc. Natl. Acad. Sci. USA Vol. 88, pp. 4333-4337, May 1991 Biochemistry Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors (signal transduction/transducin/okadaic acid) FuMIo HAYASHI*t, GRACE Y. LIN*, HIROYUKI MATSUMOTOt, AND AKIO YAMAZAKI*§ *Cellular and Molecular Biology Group, Life Sciences Division, Los Alamos National Laboratory, University of California, Los Alamos, NM 87545; and tDepartment of Biochemistry and Molecular Biology, College of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 Communicated by Floyd Ratliff, February 19, 1991 ABSTRACT An inhibitory subunit (Py) of cGMP phos- 11,000-13,000) and II (Mr 8,000-12,000) (9-14) in vertebrate phodiesterase from vertebrate rod photoreceptors (frog, toad, rod photoreceptors, and an S-antigen-like protein in inver- and bovine) was phosphorylated by cytosolic protein kinase(s) tebrate photoreceptors (15). Phosphate incorporation to derived from intact frog rod outer segments. The phosphory- phosducin was detected during dark incubation in a cyclic lation offrog Py was stimulated by phosphatidylinositol but not nucleotide-dependent manner, and the phosphorylation is by cAMP or cGMP. One- and two-dimensional gel electropho- reduced during subsequent illumination (7, 8). The levels of resis revealed that 70-80% of Py was phosphorylated with 1 phosphorylation of components I and II also appear to be mol of phosphate per frog Py under optimal conditions. A modulated by light as well as by cyclic nucleotide (9, 10). peptide that derived from an active domain of bovine Py was Moreover, the phosphorylation of components I and II was also phosphorylated. Phosphorylation offrog Py was inhibited also facilitated by phosphatidylinositol (PtdIns) (11), phos- by addition of the peptide to the reaction mixture. Phospho- photidylglycerol (12), and protein kinase C activators (13). rylation of frog Py was also inhibited by addition of transducin The nonspecific nature of the regulation suggests that com- subunits or active (Py-less) cGMP phosphodiesterase. Okadaic ponents I and II may be a mixture ofseveral proteins. Indeed, acid, on the other hand, enhanced Py phosphorylation, sug- component II has been shown to be the mixture of three gesting the presence of protein phosphatase(s) in the cytosolic proteins with different isoelectric points (12). The phospho- fraction. These data suggest another mechanism for the regu- rylation of the S-antigen-like protein in Drosophila photore- lation of cGMP phosphodiesterase in vertebrate rod photore- ceptors (15) suggests the possibility of phosphorylation of ceptors. S-antigen in vertebrate rod photoreceptors. To explore the roles of each Py in the regulatory mecha- The hydrolysis of cGMP by phosphodiesterase (PDE) ap- nism of PDE activation in vertebrate rod photoreceptors, we pears directly involved in the process of visual transduction have attempted to characterize the heterogeneity of Py. In in vertebrate photoreceptor rod outer segments (ROS) (1-3). the present study, we describe PtdIns-stimulated phospho- Illumination of rhodopsin leads to activation of transducin, a rylation of Py by kinase(s) derived from intact frog ROS. We G protein in ROS. Transducin, in turn, activates PDE. The also describe the effects of interactions with other compo- resulting fall in cytoplasmic cGMP concentration leads to the nents of the PDE cascade on the Py phosphorylation and the closure of cGMP-sensitive cation channels, causing the hy- possibility of protein phosphatase(s) in rod photoreceptors. perpolarization of rod plasma membranes. PDE is composed of catalytic subunits (Pa, ,3) with Mr values of 89,000 and MATERIALS AND 87,000 and two inhibitory subunits (Pys) with Mr of 13,000 METHODS (1-3). The activation cascade offrog PDE has been described Materials. Frozen bovine retinas were purchased from (4, 5). In an in vitro system, frog PDE activation by trans- Hormel (Austin, MN). TSK DEAE-5-PW (7.5 x 75 mm) and ducin depends upon the physical release of a PTy from PDE TSK 250 (7.5 x 30 mm) columns were obtained from Bio- through an interaction with GTP and the a subunit of trans- Rad. Percoll was purchased from Pharmacia LKB. Chemical ducin (Ta). After GTP hydrolysis on Ta, Py remains tightly reagents were obtained from the following sources: [y_32p]_ bound to GDP-Ta, and the Py release from GDP-Ta (and ATP from Du Pont/NEN; ATP, GTP, GDP, and guanosine subsequent inhibition of Py-free PDE) requires additional 5'-[y-thio]triphosphate (GTP[ys]) were from Boehringer subunits of transducin, T,8,y. However, previous data have Mannheim; PtdIns, aprotinin, leupeptin, pepstatin, dibutyryl not demonstrated differences in the characters ofeach Py nor cGMP, and phenylmethylsulfonyl fluoride were from Sigma; the roles of each Py in the activation mechanism of PDE in okadaic acid was from Wako Biochemicals (Richmond, VA). rod photoreceptors. A peptide corresponding to bovine Py residue 31-45 (16) was Phosphorylation ofproteins is one ofthe most fundamental synthesized by K. W. Jackson, who was supported by The mechanisms regulating cellular metabolism and function. In Saint Francis Hospital of Tulsa Medical Research Institute. vertebrate rod photoreceptors, rhodopsin phosphorylation Preparation of Py from Amphibian and Bovine ROS Mem- has been studied extensively as a mechanism terminating the branes. Purification offrog (Rana catesbiana) P'y was done as light-activated biochemical processes (1-3, 6). In addition, described (5). Recently, we found that toad (Bufo marinus) phosphorylation of some proteins has been investigated, although little is known about the molecular roles of these Abbreviations: PDE, cGMP phosphodiesterase; ROS, rod outer proteins in the process of visual transduction. These proteins segments; Pa, f3, catalytic subunits of PDE; Py, inhibitory subunit of included phosducin (Mr 33,000) (7, 8), components I (Mr PDE; Ptdlns, phosphatidylinositol; Ta, TJ, and Ty, a, /3, and y subunits of transducin; GTP[ys], guanosine 5'-[y-thio]triphosphate. tPresent address: Department of Biology, College of General Edu- The publication costs of this article were defrayed in part by page charge cation, Kobe University, Kobe 657, Japan. payment. This article must therefore be hereby marked "advertisement" §To whom reprint requests should be addressed at: Los Alamos in accordance with 18 U.S.C. §1734 solely to indicate this fact. National Laboratory, MS M888, Los Alamos, NM 87545. 4333 4334 Biochemistry: Hayashi et al. Proc. Natl. Acad. Sci. USA 88 (1991) P)y is also complexed and released with GTP'Ta when ROS leupeptin, and PtdIns at 100 Mg/ml. In some experiments, 100 membranes are washed with an isotonic buffer containing GTP MM cAMP or dibutyryl cGMP was added as a substitute for (A.Y., unpublished data). Based on these specific properties PtdIns. Phosphorylation was initiated by adding the crude of amphibian Py, toad Py was also purified by applying the kinase (0.5 to =1 Mug of protein), and the mixtures were same procedures used for the purification of frog PTy. incubated for 60 min at 330C. Addition of an ATP- Because bovine Py was barely released to the supernatant regenerating system (15 mM phosphocreatine and creatine with GTP*Ta even in hypotonic buffers (A.Y., unpublished phosphokinase at 0.1 mg/ml) had no effect on Py phospho- data), bovine Py was prepared as follows. After preparation rylation. The reaction was terminated by adding a concen- of ROS from frozen retinas and extensive washing of ROS trated (xS) sample buffer for SDS/PAGE, and the mixture membranes, crude PDE preparations were extracted by was heated at 900C for 5 min. Samples were analyzed by further washing ROS membranes as described (17). The SDS/polyacrylamide gradient (5-20%) gel electrophoresis. crude PDE preparation was applied to a TSK DEAE-5PW After cutting gels above the dye front to avoid contamination column that had been equilibrated with buffer A (10 mM of free-radioactive ATP, the gels were stained for proteins Tris HC1, pH 7.5/1 mM dithiothreitol/5 mM MgSO4). The (silver or Coomassie blue), dried, and subjected to autorad- column was washed with 20 ml of buffer A, and PDE was iography and densitometric scanning for quantitation. In eluted with a NaCl gradient (0-0.6 M) in buffer A (flow rate some experiments, protein bands were excised from gels and of 1 ml/min, fraction volume 0.5 ml). The purified PDE was incubated (overnight at 700C) with 500 Mul of 30% hydrogen concentrated with a Centricon 30 microconcentrator (Ami- peroxide in sealed containers. The radioactivity of the dis- con) at 4TC. Concentrated PDE (.5-10 mg/ml) was added solved gels was then quantitated. To measure phosphoryla- dropwise to an equal volume of 0.2 M formic acid (pH 2.4). tion of a peptide, the gels were stained to avoid diffusion of The mixture was heated at 800C for 5 min, quickly cooled at the peptide from gels. Peptide was stained for 10 min at 90°C 0C, and then centrifuged at 40C for 30 min (120,000 x g). The in the solution containing 0.25% Coomassie blue R250, 50% resulting supernatant was applied to a TSK 250 column that methanol, and 10%o acetic acid. had been equilibrated with 0.2 M formic acid (pH 2.4). After Analytical Procedures. Protein concentration and PDE column chromatography (flow rate 0.5 ml/min, fraction vol- activity were measured as described (5). SDS/PAGE was ume 0.5 ml), an aliquot ofeach fraction was diluted with water done as described (5). Two-dimensional gel electrophoresis, and lyophilized; the inhibitory activity was measured by using including either an isoelectrofocusing or a nonequilibrium pH frog ROS membranes containing active (PTy-less) PDE as gradient electrophoresis, and an SDS/polyacrylamide gradi- described (5). Purity and protein concentration of Py were ent (5-20%) gel electrophoresis were done as described (20).
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