Proc. Natd. Acad. Sci. USA Vol. 88, pp. 2568-2572, March 1991 Biochemistry
Regulation of rhodopsin kinase by autophosphorylation JANINA BUCZYtKOt, CAROLINE GUTMANN,
AND
KRZYSZTOF PALCZEWSKIt
R. S. Dow Neurological Sciences Institute of Good Samaritan Hospital and Medical Center, 1120 N.W. 20th Avenue, Portland, OR 97209
Communicated by Edwin G. Krebs, December 18, 1990
then noted that the autophosphorylation reaction is not affected by the binding of RK to Rho*. However, it was not determined whether autophosphorylation alters the properties of RK. Herein we describe the isolation and characterization of fully phosphorylated (a-RK), intermediately phosphorylated (P-RK), and unphosphorylated (y-RK) forms of RK that can be resolved on the basis of their phosphorylation. We found that a-RK binds with lower affinity to phosphorylated Rho* than to Rho*, whereas 'y-RK binds tightly to both Rho* and phosphorylated Rho*. The effect of arrestin on the phosphorylation of Rho* was investigated in the context of these properties of RK.
Rhodopsin kinase (RK) catalyzes the phosABSTRACT phorylation of rhodopsin (Rho) as one of the steps in quenching photoactivated Rho. In this work, we investigated the autophosphorylation of RK and how it affects the interaction between RK and Rho. RK undergoes intramolecular phosphorylation, resulting in the incorporation of three or four phosphates per RK molecule. Phosphorylated RK subsequently is a substrate for protein phosphatases 2A and 2B. We isolated three forms of RK based on their differential interactions with heparin-Sepharose. Fully phosphorylated RK (orRK) binds tightly to Rho but has significantly lower affinity to phosphorylated Rho, whereas unphosphorylated RK (r-RK) binds avidly to both forms of Rho. The heterogenous intermediately phosphorylated RK (18-RK) was not studied. Our data support the hypothesis that RK dissociates from Rho when both Rho and RK become phosphorylated, thereby allowing the binding of arrestin to phosphorylated Rho. These results suggest that autophosphorylation plays an important role in regulating the binding of RK to Rho and that the binding sites of RK and arrestin overlap at least partially.
MATERIALS AND METHODS Isolation of Bovine and Human Rod Outer Segments (ROS). Bovine ROS were prepared from fresh retinas according to the procedure of Wilden and Kuhn (11). Human ROS were prepared from frozen retinas according to the same procedure. Protein Preparations. Bovine RK was purified as follows. ROS from 100 retinas were suspended in 100 ml of 10 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP; pH 7.5) containing 10 mM MgCI2 and the protease inhibitors aprotinin (30 pg), benzamidine (30 pg), and leupeptin (30 ,Ag). The suspension was homogenized and then illuminated at 0°C for 6-10 min under a 150-W floodlamp. ROS membranes containing RK were collected by centrifugation at 46,000 x g for 20 min, then mixed with a 5-ml suspension of DEAEcellulose (Whatman DE-52), and loaded on a prepared DEAE-cellulose column (1.6 x 10 cm) that had been equilibrated with 10 mM BTP (pH 7.5). The column was washed extensively under room light until the absorbance of the eluant at 280 nm dropped below 0.01. The column was wrapped with aluminum foil to shield it from light, and RK was eluted with 110 mM NaCl/10 mM BTP, pH 7.5 at a flow rate of 12 ml/hr. The fractions (1.5 ml) were collected and assayed for RK activity. The fractions containing RK activity were combined, diluted 1:1 with 10 mM BTP (pH 7.5), and then loaded on a heparin-Sepharose column (1.0 x 12 cm) equilibrated with 10 mM BTP (pH 7.5) at a flow rate of 12 ml/hr. RK species were eluted with a linear gradient of 0-500 mM NaCl in 10 mM BTP (pH 7.5) (total volume, 64 ml). All procedures were performed at 0-4°C. The catalytic subunits of protein phosphatases (PrPs) 1 and 2A were purified to near homogeneity from rabbit skeletal muscle (12, 13). The activities of the PrPs were determined as described (14). PrP 2B (calcineurin) was purchased from Sigma.
Rhodopsin (Rho) is the light-sensitive receptor of the retinal rod cell. Light triggers changes in the conformation of Rho, leading to the formation of metastable photoproducts. When photolyzed rhodopsin (Rho*) activates transducin, a GTPbinding protein (G protein), it initiates an amplification cascade of chemical reactions that result in the closing of Na+ channels in the plasma membrane of the rod outer segments [for reviews, see Stryer (1) and Hurley (2)]. Rhodopsin kinase (RK) catalyzes the multiple phosphorylation of Rho* at serine and threonine residues [for review, see Kuhn (3)]. These phosphorylated residues, along with other residues on the cytosolic surface of Rho are putative binding sites for arrestin (4, 5). The complex that forms between phosphorylated Rho and arrestin no longer activates transducin. Thus, phosphorylation of Rho* and binding of the regulatory protein arrestin is one of the mechanisms for quenching Rho*. The homologous proteins /3-arrestin and /3-adrenergic receptor kinase have been found to function in a similar manner during signal transduction by the p-adrenergic receptor (6). Presumably, RK and /3-adrenergic receptor kinase are representatives of a family of protein kinases that phosphorylate G protein-coupled receptors. Although our understanding of these reactions is limited to Rho and the /3-adrenergic receptor, the mechanism is expected to be similar in other G-protein-coupled receptors. In spite of the extensive characterization of RK during the last few years, the regulation of this enzyme in vivo is unknown. Recent studies revealed that RK acts independently of second messengers and requires Rho* and MgATP for its full activity (7). Like most protein kinases, RK undergoes autophosphorylation (8). We showed that this reaction is intramolecular and suggested that RK undergoes multiple phosphorylations (9). Kelleher and Johnson (10)
Abbreviations: BTP, 1,3-bis[tris(hydroxylmethyl)methylamino]propane; PrP, protein phosphatase; RK, rhodopsin kinase; a-, /3-, and y-RK, fully phosphorylated, intermediately phosphorylated, and unphosphorylated RK, respectively; Rho, rhodopsin; Rho*, photolyzed rhodopsin; ROS, rod cell outer segment(s). tPresent address: Technical University of Wroclaw, Division of Biochemistry, Wroclaw, Poland. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2568
Biochemistry: Buczylko et al. Phosphorylated Rho was prepared from urea-washed ROS as described (7). Regeneration of phosphorylated opsin with 11-cis-retinal was performed as described (15). Bovine arrestin was prepared from fresh dark-adapted retinas obtained from a local slaughterhouse. Purification was achieved by the specific binding of arrestin to phosphorylated Rho*, followed by two chromatographic separations: the first on a DEAE-cellulose column and the second on a Mono Q column (HR 5 x 50 mm, Pharmacia; for details, see refs. 5, 16, and 17). Arrestin (95-100% pure) was dialyzed against 10 mM Hepes (pH 7.5) containing 100 mM NaCI. Assay for RK Activity. RK activity was measured using urea-washed ROS membranes as the substrate. Typically, the 140-/.l reaction mixture contained 20 AM urea-washed ROS, 5 mM MgCl2, and 100 ,M [y-32P]ATP (100-500 cpm/ pmol; 1 Ci = 37 GBq; New England Nuclear) in 20 mM BTP (pH 7.5). The reaction was initiated by adding radioactive ATP, was performed under constant illumination for 7.5 min in a water bath (300C), and was terminated by adding 10% (wt/vol) trichloroacetic acid (7). The specific activity of RK was 500-800 nmol of phosphate transferred per min per mg of RK. Under these experimental conditions, autophosphorylation of RK is insignificant compared to phosphorylation of Rho*. Stoichiometry of RK Autophosphorylation. Known amounts of purified y-RK (5-25 ,ug) were incubated with 10 ,M [y32P]ATP (3000-6000 cpm/pmol) in BTP buffer containing 1 mM MgCl2, 100 mM NaCl, and bovine serum albumin (0.1 mg/ml) at 30°C. At various times, samples of RK were tested for stoichiometry of autophosphorylation by one of three methods. (i) Samples of RK were mixed with equal amounts of 10o (wt/vol) SDS, and SDS/PAGE was performed. The radioactive bands were visualized by autoradiography, excised, dissolved in 30%o (vol/vol) H202, and mixed with scintillation fluid. Radioactivity was measured with a scintillation counter. (ii) Samples were mixed with 1 ml of 20% trichloroacetic acid, and 1 mg of bovine serum albumin was added. Proteins were collected by centrifugation (14,000 X g for 10 min). The pellet was washed extensively with 20%o trichloroacetic acid and then dissolved in 1 ml of 1O0o formic acid. The solution was mixed with scintillation fluid, and radioactivity was measured with a scintillation counter. (iii) The reaction mixture (50-100 ,l) was spotted on methanoltreated Immobilon (Millipore) using a slot blot apparatus (PR 600, Hoefer). The membrane was washed extensively with 75 mM H3PO4 (about 30 ml per trace), and traces containing proteins were excised. The membrane was moistened in 100%6 methanol, mixed with scintillator fluid, and radioactivity was measured with a scintillation counter. These three methods of measuring the stoichiometry of RK autophosphorylation gave similar results within experimental error. Binding of RK to Phosphorylated Rho*. Urea-washed ROS membranes (9 ,M) or phosphorylated and regenerated Rho (9 ,AM; 3.2 phosphates per molecule of Rho) in 10 mM Hepes (pH 7.5) containing 100 mM NaCl, 5 mM MgCl2, bovine serum albumin (0.25 mg/ml), and 0.06 ,M purified a-RK were incubated in the dark or under illumination. The total volume of the sample was 180 ,ul. After 15 min at 30°C, the sample was centrifuged for 2 min at 180,000 x g using an Airfuge (Beckman). After separation of the membranes, 150 ,ul of the supernatant was used to determine kinase activity. The binding also was performed in the presence of 10 ,AM arrestin. The identical experiment was performed employing -RK instead of a-RK. Protein Determinations. The concentration of RK was determined by the micro-Bradford method (18) and amino acid analysis. A molecular mass of 67 kDa for RK was assumed to deduce the concentration of RK from amino acid analysis. The concentration of purified' arrestin was determined spectrophotometrically at 278 nm by assuming an
2569
Proc. Natl. Acad. Sci. USA 88 (1991)
absorption coefficient of E
Regulation of rhodopsin kinase by autophosphorylation JANINA BUCZYtKOt, CAROLINE GUTMANN,
AND
KRZYSZTOF PALCZEWSKIt
R. S. Dow Neurological Sciences Institute of Good Samaritan Hospital and Medical Center, 1120 N.W. 20th Avenue, Portland, OR 97209
Communicated by Edwin G. Krebs, December 18, 1990
then noted that the autophosphorylation reaction is not affected by the binding of RK to Rho*. However, it was not determined whether autophosphorylation alters the properties of RK. Herein we describe the isolation and characterization of fully phosphorylated (a-RK), intermediately phosphorylated (P-RK), and unphosphorylated (y-RK) forms of RK that can be resolved on the basis of their phosphorylation. We found that a-RK binds with lower affinity to phosphorylated Rho* than to Rho*, whereas 'y-RK binds tightly to both Rho* and phosphorylated Rho*. The effect of arrestin on the phosphorylation of Rho* was investigated in the context of these properties of RK.
Rhodopsin kinase (RK) catalyzes the phosABSTRACT phorylation of rhodopsin (Rho) as one of the steps in quenching photoactivated Rho. In this work, we investigated the autophosphorylation of RK and how it affects the interaction between RK and Rho. RK undergoes intramolecular phosphorylation, resulting in the incorporation of three or four phosphates per RK molecule. Phosphorylated RK subsequently is a substrate for protein phosphatases 2A and 2B. We isolated three forms of RK based on their differential interactions with heparin-Sepharose. Fully phosphorylated RK (orRK) binds tightly to Rho but has significantly lower affinity to phosphorylated Rho, whereas unphosphorylated RK (r-RK) binds avidly to both forms of Rho. The heterogenous intermediately phosphorylated RK (18-RK) was not studied. Our data support the hypothesis that RK dissociates from Rho when both Rho and RK become phosphorylated, thereby allowing the binding of arrestin to phosphorylated Rho. These results suggest that autophosphorylation plays an important role in regulating the binding of RK to Rho and that the binding sites of RK and arrestin overlap at least partially.
MATERIALS AND METHODS Isolation of Bovine and Human Rod Outer Segments (ROS). Bovine ROS were prepared from fresh retinas according to the procedure of Wilden and Kuhn (11). Human ROS were prepared from frozen retinas according to the same procedure. Protein Preparations. Bovine RK was purified as follows. ROS from 100 retinas were suspended in 100 ml of 10 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP; pH 7.5) containing 10 mM MgCI2 and the protease inhibitors aprotinin (30 pg), benzamidine (30 pg), and leupeptin (30 ,Ag). The suspension was homogenized and then illuminated at 0°C for 6-10 min under a 150-W floodlamp. ROS membranes containing RK were collected by centrifugation at 46,000 x g for 20 min, then mixed with a 5-ml suspension of DEAEcellulose (Whatman DE-52), and loaded on a prepared DEAE-cellulose column (1.6 x 10 cm) that had been equilibrated with 10 mM BTP (pH 7.5). The column was washed extensively under room light until the absorbance of the eluant at 280 nm dropped below 0.01. The column was wrapped with aluminum foil to shield it from light, and RK was eluted with 110 mM NaCl/10 mM BTP, pH 7.5 at a flow rate of 12 ml/hr. The fractions (1.5 ml) were collected and assayed for RK activity. The fractions containing RK activity were combined, diluted 1:1 with 10 mM BTP (pH 7.5), and then loaded on a heparin-Sepharose column (1.0 x 12 cm) equilibrated with 10 mM BTP (pH 7.5) at a flow rate of 12 ml/hr. RK species were eluted with a linear gradient of 0-500 mM NaCl in 10 mM BTP (pH 7.5) (total volume, 64 ml). All procedures were performed at 0-4°C. The catalytic subunits of protein phosphatases (PrPs) 1 and 2A were purified to near homogeneity from rabbit skeletal muscle (12, 13). The activities of the PrPs were determined as described (14). PrP 2B (calcineurin) was purchased from Sigma.
Rhodopsin (Rho) is the light-sensitive receptor of the retinal rod cell. Light triggers changes in the conformation of Rho, leading to the formation of metastable photoproducts. When photolyzed rhodopsin (Rho*) activates transducin, a GTPbinding protein (G protein), it initiates an amplification cascade of chemical reactions that result in the closing of Na+ channels in the plasma membrane of the rod outer segments [for reviews, see Stryer (1) and Hurley (2)]. Rhodopsin kinase (RK) catalyzes the multiple phosphorylation of Rho* at serine and threonine residues [for review, see Kuhn (3)]. These phosphorylated residues, along with other residues on the cytosolic surface of Rho are putative binding sites for arrestin (4, 5). The complex that forms between phosphorylated Rho and arrestin no longer activates transducin. Thus, phosphorylation of Rho* and binding of the regulatory protein arrestin is one of the mechanisms for quenching Rho*. The homologous proteins /3-arrestin and /3-adrenergic receptor kinase have been found to function in a similar manner during signal transduction by the p-adrenergic receptor (6). Presumably, RK and /3-adrenergic receptor kinase are representatives of a family of protein kinases that phosphorylate G protein-coupled receptors. Although our understanding of these reactions is limited to Rho and the /3-adrenergic receptor, the mechanism is expected to be similar in other G-protein-coupled receptors. In spite of the extensive characterization of RK during the last few years, the regulation of this enzyme in vivo is unknown. Recent studies revealed that RK acts independently of second messengers and requires Rho* and MgATP for its full activity (7). Like most protein kinases, RK undergoes autophosphorylation (8). We showed that this reaction is intramolecular and suggested that RK undergoes multiple phosphorylations (9). Kelleher and Johnson (10)
Abbreviations: BTP, 1,3-bis[tris(hydroxylmethyl)methylamino]propane; PrP, protein phosphatase; RK, rhodopsin kinase; a-, /3-, and y-RK, fully phosphorylated, intermediately phosphorylated, and unphosphorylated RK, respectively; Rho, rhodopsin; Rho*, photolyzed rhodopsin; ROS, rod cell outer segment(s). tPresent address: Technical University of Wroclaw, Division of Biochemistry, Wroclaw, Poland. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2568
Biochemistry: Buczylko et al. Phosphorylated Rho was prepared from urea-washed ROS as described (7). Regeneration of phosphorylated opsin with 11-cis-retinal was performed as described (15). Bovine arrestin was prepared from fresh dark-adapted retinas obtained from a local slaughterhouse. Purification was achieved by the specific binding of arrestin to phosphorylated Rho*, followed by two chromatographic separations: the first on a DEAE-cellulose column and the second on a Mono Q column (HR 5 x 50 mm, Pharmacia; for details, see refs. 5, 16, and 17). Arrestin (95-100% pure) was dialyzed against 10 mM Hepes (pH 7.5) containing 100 mM NaCI. Assay for RK Activity. RK activity was measured using urea-washed ROS membranes as the substrate. Typically, the 140-/.l reaction mixture contained 20 AM urea-washed ROS, 5 mM MgCl2, and 100 ,M [y-32P]ATP (100-500 cpm/ pmol; 1 Ci = 37 GBq; New England Nuclear) in 20 mM BTP (pH 7.5). The reaction was initiated by adding radioactive ATP, was performed under constant illumination for 7.5 min in a water bath (300C), and was terminated by adding 10% (wt/vol) trichloroacetic acid (7). The specific activity of RK was 500-800 nmol of phosphate transferred per min per mg of RK. Under these experimental conditions, autophosphorylation of RK is insignificant compared to phosphorylation of Rho*. Stoichiometry of RK Autophosphorylation. Known amounts of purified y-RK (5-25 ,ug) were incubated with 10 ,M [y32P]ATP (3000-6000 cpm/pmol) in BTP buffer containing 1 mM MgCl2, 100 mM NaCl, and bovine serum albumin (0.1 mg/ml) at 30°C. At various times, samples of RK were tested for stoichiometry of autophosphorylation by one of three methods. (i) Samples of RK were mixed with equal amounts of 10o (wt/vol) SDS, and SDS/PAGE was performed. The radioactive bands were visualized by autoradiography, excised, dissolved in 30%o (vol/vol) H202, and mixed with scintillation fluid. Radioactivity was measured with a scintillation counter. (ii) Samples were mixed with 1 ml of 20% trichloroacetic acid, and 1 mg of bovine serum albumin was added. Proteins were collected by centrifugation (14,000 X g for 10 min). The pellet was washed extensively with 20%o trichloroacetic acid and then dissolved in 1 ml of 1O0o formic acid. The solution was mixed with scintillation fluid, and radioactivity was measured with a scintillation counter. (iii) The reaction mixture (50-100 ,l) was spotted on methanoltreated Immobilon (Millipore) using a slot blot apparatus (PR 600, Hoefer). The membrane was washed extensively with 75 mM H3PO4 (about 30 ml per trace), and traces containing proteins were excised. The membrane was moistened in 100%6 methanol, mixed with scintillator fluid, and radioactivity was measured with a scintillation counter. These three methods of measuring the stoichiometry of RK autophosphorylation gave similar results within experimental error. Binding of RK to Phosphorylated Rho*. Urea-washed ROS membranes (9 ,M) or phosphorylated and regenerated Rho (9 ,AM; 3.2 phosphates per molecule of Rho) in 10 mM Hepes (pH 7.5) containing 100 mM NaCl, 5 mM MgCl2, bovine serum albumin (0.25 mg/ml), and 0.06 ,M purified a-RK were incubated in the dark or under illumination. The total volume of the sample was 180 ,ul. After 15 min at 30°C, the sample was centrifuged for 2 min at 180,000 x g using an Airfuge (Beckman). After separation of the membranes, 150 ,ul of the supernatant was used to determine kinase activity. The binding also was performed in the presence of 10 ,AM arrestin. The identical experiment was performed employing -RK instead of a-RK. Protein Determinations. The concentration of RK was determined by the micro-Bradford method (18) and amino acid analysis. A molecular mass of 67 kDa for RK was assumed to deduce the concentration of RK from amino acid analysis. The concentration of purified' arrestin was determined spectrophotometrically at 278 nm by assuming an
2569
Proc. Natl. Acad. Sci. USA 88 (1991)
absorption coefficient of E
