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Light-Stimulated Phosphorylation of Rhodopsin in the Retina

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Light-Stimulated Phosphorylation of Rhodopsin in the Retina Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 381-385, January 1975 Light-Stimulated Phosphorylation of Rhodopsin in the Retina: The Presence of a Protein Kinase That Is Specific for Photobleached Rhodopsin (protein phosphorylation/cAMP/cGMP) MALCOLM WELLER, NOELLE VIRMAUX, AND P. MANDEL Centre de Neurochimie, 11 Rue Humann, 67085 Strasbourg Cedex, France Communicated by Seymour S. Kety, September 30, 1974 ABSTRACT A protein kinase has been extiacted from Preparation of Tris Extracts from ROS (unless otherwise bovine rod outer segments by a mild procedure. The en- stated all extractions were performed at 40 in dim red light). zyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of ROS were homogenized by hand at a concentration of about histones, phosvitin, or casein. We propose the name 0.5 mg of protein per ml in 0.01 M Tris*HCl (pH 7.0) and "opsin kinase" for the enzyme, which is not affected by centrifuged at 100,000 X g for 60 min. cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, Preparation of "Purified Rhodopsin" by Differential Extrac- appear to contain only low concentration of opsin phos- tion of ROS with Sodium Dodecyl Sulfate. The pellet remaining phatase activity. after extraction with Tris - HCl was extracted twice with 0.1%o It has recently been shown in several laboratories that when sodium dodecyl sulfate in 10 mM Tris*HCl (pH 7.0). The rod outer segments (ROS) prepared from frog (1, 2) or ox insoluble material was washed three times with 0.066 M Na (3-5) retinas are incubated with ATP and Mg2+ rhodopsin is phosphate buffer, pH 7.0. This procedure removes not only phosphorylated and the reaction is markedly stimulated by nonrhodopsin proteins but also bleached rhodopsin. On the light. basis of polyacrylamide gel electrophoresis opsin appears to be There seem to be three ways in which light could act. First, the only protein in the preparation (14). by directly stimulating the activity of the kinase, second, by Protein Determination was by the method of Lowry et al. altering the conformation of the rhodopsin so that it becomes a (15) using bovine plasma albumin as standard. substrate for the kinase, and third, by altering the concentra- tion of some cofactor which changes the activity of the kinase. Radioactive A TP 7y-labeled [32P]ATP was prepared (16) and In support of the third possibility, it has been observed that purified (17) by previously described methods. the rate of production of 3': 5'-cAMP and cGMP in ROS is Measurement of Protein Phosphorylation. All incubations lowered on exposure to light (6-10) and, since rates of protein were carried out in the presence of 50 mM Tris * HCl pH 7.4, 1 phosphorylation are, in so many cases, controlled by cyclic mM [a2PIATP and 1 mM MgCl2 (unless otherwise stated) at nucleotides, it was at first thought that the effect of light on 370 in a volume of 0.5 ml with 100-200 ltg of protein. Reactions cyclic nucleotide concentration could be correlated with the were terminated by the addition of 2 ml of ice-cold 10% effect on protein phosphorylation (3, 4). A decrease in cyclic trichloroacetic acid (or 20% to precipitate histones) and 0.1 nucleotide concentration could increase the net rate of protein mg (0.1 ml) of bovine serum albumin added as protein carrier. phosphorylation if the cyclic nucleotide inhibited a protein The protein precipitate was washed twice with 10% trichloro- kinase or, alternatively, stimulated a protein phosphatase. acetic acid, M orthophosphoric acid, then resuspended in 0.5 Cyclic-AMP-inhibited protein kinases have been discovered ml of 0.1 N NaOH and incubated for 10 min at 37°, after which in the slime mold (11) and cAMP-stimulated protein phos- 2 ml of 10 or 20% trichloroacetic acid was added and the pro- phatases in the toad bladder cell membrane (12). tein was spun down. The protein precipitates were washed once In this paper, we show that neither cAMP nor cGMP have in 1 ml of ethanol-ether (1/1) and dissolved in 2 ml of 0.1 N any effect on the phosphorylation of ROS protein. The protein NaOH at 1000 and the Cerenkov radiation was measured (18). kinase in ROS is specific to photobleached, not unbleached rhodopsin, and the increase in substrate concentration which Measurement of Protein Dephosphorylation. Samples of ROS results from exposure of ROS to light is alone sufficient to were incubated with 1 mM MgCl2, 1 mM [a2P]ATP and 50 account for the increase in protein phosphorylation. mM Tris HCl (pH 7.4) at a protein concentration of about 0.4 mg/ml for 30 min at 37°. The suspension was then centri- MATERIALS AND METHODS fuged at 100,000 X g for 10 min and the pellet was washed Preparation of Rod Outer Segments from Bovine Retinas was twice by resuspension and centrifugation from 50 mM sodium as previously described (13). The segments routinely showed a phosphate buffer, pH 7.0, before final resuspension at a con- ratio of absorbance at 280 to 500 nm of about 2.2. Incubation centration of 1-2 mg of protein per ml in the same buffer. with 1 1-cis-retinaldehyde only increased the absorption at 500 Aliquots of the [82P]ROS were then incubated with 50 mM nm by 5-10%, indicating that the material was only 5-10% Tris-HCl (pH 7.4) at a protein concentration of about 0.4 bleached. mg/ml with the additions stated in the text. Aliquots (0.5 ml) were then taken at different times and mixed with 2 ml of ice- cold 10% trichloroacetic acid, the denatured protein being Abbreviation: ROS, rod outer segments. washed and its radioactivity measured as described above. 381 Downloaded by guest on September 28, 2021 382 Biochemistry: WeHer et al. Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 1. The effect of phosphodiesterase inhibitors and cyclic nucleotides on the o a0% bleach intrinsic protein kinase activity of ROS 0~ Va- -0 0 I 0 rC12 0 Phosphodiesterase Cyclic % of control L I L inhibitor nucleotide L 4 /%50/ bleach (no additions) 0 White light E SQ 20009 - 61 i 2.4 (6) m It c0 SQ20009 1 mM cAMP (I mM) 58.5 ±4(6) o A~~~~24%blech mI SQ 20009 cGMP (0. 1 mM) 55.5 i 3.8 (4) t A _ ----4 _ I ._.__. C Theophylline - 61. 5 ± 7.1 (6) X A Dark ° 10 30 50 70 90 110 10 mM cAMP 6 12 18 24 30 c Relative percentage oF Theophylline (1 mM) 61.8 ± 6 (4) Time (min) unbleached rhodopsin Theophylline cGMP (0.1 mM) 60, 70 FIG. 1. The effect of bleaching on the posphorylation of Papaverine - 89 ± 8.2 (7) rhodopsin. (a) Samples of purified rhodopsin were suspended at Papaverine 2 mM cAMP (1 mM) 79 i 12 (4) about 1 mg of protein per ml in 50 mM sodium phosphate buffer Papaverine cGMP (0. 1 mM) 85, 91 (pH 7.0) and bleached to various extents by exposure to white cAMP (I mM) 94.8 ±i 8 (6) light. Aliquots were taken to determine the extent of bleaching by - cGMP (0. I mM) 93 i 4.6 (3) measuring the decrease in absorbance at 500 nm and the remain- - Dibutyryl cAMP 77.2 i 11.6 (10) ing material was incubated under the phosphorylation conditions Dibutyryl cGMP 102. 5 i 4. 1 (4) described in the text. Broken lines show the time course of phos- Dim red light phorylation of intact ROS, A in dim red light; A or in white light. 20009 Similar results were obtained with three separate experiments. SQ 95 ±t 5 (4) (b) The maximum amount of phosphate which could be in- SQ 20009 1 mM cAMP (I mM) 91.2 i 4.7 (6) corporated into rhodopsin which had been bleached to various SQ 20009 cGMP (0. I mM) 102 extents was plotted against the amount of unbleached rhodopsin Theophylline 86. 7 ± 13.1 (6) in the sample, shown as a percentage of the amount in the Theophylline 10 mM cAMP (1 mM) 99. 3 i 1. 2 (3) material before exposure to light (relative percentage of un- Theophylline cGMP (0.1 mM) bleached rhodopsin). Results are shown as means standard Papaverine - 106.25 ±- 4.8 (4) deviations. Papaverine 2 mM cAMP (1 mM) 114.3 t 27 (4) Papaverine cGMP (0.1 -mM) RESULTS - cAMP (I mM) 104 ± 9 (5) 1 In agreement with the results of other workers (1-5), we found cGMP (0. mM) 97.7 ± 7.9 (4) Dibutyryl cAMP 98.9 ± 11.6 (10) that isolated ROS-on incubation with a [32P]ATP show light- Dibutyryl cGMP 99.3 ± stimulated incorporation of 32p (Fig. 1). The effect of light is 15.5 (4) much more pronounced than that observed by Frank et al. (4) who, after Protein phosphorylation was carried out as described in the 2-min incubation, only obtained a stimulation of text for 2 min either in white or dim red light. Results are shown as about 1.3 times while we obtained a stimulation of 10-20 means ± standard deviations with the number of observations times, in agreement with other workers (1, 3, 5). shown in parentheses. The incorporated radioactivity is bound to protein and not lipid, since it could neither be extracted into chloroform- was slightly inhibitory in the light (Table 1). The effect of methanol (2/1) nor into chloroform-methanol-concentrated phosphodiesterase inhibitors could be due to an increased HCl (300/200/1).
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