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Originally published In Press as doi:10.1074/jbc.M701974200 on June 14, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23613-23621, August 10, 2007
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Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination*

Hongman Song, Marycharmain Belcastro, E. J. Young, and Maxim Sokolov1

From the Departments of Ophthalmology and Biochemistry, West Virginia University School of Medicine and West Virginia University Eye Institute, Morgantown, West Virginia 26506

Received for publication, March 7, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gbeta{gamma} subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosducin (Pdc)2 was originally identified in the retina as an abundant 33-kDa cytosolic phosphoprotein phosphorylated in the dark and dephosphorylated in the light (1). Within the retina Pdc is expressed in both rod and cone photoreceptors (2, 3). The most distinguished feature of Pdc is its ability to form a specific complex with the beta{gamma} subunits of visual heterotrimeric G protein, transducin (4, 5), and other heterotrimeric G proteins (6-8). Affinity of Pdc toward Gbeta{gamma} is down-regulated by multiple phosphorylation and probably consequent binding of 14-3-3 protein (9-10). Although the identity of Pdc kinase and phosphatase in photoreceptors remains unknown, the analysis of Pdc phosphorylation in vitro and ex vivo revealed that Pdc possesses multiple cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II phosphorylation sites (10-12) and identified protein phosphatase 2A (PP2A) as the putative Pdc phosphatase (13).

Despite the obvious progress in understanding the molecular basis of Pdc/Gbeta{gamma} interactions, the role of Pdc and its light-dependent phosphorylation in photoreceptors is poorly understood. Originally it was proposed that, upon activation by light, Pdc scavenges transducin beta{gamma} subunits from phototransduction and by doing so reduces photoreceptor light sensitivity (14-16). This hypothesis, however, is yet to be substantiated by physiological data. The first clues into in vivo Pdc functions were obtained from the analysis of Pdc-null mice (17). The analysis of long term light adaptation in this mutant revealed that Pdc significantly increases light-driven transducin translocation, another adaptive mechanism regulating the amount of transducin engaged in phototransduction (for a recent review, see Ref. 18). It was also noticed that the deletion of Pdc gene reduced the level of transducin beta subunit by 30%.

To assess the individual contributions of two known light-regulated phosphorylation sites of Pdc, serine 54 and serine 71 (11, 19), to the regulation of transducin translocation, we quantitatively analyzed their status and subcellular localization in free-running mice under physiologically relevant levels of illumination. We found that Pdc phosphorylated at serine 54 is present predominantly in the ellipsoid and rod outer segment, whereas phosphorylation of serine 71 occurs throughout the entire rod. We also found that rapid and reversible dephosphorylation of both sites was triggered by a very dim light below the threshold of transducin translocation. Prolong exposure to moderate ambient light initiated light-driven transducin translocation and also caused massive phosphorylation of serine 71 in the rod outer segments. Our results provide evidence for Pdc playing potential roles in both light adaptation and light protection of rod photoreceptors and point to the distinct functions of phosphorylation sites serine 54 and 71.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The phospho-specific antibodies against Pdc were generated by 21st Century Biochemicals (Marlboro, MA) as follows. Peptides corresponding to amino acids 50-59 and 67-77 of mouse Pdc containing phosphoserine at positions 54 and 71 (peptide sequences LRQMpSSPQSR (pS, Ser(P)-54) and SRKMpSIQEYEL (Ser(P)-71)) were synthesized and used to immunize rabbits according to the standard procedure. Phospho-specific antibodies against Pdc, designated as Pdc54p and Pdc71p, were affinity-purified from the immune serum using immobilized specific peptides. To eliminate binding to dephosphorylated Pdc, each antibody was depleted against LRQMSSPQSR and SRKMSIQEYEL peptides. Antibody against full-length Pdc was described previously (17). Antibody against rod transducin {alpha} subunit was sc-389 from Santa Cruz Biotechnology. Antibody against subunit I of cytochrome c oxidase, was MS404 from MitoSciences. Monoclonal 4D2 antibody against rhodopsin was a gift from Dr. Robert S. Molday (University of British Columbia).

Light Conditioning of Animals—All experiments involving animals were performed according to the procedures approved by the West Virginia University Animal Care and Use Committee. Wild type pigmented Long Evans rats and 129SV mice were purchased from Charles River Laboratories. Pdc knock-out mice described previously (17) were back-crossed into 129SV background for three generations. Animals were maintained in standard cage rooms with cyclic light (12 h light/12 h dark) until used. Before all experiments animals were dark-adapted overnight. Light conditioning of mice was performed in a 23 x 15 x 20-cm white box illuminated from a white diffuser embedded into the lid and connected to the light guide of an adjustable light source ACE I (Schott). Light conditioning of rats was carried out in 40 x 50 x 30-cm white box evenly illuminated from the top by the bench light source. The levels of illumination in the box were measured using a Traceable light meter (Fisher) (units, photopic lux) and a calibrated photodiode attached to a PDA-750 amplifier (Terahertz Technologies) (units, microamperes, µA). The light-collecting surface of the photodiode was covered by a blue glass filter (BG 39, Newport Franklin Inc., Franklin, MA) with a spectral sensitivity closely matching that of rhodopsin. The luminance of the walls was calculated assuming the white box to be a cube according to the formula 1 cd/m2 = 2{pi}/3 lux. At the end of the experiment animals were euthanized by flashing the box with CO2 followed by cervical dislocation, and their eyes were harvested and either frozen on the dry ice or dissected to obtain the retinas.

The rate of rhodopsin activation in the photoreceptors during light conditioning in the box was determined experimentally. Dark-adapted 129SV mice were anesthetized to reduce rhodopsin regeneration (20), and their corneas were protected by applying the methylcellulose ophthalmic lubricant, Murocel (Bausch and Lomb). Mice were exposed to 103 lux (240 scotopic cd/m2) light in the center of the box for various durations of time. After the exposure, mice were sacrificed, and their eyes were harvested and frozen on dry ice. The rhodopsin content of the eyes was determined as described below. We found that during the first 15 min of exposure the bleaching of rhodopsin could be approximated as a linear process with a rate of 0.0072% s-1. Assuming that the rod cell has 7 x 107 molecules of rhodopsin (21), this rate corresponds to activation of 5 x 104 rhodopsin rod-1 s-1. The rates of rhodopsin activation under all other levels of illumination were calculated from this value.

Determination of the Levels of Rhodopsin—A protocol modified from Sokolov et al. (22) was used as follows. All procedures were carried out under dim red light. Two retinas were harvested from a dark-adapted mouse and homogenized in 0.6 ml of water containing 2.5% n-octyl beta-D-glucopyranoside (O3757, Sigma) and 1.25% cetyltrimethylammonium chloride (292737, Sigma) by short ultrasonic pulses delivered from a Microson ultrasonic cell disruptor equipped with a 3-mm probe (Misonix, Farmingdale, NY). When required, 5 µM microcystine LR (Sigma) and 50 mM sodium EDTA were added to inhibit protein phosphatase and kinase activities in the extracts. The extract was cleared by centrifugation on a Centrifuge 5415 D (Eppendorf) at 16,000 rpm for 3 min. The supernatant was divided into two equal aliquots; one was kept in the dark, and the second one was exposed to bright light for 1 min to bleach all rhodopsin. The absorption 400-700 nm spectrum of the dark aliquot was then obtained in a UV-Mini 1240 spectrophotometer (Shimadzu) using the bleached aliquot as a base line. Rhodopsin concentration was determined from a 500-nm peak using a molar extinction coefficient of 40,500 (23). When required, the same protocol was used to determine rhodopsin content of the whole eye.

Determination of the Levels of Pdc—The amounts of Pdc in the retinas were determined by quantitative Western blotting with a standard curve according to the protocol modified from Sokolov et al. (17) as follows. The retinas were harvested from dark-adapted 129SV mice, and the whole retina extracts containing known amounts of rhodopsin were prepared as described above. The extracts were mixed with SDS-PAGE sample buffer and analyzed by Western blotting together with the standard curve comprised of various amounts of purified recombinant Pdc added to the Pdc-null retinal extracts containing the same amount of rhodopsin as the analyzed samples. Phospho-Pdc standards were generated by in vitro phosphorylation with an excess of either Ca2+/calmodulin-dependent protein kinase II or cAMP-dependent protein kinase, which was previously reported to cause virtually complete phosphorylation of serine 54 and serine 71, respectively (19). Serine 54-phosphorylated Pdc standard was obtained by incubating 100pmol of recombinant Pdc with 500 units of Ca2+/calmodulin-dependent protein kinase II (P6060S, New England Biolabs) using buffers, reagents, and protocols provided by the manufacturer. Serine 71-phosphorylated Pdc standard was obtained by incubating 400 pmol of Pdc with 100 units of bovine cAMP-dependent protein kinase catalytic subunit (Sigma) in phosphate-buffered saline buffer, pH 6.5, containing 2.5 mM ATP, 10 mM MgCl2, for 1 h at 30 °C. Blots were probed with the pan- and phospho-specific antibodies against Pdc, and the amounts of total- and phosphorylated Pdc in the mouse retinas were calculated from the standard curves and presented as molar ratios with rhodopsin.

Pdc Phosphorylation Assays—The eyes were harvested from dark-adapted and light-conditioned 129SV mice of the same age and frozen on dry ice. An eye was homogenized in 0.2 ml of buffer containing 125 mM Tris/HCl, pH 6.8, 4% SDS, 6 M urea, and 10 mg/ml dithiothreitol by short ultrasonic pulses resulting in complete disintegration of the eye tissue. The extract was cleared by centrifugation. 15-µl aliquots were separated on 18-well 10% Tris-HCl gels (Bio-Rad), transferred to polyvinylidene difluoride membrane Immobilon FL (Millipore), and probed with phospho-specific Pdc54p and Pdc71p antibodies. Total Pdc was determined after diluting original extracts 100 times. Fluorescence values of phosphorylated Pdc bands were divided by those of total Pdc bands, and then the amounts of phosphorylated Pdc in the light-adapted samples were normalized to those in the dark-adapted samples on the same gel. The time course data were fit to a first order rate equation, ln[A] = -kt + ln[A0] (exponential rise to maximum, two parameters), where [A] and [A0] are the amounts of phosphorylated Pdc at times t and 0 min, respectively, and k is the first order rate constant using Sigma Plot software. Half-life value (t1/2) was calculated from the rate constant k by the equation t1/2 = ln(2)/k.

Immunoprecipitation—Two mouse retinas were homogenized into 0.4 ml of radioimmune precipitation assay buffer (R0278, Sigma) containing 10 mM sodium EDTA, 5 µM microcystine LR (Sigma), 5 µM okadaic acid (Sigma), and protease inhibitor mixture (#539131, Calbiochem) by several short ultrasonic pulses. Homogenates were cleared by centrifugation and incubated with 10 µl of protein G-Sepharose beads (Pierce) and 20 µg of Pdc71p antibody for 1 h at room temperature with gentle rocking. Beads were separated from supernatant by centrifugation and washed 3 times with 1.0 ml of radioimmune precipitation assay buffer, and then bound Pdc was eluted from the beads with 0.1 ml of 0.5% trifluoroacetic acid and vacuum-dried in a Vacufuge (Eppendorf). The supernatant was mixed with 9 volumes of 10% trichloroacetic acid, 50% acetone and incubated on ice for 30 min. Precipitates were collected by centrifugation, washed 3 times with 1.0 ml of cold acetone, and vacuum-dried. Both bound and unbound fractions were resuspended in 50 µl of SDS-PAGE sample buffer containing 6 M urea, 125 mM Tris-HCl, pH 6.8, 4% SDS, bromphenol blue tracking dye, and 10 mg/ml dithiothreitol for the subsequent Western blot analysis.

Serial Tangential Sections of the Retina—Tangential sectioning of the rat retina was carried out as previously described (17) with several optimizations. Eyes were dissected under a stereo microscope in HEPES-Ringer solution containing 130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 0.02 mM EDTA, 10 mM HEPES-NaOH, pH 7.4, osmolarity adjusted to 313 mosM. When necessary all tissue manipulations were conducted in the dark under a Stemi 2000-C stereomicroscope (Zeiss) equipped with OWL Gen 3+ intensifiers (B.E. Meyers and Co. Inc.) and an infrared light source. The anterior portion of the eye was cut away, and the lens was removed. The eye cup with the retina attached to it was cut into four pieces of the same size. Each piece was transferred to the flattening chamber filled with HEPES-Ringer, where the retina was gently pulled away from the eyecup and mounted photoreceptors up onto a supporting polyvinylidene difluoride membrane, which was positioned on top of a flat glass capillary array (GCA 09/32/25/0/20 LM, BURLE Electro-Optics, Sturbridge, MA). The retina was flattened by first applying a suction force from underneath the filter slowly removing all solution from the flattening chamber and then by clamping the retina on the supporting membrane between two glass slides separated by 0.5-mm spacers. The assembly was frozen on dry ice. To align the retina with the cutting plane of the cryostat blade, optimal cutting temperature compound was allowed to freeze at -20 °C on the specimen holder and then sectioned through to create a flat surface large enough to accommodate the glass slide. The clamps, the cover glass, and the spacers were removed; the base slide with the retina on the supporting membrane attached to it was gently pressed against the optimal cutting temperature compound surface and secured by the addition of water drops to the sides of the glass base. The retina and its supporting membrane were generously trimmed around the perimeter to remove uneven and folded parts and 5-µm-sectioned. When the first sizeable section was cut out, all other parts of the retina not included in this section were trimmed away, and then sectioning was resumed. Each section was collected and thawed in 50 µl of SDS-PAGE sample buffer containing 6 M urea, 125 mM Tris-HCl, pH 6.8, 4% SDS, bromphenol blue tracking dye, and 10 mg/ml dithiothreitol. To assess the quality of the sectioning, 0.5-µl aliquots were applied on dry nitrocellulose membrane, and the dot blots were probed with anti-rhodopsin antibody. Only the sets containing rhodopsin in the upper 4-6 sections were selected and stored at -80 °C until analyzed.

15-µl aliquots were analyzed by Western blotting using a Criterion Cell and Blotter system and 26-well 10% Tris-HCl gels (Bio Rad). Polyvinylidene difluoride membrane Immobilon FL (Millipore) was used. For the detection of rhodopsin the original extracts were diluted 100 times. For the detection of Pdc and transducin subunits, the original extracts were diluted 25 times.

The profiles of protein distribution in rods were obtained as previously described (22). In brief, fluorescence of a specific band in each section was plotted as a percentage of combined fluorescence in all sections. To determine the degree of Pdc phosphorylation in different rod compartments, the fluorescence value of the phosphorylated Pdc band in each section was divided by that of total Pdc. The experiments were repeated five times with dark-adapted animals and four times with light-adapted animals.

Western Blotting—Quantification of the specific bands was performed on an Odyssey Infrared Imaging System (LI-COR Biosciences) according to the manufacturer's protocols and using specific primary antibodies and anti-rabbit, anti-sheep, and anti-mouse secondary antibodies conjugated to either Alexa Fluor 680 (Invitrogen) or IRDye 800 (LI-COR Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Properties of Phospho-specific Antibodies Against Pdc—Phospho-specific antibodies against two known light-regulated phosphorylation sites of Pdc, serine 54 and 73 (11, 19), were generated and affinity-purified. The antibodies were designated as Pdc54p and Pdc71p according to the mouse Pdc sequence. The ability of the phospho-specific antibodies to discriminate phosphorylated and dephosphorylated Pdc is illustrated in Fig. 1. In this experiment, recombinant rat Pdc was phosphorylated in vitro with either Ca2+/calmodulin-dependent protein kinase II (Fig. 1, upper blot) or cAMP-dependent protein kinase (Fig. 1, lower blot) to introduce phosphate in position 54 or 71, respectively. This approach was previously validated using mass spectrometry (19). Non-phosphorylated Pdc was used as a control. As evident from the data in Fig. 1, each antibody recognizes only phosphorylated Pdc and was blocked by the phosphopeptide corresponding to its immunization antigen but not by the other phosphopeptide. Consistent with a previous report (19), each Ca2+/calmodulin-dependent protein kinase II or cAMP-dependent protein kinase in vitro phosphorylates Pdc at multiple sites, resulting in the appearance of several bands on an SDS-PAGE. We utilized phospho-specific Pdc54p and Pdc71p antibodies to detect site-specific Pdc phosphorylation by Western blotting.


Figure 1
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  		FIGURE 1.
Characterization of phospho-specific antibodies against Pdc. Aliquots containing 2.0 pmol of Ca2+/calmodulin-dependent protein kinase II-phosphorylated and non-phosphorylated purified recombinant Pdc (upper blot) and 0.75 pmol of cAMP-dependent protein kinase-phosphorylated and non-phosphorylated Pdc (lower blot) were analyzed by Western blotting using Pdc54p and Pdc71p antibodies. When stated, 1 mM concentrations of the corresponding phosphopeptide described under "Experimental Procedures" was present during incubation of the blots with primary antibodies.

 
Phosphorylation of Pdc in the Dark-adapted Rods—It is well documented that in the retinas of dark-adapted animals Pdc undergoes phosphorylation; however, the degree of Pdc phosphorylation is unknown. To obtain this information, which is crucial for the understanding of Pdc function, we determined the amounts of serine 54- and serine 71-phosphorylated Pdc in the retinas of dark-adapted 129SV mice using three different experimental approaches.


Figure 2
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  		FIGURE 2.
Quantification of the levels of serine 54- and serine 71-phosphorylated Pdc in the dark adapted retina. Retinas were harvested from dark-adapted mice under a dim red light. A, SDS extracts of the whole retina (open circles) containing the indicated amounts of rhodopsin (Rho) were analyzed by Western blotting together with a calibration curve comprised of various known amounts of purified recombinant Pdc premixed with the retinal extracts of Pdc knock-out mice containing the same amount of rhodopsin (closed circles). When indicated, Pdc standards were phosphorylated at serine 54 and 71, with Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP-dependent protein kinase (PKA), respectively, as described under "Experimental Procedures." Blots were probed with Pdc, Pdc54p, and Pdc71p antibodies. The amounts of Pdc in the retinal extracts were calculated from the calibration curve, which was obtained by plotting the fluorescence values of the specific bands (1-5) against the amounts of Pdc in that standard. The amounts of total-, serine 54-, and serine 71-phosphorylated Pdc were determined to be 26 ± 0.6 (100%), 1 ± 0.03 (4%), and 9 ± 3 (35%) molecules per 100 molecules of rhodopsin, respectively (S.E., n = 6). B, each retina was extracted in radioimmune precipitation assay buffer, and then serine 71-phosphorylated Pdc was immunoprecipitated with an excess of immobilized Pdc71p antibody. The amounts of Pdc in the unbound and bound fractions were analyzed by Western blotting using Pdc71p (upper blot) and Pdc antibody (lower blot). Comparison of fluorescence values of the Pdc bands in the unbound and bound fractions revealed that 44 ± 4% (S.E., n = 5) of total Pdc in the retina was phosphorylated at serine 71 under these conditions. C, retinas were incubated in HEPES-Ringer solution containing 10 mM glucose, with (+) or without (-) PP inhibitors, including 10 µM microcystine LR and 10 µM okadaic acid for 20 min in the dark. The amounts of serine 54- and serine 71-phosphorylated Pdc was determined by Western blotting. Comparison of fluorescence values of the phospho-Pdc bands in the PP (+) and (-) retinas, shown as a bar graph, indicated that 11 ± 4 and 37 ± 10% (S.E., n = 3) of total Pdc is phosphorylated at serine 54 and 71, respectively.

 
In the first approach the amounts of phosphorylated Pdc in the retina were determined by quantitative Western blotting with a calibration curve (Fig. 2A). The retinas were harvested under dim red light, and then the SDS extracts of the whole retina containing known amounts of rhodopsin were prepared as described under "Experimental Procedures." To quantify the total amount of Pdc, three different extracts were analyzed together with the calibration curve comprised of various known amounts of recombinant Pdc (Fig. 2A, left panel). To create an identical environment in the extracts and calibration curve, which may potentially affect protein transfer from gel to membrane and exposure to antibody, Pdc standards were premixed with the whole retinal extract of Pdc knock-out mice. We found that on average dark-adapted mouse retina contains 26 ± 0.6 molecules of Pdc per 100 molecules of rhodopsin. This number represents the protein level of Pdc in rods because Pdc expression in the retina is limited to photoreceptors, and cones comprise only 3% of the total photoreceptor pool in the mouse retina (24). Next we determined the amounts of Pdc phosphorylated at serine 54 and 71. In these assays Pdc standards were phosphorylated in vitro by either Ca2+/calmodulin-dependent protein kinase II at serine 54 or cAMP-dependent protein kinase at serine 71, and specific bands were visualized with phospho-specific Pdc54p and Pdc71p antibodies. On average 1 ± 0.03 molecules of serine 54-phosphorylated Pdc and 9 ± 3 molecules of serine 71-phosphorylated Pdc per 100 molecules of rhodopsin were detected in the extracts, respectively. Thus, 4 ± 0.1 and 35 ± 12% of total Pdc in dark-adapted rods appeared to be phosphorylated at serine 54 and serine 71, respectively.


Figure 3
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  		FIGURE 3.
Light dependence of Pdc phosphorylation. Dark-adapted mice were exposed to the designated levels of constant illumination for 10 min and sacrificed, and their eyes were harvested and frozen. Each eye was homogenized in SDS-containing buffer. A, 15-µl aliquots of the whole eye extracts were analyzed alongside each other by Western blotting with Pdc54p and Pdc71p antibodies. For the detection of total Pdc, original extracts were diluted 100 times. B, fluorescence values of Pdc54p (black) and Pdc71p (white) bands were divided by fluorescence of corresponding total Pdc band, and then the amount of phosphorylated Pdc in the light was normalized to the dark-adapted value, designated as 0 lux (S.E., n = 6). Note that the cluster of Pdc71p bands of reduced electrophoretic mobility that appears in the 104 lux light-conditioned samples was excluded from the analysis, and only the 33-kDa band (*) was analyzed.

 
The second approach was to immunoprecipitate all phosphorylated Pdc in the retinal extracts with an excess of immobilized phospho-specific antibody and then to determine the degree of Pdc phosphorylation by comparing the amounts of Pdc in the bound, phosphorylated, and unbound, dephosphorylated fractions. As illustrated in Fig. 2B, after immunoprecipitating almost all serine 71-phosphorylated Pdc in the extract with Pdc71p antibody (upper blot), we found that it composed only 44 ± 4% of the total amount of Pdc in the retina (lower blot). Unfortunately, we could not estimate the amounts of serine 54-phosphorylated Pdc by this approach due to the poor performance of Pdc54p antibodies in the immunoprecipitation assay.

The third approach was to determine whether or not additional phosphorylation of Pdc in the dark could be achieved by suppressing the activity of protein phosphatase pharmacologically. We reasoned that this would allow endogenous protein kinase to phosphorylate all available Pdc in the retina, thus providing the estimation of the highest achievable level of Pdc phosphorylation in rods. As shown in Fig. 2C, incubation of freshly obtained dark-adapted mouse retinas with phosphatase inhibitors microcystine LR and okadaic acid resulted in the robust increase of the levels of phosphorylated Pdc, providing yet another estimation that on average only 11 ± 4 and 37 ± 10% of total Pdc are phosphorylated at serine 54 and 71, respectively.

Combined these data demonstrate that even during prolonged overnight dark adaptation, only a small 4-10% fraction of total Pdc in the rods of living animals becomes phosphorylated at serine 54, whereas approximately 40% of total Pdc becomes serine 71-phosphorylated.

Light Dependence of Pdc Phosphorylation—To get a better understanding of the role of Pdc phosphorylation in rod visual function, we monitored the phosphorylation status of serine 54 and serine 71 under various levels of illumination. Seeking physiologically relevant data, we exposed non-anesthetized pigmented mice to the controlled steady illumination in a white box. The levels of rhodopsin activation in the retinas under different levels of illumination in the box were determined experimentally, as described under "Experimental Procedures." After 10 min of light conditioning, mice were sacrificed, and their eyes were promptly frozen on dry ice. The whole procedure of enucleation and freezing was completed within a minute and was easy to reproduce. The levels of serine 54 and 71 phosphorylation were determined in the whole eye extracts by Western blotting after homogenizing the entire eye in SDS-containing buffer (Fig. 3). We found that exposure of dark-adapted mice to a very dim 0.08 lux background light resulted in a reproducible increase in the level of phosphorylation of each site, and was insufficient to trigger dephosphorylation. Increasing the illuminance level to a modest 0.8 lux triggered dephosphorylation of serine 54 and serine 71 by more than 80%. Further increasing background illumination to 8, 103, and 104 lux illuminance essentially eliminated phosphorylation of serine 54. In contrast, a dramatic increase in the level of serine 71 phosphorylation was observed during exposure to 104 lux light. Different from the dark, phosphorylation of serine 71 in the light was accompanied by a shift of Pdc electrophoretic mobility and the appearance of slower running bands, similar to those observed in in vitro phosphorylated Pdc (Fig. 1), suggesting the presence of additional phosphorylation sites. These results demonstrate that phosphorylation sites serine 54 and serine 71 of Pdc are regulated differently by light.

Time Course of Pdc Phosphorylation—Using a similar experimental approach we determined the time course of Pdc phosphorylation. A group of dark-adapted mice was exposed to 8 lux constant light, which saturates Pdc dephosphorylation (Fig. 3), and then placed in the dark again. Mice were sacrificed at different time points in the experiment, and the levels of serine 54 and 71 phosphorylation were determined in the whole eye extracts by Western blotting (Fig. 4A) and compared with the dark-adapted levels (Fig. 4B). We found that the levels of serine 54 and serine 71 phosphorylation were reduced by ~50% after 1 min of light exposure (Fig. 4, compare time points -10 min and -9 min). After 10 min of the exposure, dephosphorylation of ~97% of serine 54 and ~90% of serine 71 was completed (Fig. 4, 0 min). The process was rapidly reversed in the dark and appeared to be biphasic. When approximated as a first order reaction, the initial phase of serine 54 phosphorylation had a rate constant of 0.44 ± 0.04 s-1 and a t1/2 value of ~57 s. Respective parameters of serine 71 phosphorylation were found to be 0.72 ± 0.13 s-1 and ~94 s, respectively. The complete recovery of the dark levels of serine 54 and 71 phosphorylation on average occurred after 20 min of dark adaptation. Thus, light-dependent Pdc phosphorylation appeared to be a moderately fast reversible process operating in the minutes time scale.


Figure 4
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  		FIGURE 4.
Time course of Pdc phosphorylation. Dark-adapted mice were exposed to 8 lux light from -10 min to 0 min and then placed in the dark again. Mice were sacrificed at different time points of the experiments, and their eyes were harvested and frozen. Each eye was homogenized in SDS-containing buffer. A, Western blots showing the levels of Pdc phosphorylated at serine 54 (Pdc54p), serine 71 (Pdc71p), and total Pdc in the extracts at different time points of the experiment. B, fluorescence values of Pdc54p (closed circles) and Pdc71p (open circles) bands were divided by fluorescence of corresponding total Pdc band, and then the amounts of phosphorylated Pdc in each sample were normalized to the dark-adapted value, designated as -10 min (S.E., n = 6). The initial rate of Pdc dephosphorylation is shown as a straight dotted line. The lines represent fits of time points 0, 1, 3, 5, and 10 min to the pseudo-first order rate equation for phosphorylation.

 
Compartmentalization of Phosphorylated Pdc in Rod Photoreceptors—In our previous studies we successfully utilized an experimental approach combining serial tangential sectioning of the flat mounted frozen retina with the analysis of specific protein content in individual sections (22). This technique takes advantage of the aligned, layered structure of the retina, with the major cellular compartments of the rod present in parallel layers (Fig. 5A). Combined with Western blot detection, this technique allowed us to describe protein localization within rod photoreceptors quantitatively. We have applied this technique for determining compartmentalization of Pdc phosphorylated at serine 54 or serine 71 under different conditions of illumination. Retinas were obtained from either dark-adapted rats or rats that were allowed to run in moderately bright ambient 103 lux light for 1 h. The retinas were flat-mounted, frozen, and serially sectioned. Each set of serial sections was then analyzed by Western blotting (Fig. 5, B and D). The rod outer segments were identified using antibodies against rhodopsin confined in this cellular compartment. A mitochondrial marker subunit I of cytochrome c oxidase was used to highlight the ellipsoid, a subcellular compartment of the inner segment, densely packed with mitochondria and adjacent to the outer segment. Total Pdc and phosphorylated Pdc were determined using pan- and phospho-specific antibodies and expressed as a ratio of phosphorylated Pdc and total Pdc in each section (Fig. 5, C and E). We found that site-specific phosphorylation of Pdc in rod photoreceptors is strongly compartmentalized. As shown in Fig. 5B, in the retinas of dark-adapted animals, Pdc phosphorylated at serine 54 was specifically enriched in the ellipsoid of the inner segment, and from there it appeared to spread into the outer segment, but it was virtually undetectable in the sections corresponding to rod nuclei and synaptic termini. Under these conditions Pdc phosphorylated at serine 71 was readily detectable in all cellular compartments of rods, with some enrichment also observed in the inner segments. When rat was exposed to a moderate 103 lux light for 1 h (Fig. 5D), phosphorylation of residue 54 was efficiently eliminated in all cellular compartments, whereas significant amounts of Pdc phosphorylated at serine 71, apparently exceeding those in the dark adapted retinas, appeared in the rod outer segments. The light-induced phosphorylation of serine 71 in the outer segment was accompanied by the appearance of multiple bands, indicating that a 1-h exposure to 103 lux light has the same effect on Pdc phosphorylation as a shorter 10-min exposure to 104 lux light (Fig. 3), which was further confirmed experimentally (data not shown). The gradient distribution of serine 71-phosphorylated Pdc within rod outer segment, as in Fig. 5D, was not common for all preparations and, therefore, not discussed.

To determine whether compartment-specific phosphorylation of Pdc coincides with its redistribution within rods, we compared the fractions of Pdc present in the outer segment compartments in dark and light. A similar analysis was previously utilized to study light-driven transducin translocation in rods (22). In brief, the specific fluorescence in each section was expressed as a percent fraction of the combined fluorescence in all sections. Then the fraction of the Pdc confined in sections containing rhodopsin and free from subunit I of cytochrome c oxidase was calculated and normalized to the fraction of rhodopsin present in these sections. For example, in the experiment shown in Fig. 5B, sections 1-5 contain 81% of rhodopsin, 35% of Pdc and are essentially free from subunit I of cytochrome c oxidase. Thus, the fraction of Pdc in the outer segment, calculated as 35/85 x 100, is equal to 43%. Our analysis of five dark-adapted and four light-conditioned rats revealed that similar fractions of Pdc, 44 ± 9 and 39 ± 8% (S.E., t test p > 0.7), respectively, are present in the outer segments of rods under these conditions, which suggests that, contrary to the early report (25) and in good agreement with the most recent reports (17, 26), no significant translocation of Pdc from the outer segment has occurred as a result of prolonged light-conditioning.

Using the same analysis, we monitored light-driven translocation of rod transducin. During prolonged dark adaptation transducin is accumulated in the membranes of the rod outer segment. For example, in the experiment shown in Fig. 5B, 83% of the total transducin {alpha} subunit was found to be present in the rod outer segment. Exposure of free-running rats to a moderate ambient 103 lux light for 1 h resulted in reproducible transducin translocation from the rod outer segment, and on average only 53 ± 1% (S.E., n = 4) of transducin {alpha} subunit remained in this cellular compartment. These data indicate that phosphorylation of Pdc at serine 71 in the outer segments coincides with translocation of transducin from this cellular compartment; thus, dephosphorylation of serine 71 is unlikely to be a mechanism triggering transducin translocation.


Figure 5
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  		FIGURE 5.
Analysis of the distribution of phosphorylated Pdc in the retina by Western blotting of serial sections. Retinas were obtained from either dark-adapted rat (B) or rat exposed to 1000 lux light for 1 h (D), flat-mounted, frozen, and serially 5-µm-sectioned (1-21). Each section was dissolved in SDS sample buffer and analyzed by Western blotting with antibodies against proteins indicated on the right of each blot. A, cross-section of rat retina stained with toluidine blue and a schematic drawing of a rod photoreceptor, shown to juxtapose rod morphology and the appearance of protein bands in serial sections. The following abbreviations are used to denote rod subcellular compartments: OS, outer segment; IS, inner segment; N, nucleus; S, synapse. B and D, blots showing distribution of the outer segment marker rhodopsin (Rho), inner segment marker (COX I), total phosducin (Pdc), phosducin phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p), and rod transducin {alpha} subunit (G{alpha}t) in serial tangential sections of the retina. C and E, the fluorescence values of each phosphorylated Pdc band was divided by the fluorescence value of the corresponding total Pdc band, and the resulting normalized Pdc54p (closed circles) and Pdc71p (open circles) amounts were plotted against the section number to show the relative abundance of phosphorylated Pdc in different cellular compartments. Data from blots B and D are shown on C and E, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To complement previous studies conducted in isolated retina preparations (1, 11, 19) and to obtain clues crucial to our understanding of the role of Pdc in rod dark and light adaptation, we have quantitatively analyzed phosphorylation of two known light-regulated phosphorylation sites of Pdc, serine 54 and serine 71, in free-running animals exposed to physiologically relevant levels of illumination. Our data demonstrate that in rods phosphorylation of Pdc at serines 54 and 71 has distinct spatial and temporal distribution, which is governed by the intensity of background illumination as follows.

Phosphorylation of Pdc in the Dark—Pdc phosphorylated at serine 71 is distributed throughout the entire rod, whereas phosphorylation of serine 54 appears predominantly in the ellipsoid and the outer segment. The most important observation was that even during prolonged dark adaptation, the percent fraction of phosphorylated Pdc in any cellular compartment of rod is unlikely to exceed 40%, with the exception of the ellipsoid, where the degree of Pdc phosphorylation may be higher. This suggests that, contrary to the commonly accepted notion, significant amounts of dephosphorylated Pdc are available for interaction with the beta{gamma} subunits of transducin or other heterotrimeric G proteins in the dark-adapted rods. It also refutes the idea that light-evoked dephosphorylation of either serine 54 or 71 is setting up an activation threshold for rod transducin translocation, which most recently was demonstrated to be determined primarily by the capacity of the GTPase-activating complex (27, 28).

Dephosphorylation of Pdc in Operating Rods—Exposure of animals to dim and moderate light triggers massive and reversible dephosphorylation of Pdc, reducing the levels of serine 54 and 71 phosphorylation nearly 10-fold, and operating in the minutes time scale. The light sensitivity threshold of Pdc dephosphorylation was estimated to be near 0.8 lux (27 rhodopsin rod-1 s-1) of steady light, which is within rod operational range. Indeed, even 3-fold higher 2.5 lux background light was reported to be still comfortable for rod-meditated vision in living pigmented mice (21). The low light sensitivity threshold of Pdc dephosphorylation suggests its potential role in the regulation of rod visual signaling.

Hyperphosphorylation of Pdc in the Outer Segments of Saturated Rods—A surprising observation of this study was that phosphorylation of Pdc at serine 71 is significantly increased rather than decreased during prolong exposures to saturating light. In our experiments massive Pdc phosphorylation was evident when free-running animals were exposed to a moderate ambient 103 lux steady light estimated to activate 5·104 rhodopsin rod-1 s-1, which would completely saturate rod responses in electrophysiological experiments (29). Most remarkably, virtually all serine 71-phosphorylated Pdc was present in the rod outer segment, whereas serine 54 remained completely dephosphorylated. An obvious hallmark of light-induced Pdc phosphorylation, a multiple banding pattern on SDS-PAGE that is absent in dark-adapted rods, suggests the presence of additional phosphorylation sites or other covalent modifications on Pdc.


Figure 6
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  		FIGURE 6.
Phosphorylation cycle of Pdc under different conditions of illumination. Dark-adapted rod, comparable levels of dephosphorylated and phosphorylated Pdc are due to the equal rates of its phosphorylation by protein kinase (PK) and dephosphorylation by protein phosphatase. No appreciable phosphorylated rhodopsin (Rho) is present. Operating rod, activation of rhodopsin (Rho*) and phototransduction cascade by dim background light suppresses activity of protein kinase via negative Ca2+ feedback, resulting in Pdc dephosphorylation. The amount of phosphorylated rhodopsin is insignificant. Saturated rod, large amounts of phosphorylated rhodopsin exceeding those of phosphorylated Pdc competitively inhibit dephosphorylation of Pdc by protein phosphatase. As a result, protein kinase activity prevails, and Pdc becomes phosphorylated.

 
Model—A putative mechanism of Pdc phosphorylation under three different levels of background illumination, which is consistent with our data, is presented in Fig. 6. This model is based on the assumption that the same protein phosphatase catalyzes dephosphorylation of Pdc and rhodopsin (30, 13). In dark-adapted rods (Fig. 6, upper panel), the near 1:1 molar ratio of phosphorylated and dephosphorylated Pdc is due to the comparable rates of Pdc phosphorylation by protein kinase and dephosphorylation by protein phosphatase. Under these conditions phosphorylation of rhodopsin is negligible. In the operating rods (Fig. 6, middle panel), closure of cyclic nucleotide gated channels causes reduction in intracellular Ca2+ (31, 32). As a result, suppression of Ca2+-dependent Pdc kinase, protein kinase (10, 33), shifts the equilibrium toward Pdc dephosphorylation. Under these conditions, phosphorylated Pdc is a primary substrate for protein phosphatase because the amounts of phosphorylated rhodopsin in the cell are still modest. In the saturated rods (Fig. 6, lower panel), significant amounts of phosphorylated rhodopsin competitively inhibit the reaction of Pdc dephosphorylation by protein phosphatase, shifting the equilibrium toward Pdc phosphorylation. Translocation of protein phosphatase activity between cytoplasm and membrane originally described by Brown et al. (13) may occur under these conditions.

In summary, our studies provide the first quantitative account of compartment-specific phosphorylation of two principal light-regulated phosphorylation sites of Pdc, serine 54 and serine 71, in rod photoreceptors of living animals exposed to different levels of illumination. Our data support Pdc playing plural roles in a photoreceptor dark/light adaptation and in light protection. Analysis of Pdc phosphorylation mutants lacking serine 54 and serine 71 phosphorylation sites is required to get a better understanding of the physiological role of light-dependent Pdc phosphorylation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NCRR 2P20 RR15574-06 COBRE in Sensory Neuroscience, Subproject 5 (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: WV University Eye Institute, 1 Stadium Dr., Morgantown, WV 26506. Tel.: 304-598-6958; Fax: 304-598-6928; E-mail: sokolovm{at}rcbhsc.wvu.edu.

2 The abbreviations used are: Pdc, phosducin; Gbeta{gamma}, the beta{gamma} subunits complex of heterotrimeric G protein; Pdc54p and Pdc71p, phospho-specific antibodies recognizing phosducin phosphorylated at residue 54 and 71, respectively; PP2A, protein phosphatase 2A. Back


   ACKNOWLEDGMENTS
 
We thank Norman A. Michaud, Massachusetts Eye and Ear Infirmary, for providing microphotographs of rat retina cross-sections and Dr. Visvanathan Ramamurthy, West Virginia University Eye Institute, for critically reading this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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C. M. Krispel, M. Sokolov, Y.-M. Chen, H. Song, R. Herrmann, V. Y. Arshavsky, and M. E. Burns
Phosducin Regulates the Expression of Transducin {beta}{gamma} Subunits in Rod Photoreceptors and Does Not Contribute to Phototransduction Adaptation
J. Gen. Physiol., August 27, 2007; 130(3): 303 - 312.
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