|Molecular Vision 2002;
Received 3 September 2002 | Accepted 27 November 2002 | Published 19 December 2002
Presence of phosducin in the nuclei of bovine retinal cells
Alexander Margulis,1 Loan Dang,1 Sadhona
Pulukuri,1 Rehwa Lee,2 Ari
1Eye Research Institute, Oakland University, Rochester, MI; 2Department of Neurobiology, UCLA School of Medicine, Los Angeles, CA
Correspondence to: Ari Sitaramayya, Eye Research Institute, 423DHE, Oakland University, Rochester, MI, 48309-4481; email: email@example.com
Purpose: During the course of our investigations on cyclic nucleotide dependent-phosphorylation of membrane proteins, we observed that phosducin was present in both the cytosolic and nuclear fractions. It has been suggested that phosducin might have a role in regulating transcription, but its presence in the nucleus has not been previously reported. We therefore attempted to purify nuclei from bovine retina and determine whether the purified preparation contained phosducin. Cyclic nucleotide-dependent phosphorylation of the protein was also investigated in the homogenate and subcellular fractions of bovine retina.
Methods: Freshly obtained bovine retinas were homogenized and fractionated in an isotonic buffer. The homogenate and subcellular fractions were subjected to phosphorylation in the presence of γ-32P ATP and the presence and absence of cyclic GMP. The phosphorylated proteins were identified by 1- or 2-dimensional electrophoresis and autoradiography. Phosducin was detected in the fractions by western blotting. A nuclear preparation was obtained from the homogenate by sucrose gradient centrifugation, its purity was determined with a nuclear stain, and the presence and phosphorylation of phosducin was investigated as above.
Results: Phosducin was found in the cytosol, 100,000x g membrane fraction, as well as in the 120x g pellet of a fresh, isotonic retinal homogenate, but cyclic nucleotide-dependent phosphorylation of the protein was observed only in the cytosolic fraction. Western blotting on a highly purified nuclear fraction showed that phosducin was present in the nuclei. The protein could be phosphorylated in a cyclic GMP-dependent fashion in a nuclear preparation permeabilized by freezing and thawing.
Conclusions: It has been suggested that a C-terminal fragment of phosducin might be transported into the nucleus where it might have a role in the regulation of transcription. Phosducin localization in the nucleus was shown in COS cells cotransfected with genes of phosducin and a transcription factor. However, the presence of phosducin in the nuclei of retina has not been demonstrated hitherto. The present results show that phosducin is present in the nuclei in bovine retina. It could be phosphorylated in the nuclei in a cyclic GMP-dependent fashion. These results support a possible role for phosducin in the regulation of transcription.
Phosducin, a soluble protein in vertebrate retina, was originally identified as a 33 kDa phosphoprotein in retinal photoreceptor cells. It is rapidly dephosphorylated in response to light activation and a decrease in the intracellular concentrations of cyclic nucleotide or calcium [1-5]. The protein was found to bind and sequester βγ subunits of G proteins with implications for regulation of the availability of transducin for light activation via rhodopsin as well as other G protein-mediated signaling pathways [2,6-9]. Recently, phosducin was shown to interact with 14-3-3 protein. This property might limit the availability of phosducin to interact with a G-protein in the photoreceptor synapses that play a role in glutamate release [10,11].
In addition to such roles, phosducin degradation products and phosducin-like proteins have been suggested to interact with CRX, a transcription factor, and play a role in regulating its activity [12,13]. However, phosducin has never been shown to be present in the nucleus of photoreceptor or other retinal cells. In our effort to identify retinal proteins phosphorylated in response to cyclic GMP or cyclic AMP, we found that phosducin is not only present in retinal cytoplasm, but also in the nucleus. In this paper we describe evidence in favor of nuclear localization of this protein and discuss its implications for a role for phosducin as a transcription factor.
Preparation of retinal homogenate
Fresh bovine eyes were dark-adapted for 3.5 h on ice in a light-tight container. All subsequent operations were conducted under infrared light with the aid of an image converter. Retinas were extracted from the eyes, washed once and homogenized in a buffer containing 0.32 M sucrose, 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL trypsin inhibitor, and 50 μg/mL benzamidine, The suspension was filtered through a 200 μm nylon mesh, supplemented with 2 mM DTT, and immediately used in phosphorylation assays or stored at -70 °C in aliquots.
Purification of nuclear fraction
Purified nuclei were obtained according to previously published procedures with slight modifications [14,15]. Briefly, fresh retinal homogenate (before or after phosphorylation) was centrifuged at 120x g for 30 min at 4 °C, supernatant was saved for further subcellular fractionation, and the pellet was resuspended in homogenization buffer and centrifuged again at 120x g for 30 min. The washed pellet was suspended in 56% sucrose containing 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM EGTA and 1 mM PMSF, and centrifuged at 70,000x g for 1 h at 4 °C in a swinging-bucket rotor. The resulting pellet was resuspended in the homogenization buffer.
Preparation of retinal subcellular fractions
The 120x g supernatant of the homogenate obtained as above was centrifuged at 100,000x g for 1 h at 4 °C to obtain cytosolic (supernatant) and membrane (pellet) fractions. The membrane fraction was suspended in the homogenization buffer.
The assay mixtures (200 μL) contained about 400 μg of protein, 0.32 M sucrose, 100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 0.2 mM ATP, and 20 μCi of γ-32P ATP. When studying the effects of cyclic nucleotides, 0.1 mM 8-Br-cAMP or 8-Br-cGMP were included in the assay. The reactions were initiated with the addition of ATP. After 5 min at 30 °C, the reactions were terminated by the addition of 100 μL of a 3-fold concentrated electrophoresis sample buffer containing SDS.
Gel electrophoresis and western blotting
The samples were electrophoresed in either a 10 to 20% gradient polyacrylamide gel, 12.5% acrylamide mini gel, or on a 2-D gel. For the 2-D gel isoelectrofocusing was done on the IPGphor system (Amersham Biosciences) utilizing 13 cm Immobiline DryStrip gel, pH 3-10, and a 5-step protocol: 100 V for 2 h, 300 V for 1 h, 600 V for 0.5 h, 1000 V for 2 h, and 8000 V for 8 h. For the second dimension, electrophoresis was done on a 10-20% acrylamide gradient gel.
Following electrophoresis, proteins were transferred from the gel to a PVDF membrane either for 24 h at 100 mA (regular size gel), or for 2 h at 100 V (mini gel) in a transfer buffer containing 25 mM Tris, 192 mM Glycine, 10% Methanol, and 0.05% SDS. The membrane was blocked for 1 h in 5% non-fat dry milk in TTBS (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% Tween-20), washed with TTBS, and incubated for 1 h with a polyclonal anti-phosducin antibody diluted 1:50,000 or with a monoclonal anti-rhodopsin antibody diluted 1: 1000 in 1% BSA in TTBS. After washing with TTBS, membranes were incubated for 1 h with respective alkaline phosphatase-conjugated secondary antibodies followed by two TTBS washes. Blots were developed with Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad; Hercules, CA), dried and put on a Kodak BioMax film for autoradiography.
Light and fluorescent microscopy
The purified nuclear pellet was suspended in 2.5% glutaraldehyde in PBS, pH 7.4, incubated for 30 min, washed with PBS, spread on a glass slide and air dried. The dried sample was stained with Hoechst dye, cover slipped, and digitally photographed either under light or fluorescent microscope at 40x.
Bovine eyes were purchased from Wolverine Packing Company. (Detroit, MI). γ-32P ATP was from PerkinElmer (Boston, MA). Nylon mesh was from Spectrum Laboratory Products, Inc., (New Brunswick, NJ). Immobiline DryStrip gels were from Amersham Biosciences (Piscataway, NJ). PVDF membranes were from Bio-Rad. All chemicals used were from Sigma Chemical Co. (St. Louis, MO), except for the Alkaline Phosphatase Conjugate Substrate Kit, which was from Bio-Rad (Hercules, CA). Hoechst stain was from Molecular Probes, Inc. (Eugene, OR). Polyclonal anti-phosducin antibody was raised against phosducin purified as described earlier . This protein also served as a standard as shown in electrophoresis. Anti-rhodopsin monoclonal antibody was a generous gift from Dr. G. Adamus (Oregon Health Sciences University, Portland, OR). Bovine rod disk membranes washed free of peripheral and soluble proteins  were used as rhodopsin standard. Protein content of preparations was measured with Bio-Rad Protein Assay dye reagent using bovine serum albumin as standard.
Results & Discussion
Cyclic nucleotides, both cyclic AMP and cyclic GMP, stimulated phosphorylation of a number of proteins when a homogenate of dark-adapted retinas was incubated in the presence of γ-32P ATP. A 33 kDa protein whose phosphorylation was most prominently influenced by the cyclic nucleotides was chosen for further investigation. There are several known proteins of approximately the same molecular weight whose properties are regulated by phosphorylation. In order to establish the identity of the 33 kDa protein, phosphorylated retinal proteins in the homogenate were electrophoresed on a polyacrylamide gradient gel, transferred to a PVDF membrane, and probed with antibodies against 14-3-3, DARP-32, and phosducin. Only the anti-phosducin antibody reaction coincided with the 32P labeled protein (Figure 1).
DARP-32 antibody did not show any reaction with retinal proteins, while immunological reaction with 14-3-3 antibody did not match the radioactive band (data not shown). Further confirmation that the 33 kDa phosphorylated protein was indeed phosducin was obtained when a soluble fraction of phosphorylated retinal homogenate was subjected to 2-dimentional electrophoresis, autoradiography and western blotting with anti-phosducin antibody. Again, as on the one-dimensional gel, radioactivity coincided with immunoreaction (Figure 2).
We have been interested in membrane proteins that exhibit enhanced phosphorylation in the presence of cyclic nucleotides. To our surprise, when phosphorylated retinal homogenate was subjected to subcellular fractionation and western blotting with anti-phosducin antibodies, immunological reaction was detected in the nuclear fraction (120x g pellet) along with the cytosolic (100,000x g supernatant) and membrane (100,000x g pellet) fractions, though cyclic nucleotide-dependent phosphorylation of the protein was observed only in the cytosolic fraction (Figure 3).
Presence of phosducin in the retinal nuclear fraction has not been reported previously, and the present observation could potentially support a role for this protein in regulating gene expression. The first task was to make sure the observed immunoreactivity in the nuclear fraction was not due to contaminating cellular structures. We therefore subjected the crude nuclear fraction to a purification protocol according to a standard procedure [14,15] and reinvestigated the presence of phosducin immunoreactivity. Figure 3 shows phosducin immunoreactivity and autoradiogram of 40 μg of proteins of purified nuclei, 100,000x g pellet and 100,000x g supernatant from a homogenate phosphorylated in the absence and presence of cyclic GMP. As evident, phosducin is present in the purified nuclear fraction and is, in fact, more abundant than in the 100,000x g pellet (Figure 3).
The purity of the nuclear preparation was assessed by microscopy (Figure 4) after treatment with Hoechst dye to highlight the nuclei. The figure shows that the vast majority of material is indeed due to nuclei.
Since phosducin is a cyclic nucleotide-dependent phosphoprotein with a role in phototransduction, we have investigated the possibility that contamination of nuclear preparation with rod outer segment material might account for the nuclear localization of the protein. Rhodopsin was used as a marker for rod outer segments, and rhodopsin and phosducin were estimated in the homogenate and the purified nuclear fraction by western blotting utilizing specific antibodies. The results are presented in Figure 5.
Comparing the intensity of rhodopsin immunostaining in the retinal homogenate with that of 100 ng of rhodopsin standard, we estimated that the amount of rhodopsin in the retinal homogenate is about 2.5 pmol/μg of protein. Comparing the intensity of rhodopsin immunostaining in the retinal homogenate and the nuclear preparation, we estimated that the relative amount of rhodopsin in the nuclei is about 100 times lower than in the retinal homogenate, amounting to about 0.025 pmol/μg of total protein. In contrast to that, the relative intensity of phosducin immunostaining in the retinal homogenate and the nuclear preparation is about the same, and when compared to the intensity of immunostaining of 100 ng of phosducin standard, the relative amount of phosducin in both the retinal homogenate and the nuclear preparation is calculated to be 0.156 pmol/ μg of protein. These estimates showed that there was one phosducin for every 16 rhodopsins in the retinal homogenate, in close agreement with one phosducin for 21 rhodopsins reported by Thulin et al. . For the nuclear preparation, we estimated that there was one phosducin for every 6 rhodopsins. There was no earlier report of phosducin in the nucleus, and given the report by Thulin et al.  that there was one phosducin for every 228 rhodopsins in rod outer segments, it is quite unlikely that contamination by rod outer segments could account for phosducin in our purified nuclear preparation. The purpose of this investigation was not to get a precise estimate of phosducin in the nucleus, but to assess the possibility that its very presence is due to contamination. The above estimates show that phosducin is present in the nucleus, possibly at a high concentration.
The fact that the protein was present in a large amount in the nucleus, but that it was not phosphorylated in our assay, was surprising. Possible reasons for the failure to phosphorylate are that nuclei were intact in our phosphorylation assays and that either cyclic GMP, γ-32PATP, or both, did not enter nuclei during the period of our assays to phosphorylate phosducin. It is also possible that the nuclei did not harbor cyclic-nucleotide dependent kinases. To test these hypotheses, we isolated intact nuclei from retina, subjected them to freezing and thawing to make them permeable, and incubated them with (γ-32P)ATP in the absence and presence of cyclic GMP. Following phosphorylation, nuclear proteins were electrophoresed and transferred to a PVDF membrane, and probed with phosducin antibody and exposed to an x-ray film. The results presented in Figure 6 clearly show that after disruption of pure nuclei, phosducin could be phosphorylated by endogenous kinases in a cyclic nucleotide-dependent fashion. It may also be noted that there was little phosphorylation of any protein in the intact nuclei (Figure 3), whereas numerous proteins were phosphorylated in the permeabilized nuclear preparation (Figure 6). The data presented here do not, however, reveal the extent to which nuclear phosducin is already phosphorylated or what fraction of the protein is phosphorylated in assays on permeabilized nuclei.
Zhu and Craft have shown recently that phosducin remains a cytoplasmic protein in COS-7 cells transfected with a plasmid bearing the phosducin gene, but in cells cotransfected with CRX, a transcription factor, it is found both in the cytoplasm and the nucleus . Based on this observation and the ability of phosducin to bind CRX, these authors suggested that phosducin might restrict the availability of CRX and thus influence gene expression . Zhu and Craft hypothesized that intact phosducin might be proteolyzed in the cytoplasm and that the C-terminal fragments interact with CRX and migrate to nucleus . Our results show that phosducin, not its breakdown product, is present in the nuclei quite abundantly (Figure 3). Our results also show that the protein in the nucleus is capable of being phosphorylated in a cyclic nucleotide-dependent fashion indicating that phosducin is not fully phosphorylated as a requirement to be transported to the nucleus. However, our results do not rule out the possibility that a fully phosphorylated phosducin enters the nucleus and gets dephosphorylated subsequently.
In conclusion, our results demonstrate for the first time an abundant presence of phosducin in its intact form in the nucleus and support a role for it in the regulation of gene expression as hypothesized by Zhu and Craft .
This work was supported by the National Eye Institute (grants EY 07158 and EY 05230) and the Department of Veterans Affairs Medical Service.
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