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J. Biol. Chem., Vol. 277, Issue 45, 43319-43326, November 8, 2002

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In Vivo Regulation of Phosphoinositide 3-Kinase in Retina through Light-induced Tyrosine Phosphorylation of the Insulin Receptor -Subunit^*

Raju V. S. Rajala^§¶, Mark E. McClellan^§, John D. Ash^§, and Robert E. Anderson^§**

From the Departments of  Cell Biology,  Ophthalmology, and ^** Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and the ^§ Dean A. McGee Eye Institute, Oklahoma City, Oklahoma 73104

Received for publication, June 26, 2002, and in revised form, August 18, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110-3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the -subunit of the insulin receptor (IR) in ROS membranes, which leads to the association of PI3K enzyme activity with IR. Retinas from light- or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P₂ as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P₃. We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2)¹ domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5). The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).

One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ~85-kDa regulatory subunit (p85) and a ~110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyrosine-phosphorylated sites on the receptor (12, 13). PI3K activity is also regulated by activated receptor-tyrosine kinase substrates such as the insulin receptor substrate-1 (5). Thus, substantial evidence exists suggesting that the Class I p85-p110 complex of PI3K is a common element of numerous signaling pathways involving a large number of tyrosine kinases (14-16) and that a variety of stimuli can trigger distinct and specific biological responses in different cell types.

We have reported that bovine rod outer segments (ROS) contain a Class I p85-p110 enzyme complex that is more active in light-adapted retinas in vitro (17) and can be activated by tyrosine phosphorylation of proteins in these membranes (18). We have identified a mechanism for regulation of PI3K activity in bovine photoreceptor cells through tyrosine phosphorylation of the insulin receptor -subunit (IR) in vitro (19). The activation is mediated through direct binding of the N-terminal SH2 domain of p85 subunit of PI3K to the tyrosine-phosphorylated C terminus of the insulin receptor (19). The role of light in PI3K activation in vivo has not been investigated, although we have found that light stimulates tyrosine phosphorylation of several proteins in rat ROS in vivo (20). In addition, in vivo light-dependent association of Src with ROS (21) and light-mediated activation of diacylglycerol kinase in rat ROS have been reported (22). In the present study, we demonstrate that light-induced tyrosine phosphorylation of the insulin receptor in ROS in vivo is involved in the regulation of PI3K activity in retina.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Polyclonal antisera to the p85 regulatory subunit of PI3K, and 1- and 3-Na⁺-K⁺-ATPase were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-IR and anti-Tyr(P) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-opsin antibody (Rho 4D2) was a gift from Dr. Robert Molday (University of British Columbia). Mouse anti-opsin was a gift of Dr. Colin Barnstable (Yale University). Anti-arrestin antibody was a gift from Dr. James McGinnis (University of Oklahoma Health Sciences Center). PY-20 antibody was from Transduction Laboratories (Lexington, KY). [-³²P]ATP was from PerkinElmer Life Sciences. Echelon Research Laboratories Inc. (Salt Lake City, UT) provided D-myo-phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂). All other reagents were of analytical grade from Sigma.

Preparation of Rat ROS-- Albino Sprague-Dawley rats were dark-adapted overnight and sacrificed either under dim red light or following 30 min of light exposure (300 lux). ROS were prepared on a discontinuous sucrose gradient from either dark- or light-adapted retinas as previously described (23) with some minor modifications (21). Retinas were homogenized in 4.0 ml of 47% sucrose solution containing 100 mM NaCl, 1 mM EDTA, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris-HCl (pH 7.4) (buffer A). Retinal homogenates were transferred to 15-ml centrifuge tubes and sequentially overlaid with 3.0 ml of 42%, 3.0 ml of 37%, and 4.0 ml of 32% sucrose dissolved in buffer A. The gradients were spun at 82,000 × g for 1 h at 4 °C. The 32%/37% interfacial sucrose band containing ROS membranes was harvested; diluted with 10 mM Tris-HCl (pH 7.4) containing 100 mM NaCl, 1 mM EDTA, 1 mM orthovanadate (buffer B); and centrifuged at 27,000 × g for 30 min. The ROS pellets were resuspended in buffer B and stored at 20 °C. The non-ROS band designated Band II (37%/42%) was also saved for comparison with ROS. All procedures were carried out under dim red light for dark-adapted retinas and in room light for light-adapted retinas. All protein concentrations were determined by the BCA reagent from Pierce, following the manufacturer's instructions.

Preparation of Bovine ROS and Retinal Pigment Epithelium-- Fresh bovine eyes were obtained from a local abattoir and placed on ice and retinas were dissected within 2 h. ROS were prepared from fresh retinas on a continuous sucrose gradient (25-50%) as previously described (19). Bovine retinal pigment epithelial cells (RPE cells) were prepared according to the method described by Saari et al. (24).

Immunoprecipitation (IP)-- Retinal lysates or ROS were solubilized for 30 min at 4 °C in a lysis buffer containing 1% Triton X-100, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EGTA, 1 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM Na₃VO₄, 10 µg/ml leupeptin, and 1 µg/ml aprotinin. Insoluble material was removed by centrifugation at 17,000 × g for 20 min, and the solubilized proteins were precleared by incubation with 40 µl of protein A-Sepharose for 1 h at 4 °C with mixing. The supernatant was incubated with either anti-p85 (1:300), anti-Tyr(P) (4 µg), or anti-IR (4 µg) antibodies overnight at 4 °C and subsequently with 40 µl of protein A-Sepharose for 2 h at 4 °C. Immune complexes were washed twice with modified solubilization buffer (the 1% Triton X-100 was reduced to 0.1%, and glycerol was removed) and once with phosphorylation buffer without ATP (19). Precipitates were assayed for PI3K activity or subjected to immunoblot analysis.

SDS-PAGE and Western Blot Analysis-- Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes, and the blots were washed two times for 10 min with TTBS (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.1% Tween 20) and blocked with 10% bovine serum albumin in TTBS overnight at 4 °C. Blots were then incubated with anti-p85 (1:4000), anti-IR (1:1000), or anti-Tyr(P) (1:1000) antibodies for 2 h at room temperature. Following primary antibody incubations, immunoblots were incubated with horseradish peroxidase-linked secondary antibodies (either anti-rabbit, anti-mouse, or anti-Goat IgG) and developed by ECL according to the manufacturer's instructions.

PI3K Assay-- Enzyme assays were carried out essentially as previously described (25). Briefly, assays were performed directly on immunoprecipitates in 50 µl of the reaction mixture containing 0.2 mg/ml PI-4,5-P₂, 50 µM ATP, 0.2 µCi of [-³²P]ATP, 5 mM MgCl₂, and 10 mM HEPES buffer (pH 7.5). The reactions were carried out for 15 min at room temperature and stopped by the addition of 100 µl of 1 N HCl followed by 200 µl of chloroform/methanol (1/1, v/v). Lipids were extracted and resolved on oxalate-coated TLC plates (silica gel 60) with a solvent system of 1-propanol, 2 M acetic acid (65/35, v/v). The plates were coated in 1% (w/v) potassium oxalate in 50% (v/v) and then baked in oven at 100 °C for 1 h prior to use. TLC plates were exposed to x-ray film overnight at 70 °C, and radioactive lipids were scraped and quantified by liquid scintillation counting.

GST-p85 Fusion Proteins and Pull-down Experiments-- GST-p85 fusion proteins were generated by PCR amplification of the indicated p85 regions and cloned into pGEX2T (Amersham Biosciences). The amino acids of bovine p85 present in each fusion protein are N-SH2 (residues 314-446) and C-SH2 (residues 614-724), based on the sequence published by Otsu et al. (26). The sequence of each clone was verified by DNA sequencing. All inductions yielded proteins of the expected size as judged by Coomassie staining. Pull-down experiments were carried out as described (19) using 5 µg of GST fusion proteins that had been adsorbed onto glutathione-Sepharose 4B matrix. ROS from dark- and light-adapted rats were incubated with GST/GST-p85 fusion proteins at 4 °C for 1.5 h, with continuous mixing. The Sepharose beads were washed three times in 500 µl of HNTG buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) and centrifuged at 5,000 rpm for 30-60 s at 4 °C. GST-p85 fusion proteins and bound proteins were eluted by boiling in 2× SDS sample buffer 5 min prior to 10% SDS-PAGE. After the SDS-PAGE, the gels were subjected to Western blot analysis with anti-IR antibody.

Site-directed Mutagenesis-- Site-directed mutagenesis was carried out using a QuikChange site-directed mutagenesis kit (Stratagene Inc., La Jolla, CA), using a PTC 100 programmable thermal controller (MJ Research, Inc., Watertown, MA). The reaction mixture contained site-directed mutagenesis buffer (200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM NH₄SO₄, 20 mM MgSO₄, 1% Triton X-100, 1 mg/ml nuclease-free bovine serum albumin), 1 mM deoxynucleotide mix (dATP, dCTP, dTTP, and dGTP), 50 ng of GST-pGEX vector containing either p85 (N-SH2) or p85 (C-SH2) fusion proteins, and 125 ng of sense and antisense primers with mutations, in a total volume of 50 µl, followed by the addition of 2.5 units of Pfu DNA polymerase. The mutant primers were R358A (sense, ACC TTT TTG GTA GCA GAC GCA TCT ACT AAA; antisense, TTT AGT AGA TGC GTC TGC TAC CAA AAA GGT) and R649A (sense, ACT TTT CTT GTC GCG GAA AGC AGT AAA CAG; antisense, CTG TTT ACT GCT TTC CGC GAC AAG AAA AGT). The extension parameters of site-directed mutagenesis were as follows: initial denaturation at 95 °C for 30 s, followed by 16 cycles at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 12 min (2 min/kb of plasmid length). Following temperature cycling, the reaction was placed on ice for 2 min, after which 10 units of DpnI restriction enzyme were added, mixed, and incubated at 37 °C for 60 min. Transformation was carried out using 1 µl of the DpnI-treated reaction to Epicurean XL-blue supercompetent cells, and the reaction was placed on LB/Amp (100 µl/ml) plates. The cDNAs of all mutants were sequenced after PCR; the only mutations observed were those intentionally introduced to create each desired mutation. The clones were induced with isopropyl-1-thio--D-galactopyranoside (0.1 mM), and the expressed fusion proteins were purified through GST-Sepharose 4B matrix.

In Vivo Light Experiments on Rats and Mice-- Albino Sprague-Dawley rats (150-200 g) purchased from Harlan Sprague-Dawley (Indianapolis, IN) were maintained for at least 2 weeks in dim cyclic light (12 h on; 12 h off; 5-10 lux). A breeding colony of FVB/N mice is maintained in our vivarium in cyclic light (12 h on; 12 h off; ~400 lux). Light absorption by rhodopsin activates transducin, a G-protein, which in turn promotes cGMP hydrolysis by cGMP-phosphodiesterase, leading to hyperpolarization of rod photoreceptor cells (27). FVB/N mice are homozygous for the Pdeb^rd1 mutation (formally known as rd1) in the cGMP-phosphodiesterase -subunit. As a result, these mice undergo rapid photoreceptor degeneration beginning at postnatal day 9 (28-31). The retinas from adult FVB/N mice completely lack photoreceptors. On the day of an experiment, rats or mice were dark-adapted overnight, and half were subjected to normal room light (~300 lux) for 30 min. Eyes were enucleated, and the retinas were quickly removed and homogenized by hand in homogenizing buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% sucrose, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The retina lysates were either used directly or for the preparation of ROS. The retina lysate or ROS was immunoprecipitated with anti-IR antibody, followed by either Western blotting with anti-IR or anti-Tyr(P) antibodies or measuring the PI3K activity using PI-4,5-P₂ as substrate. All animal work was in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the IACUC of the University of Oklahoma Health Sciences Center and the Dean A. McGee Eye Institute. The data were analyzed by Student's t test, and statistical significance was set at p < 0.05.

Immunolabeling of ROS and Whole Mount Preparations-- Intact ROS, some containing blebs of inner segments attached through the connecting cilium, were prepared by mechanical detachment from freshly dissected bovine retinas, as a suspension in Hanks' balanced salt solution (Sigma) buffered with 25 mM HEPES (pH 7.4) (32). After gentle homogenization by five passes through a Pasteur pipette, cell fragments in suspension were allowed to adsorb for 3 min to a Vectbond^TM (Vector Laboratories, Burlingame, CA)-treated glass slide. Adhered cell fragments were fixed for 5 min in methanol at 20 °C and washed three times with PBS before processing for immunostaining. To detect IR in the dissociated photoreceptors, slides were incubated with a mixture of mouse anti-IR (GR36; Calbiochem) and rabbit-anti-transducin  (SC-389; Santa Cruz Biotechnology) diluted 1:1000 and 1:800, respectively, in PBS containing 10% horse serum. The transducin  antibody was included to clearly identify intact ROS. To determine whether the anti-IR was specific, an identical antibody mixture was incubated with a blocking IR peptide antigen (SC-711P; Santa Cruz Biotechnology) for 24 h at 4 °C before incubation on the isolated photoreceptors. To demonstrate that the blocking peptide was specific to IR, additional slides were incubated with an antibody mixture containing mouse anti-opsin, rabbit anti-transducin , and the IR blocking peptide, diluted 1:100, 1:800, and 1:200, respectively. Antibodies were incubated overnight at 4 °C and then washed with PBS. For fluorescent detection, slides were incubated with a mixture of Texas Red-anti-mouse and fluorescein isothiocyanate-anti-rabbit antibodies (Vector Laboratories), each diluted 1:200 in PBS with 10% horse serum. Following incubation for 1 h at room temperature, the slides were washed with phosphate-buffered saline and cover-slipped in 50% glycerol in phosphate-buffered saline. Antibody-labeled complexes were examined on a Nikon Eclipse E800 microscope equipped with a digital camera, and images were captured using Metamorph (Universal Imaging, West Chester, PA) image analysis software. For quantitation, all images were captured using identical microscope and camera setting, so that intensities of the digital images quantitatively reflect antibody binding.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Increased PI3K Activity Associated with Anti-Tyr(P) but Not with Anti-p85 IPs-- To determine whether light has an effect on PI3K activity and the phosphorylation of the insulin receptor, rats were dark-adapted overnight, and half were subjected to normal room light for 30 min. Retinas were removed quickly and homogenized, and lysates were immunoprecipitated with anti-Tyr(P) or anti-p85 antibodies. PI3K enzyme activity was higher in anti-Tyr(P) IPs from whole retinas (Fig. 1A) and ROS (Fig. 1B) from light-adapted (L) rats, compared with those from dark-adapted (D) animals. In contrast, only a marginal increase in PI3K activity was observed in anti-p85 IPs of light-adapted retinas over dark-adapted retinas (Fig. 1C), suggesting that light may induce the translocation of PI3K from cytoplasm to tyrosine-phosphorylated proteins in the disk membrane.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   PI3K enzyme activity was measured in the anti-Tyr(P) (A) or anti-p85 (C) immunoprecipitates (50-100 µg of protein) from dark- or light-adapted rat retina homogenates or ROS membranes immunoprecipitated with anti-Tyr(P) (PY) antibodies (B). PI3K activity was measured using PI-4,5-P2 and [32P]ATP as substrates. The radioactive spots of PI-3,4,5-P3 were scraped from the TLC plate and counted. Data are means ± S.D. (n = 3). L, light; D, dark.

Increased IR Phosphorylation, p85 Association, and PI3K Enzyme Activity Associated with anti-IR IPs of Light-adapted Rat Retinas-- When retinal homogenates were immunoprecipitated with anti-IR antibodies, PI3K activity was more than 4-fold higher in IPs from light-adapted rats compared with those from dark-adapted animals (Fig. 2, D and E). Western blot analysis of anti-IR IPs probed with anti-IR antibody indicated an equal amount of IR in both light- and dark-adapted rat retinas (Fig. 2A). However, anti-IR IPs probed with anti-Tyr(P) (Fig. 2B) and anti-p85 (Fig. 2C) antibodies showed almost 2-fold greater phosphorylation of IR (quantitative analysis of bands was carried out using Image software 1.62 (National Institutes of Health; available on the World Wide Web at rsb.info.nih.gov/nih-image/download.html) in light-adapted rat retinas and binding of p85, respectively. These results demonstrate light-induced phosphorylation of the IR, as well as light-induced binding of p85 to the insulin receptor.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   In vivo light-dependent phosphorylation of IR. Rats (six per group) were either dark- or light-adapted as described under "Experimental Procedures." Retinas from each rat were pooled, homogenized, immunoprecipitated with anti-IR antibodies, and immunoblotted with anti-IR (A), anti-Tyr(P) (B), or anti-p85 antibodies (C) or measured for PI3K activity using PI-4,5-P2 and [-32P]ATP as substrates (D). E, the radioactive spots of PI-3,4,5-P3 were scraped from the TLC plate and counted. Data are means ± S.E. (n = 6). The difference between light and dark PI3K activity is significant at p < 0.01. WB, Western blot.

Light-dependent Association of p85 with ROS Membranes-- ROS membranes that were prepared on a discontinuous sucrose density gradient showed a greater PI3K enzyme activity in light-adapted rats compared with dark-adapted rats (Fig. 3D). Western blot analysis indicated an increased amount of p85 in light-adapted ROS compared with dark-adapted ROS (Fig. 3A). The photoreceptor-specific protein opsin was used as internal control and indicated that an equal amount of protein was used in these analyses (Fig. 3C). Arrestin is known to move from the inner segment to outer segment in light (33, 34), and its accumulation in the ROS was a positive control to verify that the retinas responded to light in a predictable manner (Fig. 3B). These in vivo studies demonstrate the light-induced tyrosine phosphorylation of IR and the subsequent association of PI3K in ROS.

View larger version (54K): [in this window] [in a new window]   Fig. 3.   PI3K activity and expression of p85 (A), arrestin (B), and opsin (C) in dark- and light- adapted rat ROS (20 µg of protein). PI3K activity was measured in 50 µg of light- or dark-adapted ROS protein. The radioactive spots of PI-3,4,5-P3 were scraped from the TLC plate and counted (D). Data are means ± S.D. (n = 3).

Binding of Light-induced Tyrosine-phosphorylated Insulin Receptor to GST-p85 (N-SH2) Fusion Proteins-- Rats were dark-adapted overnight, and half were subjected to normal room light for 30 min. Retinas were quickly removed and homogenized, and ROS were prepared by discontinuous sucrose density gradient centrifugation. Solubilized light- and dark-adapted rat ROS were incubated with GST-p85 (N-SH2) or mutant p85 (N-SH2, R358A) fusion proteins and subjected to GST pull-down assays. The bound proteins were run on 8% SDS-PAGE and probed with anti-IR antibodies. IR did not bind to the GST controls (Fig. 4, lanes 1 and 2) but did bind to the GST-p85 (N-SH2) fusion protein. Quantitatively, more IR was pulled down from light-adapted ROS than from dark-adapted ROS (lanes 3 and 4). Mutating Arg-358 in p85 to Ala-358 abolished all binding (lanes 5 and 6), demonstrating the requirement for the native N-SH2 motif. Western blots of the ROS preparations using the anti-IR antibody showed the presence of the insulin receptor in the membranes (lanes 7 and 8). Since p85 only interacts with phosphorylated IR, these results further confirm the light-induced tyrosine phosphorylation of IR in vivo. Since our previous studies had shown that C terminus SH2 and its mutant version (R649A) did not interact with bovine retinal IR in vitro (19), we did not test these fusion proteins in vivo.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   GST pull-down experiments. Light- and dark-adapted ROS (50 µg each) were incubated with GST, GST-p85, or GST-p85 (R358A) fusion proteins, followed by Western blot analysis of the bound proteins with anti-IR antibody. Ten µg of light- or dark-adapted ROS were directly loaded to assess equal amounts of IR in light- and dark-adapted conditions. Lanes 1, 3, 5, and 7, light-adapted; lanes 2, 4, 6, and 8, dark-adapted.

Role of Insulin in the Activation of the Retinal Insulin Receptor-- To assess the role of insulin in retinal IR phosphorylation, we measured IR activation in rats that were made insulin-deficient by the injection of streptozotocin. The state of diabetes was confirmed by measuring blood glucose levels (35). Retinas were removed from hyperglycemic (insulin-deficient animals) and control animals in normal room light. Samples were lysed in homogenizing buffer and subjected to immunoprecipitation with anti-IR antibody. PI3K activity and the phosphorylation of IR were examined in the IPs. There was no difference in the PI3K activity (Fig. 5B) or the degree of IR phosphorylation (Fig. 5A) in control and insulin-deficient rats. These studies suggest that whereas insulin may be sufficient to activate insulin receptors in isolated ROS in vitro (19, 36, 37), it may not be required for the light-dependent activation of IR in vivo.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   PI3K activity and phosphorylation of IR in control and diabetic rat retinas. One hundred µg of protein was immunoprecipitated with anti-IR antibody and subjected to either Western blotting (WB) analysis with anti-Tyr(P) antibody (A) or measuring PI3K activity (B) using PI-4,5-P2 and [-32P]ATP as substrates. The radioactive spots of PI-3,4,5-P3 were scraped from the TLC plate and counted (B). Data are mean ± S.D. (n = 4).

Location of Insulin Receptors in the Retina-- We verified the presence of the insulin receptor in ROS by several approaches, to eliminate the possibility that our ROS preparations were contaminated with inner segment or RPE membranes. RPE cells were obtained from fresh bovine eyes, and ROS and Band II retinal membranes were prepared from light-adapted rat retinas by discontinuous sucrose gradient centrifugation. Western blots of the three preparations were probed with two marker enzymes, the 1 and 3 isoforms of Na⁺-K⁺-ATPase. The 1 isoform is mainly present in RPE cells, whereas 3 is mainly found in inner segments; neither is present in ROS (38). As shown in Fig. 6, the 1 isoform was present in RPE (lane 1) and Band II (lane 3) but not in the ROS fraction (lane 2). The 3 isoform was absent from RPE (lane 1) and rat ROS (lane 2) but was present in Band II from rat retina (lane 3). Similar blots probed with the anti-IR antibody indicated the presence of insulin receptors in all fractions (Fig. 6).

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Western blot (WB) analysis of 1-Na+-K+-ATPase, 3-Na+-K+-ATPase, and IR expression in bovine RPE and rat ROS. Twenty µg of bovine RPE and 20 µg of rat ROS membranes prepared on a discontinuous sucrose density gradient from light- and dark-adapted retinas were probed for 1-Na+-K+-ATPase, 3-Na+-K+-ATPase, and IR expression employing respective antibodies. Lane 1, bovine RPE; lane 2, rat ROS; lane 3, rat non-ROS membranes (Band II).

To further demonstrate that insulin receptors are present in photoreceptor outer segments, we immunolabeled bovine ROS (containing some attached inner segment) with an anti-IR antibody. Immunostaining was found in both outer and inner segments (Fig. 7A). Inclusion of the IR-blocking peptide inhibited the immunoreactivity of IR (Fig. 7B). Photoreceptors were identified by the expression of transducin  (Fig. 7, D-F). The blocking peptide did not block the binding of the anti-opsin antibody (Fig. 7C) or the anti-transducin antibody (Fig. 7, E and F). These results provide strong evidence that the insulin receptor is present in ROS membranes.

View larger version (90K): [in this window] [in a new window]   Fig. 7.   Immunolocalization of IR in dissociated ROS. Bovine ROS were prepared on glass slides as described under "Experimental Procedures." Immunolabeling with transducin  (D-F) was used to identify ROS. The co-localization of IR (A) and transducin  (D) clearly demonstrates the presence of IR in rod photoreceptors. The immunostaining of IR was significantly blocked using the peptide from which the antibody was generated (B). The same peptide did not block transducin  staining (E and F), nor did it block the anti-opsin antibody (C).

Co-localization of IR with Opsin-- A third approach was taken to demonstrate the presence of the insulin receptor in rod outer segments. Bovine ROS were prepared on a continuous sucrose density gradient and further purified on a second gradient (18). Serial 1.0-ml fractions were collected and analyzed for opsin (Fig. 8D), p85 (Fig. 8B), and IR (Fig. 8A). PI3K enzyme activity was measured in anti-IR immunoprecipitates (Fig. 8C). Western blot analysis of IR indicated its presence predominantly in the fractions containing the largest concentration of opsin (ROS fractions). PI3K activity in the immunoprecipitates of anti-IR was also highest in the major opsin and IR fractions. These results provide additional evidence that insulin receptors are localized in the rod outer segments.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Co-elution of ROS protein from continuous sucrose density gradient centrifugation. Bovine ROS were further purified by a second sucrose density gradient centrifugation. Each fraction was subjected to SDS-PAGE followed by silver staining for the detection of opsin (D). Individual fractions were subjected to either immunoprecipitation with anti-IR antibody followed by measurement of PI3K enzyme activity (C) or subjected to Western blotting analysis for the detection of IR (A) and p85 (B).

Role of Photobleachable Visual Pigments in the Activation of PI3K-- Experiments were carried out on wild type and mutant mice to investigate the involvement of photobleachable visual pigments in the regulation of PI3K activity. As previously shown in rat retinas (Fig. 1-3), PI3K activity was significantly higher in anti-IR IPs of light-adapted wild type mouse retinas compared with dark-adapted mouse retinas (Fig. 9A). However, there was no difference in PI3K activity in light- and dark-adapted Pdeb(rd) mutant mice (FVB), which lack photoreceptors (Fig. 9B). Lack of photoreceptors was confirmed by probing the retina lysates with an anti-opsin antibody, where no detectable opsin was observed (Fig. 9D), compared with wild type mouse retina (Fig. 9C). Probing the FVB mouse retinas for IR expression indicated the presence of IR (Fig. 9E), demonstrating that IR is also present in retina cells other than photoreceptors. The results suggest that the observed light/dark differences in IR phosphorylation and subsequent binding of PI3K are photoreceptor-specific phenomenon that are probably mediated by photon capture in the ROS. In Fig. 9E, we have observed two IR bands in mouse retina lysate. The bottom band could be a proteolytic degradation of 97-kDa IR (top band) or an IR isoform.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   PI3K activity, opsin, and IR expression in retinas of wild type and FVB mice. PI3K activity was measured in the immunoprecipitates of IR from 100 µg of (A) wild type (n = 9) and (B) FVB (n = 4) mouse retinas. Data are means ± S.E. *, p < 0.05. D, dark; L, light. Opsin expression was examined with 10 µg of protein from light- and dark-adapted wild type (C) and FVB mice (D). IR (E) expression (10 µg) was examined only from light-adapted wild type and FVB mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the current study, we have made two novel findings that are likely to be functionally significant in photoreceptors. We have demonstrated light-dependent tyrosine phosphorylation of IR in ROS, and we have shown the association of PI3K enzyme activity with the activated receptor only in rod outer segments stimulated by light. The association of PI3K and IR is dependent on tyrosine phosphorylation. We have previously reported that light stimulates tyrosine phosphorylation of multiple proteins in ROS in vivo (20). In the current study, we have demonstrated that one of the targets is the IR and that this phosphorylation leads to the association of PI3K activity with ROS membranes. The increased PI3K activity could have been due to increased p85 expression or to translocation of PI3K from the cytoplasm to the ROS membranes. To distinguish between these possibilities, we measured PI3K activity in anti-p85 immunoprecipitates of retinal homogenates and in isolated ROS membranes. Total PI3K activity was not significantly different between light- and dark-adapted anti-p85 precipitates from homogenates but was increased in isolated ROS. Therefore, the increased activity of PI3K associated with IR in light-adapted ROS is the result of PI3K translocation from the cytoplasm to the membrane. Previous studies from our laboratory using radiolabeled inositol have shown that the enzymes for PI-4,5-P₂ synthesis are present in bovine ROS. Further, light adaptation of bovine retinas in situ stimulates phosphatidylinositol (PI) synthesis in rod outer segments (39). These studies indicate an active PI cycle in rod outer segments that can provide the substrate PI-4,5-P₂ for phosphorylation by PI3K.

We have localized the activation of IR to retinal photoreceptors using biochemical, genetic, and immunolocalization approaches. First, we identified IR in highly enriched ROS that lack markers for rod inner segments and RPE. Second, in adult mice homozygous for the Pdeb (RD) mutation that lack rods and cones, we failed to observe light-dependent activation of IR or increased PI3K activity. These experiments also indicate that the light response requires functional photoreceptor cells. This result excludes the possibility that the light-dependent activation occurs in photopigment-expressing ganglion cells, inner retinal neurons, or RPE (40, 41). Third, we have immunolocalized the expression of IR to rod photoreceptor cells. Combined, these data provide compelling evidence that the light-dependent activation of IR is occurring in photoreceptor outer segments.

Our results demonstrate that light stimulates tyrosine phosphorylation of ROS proteins including IR, which promotes the binding of the p85 regulatory subunit of PI3K to the ROS membranes. The mechanism for light-induced phosphorylation of IR and subsequent activation of PI3K is not known. Several studies have shown that the -subunit of the insulin receptor is present in ROS and can be phosphorylated in response to insulin (19, 36, 37). Insulin activates PI3K in many other tissues (42), including cultured retinal neurons (43). However, it appears unlikely that insulin is involved in IR activation in the retina in vivo, since insulin does not cross the blood-retinal barrier (44), and our studies on diabetic animals indicate that light-dependent phosphorylation of IR is not insulin-dependent.

We speculate that there could be two possible mechanisms that trigger the phosphorylation of IR. One could involve ligand(s) other than insulin, which are induced in response to light and bind to IR. There are no data to support this notion, although this does not preclude activated transducin or some other participant in the visual transduction cascade playing an important role in this process. This point will be addressed in subsequent studies using mice with specific mutations in genes expressing proteins involved in visual transduction. The second mechanism involves the activation of a nonreceptor tyrosine kinase(s) in response to light. It has been shown that the nonreceptor tyrosine kinase Src phosphorylates insulin- and insulin-like growth factor receptors on autophosphorylation sites (45, 46). Thus, the Src kinase has been shown to substitute for the ligand-dependent receptor activation (44, 45). It has recently been shown that c-Src associates with light-activated opsin (21). Together, these results suggest a model where the light activation of opsin results in its association with c-Src, which in turn associates with and activates the insulin receptor. Consistent with this mechanism, we have also reported previously the in vitro phosphorylation of IR by c-Src in ROS (19).

In addition to activation of the phototransduction cascade, light is known to cause the activation and regulation of several genes, including those that regulate circadian rhythms (47). Light has been shown to regulate retinal dopamine biosynthesis and tyrosine hydroxylase activity (48), apoptosis (49), and intracellular trafficking of photoreceptors in plants (50). Increasing evidence from several laboratories suggests that normal light triggers the tyrosine phosphorylation of several proteins. Nakahata et al. (51) have reported the light-induced tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs) protein in rat suprachiasmatic nucleus, which resulted in its association with nonreceptor tyrosine phosphatases, SHIP-1 and SHIP-2, which have two SH2 domains (51). Tyrosine phosphorylation of MAP kinase-like protein kinase has been activated by light in soybean cell cultures (52). The mitogen-activated protein kinase cascade can be activated by brief exposure to light during subjective night in the mouse suprachiasmatic nucleus (53). It has also been reported that light-induced photoreceptor apoptosis in vivo through the involvement of neuronal nitric-oxide synthase and guanylate cyclase activity independent of caspase-3 (54).

The functional consequence of light-dependent activation of the IR and PI3K in photoreceptor cells is not known. Activation of the IR signaling pathway has been shown to have complex physiological roles in a variety of cell types (55). In rods, IR activity is regulated by light, which suggests that its function could be related to rod-specific activities that are driven by light such as shedding of photoreceptor tips (56), biogenesis of new ROS membranes through the addition of newly synthesized membranes at the base of the ROS (57), or light adaptation (58). Alternatively, it is possible that light-induced PI3K activity may mediate an innate self-protection mechanism. In some neuronal cell types, such as cerebellar granular neurons (59) and PC-12 cells (60), receptor activation of PI3K has been shown to protect these cells from stress-induced neurodegeneration. It has been well established that PI3K/c-Akt is crucial in mediating cell survival. Since bright light stimulation can cause the death of rod and cone photoreceptor cells (61, 62), activation of the PI3K/c-Akt pathway by relatively modest light levels may serve to prevent death of photoreceptors. Consistent with this hypothesis, we have recently observed that biotinylated 3,4,5-PIP₃ could pull down Akt from bovine ROS,² suggesting that light-induced PI3K activity could regulate downstream effector molecules. Understanding the regulation of PI3K in photoreceptor cells will greatly facilitate studies on the regulation of downstream targets that may reveal a role for PI3K in protection from retinal degeneration. Whether PI3K activation protects the retina from stress is currently under study in our laboratory.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Yashige Kotake (Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma) for providing diabetic rats. We thank Mark Dittmar for outstanding assistance in managing and maintaining the animal colonies.

	FOOTNOTES

^* This work was supported by NEI, National Institutes of Health, Grants EY00871, EY04149, and EY12190; Research to Prevent Blindness Inc. (New York, NY); The Foundation Fighting Blindness (Baltimore, MD); the Samuel Roberts Nobel Foundation, Inc. (Ardmore, OK); Presbyterian Health Foundation (Oklahoma City, OK); the Oklahoma Center for Advancement of Science and Technology (Oklahoma City, OK); and the Knights Templar Eye Foundation (Chicago, IL).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: 608 Stanton L. Young Blvd., Rm. 409, Oklahoma City, OK 73104. Tel.: 405-271-8255; Fax: 405-271-8128; E-mail: raju-rajala@ouhsc.edu.

Published, JBC Papers in Press, September 3, 2002, DOI 10.1074/jbc.M206355200

² R. V. S. Rajala and R. E. Anderson, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: SH2 and SH3, Src homology 2 and 3, respectively; PI3K, phosphoinositide 3-kinase; GST, glutathione S-transferase; IR, insulin receptor  subunit; ROS, rod outer segment(s); IP, immunoprecipitate; PI-4, 5-P₂, phosphatidylinositol 4,5-bisphosphate; PI-3, 4,5-P₃, phosphatidylinositol 3,4,5-trisphosphate; RPE, retinal pigment epithelium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES