Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: Evidence of a light-dependent mechanism

Experimental Eye Research 83 (2006) 202e211 www.elsevier.com/locate/yexer

Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: Evidence of a light-dependent mechanism Gabriela A. Salvador, Norma M. Giusto* Instituto de Investigaciones Bioquı´micas de Bahı´a Blanca, Universidad Nacional del Sur and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, C.C. 857, B8000FWB Bahı´a Blanca, Argentina Received 15 September 2005; accepted in revised form 19 December 2005 Available online 21 April 2006

Abstract Photoreceptor cells contain rod outer segments (ROS) which are specialized light-sensitive organelles. The biological function of ROS is to generate a photoresponse, which occurs via the classic transducin-mediated pathway. Moreover, ROS undergo light-regulated membrane turnover and protein translocation whose mechanisms have not been fully elucidated to date. Phospholipase D (PLD) is a key enzyme involved in lipid signal transduction and membrane trafficking. We have previously reported that PLD activity is present in purified ROS (Salvador, G.A., Giusto, N.M., 1998. Characterization of phospholipase D activity in bovine photoreceptor membranes. Lipids 33, 853e860). We now demonstrate that ROS PLD activity is enhanced by phosphatidylinositol bisphosphate (PIP2) and cytosolic factors in a GTP dependent-manner. Western blot analysis demonstrates the presence of PLD1 isoform in purified ROS. In ROS obtained from dark-adapted retinas (DROS), PIP2-dependent PLD activity was higher than that observed in ROS obtained from light-adapted retinas (LROS). In addition, experiments carried out in the presence of C3 toxin inhibited PLD activity from DROS whereas pertussis toxin did not affect the enzyme activity. Western blot analysis demonstrates the presence of RhoA, a PLD upstream-regulator. Moreover, RhoA levels were higher in DROS with respect to those in LROS. The present study reports evidence of the involvement of the small G-protein, RhoA, in ROS PLD regulation. Our data strongly suggest that RhoA regulates ROS PLD activity under a light-dependent mechanism. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: retina; rod outer segments; phospholipase D; RhoA; phosphatidic acid; protein kinase A; protein kinase C

1. Introduction Phospholipase D plays an important role in signal transduction of a variety of cells. PLD hydrolyses phosphatidylcholine (PC) in order to produce phosphatidic acid (PA) and choline (Exton, 2002; Liscovitch et al., 2000). PA is a biologically active molecule and can also be hydrolyzed by PA phosphatase (PAP) to yield diacylglycerol (DAG) (McDermott et al., 2004). The DAG from PC is a known activator of PKC (Exton, 2002; Liscovitch et al., 2000). In previous studies we have shown the presence of PLD activity in isolated rod outer segments (ROS) from bovine retina (Salvador and Giusto, 1998). PLD has been

* Corresponding author. Tel.: þ54 291 4861201; fax: þ54 291 4861200. E-mail address: [email protected] (N.M. Giusto). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.12.006

involved in vesicular trafficking processes in many cellular types, and it could thus play an important role in photoreceptor renewal and phagocytosis (Exton, 2002; Liscovitch et al., 2000). Additionally, the activity of enzymes involved in photoreceptor membrane phospholipid turnover has been characterized in the last two decades (Giusto et al., 2000). However, regulation mechanisms and the physiological significance of PLD in ROS remain unclear. Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell cycle progression, and cell adhesion. Their principal mechanism of activation is the binding to their effector proteins initiating downstream signaling (Bishop and Hall, 2000; Reif and Cantrell, 1996; Ward et al., 2002; Yonemura et al., 2004). RhoA modulates several enzymes involved in the generation of lipid second messengers. RhoA and Arf

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(another small G protein), along with PKC and the phospholipid phosphatidylinositol bisphosphate (PIP2), have been shown to regulate phospholipase D (PLD) (Exton, 2002; Malcolm et al., 1996; Yokozeki et al., 1996) in several cellular systems. Although RhoA is clearly involved in GTPgSmediated phospholipase D activation, a requirement for Rho in GPCR-mediated activation of this enzyme is seen in some, but not all, systems (Malcolm et al., 1996; Yokozeki et al., 1996). Several lines of evidence over the past few years have demonstrated that light regulates the activity of several enzymes involved in ROS lipid metabolism and signal transduction, such as phosphatidylinositol 3-kinase (PI3K), diacylglycerol kinase (DAGK), phospholipase A2, PA phosphatase (PAP), and the tyrosine kinase, c-src (Ghalayini et al., 2002; Rajala et al., 2002; Huang et al., 2000; Pasquare et al., 2000; Castagnet and Giusto, 1993). Another interesting finding is the observation that light induces G-alpha transducin translocation from ROS to rod inner segment (RIS) (Sokolov et al., 2002, 2004). On the other hand, arrestin undergoes a massive light driven translocation from RIS to ROS in retinas exposed to light (Elias et al., 2004; Palczewski et al., 1992; Organisciak et al., 1991). The light-dependent redistribution and regulation of proteins in mammalian ROS constitutes an intriguing feature whose underlying mechanisms still remain unclear. In the present paper we explored whether or not light has any effect on ROS PLD activity. In ROS prepared from dark-adapted retinas (DROS), we found higher PLD activity values than in membranes prepared from light-adapted retinas (LROS). Additionally, we showed by western blot analysis the presence of the monomeric G protein RhoA, a known upstream regulator of PLD1/PLD2 isoforms. RhoA association with ROS was a light-dependent phenomenon, and DROS showed the highest levels of the small G protein. ADPribosylation with bacterial toxins demonstrated that C3 toxin inhibited PLD activity in DROS. These results allowed us to demonstrate that RhoA is involved in the regulation of ROS PLD by light.

2. Materials and methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phospho[methyl-3H]choline (43 Ci/mmol) and 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine (54.5 mCi/mmol) were obtained from New England Nuclear-Dupont, Boston, MA. All other chemicals were obtained from Sigma-Aldrich, St. Louis, MO, USA. Monoclonal anti-RhoA and anti-Gt antibodies were obtained from Santa Cruz Biotechnology and anti-PLD1 was obtained from Upstate Biotechnology. Monoclonal antibody against rhodopsin, Rho4D2, was generously supplied by Dr Robert Molday from the University of British Columbia, Vancouver, Canada. Anti-PLD2 was gently provided by Dr Dan Raben from St Louis University School of Medicine, USA.

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2.2. Rod outer segments isolation Fresh bovine eyes were obtained from a local abattoir, stored, and placed in crushed ice within 10 min of the animal’s death, and kept in the dark for 2 h. Retinas were dissected from the eyes after dark- or light-adaptation. Dark-adapted bovine ROS (DROS) were prepared under dim red light from dark-adapted retinas. Light-ROS (LROS) were prepared by exposing the eye’s cup from dark-adapted retinas to light (300 W at 30 cm) for 30 min at room temperature. Retinas were dissected and ROS were subsequently isolated. All isolation procedures were done on ice (4
C) in room light for lightadapted retinas or in a dark room under dim red light for dark-adapted retinas. Rod outer segments were detached by shaking the retinas twice in a 40% sucrose solution containing 1 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml aprotinin, and 2 mg/ml leupeptin in 70 mM sodium phosphate buffer (pH 7.2). The remainders of the retinas were sedimented at 2200  g for 4 min and the supernatants containing the ROS were diluted 1:2 with sucrose-free buffer followed by 30 min of centrifugation at 35,300  g. The crude ROS preparations were then purified by discontinuous density gradient centrifugation as described by Ku¨hn (1982). This procedure gave intact ROS (band I), retained at the 0.84/1.00 M density interface, broken ROS contaminated with mitochondria and RIS, retained at the 1.00/ 1.14 M density interface (band II) and a pellet composed of non-ROS membranes. Band I was collected and diluted with 70 mM sodium phosphate buffer (pH 7.2), and centrifuged at 35,300  g for 30 min (Krebs and Ku¨hn,1977; Ku¨hn, 1982). Electron micrographs from purified ROS (band I), obtained as described, showed intact ROS with their typical structures and no other membrane material was observed (data not shown). Purity in ROS membrane preparation was controlled also by the ratio of absorbance at 278 and 500 nm after solubilization in 70 mM potassium phosphate buffer (pH 7) containing 1% Emulphogene. Values of 2.3  0.2 were typically obtained for this ratio. In addition, sodium dodecyl sulfatepolyacrylamide gel electrophoresis was used to check the purity of membranes. Even in heavily overloaded gels, rhodopsin and its oligomers comprised 75e80% of photoreceptor membrane proteins. Moreover, thin-layer chromatography of photoreceptor membrane lipids in overloaded plates showed no cardiolipin, suggesting non-detectable contamination with mitochondria. In addition, marker enzyme activities were determined in all fractions in order to assess possible mitochondrial or microsomal contamination of ROS membranes. These results indicate that band I (purified ROS) contamination with microsomes or mitochondria was lower than 5% (Roque and Giusto, 1995). 2.3. Determination of PLD activity For the determination of PIP2-dependent PLD activity, phosphatidylcholine (PC) hydrolysis was determined by using an assay described by Brown et al. (1993) with slight

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modifications. Briefly, 50 ml of mixed lipid vesicles (phosphatidylethanolamine/PIP2/dipalmitoylphosphatidylcholine (DPPC), molar ratio 16:1.4:1) with [choline-methyl-3H]DPPC to yield 200,000 cpm per assay was added to 100 ml of ROS (100 mg of protein) in a total volume of 200 ml containing 50 mM Hepes (pH 7.5), 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2. The reaction was incubated at 37
C for 30 min and stopped by the addition of 1 ml of chloroform/methanol/concentrated HCl (50:50:0.3, v/v) and 0.35 ml of 1 M HCl/5 mM EGTA. Choline was separated from the other water-soluble products using thin layer chromatography (TLC) according to Yavin (1976). Choline was scraped off the plate and quantified by liquid scintillation spectroscopy. For measuring transphosphatidylation reaction catalyzed by PLD, assays were conducted in the presence of phosphatidylcholine, 1-palmitoyl-2 [14C]arachidonoyl-sn-glycero-3-phosphocholine, and 2% ethanol. Reactions were stopped by the addition of chloroform/methanol (2:1, v/v), mixing and centrifugation (Folch et al., 1957). Phosphatidylethanol (Peth) was separated by one-dimensional thin-layer chromatography according to a previously published method (Salvador and Giusto, 1998). Peth was scraped off the plate and quantified by liquid scintillation spectroscopy.

out either under dim red light (for DROS) or bright white light (for LROS).

2.4. SDS-PAGE and immunoblot analysis

2.7. Incubation of ROS under conditions favoring endogenous protein kinase A (PKA) activity

SDS-PAGE was performed using 7.5 or 10% gels according to Laemmli (1970). Resolved proteins were transferred to plastic backed nitrocellulose sheets (0.2 mm) or Immobilon P membranes using a Mini Trans-Blot cell electroblotter (BIO-RAD Life Science Group, California) for 1 h. Membranes were blocked overnight with Tris-buffered saline (20 mM TriseHCl, 300 mM NaCl) pH 7.5, containing 0.1% Tween 20 and 5% crystalline grade bovine serum albumin (BSA). Incubations with primary antisera were performed at room temperature for 2e3 h. Immunoreactions were detected either with horseradish peroxidase conjugated to goat antirabbit or goat anti-mouse IgG, followed by enhanced chemiluminescence substrates (ECL; Amersham Biosciences, Inc.). In some experiments, immunoblots were stripped by incubation in 200 mM TriseHCl buffer, pH 6.7, containing 100 mM b-mercaptoethanol, 2% sodium dodecyl sulfate (SDS) for 1 h at 50
C with gentle agitation. Blots were reblocked with 5% BSA in Tris-buffered saline and probed as described above. 2.5. ADP-ribosylation of ROS proteins ADP-ribosylation of bovine ROS (300 mg protein) by botulinum C3 ADP-ribosyltransferase was performed as previously described (Morelli et al., 1990) in a medium containing 50 mM TriseHCl, pH 7.4, 2 mM EDTA, 1 mM DTT, 0.5 mM ATP, and 0.5 mg purified C3 in a total volume of 200 ml at 37
C for 60 min. For ADP-ribosylation by pertussis toxin, the toxin was preactivated at 30
C for 10 min and ADP-ribosylation reaction was performed in an appropriate buffer as previously described (Pasquare´ et al., 2000; Wilde et al., 2000; Bornancin et al., 1992). Incubation was carried

2.6. Incubation of ROS under conditions favoring endogenous protein kinase C (PKC) activity The incubation mixtures for ROS phosphorylation via PKC was performed according to a method previously published by our laboratory (Roque et al., 1998). Briefly, the phosphorylation reaction contained 3 mg ROS protein, 60 mM TriseHCl buffer, pH 7.4, 10 mM KF, 0.5 mM DTT, 7.5 mM MgCl2, 2.5 mM ATP, 20 mg/ml phosphatidylserine (PS), 0.8 mg/ml 1,2-diolein, and 0.5 mM CaCl2 in a total volume of 1.72 ml. The mixture was incubated in a shaking bath at 37
C for 30 min. ROS incubated with the buffer described above but without ATP, PS, and 1,2-diolein, to which dimethyl sulfoxide (Me2SO) was added at a final concentration of 0.01%, were used as controls. To study the effect of PKC inhibitors on ROS PLD, 40 nM staurosporine instead of diolein was added to the assay medium. Staurosporine was added in Me2SO 0.1% in a volume not exceeding 0.01% of the final assay volume. The assay mixture was incubated under light conditions at 37
C for 30 min.

The incubation mixture for ROS phosphorylation by cAMP-dependent protein kinase contained 3 mg ROS protein, 60 mM Triseacetate buffer, pH 6.8, 2.5 mM ATP, 10 mM KF, 50 mM cAMP, and 2 mM MgCl2 in a total volume of 1.72 ml. The mixture was incubated at 37
C for 15 min. The corresponding controls were ROS incubated with all ingredients except ATP and cAMP to which Me2SO was added at a final concentration of 0.01%. To study the effect of protein kinase A inhibitors on ROS PLD, ROS were preincubated with 25 mM protein kinase A inhibitory protein, then cAMP was added, and the mixture was further incubated for 20 min at 37
C. Protein kinase A inhibitory protein was added in Me2SO 0.1%, in a volume not exceeding 0.01% of the final assay volume. The assay mixture was sonicated for 30 s and it was subsequently incubated for 5 min with shaking at 37
C. 2.8. Bacterial expression of GST-RhoA fusion protein pGEX III expression vector containing the mutant active form of RhoA was a gift from Dr M.J. Marinissen (Dr Gutkind’s laboratory, National Institutes of Health, Bethesda, Maryland, USA). The BL 21 Lys strain of Escherichia coli was transformed with the vector pGEX-4T3 encoding the fusion protein GST-RhoAQL. The transformed bacteria were grown in 500 ml of Luria-Bertani medium until the optical density was 0.5, at which time isopropyl-b-thiogalactopyranoside (0.1 mM final concentration) was added for 2 h. Cells were collected by centrifugation at 3000  g for 30 min and were resuspended in buffer containing 10 ml of PBS, 1% Triton X-100, 1 mM EDTA, 2 mg of aprotinin per ml, 2 mg of

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leupeptin per ml, and 1 mM PMSF. The cell suspension was sonicated for 1 min and cellular debris was removed by centrifugation at 10,000  g for 15 min. The supernatant was mixed with 300 ml of glutathione-agarose beads (Pharmacia Biotech) and was centrifuged at 3000  g for 5 min. The pellet was washed five times in a buffer containing 1 PBS, 1% Triton X-100, 1 mM EDTA, 2 mg of aprotinin per ml, 2 mg of leupeptin per ml, and 1 mM PMSF and twice in a solution containing 1 PBS, 2 mg of aprotinin per ml, 2 mg of leupeptin per ml, and 1 mM PMSF. Finally, the purified fusion protein was eluted in a buffer containing 50 mM Tris, 10 mM glutathione, 2 mg of aprotinin per ml, 2 mg of leupeptin per ml, and 1 mM PMSF. Eluates were analyzed by SDS-PAGE showing a single band coincident with the 44 kDa molecular weight. Eluates analyzed by SDS-PAGE were subjected to western blot using anti-RhoA antibody. 2.9. Statistical analysis Statistical analysis was done using Student’s t test with the values representing the mean  SD of the total number of samples indicated in each legend. 3. Results 3.1. Characterization of PLD activity from ROS and enhancement of PIP2 dependent-PLD activity by cytosolic factors In order to determine if PLD from ROS belongs to PLD isozymes family activated by small G proteins, experiments were carried out for a biochemical characterization of the enzyme. The presence of PIP2 is a requirement for small-G-protein

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activation of PLD. The dependence of PLD on PIP2 presence was examined in mixed micellar systems containing PE: PIP2: PC in a molar ratio 16:1.4:1 as published by Brown et al. (1993) using liposomes constructed with PS or phosphatidylinositol (PI) instead of PIP2 as a control. Our results demonstrate that PIP2 was effective in the activation of ROS PLD activity, stimulating the enzyme activity by 50% compared with activity levels found in the presence of acidic phospholipids such as PS or PI. When enzyme assays were carried out in the presence of oleic acid (OA), another PLD activator, no effect of this fatty acid was found on PLD activity. The specificity of PIP2 action is evidenced by the ineffectiveness of other acidic phospholipids including the closely related phosphatidylinositol (Fig. 1A). For the determination of the participation of soluble factors in ROS PLD activity regulation we followed the assay system described by Brown et al. (1993). In order to determine guaninenucleotide sensitive PLD, the enzyme activity was measured combining ROS and soluble fractions from whole retinas. Incubation of ROS resulted in a measurable accumulation of choline, in the presence of GTPgS and retinal soluble fraction. This is in accordance with previous results that show a PLD activity increased by cytosolic factors in a GTP-dependent manner (Malcolm et al., 1996; Brown et al., 1993) (Fig. 1B). 3.2. PLD activity in ROS preincubated under conditions favoring phosphorylation by endogenous PKC or PKA With the purpose of continuing with ROS PLD characterization, we subsequently explored the effect of serine/ threonine phosphorylation on enzyme activity. Whole ROS were preincubated under conditions favoring endogenous PKC and/or PKA activation. Under conditions favoring

Fig. 1. Effect of PIP2 on ROS PLD activity and enhancement by soluble factors and GTPgS. (A) The activity of PLD using as enzyme source purified ROS was determined either in the presence of 5.4 mM of the indicated phospholipids or in the presence of 1 mM oleic acid. Following incubations, water-soluble products were extracted and separated by TLC and were visualized after exposure to iodine vapor. The bands corresponding to choline were scraped and quantitated by liquid scintillation spectroscopy. The results are presented as the mean  SD of four separate experiments performed in triplicate (***p < 0.001 (PC/PIP2 condition compared with control (PC/PI)). (B) Reactions were performed with 200 mg of ROS protein for 30 min as described in Section 2, except that the concentration of MgCl2 was 1 mM. Assays also contained 50 mg of cytosolic protein and 100 mM GTPgS as indicated. The molar ratio of the lipids in the vesicles was 16:1.4:1 (PE:PIP2:PC). As 1-palmitoyl-2[14C]arachidonoyl-sn-glycero-3-phosphocholine was used as substrate, reaction mixtures contained 2% ethanol. The concentration of exogenous PC used as substrate was 8.6 mM. In this assay phosphatidylethanol (Peth) was separated and quantified as described in Section 2. Assays were performed by triplicate with at least three different ROS preparations. The results are presented as the mean  SD of four separate experiments performed in triplicate (**p < 0.01, ***p < 0.001, for comparison of the control condition vs. stimulation conditions).

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phosphorylation via endogenous PKC, phosphatidylethanol (Peth) generation was stimulated by 35% with respect to the values obtained for control conditions. The presence of the PKC inhibitor, staurosporine could completely revert the PKC stimulatory effect on PLD activity (Fig. 2A). Phosphorylation of ROS proteins via PKA inhibited PLD activity by 50% with respect to control values. This inhibition was reverted in the presence of the PKA-inhibitory protein (Fig. 2B). ROS incubated in the presence of okadaic acid (OKA) produced a 100% increase in Peth levels with respect to control conditions. These results demonstrate that the inhibition of serine/threonine phosphatases by OKA promotes the activation of ROS PLD activity (Fig. 2C). 3.3. Determination of ROS PLD activity from DROS and LROS and from ADP-ribosylated ROS by pertussis and C3 botulinum toxins Two types of bovine ROS preparations were used to test the effect of light on PLD activity. LROS and DROS were prepared from light- and dark-adapted retinas, respectively. PLD activity from DROS was 60% higher than that found in LROS. This could be indicative of the presence of any activator of PLD activity in ROS obtained from dark-adapted retinas (Fig. 3A).

For the determination of the involvement of G-protein in the enhancement of PLD activity found in DROS, we used C3 ADP-ribosyltransferase (C3) derived from Clostridium botulinum and pertussis toxin (PTx) as experimental tools. C3 toxin irreversibly inactivates all the isoforms of the small G protein, Rho, whereas PTx-induced ADP-ribosylation of transducin stabilizes this G-protein in its associated inactive state (Gtabg). Treatment of DROS with C3 resulted in a statistically significant ( p < 0.001) decrease in PLD activity in DROS (80%) whereas PLD activity from LROS was not affected by C3 toxin (Fig. 3B). The presence of GTPgS in ROS, previously ADP-ribosylated by C3 toxin, could not revert the inhibitory effect seen in PLD activity. In addition, PLD activity from PTx ADP-ribosylated DROS and LROS underwent no changes with respect to control conditions (Fig. 3C), thus indicating that transducin is not involved in the regulation of PLD in ROS from dark-adapted retinas. 3.4. Identification of the PLD1 isoform and the 24 kDa proteins as RhoA in whole bovine ROS In view of the inhibition of DROS PLD activity levels by C3 toxin, we decided to explore the type of PLD present in purified bovine ROS. We used monoclonal antibodies against

Fig. 2. ROS PLD activity under phosphorylating conditions. Purified ROS were incubated under conditions for the stimulation of PKC (A) or PKA (B) or in the presence of okadaic acid (a PP2A inhibitor) (C). PLD activity was measured in the phosphorylated ROS by measuring Peth generation as described above. The control bar represents PLD activity values obtained in ROS resuspended in buffer containing 60 mM TriseHCl, pH 7.4, 10 mM KF, 0.5 mM DTT, 7.5 mM MgCl2, and 2.5 mM ATP and lacking cofactors necessary for phosphorylation by PKC and PKA. PLD activity was assayed in the phosphorylated ROS as described in Fig. 1B. The activity of PLD is expressed as DPM Peth/mg protein. The results are presented as the mean  SD of four separate experiments performed in triplicate (*p < 0.05, **p < 0.01, ***p < 0.001).

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Fig. 3. PLD activity in LROS and DROS prepared from bovine retinas. Effect of C3 and Ptx toxins. (A) PLD activity was assayed as described in Fig. 3, using as enzyme source ROS obtained from light-exposed retinas (LROS) and from dark-adapted retinas (DROS). Peth was separated and quantitated as described in Section 2. Assays were performed in triplicate with at least three different ROS preparations. (B) DROS and LROS were ADP-ribosylated by C3 toxin as indicated in Section 2. ADP-ribosylated ROS were used for measuring PLD activity. (C) DROS and LROS were ADP-ribosylated by PTx toxin as indicated in Section 2. ADPribosylated ROS were used for measuring PLD activity. Results are shown as the mean  SD of three separate experiments performed in triplicate (*p < 0.05, **p < 0.01, ***p < 0.001).

PLD1 and PLD 2 isoforms. Rat brain microsomal lysates, where the presence of PLD1 isoform has been previously described, were used as a positive control. PLD2 antibody was not reactive with any ROS protein. We could identify a 120 kDa immunoreactive band in purified ROS in the presence of PLD1-antibody (Fig. 4A). In view of the results obtained in the presence of C3 toxin in PLD activity, we decided to investigate the presence of the small G-protein RhoA in purified ROS. In order to explore the presence of native RhoA from ROS membranes, HEK293 cells transfected with the cDNA of AU5 tagged-RhoA were used as a positive control. The presence of AU5-RhoA in HEK cell lysates was tested using an anti-AU5 antibody, and the membranes were subsequently stripped and reprobed with anti-RhoA antibody. To test the presence of RhoA in purified ROS, we resolved ROS protein lysates by SDS-PAGE and transferred to nitrocellulose, and incubated with anti-RhoA (Fig. 4B). This procedure allowed us to identify a 24 kDa immunoreactive band in purified ROS. 3.5. Localization of RhoA in DROS and in LROS Taking into account the increase in PLD activity found in DROS, we explored RhoA localization in these membranes. Immunoblot analyses of purified ROS from dark (D)- and light

(L)-adapted retinas are shown in Fig. 5. DROS were significantly enriched in the amount of RhoA over LROS. Nondetectable light-dark differences were observed between retinal homogenates (not shown). The membrane was subsequently

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Blot: anti-RhoA

Fig. 4. Presence of RhoA and PLD1 in purified ROS. (A) Immunoblot of purified ROS and a positive control (rat cerebral cortex microsomes) with antiPLD1 antibody. (B) Immunoblot with anti-RhoA (1 mg/ml) of purified ROS and transfected HEK cells (positive control). Each lane contains 80 mg of protein. (A) and (B) are representative of three separate experiments.

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A

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Fig. 5. Light-dependent association of RhoA to ROS. (A) Immunoblot of purified ROS from dark (DROS)- and light (LROS)-adapted retinas with anti RhoA (1 mg/ ml). Each lane contains 30 mg of protein. The same immunoblot was reprobed with anti-Gat (1:2000) and then reprobe with anti-opsin (1/5000). (B) Immunoblot densitometric analysis. (C) Coomassie Blue-stained gel of whole ROS obtained from dark- or light-adapted retinas.

stripped and incubated with anti-transducin (anti-Gat) antibody. Gat showed a light-driven pattern, this light-induced movement has been previously reported by several groups (Sokolov et al., 2002, 2004). The same membrane was reprobed with anti-opsin antibody showing the enrichment of opsin both in DROS and LROS (Fig. 5A). The exclusive presence of opsin both in DROS and in LROS argues in favor of the specificity of RhoA presence in ROS. Fig. 5B shows a densitometric analysis of western blots from Fig. 5A. Fig. 5C shows a Coomassie Blue-stained gel of all the fractions analyzed evidencing the presence of higher levels of arrestin in LROS with respect to DROS. The presence of arrestin was used as a marker of light adaptation. 3.6. ROS PLD activity in the presence of a recombinant constitutively active RhoA protein We evaluated the effect of a constitutively active form of RhoA protein on ROS PLD activity. We assayed the enzyme activity from DROS and LROS in the presence of the purified constitutively active RhoA (RhoAQL). RhoAQL could revert the PLD inhibition induced by light in LROS. Additionally, RhoAQL had no effect on DROS PLD activity, demonstrating that in dark-adapted retinas the native RhoA could completely activate PLD activity (Fig. 6A). In order

to determine the specificity of RhoA effect, PLD1 levels were analyzed in DROS and LROS by western blot. Fig. 6B shows that PLD1 levels were the same in DROS as well as in LROS. 4. Discussion The key biological function of ROS is generating a photoresponse, which occurs via the classic transducin-mediated pathway. In addition, there is considerable evidence about a number of other light-regulated signal transduction pathways although their complete mechanisms are not fully elucidated (Ghalayini et al., 2002; Rajala et al., 2002; Huang et al., 2000; Pasquare et al., 2000; Castagnet and Giusto, 1993; Sokolov et al., 2002, 2004). Subcellular translocation of phototransduction proteins, i.e. arrestin and transducin, in response to light has previously been detected by immunocytochemistry and by retinal serial tangential sectioning with western blotting (Sokolov et al., 2004; Elias et al., 2004; Palczewski et al., 1992). This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity (Zhang et al., 2003; Peterson et al., 2003). Our study demonstrates that the exposure of bovine retinas to light regulates RhoA association with ROS. DROS were

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Fig. 6. ROS PLD activity in the presence of a recombinant active form of RhoA. PLD activity was assayed in the presence of the fusion protein, RhoAQL (200 nM), previously loaded with GTP. PLD assay was performed using DROS and LROS as enzyme source in the presence of the GTP-loaded GST RhoA protein or in the presence of buffer loading alone (control condition). Results are shown as the mean  SD of two separate experiments performed in triplicate (***p < 0.001). (B) Immunoblot of purified ROS from dark (DROS)- and light (LROS)-adapted retinas with anti-PLD1. Each lane contains 80 mg of protein.

enriched 2.5e4-fold with RhoA over LROS. However, our results demonstrate that RhoA behaves in a manner opposite to PI3K, DAGK, and c-src as these proteins are reported to localize to ROS membranes and to be more active after light exposure (Ghalayini et al., 2002; Rajala et al., 2002; Huang et al., 2000; Giusto et al., 2000). PLD is one of the downstream effectors of RhoA signaling. We have previously described a PLD activity native to ROS membranes (Salvador and Giusto, 1998). It has been exhaustively demonstrated that PLD1 isoform is one of the downstream effectors of RhoA pathway in many tissues (Exton, 2002; Liscovitch et al., 2000; McDermott et al., 2004). In the characterization of ROS PLD activity we demonstrated the ability of exogenous PIP2 for their activation. The specificity of PIP2 action is evidenced by the ineffectiveness of other acidic phospholipids in the activation of the enzyme. It has been extensively reported that the presence of PIP2 is a strict requirement for PLD activation by small G-proteins such as ARF and RhoA (Malcolm et al., 1996; Yokozeki et al., 1996; Brown et al., 1993). Taking into account that monomeric G-proteins are soluble in its inactive state, we wondered if PIP2-dependent PLD from ROS could be activated in the presence of retinal cytosolic fractions and GTPgS (Brown et al., 1993). PLD activity was stimulated in the

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presence of GTPgS and cytosolic fraction, evidencing that the regulation of ROS PLD activity is similar to that reported for PLD1 in other experimental systems. Our western blot analyses of ROS protein using a monoclonal anti-PLD1 antibody demonstrate that PLD1 is present in bovine ROS. The presence of PLD1 in the outer segment layer from rat retina has been previously reported by immunohistochemistry (Lee et al., 2002). The distribution of Rho GTPases family proteins (Rac1, Cdc42, RhoA and RhoB) has been previously studied in chick retina (Santos-Bredariol et al., 2002). Findings from Wieland and Morelli laboratories particularly demonstrate that soluble fractions obtained from ROS may be ADP-ribosylated by C3 toxin and that exogenous recombinant RhoA has the ability of binding activated rhodopsin (Wieland et al., 1990a,b; Morelli et al., 1990). In addition, the presence of the small G protein Rac and its activation by light has been recently reported in purified ROS (Balasubramanian and Slepak, 2003). Increased PLD activity was coincident with RhoA levels in ROS obtained from dark-adapted retinas. Additionally, C3 toxin inhibited PLD activity from DROS whereas PTx did not affect enzyme activity. The same levels of PLD1 in DROS and LROS unequivocally demonstrate that decreased PLD activity in LROS is due to reduced levels of RhoA. Moreover, recombinant constitutively active RhoA could only stimulate PLD activity from LROS. Our findings demonstrate that RhoA is a positive modulator of PLD activity in DROS and that light not only diminishes PLD activity but also regulates the association of RhoA to ROS membranes. It is well known that several PKC isoforms participate in the mechanism of PLD1 activation by RhoA. The presence of a variety of kinases in ROS, such as rhodopsin kinase, PKC and PKA, has been previously reported (Thompson and Findlay, 1984; Hamm, 1990; Kapoor et al., 1987; Balasubramanian et al., 2001). Here, we show that ROS PLD is activated by PKC and that this activation is reverted by the presence of staurosporin. It has been described that phosphorylation of PLD1 by PKA inhibits the interaction between PLD and RhoA (Jang et al., 2004). In view of these findings, our further query was whether or not PKA from ROS participates in PLD modulation. The presence of cAMP inhibited ROS PLD activity whereas the PKA inhibitor peptide abolished the inhibitory action of the cyclic nucleotide. The regulation of ROS PLD activity by PKC and PKA is in accordance with the typical activation mechanism in RhoA-stimulated PLD isoforms (Exton, 2002; Liscovitch et al., 2000). Studies from other tissues involve RhoA signaling in several cellular events, such as cytoskeletal reorganization, vesicular trafficking, cell adhesion and growth factors action (Fukata et al., 2003; Nikolic, 2002; Fukata and Kaibuchi, 2001; Yuan et al., 2003). Although the physiological role of PLD1/RhoA pathway in ROS is unknown, its function could be related to the activities driven by light, such as shedding and renewal of photoreceptor membranes, light adaptation or membrane remodeling.

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