January 2006
Volume 47, Issue 1
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Retinal Cell Biology  |   January 2006
Selective Relocalization and Proteasomal Downregulation of PKCα Induced by Platelet-Activating Factor in Retinal Pigment Epithelium
Author Affiliations
  • Zahra Faghiri
    From the LSU Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana.
  • Nicolas G. Bazan
    From the LSU Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana.
Investigative Ophthalmology & Visual Science January 2006, Vol.47, 397-404. doi:10.1167/iovs.05-0290
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      Zahra Faghiri, Nicolas G. Bazan; Selective Relocalization and Proteasomal Downregulation of PKCα Induced by Platelet-Activating Factor in Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2006;47(1):397-404. doi: 10.1167/iovs.05-0290.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Protein kinases C (PKCs) are key cell-signaling mediators in retinal physiology and pathophysiology. The cellular localization of PKC isoforms is important in defining their activity and specificity; the present study investigated the modulatory potential of the proinflammatory mediator platelet-activating factor (PAF) on the subcellular distribution of PKCα, β, and δ isotypes.

methods. This study used real-time visualization of green fluorescent protein fused to PKCα, β, or δ in the human retinal pigment epithelial (RPE) cell line ARPE-19.

results. In PAF-stimulated ARPE-19 cells, PKCα translocated to the plasma membrane and then colocalized with Golgi markers p230 and GM130; PKCβ translocated to the plasma membrane but not to the Golgi; and PKCδ translocated to the Golgi. Pretreatment with PKC inhibitor calphostin C abolished the PAF-induced translocation of PKCα to the plasma membrane or to the Golgi, but the Golgi inhibitor Brefeldin A only prevented the accumulation of PKCα in Golgi, without affecting its membrane relocalization. PAF promoted depletion of PKCα and δ isoforms but not that of PKCβ. Proteasome inhibitors lactacystin and MG-132 prevented the PAF-induced depletion of PKCα, but the inhibitor of lysosomal proteolysis E-64d was ineffective in rescuing PKCα.

conclusions. These results suggest that the PAF-induced downregulation of PKCα occurs principally through the proteasomal pathway. This remarkable PAF-mediated diversity in PKC translocation and downregulation highlights the significance of isotype-specific PKC activation in signaling pathways in ARPE-19 cells. These signaling events may be critical during RPE responses to oxidative stress, inflammation, and retinal degenerations, when PAF production is enhanced.

The retinal pigment epithelial (RPE) cells, derived from the neuroepithelium, comprise a monolayer of mitotically inactive cells lying between the choroidal circulation and the outer segments of the photoreceptors in retina. RPE cells contribute to the maintenance of the blood-retinal barrier, retinal adhesion, and transport of trophic factors and nutrients to the retina. In addition, these cells phagocytize the shed distal disks of photoreceptor outer segments. 1 2 RPE cells play an active role in inflammatory diseases (e.g., proliferative vitreoretinopathy after retinal injury or detachment) and in age-related macular degeneration. The potent phospholipid messenger platelet-activating factor (PAF) is involved in cell functions and pathology, including the inflammatory response, oxidative stress, and apoptosis. 3 PAF actions are mediated through a G-protein–coupled transmembrane receptor 4 that is linked to diverse signal-transduction pathways such as phospholipases C and D. 5  
Protein kinases C (PKCs), a family of serine/threonine kinases, participate in cell signaling, neurotransmission, gene expression, and cell growth and differentiation. 6 7 8 The PKC family is classified into classical PKCs (α, βI, βII, γ), which are Ca2+-dependent and diacylglycerol (DAG)-responsive; novel PKCs (δ, ε, η, θ), which are Ca2+-independent and DAG-responsive; atypical PKCs (ζ, λ/ι), which are both Ca2+- and DAG-independent; and the PKCμ/PKD subfamily (PKD1 and -2), which possess unique structural features including a nonhomologous kinase domain. Classical and novel PKCs are cellular targets for tumor-promoting phorbol esters (e.g., phorbol myristate acetate [PMA]). 6 9 Acute exposure to PMA induces the translocation to the plasma membrane and activation of cytosolic PMA-responsive PKCs, whereas prolonged incubation results in proteolytic degradation of the responsive PKCs and their depletion from the cell. 10 11 Modulation of both activation and downregulation of PKCs is important for cell function, including differentiation and carcinogenesis. 12 13  
Expression of PKCα, -βI, -βII, and -δ in human RPE cells has been linked to both physiologic and pathophysiologic responses. 14 15 PKC-mediated signal transduction may be involved in RPE cell migration, 16 and activation of PKC by phorbol esters inhibits rod outer segment phagocytosis by rat RPE cells in culture. 17 The specificity and biological activity of PKC isoforms are regulated by their subcellular localization. Thus, after activation, PKC isoforms are often translocated to other compartments. Translocation of PKC is isoform-, cell type-, and activator-specific, and is tightly regulated by various cofactors. 18 19 20 Each PKC may, therefore, display a distinct subcellular localization and bind to intracellular proteins that serve as substrates and/or carriers, such as receptors for activated C kinases, receptors for inactive C kinases, myristoylated alanine-rich C-kinase substrate, annexins, and cytoskeletal components. 21  
In the present study, by expressing the fusion proteins green fluorescent protein (GFP) and PKCα, -β, or -δ in ARPE-19 cells, we studied the modulatory potential of the proinflammatory mediator PAF by real-time visualization. We showed that PKCα translocated to the plasma membrane and later accumulated within the Golgi and colocalized with both cis- and trans-Golgi markers. PKCβ translocated only to the plasma membrane, while PKCδ translocated to the Golgi. We also studied how the lipid mediator activates degradation of PKCα. Using specific inhibitors, we showed that PKCα downregulation mediated by PAF is proteasome-dependent in ARPE-19 cells. Both diversity in translocation targeting of PKC isoforms and the mechanism of PKCα degradation may be critical during RPE responses to oxidative stress, when PAF production is enhanced. 
Methods
Antibodies and Reagents
PAF, U73122, and calphostin C were purchased from Sigma (St. Louis, MO). Monoclonal antibodies for PKCα, -β, and -δ, and for p230 and GM130, as well as peroxidase-conjugated goat anti-mouse IgG, were obtained from Transduction Laboratories (Lexington, KY). Alexa 546-conjugated goat anti-mouse antibody and BODIPY TR ceramide were purchased from Molecular Probes (Eugene, OR). Proteasome inhibitors MG-132 and lactacystin, and calpain inhibitor E-64d, were obtained from Calbiochem (La Jolla, CA). Golgi inhibitor Brefeldin-A was obtained from Epicenter (Madison, WI). PAF antagonist BN50730 was obtained from Biomol (Plymouth Meeting, PA). 
Cell Culture and Transfections
Cultures of ARPE-19 cells were maintained at 37°C in DMEM-F12 supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2. For live-cell analysis, ARPE-19 cells were grown in 35-mm Petri dishes (Corning, Corning, NY) to 60% to 80% confluence. Cells were transfected overnight with 2 μg plasmid DNA of choice and 5 μL transfection reagent (FuGene 6; Roche, Mannheim, Germany) in 2 mL tissue culture medium. All experiments were performed after 18-hour starvation of cells in DMEM-F12 containing 0.5% FBS. 
Real-Time Cell Imaging
To maintain a constant temperature of 37°C during the experimental procedures, an open-perfusion microincubator (Model PDMI-2; Medical System Corp., Greenvale, NY) was attached to the microscope stage. Cells were grown in 35-mm Petri dishes and placed in the microincubator system set at 37°C. Images were recorded at different time points on a confocal microscope (Nikon Diphot 200; Nikon, Tokyo, Japan) with a color-chilled three–charge coupled device camera (ORCA-285 IEEE 1394; Hamamatsu, Bridgewater, NJ) driven by imaging software (Metamorph Imaging Series 4.6; Universal Imaging Corporation, Downington, PA). Movies 1 to 3, were made using the same software. 
Hoechst Staining
ARPE-19 cells were grown in 35-mm Petri dishes, transfected with PKCα-GFP, and serum-starved for 18 hours. Before addition of the stimulant, cells were stained for nuclei with 2 mL Hoechst 33258 (Sigma), 10 μM in PBS, and incubated at 37°C in darkness for at least 45 minutes. The staining solution was then replaced with 2 mL fresh PBS, and the cells were examined under the microscope. 
Golgi Staining
ARPE-19 cells expressing PKC-GFP were treated for 10 to 15 minutes at 37°C with BODIPY TR ceramide (5 nmol/mL) to stain for the Golgi apparatus. Cells were then washed with and incubated in DMEM-F12 supplemented with 0.5% FBS at 37°C for 30 minutes before addition of PAF. 
Immunochemistry
ARPE-19 cells were grown in 35-mm Petri dishes, stimulated according to the experimental design, washed once with PBS, and then fixed with methanol for 6 minutes at −20°C. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. Blocking was performed with 10% bovine serum albumin (BSA) and 1% goat serum in PBS for 30 minutes at room temperature. Cells were incubated with p230 or GM130 mouse antibody (1:200) for 1 hour at room temperature. After being washed three times with 1% BSA in PBS, cells were incubated with an Alexa 546-labeled anti-mouse IgG antibody (1:500) for 30 minutes at room temperature. Cells were then washed three times, mounted in fluorescent mounting medium (Vector, Burlingame, CA), and examined under the confocal microscope. 
Preparation of Cell Lysates
ARPE-19 cells were grown in six-well plates to 100% confluence, serum-starved for 18 hours, and stimulated according to the experimental design. Cells were washed once with ice-cold PBS and collected by scraping into 100 to 300 μL cytosolic lysis buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 10 mM EDTA; 200 μM Na3VO4;10 mM NaF; 5 μg/mL leupeptin; 1 mM phenylmethylsulfonylfluoride, and 10% glycerol). Cells were further lysed with 20 strokes of a Dounce homogenizer (Kimble-Kontes, Vineland, NJ), and supernatants were collected as a cytosolic fraction after centrifugation for 30 minutes at 50,000 rpm and 4°C. The pellets were washed once with ice-cold PBS and homogenized in membrane lysis buffer (cytosolic lysis buffer containing 1% NP-40) using 20 strokes of a Dounce homogenizer. After incubation on ice for 20 minutes with interval shaking and centrifugation for 30 minutes at 50,000 rpm and 4°C, the supernatants were collected as the membrane fraction. The whole-cell lysate was prepared by scraping the cells into 100 to 300 μL membrane lysis buffer, incubation on ice for 20 minutes with shaking every 5 minutes, and centrifugation for 30 minutes at 50,000 rpm and 4°C. 
Western Blot Analysis
The protein content was estimated with protein assay reagent (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Equal amounts of cell extract protein from different conditions (10–30 μg) were loaded onto and separated by 8% to 16% SDS-PAGE ready-made gels (Invitrogen; Carlsbad, CA). On fractionation, proteins were transferred to polyvinylidene fluoride membranes (Invitrogen) in a transblot apparatus (Novex, San Diego, CA). Membranes were blocked with 5% skim milk (Bio-Rad) in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 1 hour at room temperature or overnight at 4°C. Membranes were incubated with monoclonal antibody against PKCα (1:1000), PKCβ (1:200), PKCδ (1:200), or GAPDH (1:106) for 1 hour at room temperature or overnight at 4°C followed by three washes with PBST and incubation with HRP-conjugated anti-mouse antibody for 20 minutes at room temperature. Protein bands were visualized by ECL (Amersham; Little Chalfont, Buckinghamshire, UK) and autoradiography on X-OMART AR films (Kodak, Rochester, NY). The bands were scanned and quantified by the Gel Doc system using Quantity One software (Bio-Rad). 
Results
PAF-Stimulated Translocation of PKC Isoforms to Different Subcellular Sites
To evaluate the role of PAF in translocation of PKC isoenzymes in ARPE-19 cells, PKCα-, -β-, and -δ-GFP were transiently transfected into ARPE-19 cells. As a control, the GFP plasmid without the PKC insert was also transfected in parallel. In the absence of stimuli, all three PKCs were present in the cytoplasm, and the GFP alone appeared both in the cytoplasm and nucleus. Addition of PAF (100 ng/mL) did not change the subcellular localization of GFP (data not shown), but PKCα-GFP translocated to the plasma membrane (Figs. 1A 1B)and later accumulated in a single spot in the perinuclear region (Fig. 1B , arrows; Movie 1). PKCβ-GFP translocated to the plasma membrane after treatment with PAF without accumulating in the perinuclear area (Fig. 1C ; Movie 2). PKCδ-GFP translocated to the perinuclear area (Fig. 1D , arrows; Movie 3). Thus, stimulation with PAF resulted in distinctive translocation patterns for these PKC isoenzymes. To further confirm PKCα translocation to the membrane, cytosolic and membrane expression of PKCα was investigated by Western blot analysis. In unstimulated cells, PKCα appeared as a single band at 82 kDa, mainly in the cytosol, and could only be detected as a faint band in the membrane fraction, suggesting that it is present in a very low abundance in membranes. With PAF stimulation (100 ng/mL) there was a gradually decreasing PKCα expression in the cytosolic fraction, while it greatly increased in the membrane fraction (Fig. 1F)
The PAF precursor, lyso-PAF, which is the biologically inactive form of PAF, does not activate the PAF receptor. 22 We used lyso-PAF to define the specificity of PAF-induced translocation of PKCα to the membrane. Lyso-PAF did not stimulate translocation of PKCα to the plasma membrane or Golgi apparatus (Fig. 1E) . To confirm this finding, we also performed Western blot analysis on the cytoplasmic and membrane fractions of lyso-PAF-treated cells. There was no translocation of PKCα in the cells treated with lyso-PAF (Fig. 1G)at the same time points at which PAF induced a remarkable movement of PKCα from the cytoplasm to the membrane (Fig. 1A and 1B)
Colocalization of PKCα to the Golgi
To confirm that the final accumulation of PKCα-GFP (30 minutes after stimulation with PAF) was not in the nucleus, we first performed Hoechst staining, which clearly showed the accumulation of PKCα in the perinuclear area (Fig. 2A)
To identify the perinuclear compartment to which PKCα- and -δ-GFP were localized after PAF treatment, cells were pretreated with a red fluorescent probe, BODIPY TR ceramide, to visualize the Golgi apparatus. As shown in Figure 2B , the colocalization of accumulated PKCα- and -δ-GFP was most apparent when the Golgi was visualized by dual illumination of the cells and sequential placement of green and red fluorescence filters. PKCβ did not colocalize with the Golgi (Fig. 2B) . To further elucidate the colocalization of PKCα with the Golgi, PKCα-GFP-expressing ARPE-19 cells were stimulated with PAF for 30 minutes and immunostained for Golgi-specific markers p230 and GM130. Immunostaining showed Golgi compartment association of PKCα-GFP with both cis- and trans-Golgi markers (Fig. 2C)
PKCα Translocation and Golgi Structural Integrity
Brefeldin A (BFA) causes disassembly of the Golgi in a variety of cells, 23 24 25 blocks membrane export out of the endoplasmic reticulum in vivo, 23 24 and inhibits vesicle formation both in vivo 26 and in vitro. 27 To investigate the effect of BFA on translocation of PKCα-GFP, ARPE-19 cells were pretreated with BFA and stimulated with PAF. Translocation of PKCα was then compared with that of the cells stimulated in the absence of BFA. BODIPY TR ceramide staining was also used to visualize the Golgi compartment. Pretreatment with BFA prevented the PAF-induced accumulation of PKCα-GFP in the Golgi without affecting its membrane translocation (Fig. 2D) . This suggests that disruption of the Golgi apparatus by BFA blocks the translocation of PKCα-GFP to the Golgi. 
PAF Exertion through G-Protein–Linked Cell-Surface Receptor and Phospholipase C
PAF action is mediated by a G-protein–coupled receptor. 4 To confirm that PAF-induced translocation of PKCα in ARPE-19 cells is receptor specific, PKCα-GFP–expressing cells were pretreated with PAF-receptor antagonist, BN50730, before PAF stimulation. Results were compared to those obtained from controls that were either untreated or stimulated with PAF only (Fig. 3) . PAF antagonist prevented PKCα-GFP translocation to the plasma membrane and its accumulation in the Golgi. This indicates the involvement of the PAF receptor in the pathway of PKCα translocation in ARPE-19 cells. 
Activated phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol (DAG), which mobilizes calcium from intracellular stores and activates PKC. 10 To elucidate the involvement of the PLC-DAG pathway in the PAF-induced translocation of PKC, PKCα-GFP–expressing cells were pretreated with PLC inhibitor U73122 before being stimulated with PAF. As shown in Figure 3 , stimulation of the cells with PAF in the presence of the PLC inhibitor U73122 caused the translocation of PKCα-GFP neither to the plasma membrane nor to the Golgi. This suggests that the mechanism of action of the PAF receptor is through the PLC-DAG pathway (Fig. 4)
PKCα Activity Requirement for PAF-Induced Translocation
To investigate whether the activation of the PKCα isoenzyme is associated with its translocation from the cytosol to the plasma membrane and then to the Golgi, we studied the effect of PKC-specific inhibitor calphostin C, a perulenequinone that acts on the phorbol ester-binding site of PKC. PKCα-GFP–transfected ARPE-19 cells were pretreated with calphostin C and then induced with PAF. No translocation was observed in pretreated cells compared with the cells that were not treated with calphostin C (Fig. 3) , suggesting that the activation of PKCα is required for its translocation to the plasma membrane and Golgi. 
PAF-Induced Downregulation of PKCα and PKCδ
To investigate any effect of PAF on degradation of PKCα, -β, or -δ isotypes, ARPE-19 cells were incubated with PAF (100 ng/mL), and whole-cell lysates were prepared at different times up to 18 hours to be tested by Western blotting for the presence of PKCα, -β, and -δ isotypes. PKCα was downregulated starting a few hours after stimulation (4–8 hours) and was hardly detectable by 18 hours (Fig. 5A) . There was no downregulation of PKCβ even after 18 hours of stimulation (Fig. 5B) . The decrease in PKCδ expression was observed after only 15 minutes and was more prominent at later time points (Fig. 5C) , suggesting that chronic exposure of ARPE-19 cells to PAF downregulates PKCα and PKCδ but not the β isotype. 
Proteasome Inhibitor Suppression of PAF-Induced Depletion of PKCα
To identify the pathways involved in the downregulation of PKCα in response to PAF, we first studied the effect of proteasome inhibitors on this depletion. MG132, an inhibitor of proteasome function, 28 29 and lactacystin, which is a highly selective inhibitor of proteolysis by the proteasome, 30 almost completely prevented the PAF-induced depletion of PKCα (Figs. 6A 6B , respectively). We then examined the effect of E-64d, which also inhibits calpain protease, but not the proteasome. Pretreatment with this lysosomal inhibitor had no effect on PAF-induced PKCα depletion (Fig. 6C) . These findings suggest that the proteasomal pathway is involved in the PAF-induced downregulation of PKCα. 
To determine the requirements for the activation of proteasome degradation, we first asked whether PKC activity was important for its degradation. Therefore, we investigated the effect of the PKC inhibitor calphostin C on PAF-induced PKCα downregulation. We found that calphostin C did not prevent the downregulation of PKCα (Fig. 6D) . We also investigated the effect of BFA, which reversibly disrupts the Golgi apparatus. 31 BFA pretreatment did not affect the downregulation of PKCα (Fig. 6D) . These findings suggest that the activation of PKCα and its Golgi localization are not required for its PAF-induced degradation. 
Discussion
In the present study, we demonstrated that PAF induced differential subcellular translocation of PKC isoforms. The proinflammatory lipid mediator induced the translocation of PKCα from cytosol to the plasma membrane and then to the Golgi complex in human RPE cells, ARPE-19. PAF also induced a translocation of PKCδ to the Golgi. On the other hand, PAF caused translocation of PKCβ to the plasma membrane only, without accumulation in the Golgi. Although both PKCα and PKCδ were translocated to the Golgi complex, their patterns of terminal localization on PAF activation were different. PKCα was concentrated in a well-defined area within the Golgi complex, while PKCδ accumulated diffusely around the nucleus. We also showed that the integrity of the Golgi complex was required for translocation of PKCα to the Golgi. Taken together, these differences indicate the differential PAF-induced trafficking of PKC isoforms α, β, and δ. A diversity in the pattern of translocation has been reported for PKCε in other cells. 32 Thus, members of the PKC family play subtype- and cell-specific roles in signal-transduction pathways regulated by certain activators. 
Our findings also provided evidence that PAF induced PKCα translocation through the G-protein-coupled receptor, since PAF antagonist blocked the PKC-translocating actions. Moreover, the addition of lyso-PAF, the biologically inactive form of PAF, failed to induce PKCα translocation. PAF-induced nitric oxide release via PKCα translocation in other cells is also inhibited by PAF-receptor antagonists. 33  
Focusing on isotype-specific activation of PKC in the RPE cells allowed us to discern activation, targeting, and degradation. Questions to be addressed here included whether translocation of PKCα to the Golgi is necessary for its degradation, and whether PKCα degradation depends on its activation. Experiments with PKC inhibitor calphostin C 34 and Golgi inhibitor BFA indicate that activation and translocation of PKCα are independent of its degradation, and support the hypothesis that the proteasome-mediated downregulation of PKCα is stimulated through a different pathway. Moreover, PKCα activity and translocation are independently regulated after TNF-α stimulation in other cells. 35 Furthermore, translocation of PKCδ to the Golgi is independent of its phosphorylation. 36 Translocation of PKCδ also precedes tyrosine phosphorylation, which is essential for its activation. 37 It may, therefore, be postulated that accumulation of PKCα in the Golgi of ARPE-19 cells is associated with functions not yet defined, and is not directly related to its downregulation. It is noteworthy that PKC participates in the constitutive transport of protein through the Golgi apparatus, since calphostin C, a DAG antagonist, is a potent inhibitor of export from the endoplasmic reticulum in vivo and in vitro. 38 39 Our present results further revealed that, on stimulation, the PAF receptor activated the PLC-DAG pathway, since pretreatment of ARPE-19 cells with either calphostin C or PLC inhibitor U73122 resulted in the inhibition of PKCα translocation to the plasma membrane or Golgi apparatus (Fig. 3)
The proteasome degrades many short-lived proteins when it is triggered by external stimuli. The ubiquitin-proteasome pathway is mainly responsible for the disappearance of PKCα and -ε isoforms provoked by PMA or bryostatin 1 in human fibroblasts. 40 The cellular processes as well as molecular mechanisms involved in this regulation need to be further investigated. Moreover, we showed that treatment of the ARPE-19 cells with PAF resulted in the activation and then depletion of PKCα and -δ but not -β. Our results also suggest that the downregulation of PKCα occurs principally via the proteasomal pathway. This remarkable diversity in PKC translocation and downregulation in response to PAF indicates the importance of isotype-specific functions of PKC in signal-transduction pathways in ARPE-19 cells. A subsequent step of our studies will be to define the PAF-induced PKC-specific changes in the context of oxidative stress and pathoangiogenesis. In fact, a recent study demonstrated that a novel PAF-receptor antagonist exerts neuroprotection when the retina is confronted with light-induced damage. 41 Pharmacologic modulation of specific steps mediated by the inflammatory mediator PAF may lead to novel therapeutic developments. 
 
Figure 1.
 
Differential subcellular translocation of PKCα, -β, and -δ by PAF in ARPE-19 cells. Cells were transfected with either PKCα- (A, D), -β- (B), or -δ-GFP (C) and then serum-starved for 18 hours before being stimulated with 100 ng/mL PAF (AC) or 100 nM lyso-PAF (D). Representative images were recorded immediately before and at different time points after stimulation. In a separate series of experiments, nontransfected ARPE-19 cells were serum-starved for 18 hours and then stimulated with 100 ng/mL PAF (E) or 100 nM lyso-PAF (F) as a control. Cytosol and membrane fractions were prepared at different time points and tested by Western blotting for the presence of PKCα compared with those of untreated cells (U). Bar diagrams (F, G) represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 1.
 
Differential subcellular translocation of PKCα, -β, and -δ by PAF in ARPE-19 cells. Cells were transfected with either PKCα- (A, D), -β- (B), or -δ-GFP (C) and then serum-starved for 18 hours before being stimulated with 100 ng/mL PAF (AC) or 100 nM lyso-PAF (D). Representative images were recorded immediately before and at different time points after stimulation. In a separate series of experiments, nontransfected ARPE-19 cells were serum-starved for 18 hours and then stimulated with 100 ng/mL PAF (E) or 100 nM lyso-PAF (F) as a control. Cytosol and membrane fractions were prepared at different time points and tested by Western blotting for the presence of PKCα compared with those of untreated cells (U). Bar diagrams (F, G) represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 2.
 
(A, B) PAF activation promotes the accumulation in the Golgi of PKCα and -δ but not -β. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and stained with Hoechst reagent before being stimulated with 100 ng/mL PAF (A). ARPE-19 cells were also transfected with PKCα-, -β-, or -δ-GFP and serum-starved for 18 hours, before being stained for Golgi with BODIPY TR ceramide and then stimulated with 100 ng/mL PAF (B). Images were recorded 30 minutes after stimulation. Dual illumination was recorded by sequential exposure using the two filters for green and red fluorescence. Data are representative of three experiments. (C) Colocalization of PKCα with p230 and GM130 (trans- and cis-Golgi region, respectively). Cells were transfected with PKCα-GFP and then serum-starved for 18 hours before stimulation. After 30-minute treatment with 100 ng/mL PAF, cells were fixed and immunostained for either p230 or GM130 using an Alexa 546–labeled secondary antibody. Dual illumination was used to show the colocalization of PKCα (green) and the Golgi markers (red). Data are representative of three experiments. (D) Golgi inhibitor Brefeldin A (BFA) prevented the translocation of PKCα-GFP to the Golgi but not to the membrane. ARPE-19 cells were transfected, serum-starved for 18 hours, and pretreated with BFA (10 μg/mL) for 10 minutes before stimulation with 100 ng/mL PAF for 30 minutes. Images were recorded immediately before (uninduced) and after stimulation and compared with those recorded from cells that didn’t receive any pretreatment (control). BODIPY TR ceramide (red) staining was performed to show that, unlike the control, PKCα is not localized in the Golgi area in pretreated cells. Data are representative of three experiments.
Figure 2.
 
(A, B) PAF activation promotes the accumulation in the Golgi of PKCα and -δ but not -β. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and stained with Hoechst reagent before being stimulated with 100 ng/mL PAF (A). ARPE-19 cells were also transfected with PKCα-, -β-, or -δ-GFP and serum-starved for 18 hours, before being stained for Golgi with BODIPY TR ceramide and then stimulated with 100 ng/mL PAF (B). Images were recorded 30 minutes after stimulation. Dual illumination was recorded by sequential exposure using the two filters for green and red fluorescence. Data are representative of three experiments. (C) Colocalization of PKCα with p230 and GM130 (trans- and cis-Golgi region, respectively). Cells were transfected with PKCα-GFP and then serum-starved for 18 hours before stimulation. After 30-minute treatment with 100 ng/mL PAF, cells were fixed and immunostained for either p230 or GM130 using an Alexa 546–labeled secondary antibody. Dual illumination was used to show the colocalization of PKCα (green) and the Golgi markers (red). Data are representative of three experiments. (D) Golgi inhibitor Brefeldin A (BFA) prevented the translocation of PKCα-GFP to the Golgi but not to the membrane. ARPE-19 cells were transfected, serum-starved for 18 hours, and pretreated with BFA (10 μg/mL) for 10 minutes before stimulation with 100 ng/mL PAF for 30 minutes. Images were recorded immediately before (uninduced) and after stimulation and compared with those recorded from cells that didn’t receive any pretreatment (control). BODIPY TR ceramide (red) staining was performed to show that, unlike the control, PKCα is not localized in the Golgi area in pretreated cells. Data are representative of three experiments.
Figure 3.
 
Phospholipase C (PLC) inhibitor U73122, PKC inhibitor calphostin C, and PAF antagonist BN50730 prevent the PAF-induced translocation of PKCα-GFP. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and pretreated with U73122 (10 μM), calphostin C (100 nM), or PAF antagonist, BN50730 (10 μM), for 10 minutes before being stimulated with 100 ng/mL PAF. Images were recorded 30 minutes after stimulation with PAF and compared with controls that were either untreated (uninduced) or stimulated in the absence of inhibitors (PAF only). Data are representative of three experiments.
Figure 3.
 
Phospholipase C (PLC) inhibitor U73122, PKC inhibitor calphostin C, and PAF antagonist BN50730 prevent the PAF-induced translocation of PKCα-GFP. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and pretreated with U73122 (10 μM), calphostin C (100 nM), or PAF antagonist, BN50730 (10 μM), for 10 minutes before being stimulated with 100 ng/mL PAF. Images were recorded 30 minutes after stimulation with PAF and compared with controls that were either untreated (uninduced) or stimulated in the absence of inhibitors (PAF only). Data are representative of three experiments.
Figure 4.
 
PAF regulates intracellular trafficking of PKCα in ARPE-19 cells. PAF induces the activation and translocation of PKCα to the plasma membrane and Golgi through a G-protein–coupled receptor-specific mechanism involving the PLC-DAG pathway. Proteasome-dependent downregulation of PKCα is independent of its activation and translocation to Golgi.
Figure 4.
 
PAF regulates intracellular trafficking of PKCα in ARPE-19 cells. PAF induces the activation and translocation of PKCα to the plasma membrane and Golgi through a G-protein–coupled receptor-specific mechanism involving the PLC-DAG pathway. Proteasome-dependent downregulation of PKCα is independent of its activation and translocation to Golgi.
Figure 5.
 
PAF induces downregulation of PKCα and -δ, but not -β, in ARPE-19 cells. Cells were serum-starved for 18 hours and then incubated for different times up to 18 hours in the presence of PAF (100 ng/mL). Whole-cell lysates were prepared and tested for the presence of PKCα (A), -β (B), and -δ proteins (C) by Western blotting along with those of untreated cells (U). Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 5.
 
PAF induces downregulation of PKCα and -δ, but not -β, in ARPE-19 cells. Cells were serum-starved for 18 hours and then incubated for different times up to 18 hours in the presence of PAF (100 ng/mL). Whole-cell lysates were prepared and tested for the presence of PKCα (A), -β (B), and -δ proteins (C) by Western blotting along with those of untreated cells (U). Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 6.
 
Proteasome inhibitors, but not lysosomal, PKC, or Golgi inhibitors, preserve PKCα protein from downregulation by PAF. ARPE-19 cells were serum-starved for 18 hours and then incubated for 10 minutes with 25 μM proteasome inhibitors MG-132 (A) or lactacystin (B), 25 μM lysosomal inhibitor E-64d (C), 10 μg/mL Golgi inhibitor BFA (D), or 100 nM PKC inhibitor calphostin C (D) before the addition of PAF (100 ng/mL). Whole-cell lysates were prepared at different time points and tested for the presence of PKCα compared with those of untreated (first lane) and PAF-treated only (second lane) controls. Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 6.
 
Proteasome inhibitors, but not lysosomal, PKC, or Golgi inhibitors, preserve PKCα protein from downregulation by PAF. ARPE-19 cells were serum-starved for 18 hours and then incubated for 10 minutes with 25 μM proteasome inhibitors MG-132 (A) or lactacystin (B), 25 μM lysosomal inhibitor E-64d (C), 10 μg/mL Golgi inhibitor BFA (D), or 100 nM PKC inhibitor calphostin C (D) before the addition of PAF (100 ng/mL). Whole-cell lysates were prepared at different time points and tested for the presence of PKCα compared with those of untreated (first lane) and PAF-treated only (second lane) controls. Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Supplementary Materials
Movie 1 - 13 MB (AVI) 
Translocation of PKCα-GFP to the plasma membrane and its accumulation in a dense spot after stimulation of ARPE-19 cells with platelet-activating factor (PAF). Cells were transfected with PKCα-GFP and then serum-starved for 18 hours before being stimulated with 100 ng/ml PAF. Stimulation with PAF resulted in translocation of PKCα-GFP to the cytoplasmic membrane and its later accumulation in a single spot in the perinuclear area. Note that the nuclei in untreated cells are seen as dark and oval shapes because of the lack or low abundance of PKCα-GFP in the nucleus. After stimulation with PAF, the protein translocated to the plasma membrane covering the cells, and the green fluorescence from PKCα-GFP in the plasma membrane obscures the nuclei. The relatively uniform green fluorescence pattern indicates that PKCα-GFP is located in the plasma membrane; it later accumulates in a dense spot close to the nucleus. 
Movie 2 - 15.3 MB (AVI) 
Translocation of PKCβ-GFP to the plasma membrane after stimulation of ARPE-19 cells with platelet-activating factor (PAF). Cells were transfected with PKCβ-GFP and then serum-starved for 18 hours before being stimulated with 100 ng/ml PAF. Stimulation with PAF resulted in translocation of PKCβ-GFP to the cytoplasmic membrane. Note that the nuclei in untreated cells are seen as dark and oval shapes because of the lack or low abundance of PKCβ-GFP in the nucleus. After stimulation with PAF the protein translocated to the plasma membrane covering the cells, and the green fluorescence from PKCβ-GFP in the plasma membrane obscures the nuclei. 
Movie 3 - 19.3 MB (AVI) 
Translocation of PKCγ-GFP to the perinuclear region after stimulation of ARPE-19 cells with platelet-activating factor (PAF). Cells were transfected with PKCγ-GFP and then serum-starved for 18 hours before being stimulated with 100 ng/ml PAF. Stimulation with PAF resulted in translocation of PKCγ-GFP to the perinuclear area. 
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Figure 1.
 
Differential subcellular translocation of PKCα, -β, and -δ by PAF in ARPE-19 cells. Cells were transfected with either PKCα- (A, D), -β- (B), or -δ-GFP (C) and then serum-starved for 18 hours before being stimulated with 100 ng/mL PAF (AC) or 100 nM lyso-PAF (D). Representative images were recorded immediately before and at different time points after stimulation. In a separate series of experiments, nontransfected ARPE-19 cells were serum-starved for 18 hours and then stimulated with 100 ng/mL PAF (E) or 100 nM lyso-PAF (F) as a control. Cytosol and membrane fractions were prepared at different time points and tested by Western blotting for the presence of PKCα compared with those of untreated cells (U). Bar diagrams (F, G) represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 1.
 
Differential subcellular translocation of PKCα, -β, and -δ by PAF in ARPE-19 cells. Cells were transfected with either PKCα- (A, D), -β- (B), or -δ-GFP (C) and then serum-starved for 18 hours before being stimulated with 100 ng/mL PAF (AC) or 100 nM lyso-PAF (D). Representative images were recorded immediately before and at different time points after stimulation. In a separate series of experiments, nontransfected ARPE-19 cells were serum-starved for 18 hours and then stimulated with 100 ng/mL PAF (E) or 100 nM lyso-PAF (F) as a control. Cytosol and membrane fractions were prepared at different time points and tested by Western blotting for the presence of PKCα compared with those of untreated cells (U). Bar diagrams (F, G) represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 2.
 
(A, B) PAF activation promotes the accumulation in the Golgi of PKCα and -δ but not -β. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and stained with Hoechst reagent before being stimulated with 100 ng/mL PAF (A). ARPE-19 cells were also transfected with PKCα-, -β-, or -δ-GFP and serum-starved for 18 hours, before being stained for Golgi with BODIPY TR ceramide and then stimulated with 100 ng/mL PAF (B). Images were recorded 30 minutes after stimulation. Dual illumination was recorded by sequential exposure using the two filters for green and red fluorescence. Data are representative of three experiments. (C) Colocalization of PKCα with p230 and GM130 (trans- and cis-Golgi region, respectively). Cells were transfected with PKCα-GFP and then serum-starved for 18 hours before stimulation. After 30-minute treatment with 100 ng/mL PAF, cells were fixed and immunostained for either p230 or GM130 using an Alexa 546–labeled secondary antibody. Dual illumination was used to show the colocalization of PKCα (green) and the Golgi markers (red). Data are representative of three experiments. (D) Golgi inhibitor Brefeldin A (BFA) prevented the translocation of PKCα-GFP to the Golgi but not to the membrane. ARPE-19 cells were transfected, serum-starved for 18 hours, and pretreated with BFA (10 μg/mL) for 10 minutes before stimulation with 100 ng/mL PAF for 30 minutes. Images were recorded immediately before (uninduced) and after stimulation and compared with those recorded from cells that didn’t receive any pretreatment (control). BODIPY TR ceramide (red) staining was performed to show that, unlike the control, PKCα is not localized in the Golgi area in pretreated cells. Data are representative of three experiments.
Figure 2.
 
(A, B) PAF activation promotes the accumulation in the Golgi of PKCα and -δ but not -β. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and stained with Hoechst reagent before being stimulated with 100 ng/mL PAF (A). ARPE-19 cells were also transfected with PKCα-, -β-, or -δ-GFP and serum-starved for 18 hours, before being stained for Golgi with BODIPY TR ceramide and then stimulated with 100 ng/mL PAF (B). Images were recorded 30 minutes after stimulation. Dual illumination was recorded by sequential exposure using the two filters for green and red fluorescence. Data are representative of three experiments. (C) Colocalization of PKCα with p230 and GM130 (trans- and cis-Golgi region, respectively). Cells were transfected with PKCα-GFP and then serum-starved for 18 hours before stimulation. After 30-minute treatment with 100 ng/mL PAF, cells were fixed and immunostained for either p230 or GM130 using an Alexa 546–labeled secondary antibody. Dual illumination was used to show the colocalization of PKCα (green) and the Golgi markers (red). Data are representative of three experiments. (D) Golgi inhibitor Brefeldin A (BFA) prevented the translocation of PKCα-GFP to the Golgi but not to the membrane. ARPE-19 cells were transfected, serum-starved for 18 hours, and pretreated with BFA (10 μg/mL) for 10 minutes before stimulation with 100 ng/mL PAF for 30 minutes. Images were recorded immediately before (uninduced) and after stimulation and compared with those recorded from cells that didn’t receive any pretreatment (control). BODIPY TR ceramide (red) staining was performed to show that, unlike the control, PKCα is not localized in the Golgi area in pretreated cells. Data are representative of three experiments.
Figure 3.
 
Phospholipase C (PLC) inhibitor U73122, PKC inhibitor calphostin C, and PAF antagonist BN50730 prevent the PAF-induced translocation of PKCα-GFP. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and pretreated with U73122 (10 μM), calphostin C (100 nM), or PAF antagonist, BN50730 (10 μM), for 10 minutes before being stimulated with 100 ng/mL PAF. Images were recorded 30 minutes after stimulation with PAF and compared with controls that were either untreated (uninduced) or stimulated in the absence of inhibitors (PAF only). Data are representative of three experiments.
Figure 3.
 
Phospholipase C (PLC) inhibitor U73122, PKC inhibitor calphostin C, and PAF antagonist BN50730 prevent the PAF-induced translocation of PKCα-GFP. ARPE-19 cells were transfected with PKCα-GFP, serum-starved for 18 hours, and pretreated with U73122 (10 μM), calphostin C (100 nM), or PAF antagonist, BN50730 (10 μM), for 10 minutes before being stimulated with 100 ng/mL PAF. Images were recorded 30 minutes after stimulation with PAF and compared with controls that were either untreated (uninduced) or stimulated in the absence of inhibitors (PAF only). Data are representative of three experiments.
Figure 4.
 
PAF regulates intracellular trafficking of PKCα in ARPE-19 cells. PAF induces the activation and translocation of PKCα to the plasma membrane and Golgi through a G-protein–coupled receptor-specific mechanism involving the PLC-DAG pathway. Proteasome-dependent downregulation of PKCα is independent of its activation and translocation to Golgi.
Figure 4.
 
PAF regulates intracellular trafficking of PKCα in ARPE-19 cells. PAF induces the activation and translocation of PKCα to the plasma membrane and Golgi through a G-protein–coupled receptor-specific mechanism involving the PLC-DAG pathway. Proteasome-dependent downregulation of PKCα is independent of its activation and translocation to Golgi.
Figure 5.
 
PAF induces downregulation of PKCα and -δ, but not -β, in ARPE-19 cells. Cells were serum-starved for 18 hours and then incubated for different times up to 18 hours in the presence of PAF (100 ng/mL). Whole-cell lysates were prepared and tested for the presence of PKCα (A), -β (B), and -δ proteins (C) by Western blotting along with those of untreated cells (U). Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 5.
 
PAF induces downregulation of PKCα and -δ, but not -β, in ARPE-19 cells. Cells were serum-starved for 18 hours and then incubated for different times up to 18 hours in the presence of PAF (100 ng/mL). Whole-cell lysates were prepared and tested for the presence of PKCα (A), -β (B), and -δ proteins (C) by Western blotting along with those of untreated cells (U). Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 6.
 
Proteasome inhibitors, but not lysosomal, PKC, or Golgi inhibitors, preserve PKCα protein from downregulation by PAF. ARPE-19 cells were serum-starved for 18 hours and then incubated for 10 minutes with 25 μM proteasome inhibitors MG-132 (A) or lactacystin (B), 25 μM lysosomal inhibitor E-64d (C), 10 μg/mL Golgi inhibitor BFA (D), or 100 nM PKC inhibitor calphostin C (D) before the addition of PAF (100 ng/mL). Whole-cell lysates were prepared at different time points and tested for the presence of PKCα compared with those of untreated (first lane) and PAF-treated only (second lane) controls. Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
Figure 6.
 
Proteasome inhibitors, but not lysosomal, PKC, or Golgi inhibitors, preserve PKCα protein from downregulation by PAF. ARPE-19 cells were serum-starved for 18 hours and then incubated for 10 minutes with 25 μM proteasome inhibitors MG-132 (A) or lactacystin (B), 25 μM lysosomal inhibitor E-64d (C), 10 μg/mL Golgi inhibitor BFA (D), or 100 nM PKC inhibitor calphostin C (D) before the addition of PAF (100 ng/mL). Whole-cell lysates were prepared at different time points and tested for the presence of PKCα compared with those of untreated (first lane) and PAF-treated only (second lane) controls. Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.
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