INTRODUCTION

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Several years ago, we have shown that in a cell subpopulation of human pituitary and thyroid tumors, protein kinase C (PKC) bore a point mutation at position 294, resulting in the substitution of an aspartic acid by a glycin (2, 31). The analysis of the biochemical properties of the D294G mutant and of the phenotype of embryonic fibroblasts stably transfected with it revealed a selective loss of recognition of substrates having characteristics of anchoring proteins (32) and a dramatic decrease in the dependence on serum growth factors for proliferation (3). In Rat6 fibroblasts stably transfected with human PKC (hPKC) or its mutant and treated with phorbol 12-myristate 13-acetate (PMA) for 1 h, the D294G mutant localized in the lysosome compartment (unpublished data), whereas wild-type hPKC (hPKC-wt) localized at the plasma membrane but not selectively at cell-cell contacts (3). Fibroblasts and epithelial cells are very different in many features. We therefore changed our model to the GH3B6 epithelial pituitary cell line. In this cell line, we found that PKC is selectively targeted to the cell-cell contacts upon thyrotropin-releasing hormone (TRH) or PMA stimulation (42). To our knowledge, there is only one other study reporting on the presence of PKC at the cell-cell contacts during spontaneous or PMA-induced compaction of the embryo (28). Inhibition of PKC activity blocks compaction, meaning that preventing PKC localization at the cell-cell contacts resulted in an inappropriate cellular response (28). In view of the fact that an alteration in the cell-cell contacts is a hallmark of cell transformation and since PKC might be involved in oncogenic transformation, localization of hPKC at the cell-cell contact in GH3B6 cells, with no translocation in single cells (42), stimulated our interest. The goal of the present study was therefore to understand the mechanisms underlying the targeting of wild-type hPKC to the cell-cell contact and to analyze the incidence of the D294G point mutation on hPKC localization.

Epithelial cell-cell contacts involve extremely well-organized macromolecular structures. The transmembrane core of the adherence junction (localized at cell-cell contacts) is constituted by E-cadherin, which binds -catenin, itself bound to -catenin (4, 40). The actin cytoskeleton is linked to the adherence junction through its binding to -catenin. Recently, Vasioukhin et al. have reported on the essential role of actin polymerization in the formation of adherence junction by demonstrating its role as a driving force for epithelial cell-cell adhesion (44). PKC is not an unknown actor in this dynamic process. It has indeed been shown to upregulate intercellular adhesion of -catenin-negative human colon cancer cell variants via the induction of desmosomes (43). Several of its substrates, such as vinculin, are localized at cell-cell contacts (5, 13-15, 29, 38, 45). Glycogen synthetase kinase-3, which phosphorylates -catenin (16), is itself a PKC substrate (11). Concerning PKC, besides being localized at cell-cell contacts during compaction (28), PKC is also known to interact directly or indirectly with the F-actin network. Two PKC isoforms,  and ; possess actin-binding sites, and F-actin is able to directly stimulate PKC catalytic activity (7, 30, 39).

Localization of inactive PKC is essentially cytoplasmic. When stimulated, it interacts with membranes, including the plasma membrane, through at least two different mechanisms: a direct interaction with phospholipids (in particular phosphatidylserin) or an indirect interaction via anchoring proteins such as RACK1 (receptor for activated kinase C 1). It has been suggested that a progressive decrease in the level of RACK1 is responsible for the decrease in PKC accumulation at the plasma membrane observed in the process of aging (6). RACK1 has also been suggested to be an intracellular PKC shuttling protein for PKCII (34). Although it is generally accepted that an increase in intracellular calcium concentration ([Ca²⁺]_i) is sufficient to induce PKC translocation, we have shown that this is not the case in the GH3B6 cells since translocation occurs only in contacting cells despite the similar increase in [Ca²⁺]_i registered in all stimulated cells, whether single or apposed (42). We thus hypothesized the existence of additional levels of control that drive PKC to its targeting site and further allow its accumulation. Several candidates could be involved, including RACK1 and F-actin.

We show here that cell-cell contact targeting is highly regulated since, in the presence of the D294G point mutation, hPKC accumulates at the entire plasma membrane, including the cell-cell contacts. On the basis of the lack of colocalization or coimmunoprecipitation of RACK1 with hPKC, we think RACK1 is not involved in the cell-cell contact targeting. In contrast, we present evidence for an involvement of F-actin in wild-type hPKC accumulation. Furthermore, we show that polymerization of acting at the cell-cell contacts correlates with the accumulation of -catenin at this location upon PMA treatment or long-term TRH stimulation, both partners being thus colocalized with PKC.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. PMA, histone IIIS, phalloidin, cytochalasin D, goat anti-mouse immunoglobulin M (IgM; µ-chain specific)-agarose beads, goat anti-rabbit IgG-tetramethyl rhodamine isocyanate (TRITC), and phosphatidylserine were purchased from Sigma (Saint Quentin Fallavier, France). Restriction enzymes were from Promega (Charbonnières, France). Taq DNA polymerase, Ham F-10 and horse serum were from Eurobio (Les Ulis, France). ExGen 500 (linear polyethyleneimine) and monoclonal anti-PKC antibody were from Euromedex (Souffelweyersheim, France). Fetal bovine serum was from BioWhittaker (Walkersville, Md.). Monoclonal antibody against green fluorescent protein (GFP), 1-O-n-octyl--D-glucopyranoside, anti-mouse IgG-peroxidase, Fab fragments, and chemiluminescence detection kit were from Roche Molecular Biochemicals (Indianapolis, Ind.). pEGFP-N1 plasmid was from Clontech (Palo Alto, Calif.). The cDNA clones coding for wild type (hPKC-wt) or mutant D294G (hPKC-D294G) of PKC were provided by V. Alvaro and B. I. Weinstein from Columbia Cancer Center, New York, N.Y. Protein G-agarose and anti--catenin antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). [-³²P] ATP, sheep anti-mouse immunoglobulin horseradish peroxidase-linked antibody, and membrane Hybond C-Extra were from Amersham Pharmacia Biotech (Les Ulis, France). Monoclonal anti-RACK1 antibody was purchased from Transduction Laboratories (Lexington, Ky.). Phalloidin-TRITC was from Molecular Probes (Eugene, Oreg.). Goat anti-rabbit IgG (H+L), horseradish peroxidase conjugated, was from Pierce (Rockford, Ill.). TRH was from Calbiochem (Meudon, France). Anti-mouse IgM-TRITC was from Nordic Immunological Laboratories. Goat anti-mouse IgG-TRITC was from Jackson ImmunoResearch (Marseille, France).

Construction of plasmids encoding fusion proteins. The GFP fusion proteins used in transient-transfection experiments are schematically represented in Fig. 1A. The hPKC-wt or hPKC-D294G cDNA with an EcoRI site at their 5' terminus and a KpnI site at their 3' terminus were produced by PCR using wild-type or mutant D294G hPKCcDNA subcloned into pBabe vectors as templates.

Cell culture, transfection, and observation of fusion protein localization in living cells. GH3B6 cells were cultured in Ham F10 medium supplemented with 2.5% (vol/vol) fetal bovine serum and 15% (vol/vol) horse serum, both of which were heat inactivated at 56°C for 1 h. Transient transfection of GH3B6 cells was performed with ExGen as described previously (42). The localization of fusion proteins in living cells was examined by conventional (PMA treatment) or confocal (TRH treatment) fluorescence microscopy. The confocal laser scanning microscope was equipped with an Ar/Kr laser (Odyssey XL with InterVision 1.4.1 software; Noran Instruments, Inc., Middleton, Wis.) as described by Guérineau et al. (12). At the time of observation, the culture medium was replaced by a buffer containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, and 6 mM glucose (pH 7.4).

PKC, RACK1, F-actin, and -catenin detection by immunocytochemistry. GH3B6 cells were seeded on 20-by-20-mm² coverslips in 2.5 ml of Ham F10 medium and grown for 24 h before transfection or immunocytochemistry. Cells were washed quickly three times with phosphate-buffered saline (PBS; 140 mM NaCl, 27 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄), fixed for 1 min with 3% formaldehyde (vol/vol) in PEM buffer (80 mM PIPES, 5 mM EDTA, 2 mM MgCl₂; pH 6.5), and treated for 8 additional min with 3% formaldehyde (vol/vol) in 100 mM sodium borate at pH 11. Cells were incubated for 15 min in PBS containing 0.1% (wt/vol) sodium borohydride, washed, permeabilized by incubation in PBS supplemented with 0.2% Triton X-100, washed, incubated for 30 min with TBS (10 mM Tris, 150 mM NaCl; pH 7.6) containing 1% bovine serum albumin, and washed again. Cells were then incubated overnight at 4°C with antibodies against PKC, RACK1, or -catenin (dilution, 1:100) and washed. Cells were further incubated for 60 min with phalloidin-TRITC for F-actin labeling or with the second antibody, i.e., goat anti-mouse IgG-TRITC (diluted 1:40), goat anti-mouse IgM TRITC (diluted 1:40), or goat anti-rabbit IgG TRITC (diluted 1:125) for PKC, RACK1, or -catenin immunostaining, respectively. After being washed, cells were postfixed for 15 min with 3% formaldehyde in PBS and incubated in the presence of 50 mM NH₄Cl for 10 min. Coverslips were mounted in 1,4-diazabicyclo-[2.2.2]octane at 100 mg/ml in PBS containing 50% glycerol. Subcellular localization of fluorescence was examined by conventional fluorescence microscopy.

Immunoprecipitation of hPKC-wt-GFP and hPKC-D294G-GFP. hPKC-wt-GFP or hPKC-D294G-GFP constructs were transiently transfected into GH3B6 cells. At 48 h after transfection, cells were washed in cold PBS and incubated in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.25% sodium deoxycholate; 1 mM EGTA; 1 mM phenylmethylsulfonyl fluoride [PMSF]; 1 µg of aprotinin, 10 µg of leupeptin, and 1 µg of pepstatin per ml; 1 mM sodium orthovanadate) at 4°C by gentle rocking. Cells were then scraped and collected into microcentrifuge tubes. Lysates were precleared with 20 µl of protein G beads, incubated for 10 min at 4°C by gentle rocking, and centrifuged at 14,000 × g for 10 min at 4°C. Supernatants were collected. Then, 2 mg of the cell proteins was mixed with 2 µg of GFP antibody, and the reaction mixture was incubated at 4°C overnight. Immunocomplexes were captured by adding 20 µl of protein G beads. This mixture was gently rocked at 4°C overnight. After centrifugation and washing of the beads with 800 µl of 50 mM Tris (pH 7.4) supplemented with 1 µg of aprotinin, 1 µg of leupeptin, and 1 µg of pepstatin per ml, immunocomplexes were either resuspended in 50 µl of 50 mM Tris (pH 7.4) for further kinase activity assay or in 50 µl of Laemmli buffer (19) for Western blot analysis.

PKC catalytic activity measurement. Catalytic activity of immunoprecipitated hPKC-wt-GFP or hPKC-D294G-GFP were measured with histone IIIS as a substrate. The amount of each protein used for catalytic activity assay was estimated before the assay by Western blot analysis with a GFP antibody.

Coimmunoprecipitation. At 48 h after transfection, cells transfected with hPKC-wt-GFP or hPKC-D294G-GFP were incubated for 60 min with either fresh media or 100 nM PMA. Cells were washed in cold PBS and lysed at 4°C in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl; 1% 1-O-n-octyl--D-glucopyranoside; 1 mM EGTA; 1 mM PMSF; 1 µg of aprotinin, 10 µg of leupeptin, and 1 µg of pepstatin per ml; and 1 mM sodium orthovanadate. Lysed cells were centrifuged at 14,000 × g for 10 min at 4°C. Supernatants were collected, and immunoprecipitation with anti-GFP or anti-RACK1 antibodies was performed. Briefly, the antibodies used were cross-linked to either protein G-agarose for GFP antibody or anti-mouse IgM-agarose beads for RACK1 antibody. The cross-linked antibodies (2 µg of anti-GFP or 2.5 µg of anti-RACK1) were incubated with 2 mg of cell lysates for 90 min on ice. The beads were washed thoroughly. Proteins were removed from beads by incubation for 5 min at 95°C in 20 µl of Laemmli buffer for Western blot analysis. These samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel, and transferred onto nitrocellulose membranes. Membranes were cut at approximately the migration front of the 50-kDa proteins and probed either with anti-GFP antibody (1:500) in order to detect hPKC-GFP at 103 kDa or with anti-RACK1 antibody (1:1,000) in order to detect RACK1 at 36 kDa. After being washed, the membranes were incubated for 1 h at room temperature with anti-mouse IgG-peroxidase antibody (1:4,000) for GFP staining or with anti-mouse immunoglobulin-peroxidase (1:2,000) for the detection of RACK1. Immunoreactive bands were visualized with the chemiluminescence detection kit.

Cell fractionation and Western blot analysis. Untransfected or transiently transfected GH3B6 cells were separated into soluble and membrane fractions. Cells were washed with cold PBS followed by scraping into homogenization buffer (10 mM Tris; 2 mM EDTA; 1 mM PMSF; 1 µg of aprotinin, 10 µg of leupeptin, and 1 µg of pepstatin per ml; 1 mM sodium orthovanadate). Cells were then homogenized in a glass Dounce homogenizer and centrifuged for 30 min at 14,000 rpm. Supernatants were collected; they corresponded to the soluble fractions. Pellets were resuspended in homogenization buffer supplemented with 1% (vol/vol) Nonidet P-40 and incubated for 45 min on ice. This corresponds to the membrane fractions.

Intracellular calcium concentration changes. The cytoplasmic free calcium concentration ([Ca²⁺]_i) were measured with a real-time confocal laser scanning microscope (12). Cells were visualized with a 63-by-0.9 numerical aperture achroplan water immersion objective lens (Zeiss). The larger slit (100 µm) was used, giving bright images with a 3.1-µm axial resolution. Cells were loaded with the Ca²⁺-sensitive fluorescent probe Fluo-3 by exposure to 50 µM Fluo-3 acetoxymethyl ester (Fluo-3/AM; Molecular Probes) by incubation for 30 min at 37°C in a humidified incubator. Fluo-3 was excited through a 488-nm band-pass filter, and the emitted fluorescence was collected through a 515-nm barrier filter. [Ca²⁺]_i changes were expressed as the F/F_min ratio where F_min was the minimum fluorescent intensity measured during the recording (30 images/s). Acquired data were then processed for analysis using Igor 3.14 (Wavemetrics, Inc., Lake Oswego, Oreg.) softwares. Three separate experiments were performed, and a minimum of 10 fields per experiment with both single and contacting cells were analyzed for [Ca²⁺]_i changes.

	RESULTS

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The natural D294G mutation abolishes the specific targeting of hPKC to cell-cell contacts upon PMA stimulation. In order to determine whether PKC localization is affected by the D294G mutation, we transiently transfected GH3B6 cells with expression plasmids for the two hPKC-wt-GFP and hPKC-D294G-GFP fusion proteins (Fig. 1A). We then visualized the subcellular distribution of each fusion protein in live GH3B6 cells under basal conditions or after PMA stimulation. The pattern of GFP immunofluorescence recorded in living GH3B6 cells observed under a confocal microscope reflects the spatiotemporal dynamics of translocation of hPKC-wt-GFP and hPKC-D294G-GFP. As shown in Fig. 1B, the location of both hPKC-wt-GFP and hPKC-D294G-GFP was cytoplasmic in unstimulated cells, whether isolated or apposed. Upon stimulation with 100 nM PMA for 60 min, hPKC-wt-GFP (and endogenous PKC, results already published [42]) was selectively targeted to cell-cell contacts in apposed cells and was not translocated in isolated cells, as previously shown (42). In contrast, under the same conditions, the hPKC-D294G mutant translocated uniformly, cell-cell contacts included, to the plasma membrane of stimulated cells, whether single or apposed. Therefore, whereas the D294G mutation does not affect cytoplasmic localization under basal conditions, the specific localization at the interface of apposed cells is lost after PMA activation.

View larger version (57K): [in this window] [in a new window]   FIG. 1.   The presence of the natural D294G mutation abolishes the specific targeting of hPKC at the interface between apposed cells upon PMA stimulation. (A) Schematic representation of hPKC-wt-GFP and hPKC-D294G-GFP fusion proteins with their expected molecular weights. As described in Materials and Methods, hPKC-wt and hPKC-D294G cDNA were subcloned in frame at the 5' end of the sequence encoding GFP with EcoRI and KpnI sites. The D294G point mutation is localized in the V3 hinge region of hPKC. (B) Localization of hPKC-wt-GFP (top) and hPKC-D294G-GFP (bottom) observed in transiently transfected living GH3B6 cells. In basal conditions, hPKC-wt-GFP and hPKC-D294G-GFP are both cytoplasmic. When cells are treated with 100 nM PMA for 1 h, hPKC-wt-GFP is exclusively targeted at the interface between apposed cells. In contrast, in the same conditions, hPKC-D294G-GFP is uniformly targeted at the plasma membrane of apposed cells and of isolated cells. Bar, 5 µm. (C) The GFP tag does not affect the activity of hPKC-D294G. PKC catalytic activity was assessed by measuring the incorporation of 32P from [-32P]ATP into histone IIIS substrate in the presence of 10 µg of phosphatidylserine per ml and 10 µM PMA. The experiment was performed in the presence or in the absence of 1.2 mM calcium. Results show that histone IIIS is equally phosphorylated by hPKC-wt-GFP and hPKC-D294G-GFP. Western blot analysis of immunoprecipitated hPKC-wt-GFP and hPKC-D294G-GFP (revealed with the anti-GFP antibody) indicates the amount of each protein used for PKC activity assay. Three separate experiments gave identical results.

Different mechanisms underly translocation of the wild-type and the D294G mutant forms of hPKC. In our previous work, we observed that stimulation of GH3B6 cells by TRH induces a biphasic accumulation of hPKC at the plasma membrane (42). We also provided evidence that the mechanisms which underlie the early and transient phases of translocation induced by TRH are different from those involved in the longer, second phase that follows long-term treatment with TRH or PMA. Here, we investigated whether the presence of the D294G mutation affects hPKC translocation after short-term treatment with TRH as is the case after PMA stimulation. As shown in Fig. 2A, TRH application induced the rapid and transient translocation of the wild-type protein exclusively at the interface between cells. Under the same conditions, the D294G mutant also translocated (Fig. 2B), but its translocation was also observed in single cells (Fig. 2B), the selective translocation to cell-cell contacts being lost (data not shown), as is the case with PMA treatment, and the time the D294G mutant remained at the plasma membrane was longer (93.3 versus 22.5 s) (Fig. 2C). These results suggest that the mechanisms involved in the accumulation of the D294G mutant and the wild-type form of the enzyme at the plasma membrane are different.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Time course of plasma membrane translocation of hPKC-wt-GFP and hPKC-D294G-GFP upon TRH stimulation. (A and B) GH3B6 cells expressing hPKC-wt-GFP (A) or hPKC-D294G-GFP (B) were observed with a confocal microscope immediately before and during stimulation with 100 nM TRH. Images were recorded every 2 s for 3 min. Translocation was observed for the two proteins as soon as 8 s after the beginning of stimulation. As upon PMA stimulation, hPKC-D294G-GFP lost the selective targeting to the cell-cell contacts. Indeed, as observed in this single cell (B), hPKC-D294G-GFP translocated uniformly at the plasma membrane. Although TRH-induced translocation was reversible for both proteins, hPKC-D294G-GFP remained at the plasma membrane for a longer time than the wild-type enzyme. Bar, 5 µm. (C) Duration of the translocation of hPKC-wt-GFP and hPKC-D294G-GFP and of the increase in [Ca2+]i induced by TRH stimulation. The cytosolic variations in [Ca2+]i were recorded using real-time scanning laser confocal imaging. Cells were loaded with 50 µM Fluo-3/AM for 30 min at 37°C, and the variation in F/Fmin values was calculated from the recorded fluorescence of the cells. Values are given as the mean ± the standard deviation. Duration of wild-type enzyme translocation is statistically different from that of the mutant (P < 0.005) and different from duration of [Ca2+]i increase (P < 0.0002). The duration of mutant translocation is not statistically different from duration of the [Ca2+]i rise (P > 0.8).

RACK1 is not involved in hPKC-wt or hPKC-D294G localization. It has been proposed that localization of PKC could be in part mediated by interactions with anchoring proteins, including RACK1. In order to determine whether RACK1 is the partner involved in the translocation and/or accumulation of activated PKC, we analyzed by Western blot, immunocytochemistry, and coimmunoprecipitation whether or not RACK1 could colocalize and interact with hPKC-wt and hPKC-D294G.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   RACK1 localization upon basal condition or PMA stimulation. (A) Western blot analysis of RACK1 and PKC distribution. Soluble (s) and membrane (m) fractions of untreated or PMA-treated GH3B6 cells were subjected to SDS-PAGE and Western blot using anti-RACK1 or anti-PKC antibodies. In basal conditions, RACK1 was found in soluble and membrane fractions. PMA treatment has no significant effect on RACK1 distribution, whereas it induced the expected translocation of PKC, as evidenced by the increased PKC immunoreactivity in the membrane fraction. (B and C) GH3B6 cells transfected with hPKC-wt-GFP (B, top) or hPKC-D294G-GFP (C, top) were analyzed for RACK1 localization by immunocytochemistry with an anti-RACK1 antibody (B and C, bottom) in basal conditions (left) and after 1 h of PMA treatment (right). Under basal conditions, RACK1 immunoreactivity was found mainly at the plasma membrane but not at the cell-cell contacts. PMA stimulation did not affect RACK1 localization. As already shown in Fig. 1, hPKC-wt-GFP and hPKC-D294G-GFP were cytoplasmic in basal conditions. Upon PMA stimulation, hPKC-wt-GFP translocated at the interface of apposed cells, whereas hPKC-D294G-GFP translocated uniformly at the plasma membrane. Bars, 5 µm

View larger version (39K): [in this window] [in a new window]   FIG. 4.   RACK1 interacts neither with hPKC-wt nor with hPKC-D294G. GH3B6 cells transfected with hPKC-wt-GFP or hPKC-D294G-GFP were treated or not treated with 100 nM PMA and then lysed for a coimmunoprecipitation experiment by using anti-GFP or anti-RACK1 antibodies as described in Materials and Methods. For Western blot analysis, membranes were cut at approximately the migration front of the 50-kDa proteins and probed either with anti-GFP antibody in order to detect hPKC-GFP at 103 kDa or with anti-RACK1 antibody in order to detect RACK1 at 36 kDa. (A and B) Immunoprecipitation experiment with anti-GFP antibody or with anti-RACK1 antibody. Anti-RACK1 antibody (B) or anti-GFP antibody (C) was incubated with untreated cells (lanes 1 and 5) or PMA-treated cells (lanes 3 and 7). Protein G alone was incubated with untreated cells (lanes 2 and 6) or PMA-treated cells (lanes 4 and 8). Samples were analyzed by Western blot using anti-GFP and anti-RACK1 antibodies. When RACK1 was immunoprecipitated from untreated (B, lanes 1 and 5) or PMA-treated (B, lanes 3 and 7) cells, neither hPKC-wt-GFP nor hPKC-D294G-GFP was coimmunoprecipitated. When hPKC-wt-GFP or hPKC-D294G-GFP were immunoprecipitated from untreated (C, lanes 1 and 5) or PMA-treated (C, lanes 3 and 7) cells, RACK1 was never coimmunoprecipitated. (C) Western blot analysis of RACK1 (bottom, lanes 1 to 4), hPKC-wt-GFP (top, lanes 1 and 2), and hPKC-D294G-GFP (top, lanes 3 and 4) expression in cell extracts of untreated (lanes 1 and 3) or PMA-treated (lanes 2 and 4) cells used for coimmunoprecipitation experiment.

Wild-type hPKC accumulation at cell-cell contacts depends on the reorganization of F-actin. Since there is evidence from the literature that F-actin may interact with some PKCs and modulate their catalytic activities (7, 30, 39) and since F-actin is intimately linked with the molecular complexes present at cell-cell contacts, we investigated whether endogenous F-actin participates in the localization of hPKC. To this end, F-actin was labeled with phalloidin-TRITC in cells transfected with hPKC-wt-GFP. In the absence of PMA, F-actin was found to be uniformly distributed at the plasma membrane, whereas hPKC-wt-GFP was as expected found in the cytoplasm (Fig. 5A). After 1 h of PMA stimulation, F-actin and endogenous PKC concomitantly accumulated at the plasma membrane of apposed cells. A kinetic analysis of the effect of PMA on F-actin reorganization showed that the effect started at 10 min and lasted for at least up to 1 h of treatment (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Upon PMA stimulation, the reorganization of F-actin at the cell-cell contacts is necessary for PKC accumulation at the interface between apposed cells. (A) GH3B6 cells transfected with hPKC-wt-GFP (top) were analyzed for F-actin localization by immunocytochemistry using phalloidin-TRITC (bottom) in basal conditions (left) and after 1 h of PMA treatment (right). In basal conditions, F-actin was uniformly found at the plasma membrane, whereas hPKC-wt-GFP was cytoplasmic. Upon PMA stimulation, F-actin and hPKC-wt-GFP accumulated concomitantly at the cell-cell contacts. Merged image: green, hPKC-wt-GFP; red, phalloidin-TRITC. (B) Disorganization of F-actin network. Cells preincubated with 0.5 µM cytochalasin D for 30 min (left) were fixed and labeled with phalloidin-TRITC. As expected, the F-actin network was disrupted by this drug treatment as evidenced by the presence of spots. Cells were incubated with phalloidin-TRITC for 1 h (right). In these conditions, phalloidin-TRITC staining was seen in the cells as spots, indicating that the F-actin network has been disorganized. Bars, 5 µm. (C and D) Western blot analysis, using an anti-PKC antibody, of PKC translocation and accumulation induced by PMA stimulation in GH3B6 cells incubated or not for 1 h with 105 M phalloidin (C) or for 30 min with 0.5 µM cytochalasin D (D). In basal conditions, in the presence or in the absence of phalloidin or cytochalasin D, PKC is mainly cytoplasmic. When cells are not incubated with phalloidin or cytochalasin D, PMA treatment induced the translocation and persistent accumulation (90 min) of PKC to the membrane fraction. (C) When cells are preincubated with phalloidin before PMA treatment, the PKC amount increased in the membrane fraction before decreasing. The soluble fraction recovered its basal level after 90 min of PMA treatment. As shown in panel D, similar results were obtained when cells were preincubated with cytochalasin D prior to PMA treatment.

The D294G point mutation abolishes the F-actin dependence of hPKC accumulation. The D294G point mutation abolishes the selectivity of hPKC targeting to cell-cell contacts. Considering the colocalization of F-actin with wild-type hPKC upon PMA stimulation and the effect of the F-actin network reorganization on wild-type hPKC accumulation, we analyzed the link between the F-actin network and translocation of accumulation of the hPKC-D294G mutant.

View larger version (34K): [in this window] [in a new window]   FIG. 6.   Upon PMA stimulation, the accumulation of hPKC-D294G at the plasma membrane does not depend on the reorganization of F-actin. (A) GH3B6 cells transfected with hPKC-D294G-GFP (top) were analyzed for F-actin localization by immunocytochemistry by using phalloidin-TRITC (bottom) in basal conditions (left) and after 1 h of PMA treatment (right). In basal conditions, F-actin was uniformly found at the plasma membrane, whereas hPKC-D294G-GFP was cytoplasmic. Upon PMA stimulation, F-actin accumulated at the cell-cell contacts, whereas hPKC-D294G-GFP translocated uniformly at the plasma membrane. Merged image: green, hPKC-D294G-GFP; red, phalloidin-TRITC. Bar, 5 µm. (B) Western blot analysis, using an anti-GFP antibody, of hPKC-D294G-GFP translocation and accumulation induced by PMA stimulation in GH3B6 cells incubated or not with phalloidin. The results show that the disorganization of the F-actin network affects neither the translocation nor the accumulation of hPKC-D294G-GFP.

-Catenin accumulates at cell-cell contacts upon PMA stimulation. Cadherins are proteins specialized in cell-cell adhesion and are associated with -catenin that mediates a link between cadherins and the actin-cytoskeleton via -catenin. During embryonic compaction, it has been demonstrated that -catenin and PKC are both colocalized at the cell-cell contacts of apposed cells (28). The same study provided evidence for a role of PKC in the phosphorylation of -catenin. This led us to investigate whether -catenin was colocalized with PKC at the cell-cell contacts under PMA stimulation. The endogenous -catenin and PKC localization was determined by Western blot and immunocytochemistry in basal conditions and upon PMA treatment.

View larger version (27K): [in this window] [in a new window]   FIG. 7.   Upon PMA stimulation, -catenin accumulates at the cell-cell contacts. (A) Western blot analysis of -catenin distribution. Soluble (s) and membrane (m) fractions of untreated or PMA-treated GH3B6 cells were subjected to SDS-PAGE and Western blotting using an anti--catenin antibody. In basal conditions, -catenin was found in soluble and membrane fractions. PMA treatment induced -catenin accumulation in the membrane fraction. (B and C) GH3B6 cells transfected with hPKC-wt-GFP (B, top) or hPKC-D294G-GFP (C, top) were analyzed for -catenin localization by immunocytochemistry with an anti- -catenin antibody (B and C, bottom) in basal conditions (left) and after 1 h of PMA treatment (right). In basal conditions, -catenin immunoreactivity was found in the cytoplasm and at the plasma membrane, whereas hPKC-wt-GFP and hPKC-D294G-GFP were cytoplasmic. PMA stimulation induced the redistribution of -catenin to cell-cell contacts. Merged image: green, hPKC-wt-GFP or hPKC-D294G-GFP; red, -catenin. (D) Accumulation of -catenin at the cell-cell contacts induced by PMA treatment depends on the reorganization of F-actin at the cell-cell contacts. Immunostaining of -catenin of cells treated with cytochalasin D in the absence or in the presence of 100 nM PMA. In the presence of cytochalasin D, -catenin is no more accumulated at the cell-cell contacts upon PMA stimulation. Bars, 5 µm.

Long-term TRH stimulation induces F-actin and -catenin accumulation at the cell-cell contacts. In GH3B6 cells, TRH induces a biphasic accumulation of hPKC at the plasma membrane, the second phase being abolished by deletion of the V1 region, as is the PMA-induced translocation (42). However, in order to assess whether F-actin reorganization and -catenin relocalization at the cell-cell contacts are induced upon long-term TRH treatment, as they are after PMA treatment, GH3B6 cells were treated for 2 h with 100 nM TRH. As shown in Fig. 8, this 2-h treatment had the same effects as the PMA treatment: it induced F-actin reorganization at the cell-cell contacts and induced -catenin accumulation. Therefore, long-term physiological stimulation has effects also encountered upon treatment with PMA, which is considered a pharmacological, nonphysiological PKC activator.

View larger version (36K): [in this window] [in a new window]   FIG. 8.   After 2 h of TRH stimulation, F-actin and -catenin accumulate at the cell-cell contacts. GH3B6 cells transfected with hPKC-wt-GFP were analyzed for F-actin and -catenin localization by immunocytochemistry using phalloidin-TRITC and an anti--catenin antibody. Cells were treated for 2 h with 100 nM TRH. Merged image: green, hPKC-wt-GFP; red, either F-actin (top) or -catenin (bottom).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The present study was initiated by the observation that hPKC is selectively targeted to cell-cell contacts in TRH- or PMA-treated GH3B6 cells. Since both PKC and cell adhesion are involved in complex biological processes such as development and oncogenic transformation, we considered them to be of potential importance for improving our understanding of hPKC targeting to cell-cell contacts. The results presented here and, in particular, the fact that a natural mutation of hPKC abolishes the specific accumulation of the kinase at sites of cell-cell contact, led us to consider hPKC as an active player in the control of pituitary intercellular communication via cell adhesion.

hPKC targeting at cell-cell contacts: a regulated mechanism. Targeting of a protein is a complex phenomenon that requires an understanding of its spatiotemporal dynamic. According to our previous study (42), hPKC spatiotemporal dynamic is constituted by two translocation phases upon TRH physiological stimulation: a rapid and transient phase, followed by a slow and long-lasting phase. Both phases involve different translocation mechanisms since deletion of the V1 region of hPKC abolishes the second phase without affecting the first one. Upon PMA stimulation, there is one long-lasting translocation phase which exhibits similarities with the second phase of translocation upon TRH treatment since what affects it also affects the second translocation phase upon TRH and vice versa (42). Based on the results presented here, we now know also that the selectivity of the targeting site, which is the same whatever the translocation phase and the stimulus, is highly controlled. A single point mutation localized in the hinge region of hPKC, at position 294, is sufficient to affect it. One possible explanation is that the D294G mutation, localized in the V3 region that is not directly involved in the interaction with diacylglycerol, Ca²⁺, or phosphatidylserine, specifically abolishes the interaction of PKC with one or several cytoplasmic chaperone proteins whose mission is to bring PKC to the regions of cell-cell contacts. Indeed, we have previously shown (32) that the hPKC-D294G mutant is no longer able to interact with substrates with anchoring protein properties. Also supporting this hypothesis is the fact that the hinge V3 region is involved with the C2 calcium binding region in the cytoplasmic sequestration of hPKC, probably via binding to a cytoplasmic anchoring protein (42).

Which role for calcium in hPKC targeting? Spatiotemporal localization of a protein can be devided into two events: translocation from one subcellular compartment to another and accumulation at the targeting site. It is generally accepted that translocation of conventional PKCs, among which is the  isoform, requires calcium and that calcium is sufficient to induce translocation (1, 8, 26). We have shown that, in GH3B6 cells, this is not the case since hPKC-wt does not translocate (first translocation phase) in isolated cells despite the rise in [Ca²⁺]_i as in apposed cells (42) observed upon physiological stimulation. In the present study, we provide evidence that the accumulation of the kinase at cell-cell contacts that occurs during the first phase of translocation may be also independent of the [Ca²⁺]_i. Indeed, wild-type hPKC returns to the cytoplasm despite a still-elevated calcium concentration in the cell. Under the same conditions, the D294G mutant remains at the plasma membrane and stays there as long as the [Ca²⁺]_i is high. The mutant behaves in GH3B6 cells the same way PKC behaves in rat basophilic leukemia 2H3 cells (26): translocation follows variations in intracellular calcium concentrations. PKC is a brain-specific PKC isoform. Rat basophilic leukemia 2H3 cells may not have provided the adequate binding partners for PKC to be adequately targeted the way PKC is targeted in GH3B6 cells. As suggested above, the D294G mutant probably may no longer recognize a cytoplasmic protein involved in targeting of the wild-type enzyme and, when targeting of PKC becomes independent of isoform-specific binding partners, it might only be dependent upon variations in [Ca²⁺]_i. Also, the fact that when the [Ca²⁺]_i decreases, the mutant dissociates from the plasma membrane supports its direct interaction with phospholipids. Indeed, previous studies have shown that the interaction of PKC with phospholipidic vesicles is rapidly disrupted in the presence of a calcium chelator (25). In contrast, the fact that the wild-type enzyme returns to the cytoplasm despite elevated calcium concentrations suggests a mode of interaction of PKC with the plasma membrane at the cell-cell contacts which probably involves a direct interaction with anchoring proteins even though we cannot rule out the fact that it may still require elevated [Ca²⁺]_i.

RACK1 is not an hPKC anchoring protein in GH3B6 cells. The natural mutation of hPKC located in the V3 hinge region abolishes the specific accumulation of the kinase at sites of cell-cell contact (42) by inducing hPKC targeting to the entire plasma membrane, including cell-cell contacts. As stated above, one possible explanation for this fact is that the D294G mutation specifically abolishes the interaction of PKC with one or several cytoplasmic chaperones. To test this hypothesis, we investigated the putative role of RACK1, which was the first PKC anchoring protein to be isolated (24, 33) and was subsequently shown to bind several PKC isoforms, including II, , and  (35, 36, 46). In unstimulated and long-term TRH- or PMA-stimulated GH3B6 cells, however, we could never detect RACK1 at the cell-cell contacts. In addition, we found that RACK1 and hPKC-wt, although colocalized in the cytoplasm, never coimmunoprecipitated whether cells were treated or not with PMA. Similar results were obtained with the D294G mutant, despite its colocalization with RACK1 at plasma membrane sites other than the cell-cell contacts, upon PMA treatment of cells. On the basis of these results, we concluded that RACK1 does not mediate translocation nor accumulation of either wild-type or D294G hPKC under PMA stimulation.

F-actin network is involved in accumulation of PKC at the cell-cell contacts. In the present study, we show that upon PMA treatment or long-term TRH stimulation, there is a concentration of F-actin at the cell-cell contacts of all apposed cells, whereas the translocation of PKC occurs only in a subpopulation of these cells (42). Hence, translocation of PKC is not what causes the reorganization of the F-actin network. In GH3B6 cells, PMA does not affect the [Ca²⁺]_i (unpublished results), although it induces the growth of actin filaments at the cell-cell contacts. This indicates that, unlike the synaptic terminal of retinal bipolar cells where PMA increases the growth of actin filaments only in the presence of a Ca²⁺ influx (17), the PMA-induced F-actin network reorganization may involve activation of a calcium-independent PKC. Our present study showing that the translocation of PKC is maintained in cytochalasin D- or phalloidin-treated GH3B6 cells argues against F-actin playing an active role during translocation. This contrasts with another report showing that nuclear translocation of PKC in NIH 3T3 fibroblasts depends on the integrity of the cytoskeleton (37), but it is in line with the observed PMA-induced translocation of PKC in cytochalasin D-treated C6 glioma cells (9). Interestingly, we found that long-lasting accumulation of PKC at the plasma membrane cannot occur but that the kinase returns to the cytoplasm in phalloidin- or cytochalasin D-treated GH3B6 cells. This does indicate a role of F-actin in the mechanism by which PKC remains localized at the cell-cell contacts, in the vicinity of its substrates. The interaction between PKC and F-actin at the cell-cell contacts could be direct or indirect. Like PKC (10), PKCII, and PKC (7, 30), PKC could directly interact with F-actin at the cell-cell contacts. An example of indirect interaction with F-actin is given by the cyclic-AMP-dependent protein kinase II, which is also linked to the actin cytoskeleton in both neurons (22) and non-neuron cells (21). Rather than a direct binding to F actin, in this case the kinase is linked to the cytoskeleton by the kinase anchor protein AKAP75. Furthermore, F-actin might be required to stimulate PKC activity in order for PKC to phosphorylate its substrate at the cell-cell contacts since it has been shown to directly stimulate PKC activity (39). Since when the F-actin network is disrupted the intact PKC returns to the cytoplasm, the integrity of the F-actin network could be required for PKC to be downregulated.

Which role for PKC at the cell-cell contacts? Several examples in the literature argue in favor of a biological significance of the interaction between PKC and F-actin, hence arguing in favor of a biological significance of the interaction between PKC and F-actin at the GH3B6 cell-cell contacts. Among these examples, PKC translocates to and stabilizes the actin network after stimulation by interleukin-2 (10), and this association is also required for glutamate release in PMA-treated neuronal cells (41).