HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 15, 10174-10183, April 11, 2008

This Article Abstract Full Text (PDF) All Versions of this Article: 283/15/10174    most recent M706618200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cubells, L. Articles by Enrich, C. Search for Related Content PubMed PubMed Citation Articles by Cubells, L. Articles by Enrich, C. Social Bookmarking                      What's this?

Annexin A6-induced Inhibition of Cytoplasmic Phospholipase A₂ Is Linked to Caveolin-1 Export from the Golgi^*

Laia Cubells, Sandra Vilà de Muga, Francesc Tebar, Joseph V. Bonventre, Jesús Balsinde^¶, Albert Pol^||, Thomas Grewal^**1, and Carlos Enrich^||2

From the Departament de Biologia Cel·lular, Facultat de Medicina, and the ^||Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, 08036-Barcelona, Spain, ^**Faculty of Pharmacy, University of Sydney, Sydney, New South Wales 2006, Australia, Renal Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, and ^¶Institute of Molecular Biology and Genetics, Spanish Research Council and University of Valladolid School of Medicine, 47003 Valladolid, Spain

Received for publication, August 9, 2007 , and in revised form, January 30, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A₂ (cPLA₂). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA₂ activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca²⁺-dependent cPLA₂ inhibitor. AnxA6-mediated down-regulation of cPLA₂ activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA₂.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Annexins are a family of Ca²⁺ and membrane-binding proteins involved in membrane trafficking and various other processes including signaling, proliferation, differentiation, and inflammation (1-3). Each annexin consists of a unique N-terminal tail and a common, well conserved C-terminal core domain containing 4 (or 8 for AnxA6³) repeats of a highly homologous 70-amino acid sequence which facilitates their Ca²⁺ and phospholipid binding.

We and others showed that AnxA6 is located at the plasma membrane, in the endocytic compartment, and in caveolae. AnxA6 has been implicated in endo- and exocytic membrane trafficking pathways and regulates low density lipoprotein (LDL) receptor-mediated endocytosis (4, 5), is crucial for LDL degradation and its transport from late endosomes/pre-lysosomes to lysosomes (6), and is recruited to cholesterol-laden late endosomes (7). In addition, AnxA6 stimulates the membrane recruitment of the GTPase-activating protein p120GAP and protein kinase C to modulate the Ras signaling pathway (8, 9). These multifunctional features of AnxA6 are most probably a consequence of (a) its dynamic spatiotemporal behavior in a Ca²⁺ and/or cholesterol-dependent manner but also (b) the promiscuity to interact with a large set of other molecules (10), in particular through the flexible linker region (amino acids 320-378) which connects the N- and C-terminal AnxA6 repeats (4 repeats each).

Expression of AnxA6 and other annexins varies in cells and tissues as well as in different pathophysiological situations (1, 10). We recently compared wild type Chinese hamster ovary cells (CHOwt, contain very low AnxA6 levels) and AnxA6-deficient human epithelial carcinoma A431 cells with stable cell lines expressing high amounts of AnxA6 (CHOanx6, A431anx6). In these studies we showed that high AnxA6 levels result in an accumulation of cholesterol in the late endocytic compartment, leading to reduced amounts of cholesterol in the Golgi and the plasma membrane. This overall imbalance of cellular cholesterol is accompanied by an inhibition of caveolin export from the Golgi complex. Consequently, a significant diminution in the number of caveolae at the cell surface and a strong reduction of cholesterol efflux was observed (11). However, the molecular mechanism(s) regulating the exit of caveolin from the Golgi complex is not fully understood, and recently the participation of syntaxin 6 (12) or PTRF-cavin (13, 14) in this process has also been demonstrated.

Changes in the availability of cellular cholesterol can be directly related to the vesiculation of the Golgi apparatus. Vesiculation and tubulation events regulating membrane traffic and cargo export from the Golgi require the activity of cholesterol-dependent cPLA₂ (15). Therefore, we addressed the possibility of AnxA6 interfering with caveolin export from Golgi membranes through the inhibition of cPLA₂. In the present study we show that overexpression of AnxA6 indirectly inhibits cPLA₂ activity and its association with the Golgi complex through the reduction of cholesterol availability. In support of cPLA₂ being involved in caveolin transport, CHOwt treated with the Ca²⁺-dependent cPLA₂ inhibitor, methyl arachidonyl fluorophosphonate (MAFP), but not the Ca²⁺-independent cPLA₂ inhibitor haloenol lactone suicide substrate (HELSS), mimics the phenotype of AnxA6 overexpression and leads to an accumulation of caveolin in the Golgi complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—Nutrient Mixture Ham's F-12, DMEM, cycloheximide, water-soluble cholesterol, and saponin were from Sigma. [³H]Arachidonic acid ([³H]AA) was from Amersham Biosciences. cPLA₂ inhibitors E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), MAFP, and U18666A were from BIOMOL. Paraformaldehyde was from Electron Microscopy Sciences, and Mowiol from Calbiochem. The cloning of the green fluorescent protein (GFP)-tagged caveolin-3 (GFP-cav3) expression vector has been described previously (16). GFP-tagged T-lymphocyte maturation-associated protein (MAL; GFP-MAL) and untagged AnxA1 expression vectors were kindly provided by Dr. M. A. Alonso (Centro de Biologia Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Spain) and V. Gerke (Institute of Medical Biochemistry, University of Münster, Germany), respectively. The construction of the epidermal growth factor receptor (EGFR)-GFP fusion construct (EGFR-GFP) has been described previously (17). The construction of full-length cytosolic group IVA phospholipase A₂-GFP (GFP-cPLA₂) was described previously (18). RNAi targeting human AnxA6, GFP, and the mouse monoclonal anti-cPLA₂ (4-4B-3C; sc-454) were from Santa Cruz Biotechnology. Glutathione S-transferase (GST) and GST-AnxA6 fusion proteins were expressed in the Escherichia coli strain BL21 pLysE and purified by glutathione-Sepharose chromatography as described (6). Polyclonal anti-caveolin (C13630 [GenBank] ), anti-p120GAP, and mouse anti-GM130 were from BD Transduction Laboratories. Polyclonal anti-actin was from MP Biomedicals. Polyclonal anti--mannosidase II was kindly provided by Dr. A. Velasco (Universidad de Sevilla). Horseradish peroxidase-conjugated secondary antibodies were from Zymed Laboratories Inc.. Alexa Fluor-conjugated secondary antibodies were from Molecular Probes.

Cell Culture—CHO cells were grown in Ham's F-12, and HeLa and COS-1 cells were grown together in DMEM with 10% fetal calf serum, L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C, 5% CO₂. The generation of the stable AnxA6-overexpressing CHO cell line (CHOanx6) was described in detail (4, 6). For the intracellular accumulation of cholesterol, cells were treated for 24 h with U18666A (2 µg/ml) as described (7). For the inhibition of Ca²⁺-independent and -dependent cPLA₂ activity, cells were incubated as described with either 5 µM HELSS for 30 min or 15 µM MAFP for 60 min, respectively (15). For transient transfections, cells (40-70% confluence) were transfected with 1 µg of DNA/ml using Effectene (Qiagen) following the manufacturer's instructions.

Cycloheximide/Cholesterol Treatments—To inhibit protein synthesis in some experiments, 10 µg/ml cycloheximide (in 100 mM Hepes, pH 7.5) was added to the media for 90 min. For cholesterol addition, cells were incubated with 30 µg/ml cholesterol (premixed for 30 min in DMEM by gentle agitation) for 90 min.

RNAi-mediated Inhibition of AnxA6—To specifically knock down gene expression of human AnxA6, 1-2 x 10⁶ HeLa cells were transfected in 2 ml of medium with 10 nM AnxA6 small interfering RNA (Santa Cruz, sc-29688) and 6 µl of Lipofectamine 2000 reagent (Invitrogen) according to the instructions of the manufacturer. Experiments were conducted 72 h after transfection when the depletion of AnxA6 was more significant (see Western blot in Fig. 1C). GFP small interfering RNA was used as a negative control.

Release of [³H]AA—Cells were incubated overnight in 12 wells with 0.25 µCi/ml [³H]AA in DMEM, 0.5% fetal calf serum. Cells were washed and incubated with DMEM, 0.2% BSA for 10 min. Then the media and cells were harvested and measured by scintillation counting (15).

Pulldown Assays with Purified GST-AnxA6—Cells were incubated with and without 30 µg/ml cholesterol, placed in cold lysis buffer (50 mM Tris, 100 mM NaCl, 1% Triton X-100, 0.1 mM CaCl₂ plus protease inhibitors), scraped, and centrifuged. Then 75 µg of GST-AnxA6 and 21 µg of GST were incubated for 90 min with glutathione-Sepharose at 4 °C in phosphate-buffered saline. After washing with lysis buffer, Sepharose-bound GST-AnxA6 (and GST as control) was then incubated with 600 µg of cell extract for 2 h at 4 °C, and proteins bound to the column were collected by centrifugation and analyzed by Western blotting.

Immunoprecipitation—CHO and CHOanx6 cells grown on 100-mm dishes were transfected with pEGFP-cPLA₂. After washing in phosphate-buffered saline and solubilizing by scraping with a rubber policeman in TGH buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM HEPES, pH 7.3, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin) followed by gentle rotation for 10 min at 4 °C, lysates were then centrifuged at 14,000 x g for 10 min at 4 °C. Supernatants of transfected or not transfected cells were incubated with rabbit polyclonal anti-GFP or polyclonal anti-annexin A6 antibodies for 2 h at 4 °C and then for 60 min after the addition of protein A-Sepharose. Immunoprecipitates were washed twice in TGH supplemented with 150 mM NaCl and then once without NaCl. 10% SDS-polyacrylamide gels were used to separate proteins. Proteins were then transferred to Immobilon-P and immunoblotted using anti-annexin A6 or anti-GFP followed by the appropriate peroxidase-conjugated secondary antibody and ECL detection.

Immunofluorescence Microscopy—Cells were grown on coverslips and fixed with 4% paraformaldehyde, washed, permeabilized with 0.1% saponin, and incubated with primary and secondary antibodies as described elsewhere (19). For anti-cPLA₂ labeling, cells were fixed with cold methanol for 2 min. For double labeling with anti-cPLA₂ and anti--mannosidase II, cells were first fixed with cold methanol followed by 4% paraformaldehyde for 12 min. Finally, samples were mounted in the anti-fading media Mowiol (Calbiochem), and cells were observed using an oil immersion Plan-Apo63x/1.4 objective in an Axio-plan Zeiss microscope. Images were captured with an AxioCam HRc camera and were digitally treated with Axio-Vision 3.1 software. In some experiments a Leica TCS SL laser-scanning confocal spectral microscope (Leica Microsystems) with argon and HeNe lasers attached to a Leica DMIRE2 inverted microscope was used. Image analysis was performed with Adobe Photoshop 7.0 software. To quantify the fluorescence intensity staining, images were captured using identical contrast and exposure times. Using NIH image software (Image J), the area to be quantified (the plasma membrane) was selected, the pixel intensity was determined, and the average staining intensity (20 cells from 3 experiments) was calculated. Colocalization of cPLA₂ and -mannosidase II was quantified using the Image J program (National Institutes of Health) and the "Highlighting Colocalization" plugin (Pierre Bourdoncle, Institut Jacques Monod, Service Imagerie, Paris), which highlights the colocalized points of two images. Two points were considered to colocalize if their intensity was higher than 50%, and an image of colocalized points was generated. Then in each cell the Golgi was defined as the region of interest, and the co-localization (%) therein was calculated. In each experiment at least 50 cells per cell line were analyzed. In some experiments the colocalization of mannosidase II with transiently transfected GFP-cPLA₂ was analyzed. The transfection procedures have been described elsewhere (11).

Photobleaching Experiments and Time-lapse Confocal Microscopy—Fluorescence recovery after photobleaching (iFRAP) experiments were carried out in COS-1 cells using a Leica TCS SL laser-scanning confocal spectral microscope (Leica Microsystems) with argon and HeNe lasers attached to a Leica DMIRE2 inverted microscope equipped with an incubation system with temperature and CO₂ control (16). For visualization of GFP, images were acquired using 63x oil immersion objective lens (NA 1.32; 488 nm laser line; excitation beam splitter RSP 500, emission range detection 500-600 nm). The confocal pinhole was set at 2-3 Airy units to minimize changes in fluorescence due to GFP-tagged proteins moving away from the plane of focus. The whole cytoplasm, except the Golgi of a GFP fusion protein transfected cell, was photobleached using 50-80 scans with the 488-nm laser line at full power. Pre- and post-bleach images were monitored at 30-s intervals for 40 min. The excitation intensity was attenuated down to 5% of the half laser power to avoid significant photobleaching. The relative loss of fluorescence intensity in the unbleached region of interest and overall photobleaching in the whole cell during the time series were quantified using Image Processing Leica Confocal Software. Background fluorescence was measured in a random field outside of the cells. Fluorescence correction and normalization of GFP-tagged proteins were calculated according to Rabut and Ellenberg (20).

Isolation of Golgi Membranes—A modified method of Balch (21) described in detail by Brügger et al. (22) was used to isolate Golgi membrane fractions. All procedures were carried out at 4 °C. In brief, 4-5 x 10⁹ cells were harvested, washed twice with phosphate-buffered saline, twice with BB (breaking buffer; 0.25 M sucrose in 10 mM Tris-HCl, pH 7.4), and finally resuspended in four volumes of BB. Cells were homogenized by passing 25 times through a ball-bearing homogenizer (Balch homogenizer), which disrupts early and late endosomes, but not Golgi vesicles, and brought to a sucrose concentration of 37% (w/w) by the addition of 62% (w/w) sucrose. 14 ml of sample was overlaid with 15 ml of 35% (w/w) sucrose and 9 ml of 29% (w/w) sucrose (in 10 mM Tris-HCl, pH 7.4) and centrifuged for 2.5 h at 25,000 rpm. Typically, 2 ml of a Golgi-enriched membrane fraction was recovered at the 35-29% interphase. In some experiments, before homogenization cells were preincubated with and without cholesterol for 90 min (30 µg/ml). The purity of the isolated Golgi membranes was confirmed by Western blot analysis with markers for Golgi (TGN38, GM130, GMAP210), early and late endosomes (Rab5, Rab7), plasma membrane (Na⁺K⁺ ATPase), and endoplasmic reticulum (KDEL; data not shown).

Western Blot Analysis—CHOwt, CHOanx6, and HeLa cell lysates, samples from isolated Golgi membranes, and GST-AnxA6 pulldown assays were separated by 10% SDS-PAGE and transferred to Immobilon-P (Millipore) followed by incubation with primary antibodies and the appropriate peroxidase-conjugated secondary antibodies and ECL detection (Amersham Biosciences). Protein content was measured by the method of Lowry et al. (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
AnxA6 Expression Levels Affect Cholesterol-dependent cPLA₂ Activity—Regulation of membrane traffic and cargo export from the Golgi complex involves cPLA₂ activity (24, 25). To address whether the AnxA6-dependent reduction of cholesterol export from late endosomes (11) could be linked to cholesterol- and cPLA₂-dependent secretory pathways from the Golgi to the plasma membrane, we first compared the activity of cPLA₂ in CHOwt and CHOanx6 and measured the cellular release of radiolabeled arachidonic acid ([³H]AA) as described by Grimmer et al. (15). CHOwt express very low amounts of AnxA6 (4, 6, 8, 9), whereas AnxA6 levels in CHOanx6 are rather similar to AnxA6 expression levels in other commonly used cell lines, such as HeLa (11). As shown in Fig. 1A, both cell lines express comparable levels of cPLA₂ (Fig. 1B). Importantly, these experiments clearly showed a 1.5-2.0-fold reduction of [³H]AA release in CHOanx6 cells (p < 0.01) (Fig. 1A).

To find out whether AnxA6 down-regulation leads to increased cPLA₂ activity, HeLa cells, which express high levels of AnxA6 (11) (Fig. 1C), were transfected with RNAi targeting AnxA6 (RNAi-AnxA6), and the release of AA was measured. Upon AnxA6 knockdown, the cellular release of AA increased significantly (2.0-fold, Fig. 1C). RNAi-GFP-transfected cells were used as a control and did not show any changes in the release of AA. Then, to address if reduced cPLA₂ activity could be overcome by the addition of exogenous cholesterol in AnxA6-expressing cells, [³H]AA-labeled CHOwt and CHOanx6 cells were incubated with and without cholesterol, and the amount of released radioactivity was determined as described above (Fig. 1D). Similar to the results described in Fig. 1A, release of [³H]AA is reduced in CHOanx6 cells compared with the controls (100 ± 6 and 65 ± 7% for CHOwt and CHOanx6, respectively). In agreement with previous data (15), exogenous cholesterol increased cPLA₂ activity 1.54 ± 0.10- and 2.12 ± 0.37-fold in both cell lines, respectively (n = 3). Thus, the addition of cholesterol compensates for the inhibitory effect of AnxA6 on cPLA₂ activity in CHOanx6 cells to result in a release of [³H]AA, which is comparable with that of the controls. It is tempting to speculate that the retention of cholesterol in late endosomes of CHOanx6 cells (11) leads to an impaired supply of cholesterol for cPLA₂, thereby interfering with Golgi vesiculation.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. cPLA2 is inhibited in CHOanx6 cells. A, cells were labeled with [3H]AA overnight, washed extensively, and incubated for 10 min at 37 °C. Then the media was removed, and the radioactivity in the media and in the cells was determined. Mean values ±S.D. of three independent experiments with triplicate samples are given. **, p < 0.01 for Student's t test. B, protein levels of cPLA2 and actin were determined by Western blot analysis from CHOwt and CHOanx6 cells as indicated. C, knockdown of AnxA6 increases cPLA2 activity. HeLa cells were transfected with RNAi targeting AnxA6 (RNAi-AnxA6) as described previously (9). 72 h after transfection cells were labeled with [3H]AA overnight, washed, and incubated as described in A, and the radioactivity in the media and in the cells was determined. An increased release of [3H]AA into the media was observed after AnxA6 depletion. Western blot analysis confirmed RNAi-mediated depletion of AnxA6 at 72 h. D, cells were labeled with [3H]AA overnight as described above, washed extensively, and incubated with cholesterol for 90 min at 37 °C. Then the media was removed, and the radioactivity in the media and in the cells was determined as described above. The total [3H]AA incorporated (per 109 cells) was 0.35 ± 0.01, 0.93 ± 0.005, and 0.93 ± 0.003 nmol in HeLa, CHOwt, and CHOanx6 cells, respectively.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Cholesterol impaired translocation of cPLA2 into Golgi membranes. A, reduced cPLA2 levels in Golgi fractions isolated from CHOanx6 cells (anx6) compared with controls (wt) are shown. AnxA6 expression does not affect cPLA2 levels in whole cell lysates (WCL, A and B). B, cPLA2 levels in isolated Golgi fractions from CHOwt cells with and without U18666A were analyzed. cPLA2 is not detectable in the Golgi fraction upon U18666A treatment, indicating that cPLA2 translocation to the Golgi requires late endosomal cholesterol.

To verify and extend our findings on endogenous cPLA₂, CHOwt and CHOanx6 were transiently transfected with the full-length cPLA₂ fused to (GFP-cPLA₂) (Fig. 4). To study the localization of GFP-cPLA₂ upon elevation of Ca²⁺ or cholesterol, translocation of the GFP-cPLA₂ fusion protein was elicited by treating the cells with the Ca²⁺ ionophore ionomycin (2.5 µM for 2 min) or with cholesterol (30 µg/ml for 90 min). In untreated controls, GFP-cPLA₂ distribution was mostly cytoplasmic. However, upon Ca²⁺ increase cPLA₂ translocated from the cytoplasm to the perinuclear region including the Golgi area of both CHOwt and CHOanx6 cells. A similar pattern of translocation was previously observed (18, 26). Importantly, and similar to the endogenous cPLA₂, the addition of cholesterol also showed a translocation into the perinuclear region.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. AnxA6 inhibits translocation of cPLA2 to the Golgi. A, CHOwt (panels a-c and g-i) and CHOanx6 (panels d-f and j-l) cells were grown on coverslips, fixed, permeabilized, and immunolabeled with anti-cPLA2 and the Golgi marker anti--mannosidase II (-ManII) and analyzed by confocal microcopy as indicated. The merged images are shown. Representative differential colocalization of cPLA2 and -mannosidase II in CHOwt, but not CHOanx6, is indicated by arrows (see panels c, f, i, and l). The fluorescence intensity of the cPLA2 staining in the Golgi area (-mannosidase II-positive) of CHOwt and CHOanx6 cells was quantified. Images shown were representative for 90% of cells analyzed in each experiment (n = 3). Values are given in (%) and represent the mean ± S.D. of 3 independent experiments with 50 cells per cell line in each experiment. The same experiment was performed adding exogenous cholesterol (panels g-l). The quantification of co-localization in the Golgi region is shown in B. Areas with squares (panels h and k) indicate the increased vesiculation of the Golgi membranes. Arrows indicate colocalization in the Golgi structures. C, Western blot analysis of endogenous cPLA2 in isolated Golgi fractions from CHOwt and CHOanx6 with (+chol) or without (-chol) exogenous cholesterol. GM130 shows the enrichment of Golgi membranes in the isolated fractions compared with the whole cell lysates (WCL). Bar, 10 µm.

In support of the biochemical data (Figs. 1D and 2B), the addition of exogenous cholesterol resulted in a significant translocation of cPLA₂ to the Golgi complex (see Fig. 3A; compare panels f and l, and see quantification in Fig. 3B; Fig. 4, panels m-r), which appeared increasingly vesicular and less compact in the presence of cholesterol (compare panels b and e with h or k) in both CHOwt and CHOanx6 cells. Similarly, and as shown previously by others (27), elevation of Ca²⁺ using Ca²⁺ ionophores (ionomycin) induced translocation of cPLA₂ to the Golgi in both cell lines (data not shown). In fact, in most studies translocation is associated with increase of cPLA₂ activity (15, 28, 29). Interestingly, cholesterol-induced vesiculation of the Golgi complex most likely occurs through a mechanism involving Ca²⁺-dependent members of the cPLA₂ family (15).

However, the inhibition of cPLA₂ activity in CHOanx6 cells could also occur via a direct interaction of AnxA6 with cPLA₂ (as demonstrated for annexin A1 (30)), thereby interfering with its enzyme activity and/or translocation to Golgi membranes. To test this hypothesis, we therefore performed pulldown assays utilizing AnxA6 fused to GST (GST-AnxA6) as bait (Fig. 5A) and immunoprecipitation experiments with antibodies against AnxA6 and cPLA₂ (Fig. 5, panels B and C). In support of AnxA6 inhibiting cPLA₂ indirectly, these studies confirmed binding of established AnxA6-binding proteins, such as p120GAP and Raf-1, but did not reveal a direct interaction of AnxA6 and cPLA₂. Even the addition of exogenous cholesterol did not induce the association of cPLA₂ to AnxA6.

Protein Export from the Golgi Is Blocked in MAFP-treated Cells—Thus, in CHOanx6 cells, reduced cPLA₂ levels in the Golgi correlate with reduced cPLA₂ activity (Figs. 1 and 2) and an accumulation of cav-1 in the Golgi (11); see also Fig. 6, compare panels a and b. As mentioned above, CHOwt express only residual amounts of AnxA6 (7, 9, 11), and similar to RNAi approaches, this very low concentration of AnxA6 makes CHOwt an appropriate model system to analyze if inhibition of cPLA₂ activity per se in an AnxA6-independent manner can result in an accumulation of cav-1 in the Golgi. Therefore, CHOwt (and CHOanx6) were incubated with Ca²⁺-independent (HELSS) and Ca²⁺-dependent (MAFP) cPLA₂ inhibitors, and the staining of cav-1 was compared (Fig. 6). No differences in the subcellular distribution of cav-1 were observed in CHOwt cells after incubation with HELSS (Fig. 6, compare panels a and c). However, in CHOwt cells treated with MAFP, the cav-1 staining was more intense in the Golgi area compared with untreated CHOwt controls (98 ± 1.74% of CHOwt cells showed the accumulation of cav-1 in the Golgi) but similar to the one obtained in CHOanx6 cells (11) (Fig. 6, compare panel a with e and b).

View larger version (61K): [in this window] [in a new window]   FIGURE 4. Translocation of GFP-cPLA2 to intracellular membranes in response to Ca2+ and cholesterol in CHOanx6 cells. CHOwt and CHOanx6 cells were transfected with GFP-cPLA2. After 24 h cells were non-treated (control) (panels a-f) or treated with ionomycin (2.5 µM for 2 min) (panels g-l) or with cholesterol (30 µg/ml for 90 min) (panels m-r). Then cells were fixed and stained with anti--mannosidase II (-ManII) (in red). The rapid translocation/redistribution of GFP-cPLA2 into the Golgi membranes of cells treated with ionomycin (iono, compare panels b and h) and with cholesterol (chol, compare panels a and e with n and q) can be observed. Bar, is 10 µm.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. AnxA6 does not interact with cPLA2 but affects cholesterol-dependent cPLA2 translocation to the Golgi complex. A, pulldown experiments using GST-AnxA6 as bait with and without exogenous cholesterol (+Chol) were performed to study the potential interaction of AnxA6 with cPLA2. CHOanx6 lysates (WCL) were incubated with GST-AnxA6 for 2 h at 4 °C. Bound proteins (GST-AnxA6) were collected, separated by SDS-PAGE, and analyzed by Western blotting using antibodies to cPLA2; Raf-1 and p120GAP were used as positive control. Pulldown assays using GST alone (GST) as a negative control did not show binding of cPLA2 and p120GAP, respectively. B and C, lysates of CHOwt and CHOanx6 cells expressing GFP-cPLA2 were immunoprecipitated (IP) with anti-GFP and anti-AnxA6 antibodies. The presence of cPLA2 and AnxA6 in the immunoprecipitates was analyzed by Western blotting. NRS, normal rabbit serum.

We previously demonstrated the requirement of cholesterol for caveolin export from the Golgi (16). To validate if Ca²⁺-dependent cPLA₂ activity is involved in cav-1 export, we compared the export of caveolin from the Golgi complex of COS-1 cells with and without MAFP by inverse fluorescence recovery after photobleaching (iFRAP) experiments (16). To measure and visualize the kinetics of caveolin export and to avoid any interference of GFP-tagged cav-1 with endogenous cav-1, we monitored the localization of GFP-tagged cav-3 (GFP-cav3) (trafficking of GFP-cav-1 and GFP-cav-3 is comparable in commonly used cell lines that do not express cav-3) (16, 31). Therefore, COS-1 cells treated with and without MAFP were transfected with GFP-cav3, and the entire cytoplasm except the Golgi region of transfected cells was photobleached. Then, GFP-cav3 trafficking from the Golgi was monitored by confocal microscopy. Results from individual cells were compared, and a representative experiment is shown (Fig. 7A, for quantification see Fig. 7B). As expected, 40 min after photobleaching, 53 ± 2.23% of GFP-cav3 fluorescence disappeared from the Golgi in COS-1 control cells (mobile fraction, for quantification details see methods), but much slower kinetics for GFP-cav3 export (8 ± 2.17%, Fig. 7B) from the Golgi were observed in MAFP-treated COS-1 cells, further supporting Ca²⁺-dependent cPLA₂ proteins contributing to promote cav-1 export from the Golgi. In CHOanx6 cells, the inhibitory effect of Anx6 on Golgi export appears specific for caveolin, as kinetics for MAL, a raft-associated integral membrane protein, and EGFR, which is found in lipid rafts and clathrin-coated pits, were identical to CHOwt controls. Interestingly, in these follow-up iFRAP experiments in COS-1 cells, MAFP-mediated inhibition of cPLA₂ not only inhibited export of GFP-cav3 but also GFP-MAL and GFP-EGFR (Fig. 7B). These findings could point at cell-specific differences in CHO and COS-1 cells with respect to the regulation of Ca²⁺-dependent cPLA₂ proteins. Alternatively, cPLA₂ proteins might act as common regulators of Golgi vesiculation, and modulators, including AnxA6, might interfere only with the activity of cPLA₂ subpopulations, thereby creating specificity in the regulation of protein export from the Golgi in some cell types. However, these experiments would also allow independent pathways of AnxA6 and cPLA₂ interfering with protein transport through the Golgi complex, and future studies will have to clarify this issue.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. CHOwt (a, c, and e) and CHOanx6 cells (b, d, and f) were grown on coverslips, incubated with and without cPLA2 inhibitors (HELSS, MAFP), fixed, permeabilized, and immunolabeled with anti-cav-1 as indicated. The incubation of CHOwt cells with MAFP leads to an accumulation of cav-1 in the perinuclear region (see the arrow in e) and is similar to the cav-1 staining obtained from CHOanx6 cells under all conditions (compare e with b).

Finally, we analyzed the ability of cholesterol upon inhibition of cPLA₂ to induce vesiculation and possibly to re-establish the transport of cav-1 from the Golgi. CHOanx6 cells were treated for 2 h with MAFP with and without cholesterol for 90 min and then stained with cav-1 together with GM130 to visualize the Golgi apparatus. Fig. 8, panels a, d, and g, shows the typical pattern of cav-1 accumulation in the Golgi of CHOanx6 cells. Treatment of MAFP had no apparent effect on overall Golgi structure or the accumulation of cav-1 in the Golgi (compare panels b with e and h). The addition of exogenous cholesterol in the presence of MAFP was not sufficient to restore Golgi vesiculation (see panel h). However, when cells were preincubated with MAFP followed by extensive washing and removal of MAFP, incubation with cholesterol resulted in a dramatic vesiculation of the Golgi apparatus in CHOanx6 cells (panel k). This was accompanied by a redistribution of cav-1 from the Golgi as judged by the appearance of cav-1 staining at the plasma membrane (panel j). These results strongly suggest that the exit of cav-1 from the Golgi complex not only requires cholesterol but also functional cPLA₂. In fact, exogenous cholesterol seems to target and compete for the same population of Ca²⁺-dependent cPLA₂ proteins that is inhibited by MAFP. Therefore, the addition of cholesterol cannot re-establish the exit of caveolin from the Golgi complex when MAFP is present and Ca²⁺-dependent cPLA₂ is inhibited (Fig. 8, panel h).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Data presented in this study support our hypothesis that the concomitant sequestration of cholesterol in late endosomes together with reduced amounts of cholesterol in Golgi membranes of CHOanx6 cells (11) reduces the activity of cholesterol- and Ca²⁺-dependent cPLA₂, thereby contributing to an accumulation of cav-1 in the Golgi.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Inhibition of cPLA2 leads to an accumulation of caveolin in the Golgi. A, COS-1 cells were transfected with GFP-cav3, treated with and without MAFP as indicated, and imaged before and after photobleaching of the entire cell except for the Golgi region. The loss of fluorescence signal (iFRAP) from the Golgi was monitored for 40 min by time-lapse microscopy. Images were taken every 30 s. B, COS-1 cells were transfected with GFP-cav3, GFP-MAL, and GFP-EGFR, treated with and without MAFP as indicated, and imaged before and after photobleaching as described above. The loss of fluorescence signal (iFRAP) from the Golgi was quantified. Kinetic parameters (mobile fraction in % ±S.D.) for each protein with and without MAFP from four independent experiments are given. C, COS-1 cells were co-transfected with GFP-cav3 together with AnxA1 or empty control vector, and the export of GFP-cav3 from the Golgi was determined in iFRAP experiments as described above (for details, see "Experimental Procedures").

It has recently been identified that cholesterol-sensitive cPLA₂ activity (15, 37) correlates with the increased translocation of cPLA₂ to the Golgi apparatus in response to elevation of cellular cholesterol levels (15). Binding of cPLA₂ to Golgi membranes might then facilitate the generation of lysophospholipids, such as lysophosphatidic acid, and stimulate the vesiculation of the Golgi apparatus through the action of C-terminal-binding protein/brefeldin -ribosylated substrate (34). However, other factors like dynamin (15), the actin cytoskeleton (38), and calmodulin (39) are also involved in this process, suggesting that multifactorial protein-lipid interactions regulate Golgi vesiculation.

Taken together, mechanistically several direct/indirect events seem to converge to block the transport of caveolin in cells expressing elevated levels of AnxA6; first, AnxA6 appears to impair cholesterol transport into Golgi membranes driven by the lysosomal membrane protein NPC1 (11); second, AnxA6-induced reduction of Golgi-cholesterol leads to the loss of cPLA₂ activity through its displacement from the Golgi membranes. It should be noted that other mechanisms involving the direct binding of AnxA6 to phosphatidylserine-enriched membrane domains in lysosomes, endosomes, or the plasma membrane, thereby affecting cPLA₂ activity and/or late endosomal cholesterol transport, cannot be completely ruled out yet (40). One possible scenario might involve the membrane recruitment of AnxA6 interfering/competing with constitutive trafficking events determined by tethering and/or SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins, which eventually could arrest membrane transport (41).

View larger version (41K): [in this window] [in a new window]   FIGURE 8. Cholesterol restores the ability of cPLA2 to re-establish Golgi vesiculation in CHOanx6 cells. CHOanx6 cells were grown on coverslips and incubated with and without MAFP and cholesterol (chol) as indicated. Cells were fixed, permeabilized, and double-labeled for cav-1 and the Golgi marker GM130 (see "Experimental Procedures" for details). When cPLA2 was inhibited (panels d and g), the Golgi is compact (panels e and h), and cav-1 accumulated in the Golgi complex (panels d and g). After removing the cPLA2 inhibitor (panel j), the Golgi becomes more vesiculated (panel k), and cav-1 is not retained in the Golgi complex anymore (panel j). Relative fluorescence intensity at the plasma membrane was calculated by the Image J program (National Institutes of Health). The white and black bars relate to the average fluorescence intensity (±S.D.) of cav-1 in panel g and j, respectively. Images are representative for three independent experiments. Bar, 10 µm.

Nevertheless, for the regulation of cPLA₂ activity and localization, additional players like the lipidic environment, the actin cytoskeleton, and the direct interaction of the C2 domain of cPLA₂ with other proteins cannot be excluded. Several proteins have been shown to interact with the C2 domain of cPLA₂ in vitro, including annexins A1 and A5 (43, 44), vimentin (45), and PLIP (cPLA₂-interacting protein) (46). Nevertheless, their role in the regulation of cPLA₂ is still unclear. Future studies, in particular RNAi knockdown experiments, should help to clarify their contribution in modifying cPLA₂ activity and localization.

From the physiological point of view it is evident that the regulation of caveolin transport to the plasma membrane could have critical consequences in various cellular processes, considering the role of caveolin in the formation of caveolae at the cell surface and the importance of these structures in endocytosis, membrane microdomain formation, lipid transport (including cholesterol efflux), and cellular signaling. Most interestingly, in most cell types caveolin expression is strongly linked to the expression of PTRF-cavin, a recently identified protein that is required for caveolae formation (13, 14). In fact, caveolin is expressed in prostate cancer PC3 cells and during development of zebrafish notochord, but both model systems lack caveolae, most likely due to their lack of PTRF-cavin expression (13, 14). It is yet unknown if expression levels of other proteins are linked to caveolin function or caveolae formation. However, we recently identified a significant diminution (60%) in the number of caveolae in EGF receptors overexpressing carcinoma cells (A431) and breast cancer cell lines with high, but not low levels of AnxA6 (11). Future studies will have to clarify if levels of AnxA6 are possibly linked to caveolae formation in development and/or the potential tumor suppressor function of caveolin in some cancers such as breast cancer.

	FOOTNOTES

 
^* This study was supported by Ministerio de Educación y Ciencia, Spain Grants BFU2006-01151/BMC and GEN2003-20662 and Fellowship PR-2006-0142 (to C. E.) and National Heart Foundation of Australia, Gretl Raymond Foundation Grant G06S2559 (to T. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: tgrewal{at}pharm.usyd.edu.au.

² To whom correspondence may be addressed. E-mail: enrich{at}ub.edu.

³ The abbreviations used are: AnxA6, annexin A6; AA, arachidonic acid; AnxA1, annexin A1; cav, caveolin; CHO, Chinese hamster ovary; cPLA₂, cytoplasmic phospholipase A₂; EGFR, epidermal growth factor receptor; p120GAP, p120 GTPase-activating protein; GFP, green fluorescent protein; HELSS, E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (haloenol lactone suicide substrate); iFRAP, inverse fluorescence recovery after photobleaching; MAFP, methyl arachidonyl fluorophosphonate; wt, wild type; PTRF, polymerase I and transcript release factor; DMEM, Dulbecco's modified Eagle's medium; RNAi, RNA-mediated interference; GST, glutathione S-transferase.

	ACKNOWLEDGMENTS

 
We thank the confocal Microscopy Facility of Serveis Cientificotècmics (SCT-UB-IDIBARS) for support and advice on confocal techniques.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Raynal, P., and Pollard, H. B. (1994) Biochim. Biophys. Acta 1197, 63-93[Medline] [Order article via Infotrieve]
2. Gerke, V., Creutz, C. E., and Moss, S. E. (2005) Nat. Rev. Mol. Cell. Biol. 6, 449-461[CrossRef][Medline] [Order article via Infotrieve]
3. Grewal, T., and Enrich, C. (2006) BioEssays 28, 1211-1220[CrossRef][Medline] [Order article via Infotrieve]
4. Grewal, T., Heeren, J., Mewawala, D., Schnitgerhans, T., Wendt, D., Salomon, G., Enrich, C., Beisiegel, U., and Jackle, S. (2000) J. Biol. Chem. 275, 33806-33813[Abstract/Free Full Text]
5. Kamal, A., Ying, Y., and Anderson, R. G. (1998) J. Cell Biol. 142, 937-947[Abstract/Free Full Text]
6. Pons, M., Grewal, T., Rius, E., Schnitgerhans, T., Jackle, S., and Enrich, C. (2001) Exp. Cell Res. 269, 13-22[CrossRef][Medline] [Order article via Infotrieve]
7. de Diego, I., Schwartz, F., Siegfried, H., Dauterstedt, P., Heeren, J., Beisiegel, U., Enrich, C., and Grewal, T. (2002) J. Biol. Chem. 277, 32187-32194[Abstract/Free Full Text]
8. Rentero, C., Evans, R., Wood, P., Tebar, F., Vila de Muga, S., Cubells, L., de Diego, I., Hayes, T. E., Hughes, W. E., Pol, A., Rye, K. A., Enrich, C., and Grewal, T. (2006) Cell. Signal. 18, 1006-1016[CrossRef][Medline] [Order article via Infotrieve]
9. Grewal, T., Evans, R., Rentero, C., Tebar, F., Cubells, L., de Diego, I., Kirchhoff, M. F., Hughes, W. E., Heeren, J., Rye, K. A., Rinninger, F., Daly, R. J., Pol, A., and Enrich, C. (2005) Oncogene 24, 5809-5820[CrossRef][Medline] [Order article via Infotrieve]
10. Gerke, V., and Moss, S. E. (2002) Physiol. Rev. 82, 331-371[Abstract/Free Full Text]
11. Cubells, L., Vila de Muga, S., Tebar, F., Wood, P., Evans, R., Ingelmo-Torres, M., Calvo, M., Gaus, K., Pol, A., Grewal, T., and Enrich, C. (2007) Traffic 8, 1568-1589[CrossRef][Medline] [Order article via Infotrieve]
12. Choudhury, A., Marks, D. L., Proctor, K. M., Gould, G. W., and Pagano, R. E. (2006) Nat. Cell Biol. 8, 317-328[CrossRef][Medline] [Order article via Infotrieve]
13. Hill, M. M., Bastiani, M., Luetterforst, R., Kirkham, M., Kirkham, A., Nixon, S. J., Walser, P., Abankwa, D., Oorschot, V. M., Martin, S., Hancock, J. F., and Parton, R. G. (2008) Cell 132, 113-124[Medline] [Order article via Infotrieve]
14. Liu, L., and Pilch, P. F. (2007) J. Biol. Chem. 283, 4314-4322[CrossRef][Medline] [Order article via Infotrieve]
15. Grimmer, S., Ying, M., Walchli, S., van Deurs, B., and Sandvig, K. (2005) Traffic 6, 144-156[CrossRef][Medline] [Order article via Infotrieve]
16. Pol, A., Martin, S., Fernandez, M. A., Ingelmo-Torres, M., Ferguson, C., Enrich, C., and Parton, R. G. (2005) Mol. Biol. Cell 16, 2091-2105[Abstract/Free Full Text]
17. Llado, A., Tebar, F., Calvo, M., Moreto, J., Sorkin, A., and Enrich, C. (2004) Mol. Biol. Cell 15, 4877-4891[Abstract/Free Full Text]
18. Casas, J., Gijon, M. A., Vigo, A. G., Crespo, M. S., Balsinde, J., and Balboa, M. A. (2006) J. Biol. Chem. 281, 6106-6116[Abstract/Free Full Text]
19. Pons, M., Ihrke, G., Koch, S., Biermer, M., Pol, A., Grewal, T., Jackle, S., and Enrich, C. (2000) Exp. Cell Res. 257, 33-47[CrossRef][Medline] [Order article via Infotrieve]
20. Rabut, G., and Ellenberg, J. (2005) in Live Imaging: A Laboratory Manual (Goldman, R. D., and Spector, D. L., eds) pp. 101-126, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
21. Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416[CrossRef][Medline] [Order article via Infotrieve]
22. Brügger, B., Sandhoff, R., Wegehingel, S., Gorgas, K., Malsam, J., Helms, J. B., Lehmann, W. D., Nickel, W., and Wieland, F. T. (2000) J. Cell Biol. 151, 507-518[Abstract/Free Full Text]
23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
24. de Figueiredo, P., Drecktrah, D., Polizotto, R. S., Cole, N. B., Lippincott-Schwartz, J., and Brown, W. J. (2000) Traffic 1, 504-511[CrossRef][Medline] [Order article via Infotrieve]
25. de Figueiredo, P., Drecktrah, D., Katzenellenbogen, J. A., Strang, M., and Brown, W. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8642-8647[Abstract/Free Full Text]
26. Ghosh, M., Loper, R., Ghomashchi, F., Tucker, D. E., Bonventre, J. V., Gelb, M. H., and Leslie, C. C. (2007) J. Biol. Chem. 282, 11676-11686[Abstract/Free Full Text]
27. Evans, J. H., and Leslie, C. C. (2004) J. Biol. Chem. 279, 6005-6016[Abstract/Free Full Text]
28. Pettus, B. J., Bielawska, A., Subramanian, P., Wijesinghe, D. S., Maceyka, M., Leslie, C. C., Evans, J. H., Freiberg, J., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2004) J. Biol. Chem. 279, 11320-11326[Abstract/Free Full Text]
29. Herbert, S. P., Ponnambalam, S., and Walker, J. H. (2005) Mol. Biol. Cell 16, 3800-3809[Abstract/Free Full Text]
30. Kim, S. W., Rhee, H. J., Ko, J., Kim, Y. J., Kim, H. G., Yang, J. M., Choi, E. C., and Na, D. S. (2001) J. Biol. Chem. 276, 15712-15719[Abstract/Free Full Text]
31. Roy, S., Luetterforst, R., Harding, A., Apolloni, A., Etheridge, M., Stang, E., Rolls, B., Hancock, J. F., and Parton, R. G. (1999) Nat. Cell Biol. 1, 98-105[CrossRef][Medline] [Order article via Infotrieve]
32. Kim, S., Ko, J., Kim, J. H., Choi, E. C., and Na, D. S. (2001) FEBS Lett. 489, 243-248[CrossRef][Medline] [Order article via Infotrieve]
33. White, I. J., Bailey, L. M., Aghakhani, M. R., Moss, S. E., and Futter, C. E. (2006) EMBO J. 25, 1-12[CrossRef][Medline] [Order article via Infotrieve]
34. Brown, W. J., Chambers, K., and Doody, A. (2003) Traffic 4, 214-221[Medline] [Order article via Infotrieve]
35. Wang, Y., Thiele, C., and Huttner, W. B. (2000) Traffic 1, 952-962[CrossRef][Medline] [Order article via Infotrieve]
36. Stüven, E., Porat, A., Shimron, F., Fass, E., Kaloyanova, D., Brugger, B., Wieland, F. T., Elazar, Z., and Helms, J. B. (2003) J. Biol. Chem. 278, 53112-53122[Abstract/Free Full Text]
37. Klapisz, E., Masliah, J., Bereziat, G., Wolf, C., and Koumanov, K. S. (2000) J. Lipid Res. 41, 1680-1688[Abstract/Free Full Text]
38. Egea, G., Lazaro-Dieguez, F., and Vilella, M. (2006) Curr. Opin. Cell Biol. 18, 168-178[CrossRef][Medline] [Order article via Infotrieve]
39. Tebar, F., Villalonga, P., Sorkina, T., Agell, N., Sorkin, A., and Enrich, C. (2002) Mol. Biol. Cell 13, 2057-2068[Abstract/Free Full Text]
40. Yeung, T., Gilbert, G. E., Shi, J., Silvius, J., Kapus, A., and Grinstein, S. (2008) Science 319, 210-213[Abstract/Free Full Text]
41. Sehgal, P. B., Mukhopadhyay, S., Xu, F., Patel, K., and Shah, M. (2007) Am. J. Physiol. Lung Cell. Mol. Physiol. 292, 1526-1542[CrossRef]
42. Maxfield, F. R., and Wustner, D. (2002) J. Clin. Investig. 110, 891-898[CrossRef][Medline] [Order article via Infotrieve]
43. Solito, E., Raguenes-Nicol, C., de Coupade, C., Bisagni-Faure, A., and Russo-Marie, F. (1998) Br. J. Pharmacol. 124, 1675-1683[CrossRef][Medline] [Order article via Infotrieve]
44. Buckland, A. G., and Wilton, D. C. (1998) Biochem. J. 329, 369-372[Medline] [Order article via Infotrieve]
45. Nakatani, Y., Tanioka, T., Sunaga, S., Murakami, M., and Kudo, I. (2000) J. Biol. Chem. 275, 1161-1168[Abstract/Free Full Text]
46. Sheridan, A. M., Force, T., Yoon, H. J., O'Leary, E., Choukroun, G., Taheri, M. R., and Bonventre, J. V. (2001) Mol. Cell. Biol. 21, 4470-4481[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Monastyrskaya, E. B. Babiychuk, A. Hostettler, P. Wood, T. Grewal, and A. Draeger Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton J. Biol. Chem., June 19, 2009; 284(25): 17227 - 17242. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 283/15/10174    most recent M706618200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cubells, L. Articles by Enrich, C. Search for Related Content PubMed PubMed Citation Articles by Cubells, L. Articles by Enrich, C. Social Bookmarking                      What's this?