Epithelial Growth Factor-induced Phosphorylation of Caveolin 1 at Tyrosine 14 Stimulates Caveolae Formation in Epithelial Cells
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m512088200
ISSN1083-351X
AutoresLidiya Orlichenko, Bing Huang, Eugene W. Krueger, Mark A. McNiven,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoCaveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8–10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90–95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly. Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8–10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90–95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly. Caveolae are endocytic organelles known to participate in the internalization of specific viruses, toxins, and receptors (1Pelkmans L. Helenius A. Traffic. 2002; 3: 311-320Crossref PubMed Scopus (612) Google Scholar, 2Nichols B. J. Cell Sci. 2003; 116: 4707-4714Crossref PubMed Scopus (322) Google Scholar). The major structural components of caveolae are caveolin-1 (Cav1) 2The abbreviations used are: Cav, caveolin; MDCK, Madin-Darby canine kidney; EGF, epidermal growth factor; EGFR, EGF receptor; HRP, horseradish peroxidase; EM, electron microscopy; NRK cells, normal rat kidney cells; PBS, phosphate-buffered saline; D-PBS, Dulbecco's PBS; GFP, green fluorescent protein; Mes, 4-morpholineethanesulfonic acid; TEM, transmission electron microscopy. and caveolin-2 (Cav2) proteins, which share 38% sequence identity (3Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Both of the caveolins assemble into higher molecular weight oligomeric complexes (3Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 4Sargiacomo M. Scherer P.E. Tang Z. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (488) Google Scholar). Currently little is known about how caveolae are formed in cells and how the assembly process is regulated. Several interesting studies suggest that cells tightly regulate the assembly of caveolae from caveolin monomers/oligomers. For example, Madin-Darby canine kidney (MDCK) cells transport Cav1 and Cav2 complexes to both the apical and basolateral domains, although these proteins are assembled into caveolar vesicles only at the cell base (5Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (265) Google Scholar). Why few, if any, caveolae are observed at the apical surface in these cells is unclear. Furthermore, selective assembly of caveolae has been observed in migrating endothelial cells in which most of the caveolin protein appears at the leading cell edge, whereas the majority of assembled caveolae are found at the cell posterior (6Parat M.O. Anand-Apte B. Fox P.L. Mol. Biol. Cell. 2003; 14: 3156-3168Crossref PubMed Scopus (127) Google Scholar). Thus, cells are able to regulate caveolae formation to meet specific physiological needs for membrane trafficking or migration. Several studies have suggested that assembly of caveolae might be regulated by caveolin phosphorylation (5Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (265) Google Scholar, 6Parat M.O. Anand-Apte B. Fox P.L. Mol. Biol. Cell. 2003; 14: 3156-3168Crossref PubMed Scopus (127) Google Scholar, 7Nomura R. Fujimoto T. Mol. Biol. Cell. 1999; 10: 975-986Crossref PubMed Scopus (108) Google Scholar, 8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar). Lisanti and co-workers (9Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. Lee H. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) have demonstrated that Cav1 is phosphorylated at tyrosine 14 after stimulation of epithelial growth factor (EGF) signaling. Phosphorylation of Cav1 also appears to promote profound changes in caveolin localization as assessed by immunofluorescence in cells co-expressing Cav1 and activated c-Src (9Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. Lee H. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Consistent with these findings, phosphorylation of Cav1 in v-Src-expressing cells was shown to induce aggregation and fusion of caveolae and/or caveolae-derived vesicles (7Nomura R. Fujimoto T. Mol. Biol. Cell. 1999; 10: 975-986Crossref PubMed Scopus (108) Google Scholar). Similarly, phosphorylation of Cav1 at tyrosine 14 induced by insulin-like growth factor resulted in translocation of Cav1 in lipid raft membrane microdomains and in the formation of membrane patches on the cell surface (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar). Thus, substantial attention has been given toward testing the effects of activated receptor-tyrosine kinase cascades and downstream Src kinase activation on caveolar dynamics and Cav1 phosphorylation. Whether these activated signaling cascades actually lead to increased numbers of assembled caveolar vesicles and/or their detachment from the plasma membrane, however, remains largely undefined. In this study we report that both neoplastic and normal mammalian epithelial cells respond to EGF stimulation by altering the location of caveolin protein, resulting in a pronounced assembly of caveolae along the cell periphery. In the case of neoplastic pancreatic tumor cells possessing an elevated EGF signaling cascade, excessive caveolae assembly resulted in the formation of long caveolar towers consisting of many connecting caveolae that extended into the central cytoplasm. The vesicular structures in both normal and neoplastic cells were detected using either cholera toxin tagged to HRP or by immunogold electron microscopy (EM) using a Cav1 polyclonal antibody. Corresponding to this assembly process was a shift in the density of Cav1 protein as assessed by sucrose-gradient centrifugation and SDS-PAGE. Because phosphorylation of Cav1 at tyrosine 14 has been observed in cells treated with receptortyrosine kinase agonists (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar, 9Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. Lee H. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 10Kim Y.N. Dam P. Bertics P.J. Exp. Cell Res. 2002; 280: 134-147Crossref PubMed Scopus (40) Google Scholar), we tested to determine if preventing the phosphorylation at this specific residue might also attenuate caveolar assembly both in vivo and in vitro. Importantly, cells overexpressing a Cav1Y14F-GFP protein, when stimulated with EGF, showed little if any increase in caveolar structures compared with mock-stimulated cells. To our knowledge this is the first direct demonstration that stimulation of receptor-tyrosine kinase signaling pathways leads to the assembly of caveolar vesicles dependent upon Src-mediated phosphorylation of the Cav1 protein at tyrosine 14. Cells and Reagents—Human neoplastic pancreatic ductular epithelial cells PANC-1 (ATCC, Manassas, VA), v-Srcts-transformed MDCK epithelial cells (a gift from Dr. Fujita, Tokai University School of Medicine), and normal rat kidney (NRK) cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 15 μg/ml penicillin, and 0.05 mg/ml streptomycin. PANC-1 and NRK cells were grown at 37 °C in 5% CO2, and v-Srcts MDCK cells were maintained at restrictive 40.5 °C temperature under normal conditions and transferred to permissive 33 °C to induce Src activation. Cells were grown in plastic tissue culture dishes for biochemical analyses, on acid-washed coverslips for fluorescence microscopy, and on carbon-coated and glow-discharged grid coverslips (Bellco Glass, Inc., Vineland, NJ) for electron microscopy. Polyclonal anti-Cav1 antibodies were generated in rabbits and affinity-purified as described previously (11Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (109) Google Scholar). Monoclonal anti-Cav1 and anti-Cav1 PY14 antibodies were purchased from Transduction Laboratories (Lexington, KY). Secondary goat anti-rabbit and goat anti-mouse IgG antibodies linked to HRP were from Tago, Inc. (Burlingame, CA). ProLong antifade reagent was from Invitrogen, and HRP-cholera toxin B, protein A-Sepharose beads, LY 294002, lavendustin A, and PP2 were from Sigma. Generation of Mutant Cav1(Y14F) and Transfection—By using Cav1 in pEGFP vector as a template, a point mutation to change tyrosine 14 to phenylalanine was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The fidelity of the mutation was confirmed by direct sequencing of the plasmid. The construct and wild type cav1 in pEGFP vector further were used to transfect PANC-1 and NRK cells using GeneJammer reagent (Stratagene), according to the manufacturer's protocol. Twenty-four hours after transfection cells were cultured for 24 h in low-serum Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum. The cells were then treated with 30 ng/ml EGF for microscopy or 100 ng/ml EGF for biochemical assays for the times described in the corresponding figure legends. Pharmacological Inhibition—For TEM, cells cultured for 24 h in low serum media were pretreated with the drugs PP2 (20 μm), lavendustin A (10 μm), or LY 294002 (100 μm) for 15 min and exposed to HRP-cholera toxin B for 15 min at 4 °C to allow surface binding. Subsequently, cells were washed 3 times in Hanks'-buffered saline solution to remove unbound HRP-cholera toxin, allowed to recover for 15 min at 37 °C, and stimulated with EGF (30 ng/ml) for either 5 or 20 min. Inhibitory drugs were included in all subsequent steps before fixation and HRP treatment as described previously (14Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (637) Google Scholar). For biochemical studies, cells cultured on 10-cm dishes were pretreated with drugs for 15 min using the concentrations described above. Drugs were included during EGF-treatment (100 ng/ml) for 5 or 20 min. Cells were then washed three times in Dulbecco's PBS (D-PBS) and scraped into Eppendorf tubes, and cell lysates were prepared for SDS-PAGE and Western blot analysis using Cav1 phospho-Tyr-14 antibodies. Fluorescence Microscopy—Cells cultured for 24 h on coverslips in low serum media were fixed immediately or first treated with 30 ng/ml EGF and then fixed with 3% formaldehyde in D-PBS for 10 min. After fixation, the cells were washed three times in D-PBS and mounted on slides in ProLong antifade reagent. GFP-tagged Cav1 was visualized by fluorescence microscopy (on Zeiss Axiovert 35 microscope) using a 63× 1.4 NA lens. Immunoelectron Microscopy and Electron Microscopy—For immunolabeling of ultrathin cryosections, cells were serum-starved and stimulated with 30 ng/ml EGF for the times described in the corresponding figure legends. NRK and PANC-1 cells were fixed with 4% formaldehyde in 0.1 m sodium phosphate buffer, pH 7.4. After rinsing with phosphate buffer, the cells were scraped and then pelleted by centrifugation. Samples were embedded in 10% gelatin, cooled on ice, and cut into 1-mm3 blocks at 4 °C. The blocks were infused with 2.3 m sucrose at 4 °C overnight and frozen in liquid nitrogen. 50–60-nm-thick cryothin sections were cut at –120 °C using Ultracut FCS (Leica), and sections were picked up in a mixture of 2% methylcellulose and 2.3 m sucrose in 1:1 ratio according to Liou et al. (12Liou W. Geuze H.J. Slot J.W. Histochem. Cell Biol. 1996; 106: 41-58Crossref PubMed Scopus (443) Google Scholar). Immunogold labeling of cryothin sections was according to the protocol described by Slot et al. (13Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (744) Google Scholar). Briefly, cryothin sections were collected on Formvar-coated nickel grids and incubated with rabbit anti-Cav1 antibody, diluted 1:1000 with 10% fetal calf serum, PBS overnight at 4 °C, then incubated with goat anti-rabbit IgG coupled to 6-nm gold (Sigma) for 2 h at room temperature. After labeling, the sections were treated with 1% glutaraldehyde, counterstained with uranyl acetate, and embedded in methyl cellulose. Cryosections were examined and photographed using a Jeol 1200 electron microscope. HRP-cholera toxin B, ruthenium red labeling, and standard EM was as previously described (14Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (637) Google Scholar). Immunoprecipitation, SDS-PAGE Electrophoresis, and Western Blotting—Immunoprecipitation of Cav1 was performed as previously described (15Kim Y. Han J.M. Han B.R. Lee K.A. Kim J.H. Lee B.D. Jang I.H. Suh P.G. Ryu S.H. J. Biol. Chem. 2000; 275: 13621-13627Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) with some modifications. Briefly, cells cultured on 10-cm dishes were washed 3 times in D-PBS, scraped into Eppendorf tubes, and lysed in 0.25 ml of ice-cold hypotonic lysis buffer (10 mm Tris, pH 7.5, 1 mm EDTA, 0.5 ml EGTA, 10 mm NaCl, 1% Triton, and 1% sodium cholate) containing protease (0.5 mm phenylmethylsulfonyl fluoride) and phosphatase inhibitors (30 mm NaF, 1 mm Na3VO4). Lysates were then precleared at top speed for 15 min in an Eppendorf microcentrifuge at 4 °C. Cav1 was precipitated using polyclonal Cav1 antibody prebound to immobilized protein A-Sepharose beads (Sigma). The immunoprecipitated proteins were washed 3 times in hypotonic lysis buffer containing 150 mm NaCl. Immunoprecipitates were solubilized in an SDS-containing sample buffer and boiled for 5 min. Electrophoresis was performed in 12% acrylamide gels followed by transfer to polyvinylidene difluoride membranes. SDS-PAGE (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (214196) Google Scholar), transfer of proteins to polyvinylidene difluoride membranes (17Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (46694) Google Scholar), and Western blotting (18Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (6274) Google Scholar) were as described. For immunoblotting of sucrose gradient fractions, 100 μlof each fraction was solubilized in SDS-containing sample buffer and boiled, and 50 μl of each fraction was separated on 12% SDS-PAGE. Immunodetection of bound antibodies on nitrocellulose membrane was performed using ECL reagents (Amersham Biosciences). All procedures were carried out according to the manufacturer's instructions. Sucrose Gradient Fractionation—Caveolae membrane microdomains were purified from cultured cells using a modified carbonate method (19Kim Y. Kim J.E. Lee S.D. Lee T.G. Kim J.H. Park J.B. Han J.M. Jang S.K. Suh P.G. Ryu S.H. Biochim. Biophys. Acta. 1999; 1436: 319-330Crossref PubMed Scopus (38) Google Scholar). Briefly, serum-starved or EGF-stimulated cells were washed with D-PBS, scraped into 0.2 ml of 0.5 m Na2CO3, pH 11, containing protease and phosphatase inhibitors, and homogenized in a Dounce homogenizer. After that, cells were incubated on ice for 30 min, and membrane fractions were separated from nuclei by centrifugation of the cell homogenates at 1000 × g for 10 min. Subsequently, cell membranes were lysed with three 6-s bursts of a 100-watt Ultrasonicator (Kontes). Equal amounts of cell membranes were adjusted to 40% sucrose by mixing with 0.2 ml of 80% sucrose prepared in MBS buffer (25 mm Mes, pH 6.5, and 150 mm NaCl). This suspension was placed at the bottom of an ultracentrifuge tube and overlaid with 0.4 ml of 30% sucrose and then with 0.4 ml of 5% sucrose containing 0.25 m Na2CO3 in MBS buffer. After centrifugation at 100,000 × g for 16 h at 4 °C, a total of 12 fractions (100 μl each) was collected from the top of each gradient. Live Imaging—For live imaging, PANC-1 cells were grown in specifically designed 35-mm tissue culture dishes with glass bottoms. Live imaging of serum-starved and EGF-stimulated cells was performed every 5 s for 200 and 600 frames, respectively, using IP lab software (Scanalytics Inc., Fairfax, VA) as previously described (20Orth J.D. Krueger E.W. Cao H. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 167-172Crossref PubMed Scopus (197) Google Scholar). EGF Stimulation Induces Phosphorylation of Cav1 and Changes in Buoyant Density of Caveolar Membranes—Two different cell types were initially used in this study to test the effects of EGF on Cav1 phosphorylation and caveolae assembly. These include highly metastatic human pancreatic ductular tumor cells that we and others (21Korc M. Meltzer P. Trent J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5141-5144Crossref PubMed Scopus (181) Google Scholar) have found to overexpress EGFR (PANC-1) and NRK as non-neoplastic control cells. PANC-1 and NRK were treated with EGF for 5, 20, and 40 min, harvested, and subjected to Western blot analysis using a Cav1 monoclonal antibody as a loading control and a Cav1 phospho-tyrosine antibody (Cav1-PY14). The phosphorylation-sensitive antibody has been shown to recognize c-Src-phosphorylated Cav1 protein on tyrosine 14 (7Nomura R. Fujimoto T. Mol. Biol. Cell. 1999; 10: 975-986Crossref PubMed Scopus (108) Google Scholar, 9Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. Lee H. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). As shown in Fig. 1, EGF treatment induced a 2–3-fold increase in Cav1 tyrosine phosphorylation in PANC-1 cells and an even greater increase in the NRK cells. Similar observations have been made by others in a variety of different cell types (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar, 9Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. Lee H. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) in response to either EGF or insulin-like growth factor. To extend these studies, we next tested for alterations in the buoyant density of Cav1 protein in response to EGF stimulation. Concomitant with the phosphorylation of Cav1, agonist stimulation is believed to induce the shift of Cav1 to a less dense, more buoyant membrane fraction that can be identified at a higher fraction in the gradient tube (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar). Whether this shift represents an assembly of caveolae and/or caveolar membrane translocation is unclear, as correlative EM studies have been lacking. To this end, a widely utilized sucrose-density centrifugation assay was implemented. PANC-1 and NRK cells, either resting or EGF-stimulated, were homogenized, and total membranes were isolated and exposed to sucrose gradient centrifugation. After a 16-h centrifugation at 100,000 × g, 100-μl fractions were removed from the top of the tube and processed by SDS-PAGE and Western blot analysis with Cav1 and phospho-caveolin antibodies (Fig. 1b). Consistent with the findings of others using different agonists (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar), we observed a modest but consistent shift of Cav1 protein from a dense to a lighter fraction in both PANC-1 and NRK cells treated with EGF (a total of three distinct experiments). Additionally, in the stimulated cells, Cav1 became present in a larger number of sucrose gradient fractions with slightly different buoyant densities, suggesting that EGF stimulation induced redistribution of Cav1 in lipid raft membrane microdomains in both PANC-1 and NRK cells. The presence of Cav1 in the more buoyant density fractions after treatment with EGF also pointed to an increase in the total amount of Cav1 protein associated with the membranes. Interestingly, co-blotting with the phosphocaveolin antibody showed that the upward shifted (lighter) Cav1 band from the stimulated cells stained more intensely than the lower, less buoyant Cav1 band from the resting cells, suggesting that Cav1 phosphorylation may have played a role in a translocation of Cav1 in membrane microdomains. EGF Stimulation Induces Caveolae-like Plasma Membrane Invaginations in Normal and Neoplastic Epithelial Cells—To monitor caveolarbased membrane dynamics in EGF-stimulated epithelial cells, endogenous Cav1 in PANC-1 cells was first monitored by fluorescence microscopy and viewed under resting and serum-starved conditions or after stimulation with 30 ng/ml EGF. In resting cells Cav1 was localized largely to the periphery along the plasma membrane at cell-cell contacts (Fig. 2, a and a′, arrow) with an additional vesicle-like distribution throughout the cytoplasm. However, after 20 min of EGF stimulation, the Cav1 appeared to move away from the cell borders to a more intracellular location concomitant with the loss of cell-cell interactions (Fig. 2, b and b′). In addition, a substantial increase in Cav1 cytoplasmic puncta was observed, suggesting an active assembly and/or budding of nascent caveolae. Similar alterations in caveolae distribution have been observed in mouse fibroblasts in response to treatment with insulin-like growth factor (8Maggi M. Luconi M. Vannelli G.B. Salerno R. Sinisi A.A. Bonaccorsi L. Ferruzzi P. Barni T. Forti G. Serio M. Panetta D. Prostate. 2002; 50: 15-26Crossref PubMed Scopus (49) Google Scholar) and in v-Src-transformed NRK cells (7Nomura R. Fujimoto T. Mol. Biol. Cell. 1999; 10: 975-986Crossref PubMed Scopus (108) Google Scholar). To more closely observe the effects of EGF-treatment on caveolae and membrane dynamics in epithelial cells, resting or stimulated PANC-1 cells were fixed and prepared for EM. Consistent with the fluorescence images in Fig. 2, a and b, resting cells displayed largely intact cell borders with relatively modest numbers of associated caveolae-like structures (Fig. 2c). In marked contrast to resting cells, EGF-stimulated cells displayed a dramatic increase in the number of plasma membrane invaginations and formation of caveolae-like vesicles attached to the plasma membrane (Fig. 2, d and e). Often these vesicles appeared to fuse together, creating large membrane invaginations that were amply decorated by caveolae-like vesicles (Fig. 2, d and e). These caveolae-like towers generally formed near the sites of disrupted cell-cell borders. Coating cells with ruthenium red dye after fixation helped to identify invaginations continuous with the plasma membrane (Fig. 2, e and e′). Because PANC-1 cells are neoplastic and known to have altered signaling properties or caveolin levels, we conducted identical morphological studies on NRK cells as a control. Consistent with our observations in PANC-1 cells, endogenous Cav1 localized largely to the cell peripheries in resting NRK cells (Fig. 3, a and a′) but appeared to move inward and form larger patches after stimulation with EGF (Fig. 3, a and b). EM using ruthenium red dye again showed resting NRK cells with few caveolae-like structures (Fig. 3c), whereas 20 min of EGF treatment induced a pronounced increase in the number of caveolae-like vesicles formed (Fig. 3, d and e). Most of these vesicles appeared as discrete membrane-associated structures and did not appear to coalesce into the membrane towers seen in the neoplastic PANC-1 cells. Both cell types exhibited a marked increase in caveolae-like structures in response to acute EGF treatment. EM-based morphometry of multiple cells was performed to quantitate the number of caveolae-like structures present within 1 μm of the plasma membrane in both cell types under resting or stimulated conditions (Fig. 4). PANC-1 cells responded quickly with a 3-fold increase in the number of plasma membrane-associated vesicles by 5 min and a 4–5-fold increase by 20 min post-EGF treatment. NRK cells displayed only a modest increase in vesicle numbers at early time points but exhibited a remarkable 10–12-fold increase by 20 min after EGF addition. No noticeable changes in clathrin pits were observed in the microscopic fields examined in either cell type.FIGURE 4Quantitation of caveolae-like structures in PANC-1 and NRK cells after stimulation with EGF. a, quantitation of caveolae-like vesicle formation. Ruthenium red-stained vesicles along a 1-μm2 area of the plasma membrane were counted as detailed under "Materials and Methods." PANC-1 cells showed an increase of vesicular structures in only 5 min post-EGF treatment with a total 4–5-fold increase by 20 min post-EGF. In comparison, NRK cells took longer to respond to agonist but showed a greater (10–12-fold) increase in putative caveolae by 20 min. b, quantitation of immunogold labeling of putative caveolae in resting versus EGF-stimulated cells as shown in Supplemental Fig. S1. Numbers represent the number of gold particles at the plasma membrane representing Cav1 antigen (see "Materials and Methods"). A 6–10-fold increase in labeling was observed in both cell types when examined 20 min after EGF stimulation, suggesting an increase in caveolae formation. RR, ruthenium red.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Vesicular Invaginations Induced by EGF Stimulation Are Dynamic Caveolar Clusters That Extend and Bud from the Plasma Membrane—Although the EM images of plasma membrane-formed vesicles did not display a clathrin coat and resembled caveolae in size and shape, we pursued two additional techniques to confirm that the nascent vesicular structures were caveolae. First, during stimulation with EGF and before fixation, PANC-1 cells were inc
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