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Regulation of cAMP-mediated Signal Transduction via Interaction of Caveolins with the Catalytic Subunit of Protein Kinase A

1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês

10.1074/jbc.274.37.26353

ISSN

1083-351X

Autores

Babak Razani, Charles S. Rubin, Michael P. Lisanti,

Tópico(s)

Ion Transport and Channel Regulation

Resumo

cAMP-dependent processes are essential for cell growth, differentiation, and homeostasis. The classic components of this system include the serpentine receptors, heterotrimeric G-proteins, adenylyl cyclase, protein kinase A (PKA), and numerous downstream target substrates. Evidence is accumulating that some members of this cascade are concentrated within membrane microdomains, termed caveolae and caveolae-related domains. In addition, the caveolin-1 protein has been shown to interact with some of these components, and this interaction inhibits their enzymatic activity. However, the functional effects of caveolins on cAMP-mediated signaling at the most pivotal step, PKA activation, remain unknown. Here, we show that caveolin-1 can dramatically inhibit cAMP-dependent signaling in vivo. We provide evidence for a direct interaction between caveolin-1 and the catalytic subunit of PKA both in vitro and in vivo. Caveolin-1 binding appears to be mediated both by the caveolin scaffolding domain (residues 82–101) and a portion of the C-terminal domain (residues 135–156). Further functional analysis indicates that caveolin-based peptides derived from these binding regions can inhibit the catalytic activity of purified PKA in vitro. Mutational analysis of the caveolin scaffolding domain reveals that a series of aromatic residues within the caveolin scaffolding domain are critical for mediating inhibition of PKA. In addition, co-expression of caveolin-1 and PKA in cultured cells results in their co-localization as seen by immunofluorescence microscopy. In cells co-expressing caveolin-1 and PKA, PKA assumed a punctate distribution that coincided with the distribution of caveolin-1. In contrast, in cells expressing PKA alone, PKA was localized throughout the cytoplasm and yielded a diffuse staining pattern. Taken together, our results suggest that the direct inhibition of PKA by caveolin-1 is an important and previously unrecognized mechanism for modulating cAMP-mediated signaling. cAMP-dependent processes are essential for cell growth, differentiation, and homeostasis. The classic components of this system include the serpentine receptors, heterotrimeric G-proteins, adenylyl cyclase, protein kinase A (PKA), and numerous downstream target substrates. Evidence is accumulating that some members of this cascade are concentrated within membrane microdomains, termed caveolae and caveolae-related domains. In addition, the caveolin-1 protein has been shown to interact with some of these components, and this interaction inhibits their enzymatic activity. However, the functional effects of caveolins on cAMP-mediated signaling at the most pivotal step, PKA activation, remain unknown. Here, we show that caveolin-1 can dramatically inhibit cAMP-dependent signaling in vivo. We provide evidence for a direct interaction between caveolin-1 and the catalytic subunit of PKA both in vitro and in vivo. Caveolin-1 binding appears to be mediated both by the caveolin scaffolding domain (residues 82–101) and a portion of the C-terminal domain (residues 135–156). Further functional analysis indicates that caveolin-based peptides derived from these binding regions can inhibit the catalytic activity of purified PKA in vitro. Mutational analysis of the caveolin scaffolding domain reveals that a series of aromatic residues within the caveolin scaffolding domain are critical for mediating inhibition of PKA. In addition, co-expression of caveolin-1 and PKA in cultured cells results in their co-localization as seen by immunofluorescence microscopy. In cells co-expressing caveolin-1 and PKA, PKA assumed a punctate distribution that coincided with the distribution of caveolin-1. In contrast, in cells expressing PKA alone, PKA was localized throughout the cytoplasm and yielded a diffuse staining pattern. Taken together, our results suggest that the direct inhibition of PKA by caveolin-1 is an important and previously unrecognized mechanism for modulating cAMP-mediated signaling. protein kinase A protein kinase A anchoring protein cAMP response element cAMP response element binding protein isobutylmethylxanthine caveolin-1 glutathione S-transferase polyacrylamide gel electrophoresis phosphate-buffered saline protein kinase inhibitor Caveolae are vesicular invaginations of the plasma membrane with a characteristic Ω-shaped morphology and a diameter of ∼50–100 nm (1Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 2Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Although they are present in many cell types, caveolae are most abundant in terminally differentiated cells, such as adipocytes, endothelial cells, fibroblasts, and muscle cells (3Couet J. Li S. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar). Caveolae membranes are rich in cholesterol, glycosphingolipids, and a 21–24-kDa protein, caveolin (also known as caveolin-1). It appears that all three components are important for caveolae formation (4Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1862) Google Scholar, 5Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 6Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. 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Caveolin-2 has the same tissue distribution as and co-localizes with caveolin-1, whereas caveolin-3 is found only in cardiac and skeletal muscle cells (11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (491) Google Scholar, 12Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar). Caveolin-1 contains a 41-amino acid region that self-associates and induces the formation of caveolin-1 homo-oligomers that contain ∼14–16 individual caveolin monomers. It is thought that these caveolin oligomers are the assembly units that drive the formation of caveolae in intact cells (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (476) Google Scholar, 14Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (399) Google Scholar). Caveolae are known to participate in vesicular trafficking (i.e. endocytosis and transcytosis), as well as signal transduction processes. Biochemical and morphological experiments have shown that a variety of signaling molecules are concentrated within these plasma membrane microdomains. This is particularly true of lipid-modified signaling molecules, such as Src family tyrosine kinases, Ha-Ras, endothelial nitric-oxide synthase, and heterotrimeric G-proteins (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 16Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 17Song K.S. 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Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar). A role for the caveolin gene family in regulating such signaling cascades is emerging. Recent evidence points to a functional interaction between the caveolins and certain classes of signaling molecules. More specifically, caveolin-1 binding can functionally suppress the GTPase activity of heterotrimeric G-proteins and inhibit the kinase activity of Src family tyrosine kinases, the epidermal growth factor receptor kinase, and protein kinase C through a common caveolin domain, termed the caveolin scaffolding domain (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar,20Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 21Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 22Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Signaling via cAMP is one of the first and most rigorously studied signal transduction pathways. A wealth of information is known about the events beginning with ligand binding and the subsequent intracellular effects. Briefly, the activation of serpentine receptors by their cognate ligands causes activation of the heterotrimeric G-protein, Gs, in turn activating adenylyl cyclase and leading to the generation of cAMP. cAMP is bound by PKA1 holoenzymes, which are composed of two regulatory and two catalytic subunits. PKA catalytic subunits, the main effectors of cAMP, dissociate from cAMP-saturated regulatory subunits and phosphorylate numerous downstream targets, thereby enhancing or repressing cellular processes and gene expression (23Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar, 24Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (856) Google Scholar). Much of this cascade initially occurs at or near the plasma membrane, given that G-protein-coupled receptors and adenylyl cyclase are integral membrane proteins. Diversification and subcellular regionalization of the pathway then occurs at the level of PKA, because various protein kinase A anchoring proteins (AKAPs) can sequester PKA molecules until the arrival of liberating cAMP signals (25Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 467-479PubMed Google Scholar, 26Scott J.D. McCartney S. Mol. 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Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 20Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 32Toya Y. Schwencke C. Couet J. Lisanti M.P. Ishikawa Y. Endocrinology. 1998; 139: 2025-2031Crossref PubMed Google Scholar). Although several kinases have been shown to be either negatively or positively affected by the caveolins (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar,21Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 22Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 35Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (346) Google Scholar, 36Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 37Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Myers Jr., M. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), virtually nothing is known about the relationship of caveolins to PKA. Here, we have used both in vivo and in vitro approaches to investigate the effects of caveolin-1 expression on PKA signaling. The PKAα catalytic rabbit polyclonal IgG was purchased from Santa Cruz Biotechnology. Caveolin-1 mouse monoclonal antibody 2297 (used for immunoblotting; see Ref. 38Scherer 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 (321) Google Scholar) and monoclonal antibody 2234 (used for immunofluorescence; see Ref. 38Scherer 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 (321) Google Scholar) were the gifts of Dr. Roberto Campos-Gonzalez, Transduction Laboratories, Inc. The AV12-664 cell line was obtained from ATCC (CRL-9595). Purified bovine PKAα catalytic and truncated RIIβ subunits were as we described previously (39Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 40Li Y. Ndubuka C. Rubin C.S. J. Biol. Chem. 1996; 271: 16862-16869Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). A variety of other reagents were purchased commercially as follows: cell culture reagents were from Life Technologies, Inc., the PathDetect CRE/cis-acting reporter system was from Stratagene, and Effectene liposomal transfection reagent was from Qiagen. The cDNAs encoding caveolin-1 (full-length and deletion mutants), as well as caveolin-2 and caveolin-3, were subcloned into the pCB7 mammalian expression vector, as described previously (11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (491) Google Scholar, 12Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 29Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (860) Google Scholar, 41Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). AV12-664 cells, a tumor-derived hamster cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. Forty-eight hours post-transfection, AV12 cells were washed with PBS and incubated with lysis buffer (10 mm Tris, pH 7.5; 50 mm NaCl; 1% Triton X-100, 60 mm octyl glucoside) containing protease inhibitors (Roche Molecular Biochemicals). Where indicated, protein concentrations were quantified using the BCA reagent (Pierce). Samples were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands) followed by immunoblot analysis. All subsequent wash buffers contained 10 mm Tris, pH 8.0, 150 mmNaCl, 0.05% Tween 20, which was supplemented with 1% bovine serum albumin and 2% nonfat dry milk (Carnation) for the blocking solution and 1% bovine serum albumin for the antibody diluent. Primary antibodies (either polyclonal or monoclonal) were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution, Transduction Laboratory) were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce). To assess PKA-mediated signal transduction in vivo, we employed the PathDetect CRE cis- reporting system (Stratagene). In this assay, the luciferase reporter is driven by a promoter containing four cAMP response element (CRE) consensus sequences. Upon PKA catalytic subunit activation, endogenous CRE binding (CREB) protein is phosphorylated thereby allowing its interaction with CRE sequences and enhancing the transcription of luciferase. Transient transfections were performed using the Effectene liposome-mediated method (Qiagen) as described previously (35Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (346) Google Scholar, 36Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) with minor modifications. Briefly, 300,000 AV12 cells were seeded in 6-well plates 12–24 h prior to transfection. For stimulation experiments, each plate of cells was transfected with 0.5 μg of the caveolin-1 (pCB7-Cav-1) or empty vector (pCB7) and 0.5 μg of the pFR-Luc reporter construct. Twelve hours post-transfection, the cells were rinsed with PBS, incubated in 1% fetal bovine serum and, where indicated, supplemented with either 10 μm forskolin, 10 μm dideoxy-forskolin, or 500 μm IBMX. Twenty-four hours post-stimulation, the cells were lysed in 200 μl of extraction buffer, 50 μl of which was used to measure luciferase activity as described (42Pestell R.G. Albanese C. Hollenberg A. Jameson J.L. J. Biol. Chem. 1994; 269: 31090-31096Abstract Full Text PDF PubMed Google Scholar). For the analysis of the effects of caveolin-1, caveolin-1 deletions, and caveolins-2 and -3 directly on PKA, all points were additionally transfected with 100 ng of pFC-PKA, a vector encoding the PKA catalytic subunit (Stratagene), and the cells were not serum-starved post-transfection. Each experimental value represents the average of two separate transfections performed in parallel; error bars represent the observed standard deviation. All experiments were performed at least three times independently and yielded virtually identical results. By using this CRE assay system, we have determined that transient expression of recombinant PKA increases total PKA activity by ∼4–6-fold; thus, the ratio between exogenous and endogenous PKA activity is ∼5:1. These results are consistent with Western blot analyses showing that recombinant expression of PKA increases total PKA expression by ∼5-fold (see Fig. 1, upper panel). The construction and purification of GST-caveolin-1 fusion proteins was as we described previously (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (476) Google Scholar, 20Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 38Scherer 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 (321) Google Scholar). Briefly, full-length caveolin-1 (residues 1–178) and selected caveolin-1 regions (1–101, 1–81, 61–101, and 135–178) were subcloned into the vector pGEX-4T-1. After expression in Escherichia coli (BL21 strain; Novagen, Inc.), GST-caveolin-1 fusion proteins were affinity purified on glutathione-agarose beads, using the detergent sarcosyl for initial solubilization (43Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (829) Google Scholar). GST alone or GST-Cav-1 fusion proteins bound to glutathione-agarose beads were extensively washed first with TNET buffer (50 mm Tris, pH 8.0; 150 mm NaCl; 5 mm EDTA; 1% Triton X-100) (3×) and lysis buffer (1×), both containing protease inhibitors. SDS-PAGE followed by Coomassie staining was used to determine the approximate molar quantities of the fusion proteins per 100 μl of packed bead volume. Approximately 100 μl of equalized bead volume was incubated with precleared lysates of AV12 cells transfected with PKAα catalytic subunit by rotating overnight at 4 °C. After binding, the beads were extensively washed with TNET buffer and protease inhibitors (6×). Finally, the associated proteins were eluted with TNET buffer supplemented with 20 mm glutathione and an additional 100 mmTris-Cl, pH 8.0. The eluate was mixed 2:1 with 3× sample buffer and subjected to SDS-PAGE. The immunoblotting procedure was as described above using anti-PKAα catalytic rabbit polyclonal antibody IgG (Santa Cruz Biotechnology) as the primary antibody. The caveolin-based peptides were synthesized using standard methodology and subjected to amino acid analysis and mass spectroscopy (Massachusetts Institute of Technology Biopolymers Laboratory/Research Genetics) to confirm their purity and composition, as we described previously (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 21Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 22Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 35Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (346) Google Scholar,36Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Purified bovine PKAα catalytic subunit (5 μg/ml) was incubated with caveolin-derived synthetic peptides and the truncated (residues 1–158) PKA RIIβ subunit in 100 μl of kinase reaction buffer (30 mm KHPO4, 12.5 μm cAMP, 625 μm dithiothreitol, 12.5 mm MgCl2) at 4 °C. The reaction was initiated by the addition of 15 μCi of [γ-32P]ATP. After incubation for 15 min at 4 °C, the reaction was stopped by the addition of 3× sample buffer and boiling for 5 min. Samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Bands corresponding to phosphorylated RIIβ were visualized by autoradiography using an intensifying screen. The procedure was performed as we previously described (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). AV12 cells transfected with the PKAα catalytic subunit and/or caveolin-1 were fixed for 30 min in PBS containing 2% paraformaldehyde, rinsed with PBS, and quenched with 50 mm NH4Cl for 10 min. The cells were then incubated in permeabilization buffer (PBS; 0.2% bovine serum albumin; 0.1% Triton X-100) for 10 min, washed with PBS, and double-labeled with a 1:400 dilution of anti-caveolin-1 mouse monoclonal antibody 2234 (Transduction Laboratories, Inc.) and PKAα catalytic rabbit polyclonal antibody (Santa Cruz Biotechnology) for 60 min. After rinsing with PBS (3×), secondary antibodies (7.5 μg/ml) ((lissamine-rhodamine (LRSC)-conjugated goat anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-mouse) antibodies) were added for a period of 60 min. Cells were washed with PBS (3×), and slides were mounted with Slow-Fade anti-fade reagent (Molecular Probes). A Bio-Rad MR600 confocal fluorescence microscope was used for visualization of bound secondary antibodies. Given the ability of caveolin to inactivate a variety of signal transduction pathways (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 21Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 22Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 35Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (346) Google Scholar, 36Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), we sought to investigate its possible role in PKA signaling. It is well known that one of the important downstream effectors of PKA is cAMP response element binding (CREB) protein. CREB binds CRE sequences in promoters of cAMP-responsive genes (24Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (856) Google Scholar). PKA-catalyzed phosphorylation of Ser133 of CREB elicits a conformational change that enables CREB to promote transcription of downstream target genes. Thus, we employed a well established CRE reporter system (Stratagene, Inc.) to evaluate the in vivo effects of caveolin-1 expression on PKA signaling. In this assay, a series of consensus CRE sequences drive expression of a luciferase reporter. PKA directly activates CREB which up-regulates the transcription of the luciferase reporter. AV12-664, a tumor-derived cell line from hamsters, was chosen for this and subsequent assays because of its failure to endogenously express detectable levels of caveolin-1 (Fig.1). Therefore, overexpression of recombinant caveolin-1 in this system should allow us to assess the effect of caveolin-1 on PKA signaling. As shown in Fig. 2 A, caveolin-1 expression (driven by the cytomegalovirus promoter; pCB7-Cav-1) dramatically inhibits endogenous basal PKA signaling as compared with the empty vector (pCB7) or β-galactosidase. Most likely, this reflects the ability of caveolin-1 expression to inhibit endogenous PKA activity. However, this may still reflect the ability of caveolin-1 to inhibit signaling at the level of Gsα subunits or adenylyl cyclase, as well. To assess the effects of caveolin-1 in the presence of PKA pathway activators, 10 μm forskolin (a potent stimulator of adenylyl cyclase) and 500 μm IBMX (an inhibitor of cyclic nucleotide phosphodiesterase) were used in the same assay system. Treatment with either pharmacological agent enhanced PKA-mediated signaling nearly 3-fold; in

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