Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A

Babak Razani and Michael P. Lisanti

Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous components of the cAMP-based signaling cascade, namely G-proteins and G- protein coupled receptors, adenylyl cyclase, and protein kinase A (PKA) have been localized to caveolae and shown to be regulated by the caveolar marker proteins, the caveolins. In order to gain mechanistic insights into these processes in vivo, we have assessed the functional interaction of caveolin-1 (Cav-1) with PKA using mutational analysis. As two regions of Cav-1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants shows that Cav-1 requires the presence of either the scaffolding domain or the COOH-terminal domain (but not both) to functionally interact with and inhibit PKA. Interestingly, in contrast to the wild-type protein, these Cav-1 mutants are not localized to caveolae microdomains. However, upon coexpression with wild-type Cav-1, a substantial amount of the mutants was recruited to the caveolae membrane fraction. Using the Cav-1 double mutant with both disrupted scaffolding and COOH-terminal domains, we show that wild-type Cav-1's inhibition of PKA signaling can be partially abrogated in a dose-responsive manner; i.e., the mutant acts in a dominant-negative fashion. Thus, this dominant-negative caveolin-1 mutant will be extremely valuable for assessing the functional role of endogenous caveolin-1 in regulating a variety of other signaling cascades.

caveolae; protein kinases; signal transduction; cAMP


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE PAST FEW DECADES, intense research focused on cAMP-mediated signaling events has established these processes as paradigms for analyzing signal transduction emanating from the plasma membrane and leading to distinct end points, such as transcriptional responses (reviewed in Ref. 8). Classically, signaling commences by the binding of a diverse array of extracellular molecules (e.g., hormones and neurotransmitters) to serpentine membrane proteins known as G protein-coupled receptors (GPCRs). The activation of such receptors uncouples and stimulates associated heterotrimeric G proteins, a major function of which is the initiation of cAMP production. Specifically, the Gsalpha subunit activates adenylyl cyclases, a family of enzymes capable of converting ATP to its second-messenger form, cAMP.

cAMP-dependent processes are essential for cell growth, differentiation, and homeostasis. Perhaps the most important effector of cAMP responses is protein kinase A (PKA), a serine/threonine kinase consisting of two regulatory and two catalytic subunits (reviewed in Ref. 14). The binding of cAMP to the regulatory subunits facilitates the dissociation and enzymatic activation of the PKA catalytic domains. Numerous PKA targets have been described, most of which contain one or more canonical RRXS/T phosphorylation motifs. Examples include ion channels (Ca2+ and K+), cytoskeletal proteins, transcription factors [cAMP response element (CRE) binding protein (CREB) and CRE modulator (CREM)], and even the PKA type II regulatory subunits (13, 18, 30, 36, 50, 57).

An emerging theme for rapid, directed, and efficient signaling is the interaction and cross talk between various signaling pathways within distinct cellular compartments. This process is achieved by scaffolding and adaptor proteins (reviewed in Ref. 33). For example, Shc and Grb2 act as platforms for the binding of modules present in numerous signaling molecules (25, 27, 34). The A kinase-anchoring proteins (AKAPs) are a functionally related family of proteins that bind the PKA type II regulatory subunits and act to concentrate the available pool of PKA in distinct cellular locations (7, 41).

Recently, membrane microdomains known as caveolae, previously implicated in transcytosis and cellular cholesterol trafficking, were found to be enriched with a variety of signaling proteins (24, 43). Interestingly, many components of the cAMP-mediated signal transduction cascade have been shown to be biochemically and morphologically concentrated within caveolae. G protein-coupled receptors such as cholecystokinin, M2-acetylcholine, and beta -adrenergic receptors, Galpha and Gbeta subunits, adenylyl cyclases, and the PKA catalytic and type II regulatory subunits are a few examples (4, 12, 39, 48, 49, 51). An important facet to this compartmentalization is the role that caveolins, the structural protein components of caveolae, play in this signaling. The ubiquitously expressed caveolin-1 (Cav-1) and muscle-specific caveolin-3 (Cav-3) have been shown to act as scaffolding proteins by interacting with numerous members of the G protein-coupled cascade (3, 16, 22, 32, 49, 54). In many cases, the interactions occur via a 20-amino acid stretch characteristic of the caveolins, which has subsequently been termed the caveolin-scaffolding domain (CSD). This interaction has functional consequences, inasmuch as caveolins can dampen or inhibit cAMP-mediated signaling in vitro and in vivo. We recently delineated a novel inhibitory interaction between Cav-1 and the major cAMP effector enzyme PKA (37). We demonstrated that this interaction occurs in vitro and tentatively defined two possible regions in the Cav-1 protein that may mediate this inhibition (i.e., the CSD and a COOH-terminal domain). However, in vivo evidence for Cav-1-PKA interactions via these Cav-1 domains is lacking.

Here, we have examined the interaction of Cav-1 and PKA in cells. We show that Cav-1 can dramatically attenuate PKA-mediated CREB phosphorylation and transcriptional activity. Additionally, we demonstrate that Cav-1 can interact with the PKA catalytic subunit in vivo. This inhibitory interaction requires the presence of the scaffolding or the COOH-terminal domain, as mutation/deletion of both of these regions is required to abrogate the Cav-1-PKA interaction. Our characterization of these caveolin mutants indicates that, in contrast to the wild-type protein, they are not localized to caveolae microdomains. However, on coexpression with full-length Cav-1 (Cav-1 FL), there is a substantial recruitment of these Cav-1 mutants to the caveolae membrane fraction. We show that this recruitment occurs by interaction of wild-type Cav-1 with the respective mutant. Furthermore, we demonstrate that the Cav-1 mutant with a disrupted CSD and COOH-terminal domain acts in dominant-negative fashion when coexpressed with the wild-type protein.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials and cell culture. The anti-Cav-1 monoclonal antibody (MAb) 2297 (45) was the gift of Roberto Campos-Gonzalez (Transduction Laboratories). The anti-PKA catalytic rabbit polyclonal antibody (PAb) C-20 and anti-c-Myc MAb 9E10 were purchased from Santa Cruz Biotechnology. The anti-CREB and phosphospecific (Ser-133) anti-CREB rabbit PAbs were purchased from New England Biolabs. 293T cells were obtained from Dr. A. Koleske (Yale University). Cell culture reagents were purchased from GIBCO-BRL. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C and 5% CO2.

cDNA expression vectors and transfections. The cDNAs encoding Cav-1 FL and a COOH-terminal deletion mutant (Delta C) were tagged with Myc at the NH2 terminus and subcloned into the pCB7 mammalian expression vector, as described previously (43, 44, 52, 53). The FL and Delta C constructs then served as templates to further generate scaffolding domain alanine-scan mutants (A-scan and A-scan/Delta C) using standard PCR-based strategies. All transient transfections were performed using the calcium phosphate precipitation method, as described previously (10, 11).

Luciferase reporter assays for PKA activity. Luciferase reporter assays utilized the PathDetect CRE cis-reporting system (Stratagene), as described previously (37). Briefly, 300,000 cells were seeded in six-well plates 12-24 h before transfection. Each plate was transfected with the luciferase reporter, Cav-1 FL, the various Cav-1 mutants, or empty vector (pCB7) and pRSV-Gal (Promega, Madison, WI). At 12 h after transfection, the cells were rinsed with PBS and further incubated for 20 h in medium containing 10% fetal bovine serum. The cells were lysed in 200 µl of extraction buffer, 100 µl of which were used to measure luciferase activity, as described elsewhere (35). Luciferase activities were normalized for galactosidase activity as assayed using a galactosidase assay system (Promega). Results were expressed as the ratio of luciferase activity to galactosidase activity. Each experimental value represents the average of three separate transfections performed in parallel; error bars represent the observed standard deviation. All experiments were performed at least three times independently and yielded similar results.

Immunoblotting. At 48 h after transfection, cells were washed with PBS and incubated with lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, and 60 mM octyl glucoside) containing protease inhibitors (Roche Molecular Biochemicals). Where indicated, protein concentrations were quantified using the bicinchoninic acid 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) and subjected to immunoblot analysis. All subsequent wash buffers contained 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20, 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 (PAb or MAb) were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; Transduction Laboratory) were used to visualize bound primary antibodies with chemiluminescence substrate (Pierce).

In vivo phosphorylation experiments. 293T cells plated on 100-mm dishes were transfected with the appropriate plasmids. At 36 h after transfection, cells were lysed in RIPA-NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM NaF, 30 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 0.1 µg/ml okadaic acid, and protease inhibitors), sonicated briefly to disrupt nuclei, and subjected to immunoblot analysis with anti-CREB and phosphospecific anti-CREB PAbs.

Coimmunoprecipitation of Cav-1 with PKA. 293T cells plated on 100-mm dishes were transfected with the appropriate plasmids. At 36 h after transfection, cells were lysed in lysis buffer (see Immunoblotting), clarified by centrifugation at 15,000 g for 15 min, and precleared by incubation with protein A-Sepharose (Amersham Pharmacia) for 1 h at 4°C. Supernatants were transferred to separate 1.5-ml microcentrifuge tubes containing anti-PKAalpha -catalytic (PKAalpha -cat) PAb (Santa Cruz Biotechnology) or appropriate control antibodies (beads alone and preimmune serum) prebound to protein A-Sepharose. After incubation by rotation overnight at 4°C, immunoprecipitates were washed three times with lysis buffer and subjected to immunoblot analysis with anti-Cav-1 MAb 2297 probe.

Coimmunoprecipitation of GFP-Cav-1 with Myc-tagged Cav-1 and respective mutants. Immunoprecipitation was performed as described in detail above. Anti-green fluorescent protein (GFP) PAb (Santa Cruz Biotechnology) or preimmune serum was used to immunoprecipitate the GFP-tagged Cav-1 protein. Immunoblot analysis was performed with anti-Myc MAb (clone 9E10, Santa Cruz Biotechnology).

Purification of caveolae-enriched membrane fractions. Caveolae-enriched membrane fractions were purified essentially as previously described (43, 47). 293T cells plated on a 150-mm-diameter plate were transfected with the appropriate plasmid(s). At 36 h after transfection, the cells were washed twice in cold PBS, scraped into 2 ml of MBS (25 mM MES, pH 6.5, and 150 mM NaCl) containing 1% Triton X-100, passed 10 times through a loose-fitting Dounce homogenizer, and mixed with an equal volume of 80% sucrose (prepared in MBS lacking Triton X-100). The sample was then transferred to a 12-ml ultracentrifuge tube and overlaid with a discontinuous sucrose gradient (4 ml of 30% sucrose-4 ml of 5% sucrose, both prepared in MBS lacking detergent). The samples were subjected to centrifugation at 200,000 g (39,000 rpm in a Sorval rotor TH-641) for 16 h. A light-scattering band was observed at the 5%-30% sucrose interface. Twelve 1-ml fractions were collected, and 50-µl aliquots of each fraction were subjected to SDS-PAGE and immunoblotting.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cav-1 can interact with PKAalpha -cat and inhibit its phosphorylation of CREB in vivo. We first attempted to assess the interaction of caveolin with PKA in vivo by using coimmunoprecipitation analysis. We utilized the 293T cell line throughout these studies because of the failure of this cell line to endogenously express detectable levels of Cav-1. These cells were transfected with Cav-1 or PKAalpha -cat and subjected to immunoprecipitation using anti-PKAalpha -cat PAb or relevant negative controls (preimmune serum PAb and beads alone). A significant portion of Cav-1 (estimated at ~5-10%) is coimmunoprecipitated with PKA (Fig. 1A).


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Fig. 1.   Caveolin-1 (Cav-1) interacts with the protein kinase A (PKA)-alpha catalytic subunit (PKAalpha -cat) and inhibits its phosphorylation of cAMP response element binding protein (CREB) in vivo. A: cells were cotransfected with the cDNAs encoding Cav-1 and PKAalpha -cat. Cell lysates were prepared and immunoprecipitated with anti-PKAalpha -cat polyclonal antibody (PAb) or relevant negative controls (preimmune serum PAb and beads alone). Immunoprecipitates (IP) were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Cav-1 monoclonal antibody (MAb) 2297. Cav-1 is coimmunoprecipitated only in cells incubated with the anti-PKA PAb. B: cells were cotransfected with Cav-1, PKAalpha -cat, and/or appropriate empty vectors. Cell lysates containing phosphatase inhibitors were prepared and subjected to immunoblot analysis with phospho-Ser-133-specific anti-CREB PAb, anti-CREB PAb, or anti-Cav-1 MAb 2297. PKA can induce robust CREB phosphorylation, an effect that is completely inhibited by coexpressed Cav-1. Cav-1 FL, full-length Cav-1.

Caveolin has been shown to negatively regulate various members of the cAMP-mediated cascade (3, 16, 22, 32, 37, 49, 54). Thus we next attempted to substantiate Cav-1's regulation of PKA by assessing its effect on the phosphorylation of a major PKA substrate in vivo. Upon cAMP-mediated release from its bound regulatory subunits, PKA undergoes nuclear translocation. PKA-mediated phosphorylation of the Ser-133 residue in CREB is a pivotal step in its activation as a transcription factor (20). Using a phospho-Ser-133-specific anti-CREB antibody, we found that the expression of the catalytic subunit of PKA is sufficient to phosphorylate CREB (Fig. 1B). Coexpression of PKA with Cav-1 virtually abolished this phosphorylation event.

Construction of CSD and COOH-terminal domain mutants. By using GST-fusion pull-downs and in vitro kinase assays, it seems that two distinct regions of Cav-1 [i.e., the scaffolding domain (residues 82-101) and a COOH-terminal domain (residues 135-178)] are important in interacting with and inhibiting the enzymatic activity of PKA (37). With the use of similar in vitro assays involving synthetic peptides, mapping studies on the amino acids in the scaffolding domain implicated five mostly aromatic residues as responsible for this inhibition (37).

As a consequence, we set out to determine whether Cav-1 constructs harboring mutations in the scaffolding domain and/or the COOH-terminal domain retained the ability to abolish PKA activity in vivo (Fig. 2). Briefly, Cav-1 FL is the full-length wild-type cDNA, Cav-1 A-scan contains a conversion of the five above-mentioned scaffolding domain residues to alanine, Cav-1 Delta C lacks the COOH-terminal 38 amino acids, and Cav-1 A-scan/Delta C harbors the scaffolding domain mutations and the COOH-terminal deletion. All constructs were tagged with the Myc epitope at the NH2 terminus, a modification that has been shown not to affect Cav-1 function or its interactions with cellular constituents (52). This also served as a way to distinguish the mutants from the wild-type untagged protein in coexpression studies. Transient transfection of these constructs in 293T cells showed comparable levels of expression, as detected with antibodies directed against the Myc epitope or the NH2-terminal domain of Cav-1 (Figs. 3 and 4, bottom panels).


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Fig. 2.   Construction of Cav-1 scaffolding and COOH-terminal domain mutants. Cav-1 has been traditionally divided into 3 regions: the NH2-terminal domain (residues 1-101), the putative transmembrane domain (residues 102-134), and the COOH-terminal domain (residues 135-178) (52). The oligomerization domain (hatched bar) spans residues 61-101 and contains a subdomain (residues 82-101), known as the scaffolding domain (solid bar). Three Cav-1 mutants were constructed by standard PCR mutagenesis strategies. The Cav-1 A-scan contains the conversion of 5 residues in the scaffolding domain to alanine (dotted bar). The Cav-1 Delta C lacks the COOH-terminal 38 residues. The Cav-1 A-scan/Delta C construct is a double mutant harboring the scaffolding domain 5-residue alanine conversion, as well as the deletion of the COOH-terminal 38 amino acids. These 3 mutants harbor a disruption of one or both of the domains thought to be important for a functional interaction with PKA. TM, putative membrane spanning domain.



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Fig. 3.   Interaction between Cav-1 and PKAalpha -cat is mediated by the Cav-1 scaffolding and COOH-terminal domains in vivo. Cells were cotransfected with PKAalpha -cat and Myc-tagged full-length Cav-1 (Cav-1 FL) or the various Cav-1 mutants. Cell lysates were prepared and immunoprecipitated (IP'ed) with anti-PKAalpha -cat or relevant negative controls (preimmune serum PAb and beads alone). Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Myc MAb (9E10). Whereas Cav-1 FL and the A-scan and Delta C mutants interact to similar levels with PKA, the double mutant A-scan/Delta C fails to show an interaction. Irrel, irrelevant.



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Fig. 4.   Inhibition of PKA-mediated CREB phosphorylation by Cav-1 involves the scaffolding and COOH-terminal domains. Cells were cotransfected with CREB and a combination of PKAalpha -cat, Myc-tagged Cav-1 FL, or the Cav-1 mutants and/or appropriate empty vectors. Cell lysates containing phosphatase inhibitors were prepared and subjected to immunoblot analysis with phospho-Ser-133-specific anti-CREB PAb, anti-CREB PAb, or anti-Myc MAb (9E10). Only the double mutant A-scan/Delta C is unable to inhibit the PKA-induced CREB phosphorylation.

Functional interaction between Cav-1 and PKAalpha -cat is mediated by and requires the scaffolding and COOH-terminal domains. We first evaluated the ability of these mutants to interact with PKA by coimmunoprecipitation analysis. Cells were transfected with PKAalpha -cat and with Cav-1 FL or the various mutants and subjected to immunoprecipitation with anti-PKAalpha -cat PAb or relevant negative controls (preimmune serum PAb and beads alone). As expected, Cav-1 FL specifically interacts with PKAalpha -cat (Fig. 3). Interestingly, Cav-1 A-scan and Cav-1 Delta C also interact with PKA with levels of binding comparable to that of the full-length protein. However, the A-scan/Delta C construct harboring mutations in both putative binding domains is completely defective in its association with PKA.

We next assessed the ability of these mutants to functionally inhibit PKA-mediated phosphorylation of CREB on Ser-133. As shown in Fig. 4, cells were transfected with CREB, and a combination of PKA, the different Cav-1 cDNAs, or empty vector controls. PKA activity was assessed by a phospho-Ser-133-specific anti-CREB antibody. As expected, PKAalpha -cat alone can robustly phosphorylate CREB, while coexpression with Cav-1 FL reverts this effect to baseline. As with the immunoprecipitation studies above, Cav-1 A-scan and Delta C behave similarly to the full-length protein, while the double-mutant (Cav-1 A-scan/Delta C) is unable to inhibit PKA-mediated phosphorylation.

To corroborate these functional findings, we also assessed the inhibition of PKA signaling by using an established CRE gene reporter system (Stratagene). In this assay, a tandem of four consensus CREs drives the expression of a luciferase gene, which essentially acts as a surrogate for PKA activation. Cells were transfected with the CRE reporter and a combination of PKAalpha -cat, the different Cav-1 cDNAs, or empty vector controls. In direct support of the phospho-CREB experiments, Cav-1 FL and the single mutants (A-scan and Delta C) potently suppress this PKA-induced luciferase activity, while the double-mutant (A-scan/Delta C) completely lacks this inhibitory activity (Fig. 5).


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Fig. 5.   Inhibition of a PKA-responsive reporter by Cav-1 involves the scaffolding and COOH-terminal domains. Cells were transfected with a CREB-responsive luciferase reporter and a combination of PKAalpha -cat, Myc-tagged Cav-1 FL, or mutants and/or appropriate empty vectors. Luciferase assays were conducted on the cell lysates as described in METHODS. Luciferase activities are expressed as ratios normalized to beta -galactosidase activity, and each experimental value represented graphically is the average of 3 separate transfections performed in parallel; error bars represent the observed SD. In corroboration of the phospho-CREB experiments shown in Fig. 4, only the double mutant (A-scan/Delta C) is unable to inhibit PKA-stimulated transcriptional activity.

Wild-type Cav-1 interacts with its scaffolding/COOH-terminal domain mutant counterparts and recruits them to caveolar microdomains. The inability of the Cav-1 A-scan/Delta C mutant to inhibit PKA signaling could be due to several factors. An important possibility is the selective mislocalization of this mutant to noncaveolar membrane compartments, leading to less efficient functioning. Biochemically, we previously showed that caveolar microdomains can be separated from other cellular constituents via a sucrose gradient ultracentrifugation procedure. With this method, it is possible to concentrate Cav-1, the caveolae marker protein, by over ~2,000-fold with respect to total cellular proteins (24, 52) (Fig. 6A). By transfecting cells with the Cav-1 cDNAs, we applied this separation scheme to the various Cav-1 mutant constructs. The outputs of this centrifugation, which consist of 12 equal fractions (of which fractions 4-7 and 8-12 are considered of caveolar and noncaveolar origin, respectively) are shown in Fig. 6, A and B. Cav-1 FL selectively cofractionates in the caveolar compartment. However, the overwhelming majority of the Cav-1 mutants appear noncaveolar. This result indicates that the localization of Cav-1 to caveolae microdomains is dispensable for its inhibitory effects on PKA signaling. Perhaps more importantly, however, it also establishes that a selective mislocalization of the Cav-1 A-scan/Delta C is not the primary reason for its loss of function.


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Fig. 6.   Wild-type Cav-1 interacts with its scaffolding/COOH-terminal domain mutant counterparts and recruits them to caveolar microdomains. A: cells were transfected with Cav-1 FL and subjected to sucrose gradient centrifugation after homogenization in a buffer containing 1% Triton X-100 (see METHODS), a method that separates Triton-resistant caveolae-rich domains (fractions 4-7) from other cellular components (fractions 9-12). Immunoblot analysis with anti-caveolin MAb (2297) was used to detect the localization of Cav-1. As predicted, wild-type Cav-1 cofractionates with the caveolae microdomains. B: cells were cotransfected with the indicated Myc-tagged Cav-1 mutant cDNA and Cav-1 FL or empty vector. Centrifugation and immunoblot analysis with anti-Myc MAb (9E10) was used to detect the localization of the Cav-1 mutants. In the absence of Cav-1 FL, none of the mutants localize to caveolae microdomains. However, on cotransfection with Cav-1 FL, each mutant undergoes a dramatic shift toward the caveolar fractions. C: cells were cotransfected with the green fluorescent protein (GFP)-tagged Cav-1 FL and Myc-tagged Cav-1 FL or the various Cav-1 mutants. Cell lysates were prepared and immunoprecipitated with anti-GFP PAb or a relevant negative control (preimmune serum PAb). Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Myc MAb (9E10) (top). As predicted, Myc-tagged Cav-1 FL interacts with its GFP-tagged counterpart. The Cav-1 mutants also interact with the GFP-tagged Cav-1 FL, although the Delta C and A-scan/Delta C mutants do so to a lesser extent. Bottom: immunoblots of total cell lysates.

We next assessed whether wild-type Cav-1 could recruit these Cav-1 mutants to caveolae-enriched microdomains. For this purpose, cells were cotransfected with Cav-1 FL and a given Cav-1 mutant and subjected to subcellular fractionation. Figure 6, A and B, shows that coexpression with Cav-1 FL can recruit all three Cav-1 mutants to caveolae membranes, albeit with slightly different efficiencies. These results suggest that Cav-1 FL can interact with these Cav-1 mutants. To test this hypothesis more directly, we next used a coimmunoprecipitation approach, as outlined below.

With the use of coimmunoprecipitation and velocity sucrose gradient analyses, Cav-1 has been shown to self-associate and form high-mass homooligomers of 14-16 unit size (23, 42, 52). For example, Myc- or GFP-tagged Cav-1 can efficiently coimmunoprecipitate with the wild-type untagged protein (46; unpublished observations). Thus we were interested to see whether the Cav-1 mutants were capable of interacting with the wild-type protein. Given the localization of these mutants to noncaveolar fractions, this would allow us to determine their behavior when they are coexpressed with wild-type Cav-1. We decided to coimmunoprecipitate the Cav-1 mutants with the GFP-tagged wild-type Cav-1 to easily distinguish by size the immunoprecipitated entity from the interacting one.

Cells were cotransfected with GFP-Cav-1 FL and the desired mutant, immunoprecipitated with anti-GFP PAb, and immunoblotted with anti-Myc MAb (clone 9E10). Figure 6C shows that all three mutant constructs are capable of interacting with the wild-type GFP-tagged caveolin, albeit with different efficiencies. Although the Cav-1 A-scan mutant binds equally well to the wild-type protein, the Cav-1 Delta C and A-scan/Delta C mutants have a three- to fourfold decrease in binding efficiency to wild-type protein. These findings are in agreement with our cofractionation studies shown in Fig. 6, A and B, where these two Myc-tagged mutants are recruited by wild-type Cav-1 to caveolae microdomains, but not as efficiently as the Myc-tagged wild-type and A-scan caveolins.

CSD/COOH-terminal domain mutant acts in dominant-negative fashion to disrupt caveolin-mediated inhibition of PKA signaling. On the basis of the above-mentioned results, it appears that wild-type Cav-1 is able to interact with and sequester the Cav-1 mutants within caveolae membranes. This phenomenon led to the following attractive hypothesis: The Cav-1 A-scan/Delta C mutant, which is completely defective in inhibiting PKA-mediated signaling but retains its ability to cofractionate and interact with the wild-type Cav-1 protein, could act to disrupt the function of wild-type caveolin (i.e., exhibit a dominant-negative-type effect).

To test this hypothesis, cells were cotransfected with CREB and a combination of PKA, wild-type Cav-1, or empty vector controls. Cells expressing wild-type Cav-1 were also cotransfected with an increasing amount of the A-scan/Delta C mutant. PKA activity was assessed by a phospho-Ser-133-specific anti-CREB antibody. Figure 7 shows that, as expected, PKAalpha -cat alone can robustly phosphorylate CREB, while coexpression with Cav-1 FL reverts this effect to baseline.


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Fig. 7.   Cav-1 scaffolding/COOH-terminal domain mutant acts in dominant-negative fashion to disrupt caveolin-mediated inhibition of PKA signaling. Cells were cotransfected with CREB and a combination of PKAalpha -cat, Cav-1 FL, the Myc-tagged Cav-1 mutant A-scan/Delta C, and/or appropriate empty vectors. The level of expression for Cav-1 A-scan/Delta C was enhanced by transfecting an increasing amount of the cDNA (lanes 4-6, bottom). Cell lysates containing phosphatase inhibitors were prepared and subjected to immunoblot analysis with phospho-Ser-133-specific anti-CREB PAb. As the expression of the Cav-1 mutant A-scan/Delta C increases, the ability of Cav-1 FL to inhibit its phosphorylation is gradually abrogated. To indicate equal expression of the transfected proteins, immunoblots of the same cell lysates with anti-CREB PAb, anti-PKAalpha -cat, anti-Cav-1 MAb (2297), and anti-Myc MAb (9E10) are also shown.

Interestingly, as the amount of cotransfected Cav-1 A-scan/Delta C is gradually increased, there is a concomitant increase in phospho-CREB levels (Fig. 7). However, coexpression of this mutant is not sufficient to completely revert the suppression of CREB phosphorylation by wild-type Cav-1. This is not surprising, however, inasmuch as the interaction of the A-scan/Delta C mutant with wild-type Cav-1 is about three- to fourfold less than a wild-type-wild-type interaction (Fig. 6, B and C). Nevertheless, it is intriguing that a Cav-1 mutant harboring a disruption of the scaffolding and COOH-terminal domains (the 2 regions implicated in Cav-1's inhibition of PKA signaling) is capable of acting in dominant-negative fashion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We assessed the functional interaction of Cav-1 with PKA in vivo and used mutational analysis to gain insight into this process. We demonstrated that Cav-1 can interact with PKAalpha -cat by coimmunoprecipitation analysis. Furthermore, we showed that coexpression of Cav-1 can dramatically attenuate PKA-mediated signaling, as seen by the reduced phosphorylation levels and activity of a direct PKA substrate, CREB. Inasmuch as two regions of Cav-1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants showed that Cav-1 requires the presence of the scaffolding or the COOH-terminal domain (but not both) in its functional interaction with PKA. An analysis of the behavior of these caveolin mutants indicated that, in contrast to the wild-type protein, they are not localized to caveolae microdomains. However, each mutant maintained the ability to interact with the wild-type Cav-1 molecule, and, on coexpression with Cav-1, a substantial amount of these mutants was recruited to the caveolae membrane fraction. Finally, we showed that coexpression of the mutant with disrupted scaffolding and COOH-terminal domains can partially abrogate wild-type Cav-1's inhibition of PKA signaling in a dose-responsive manner (i.e., exhibiting a dominant-negative effect).

Numerous signaling molecules have been shown to be functionally associated with Cav-1 (reviewed in Ref. 38). A common denominator among many of these interactions seems to be the CSD, the juxtamembrane region in Cav-1 containing numerous aromatic residues. In the present study, we showed that the CSD and a second inhibitory domain, the COOH terminus, are important for binding to and inhibiting PKAalpha -cat. The possibility of a dual-domain inhibitory mechanism for caveolins had been proposed and tested in vitro for only one other signaling molecule, nitric oxide synthase (neuronal and endothelial) (19, 56). Our observation that only the dual CSD/COOH-terminal domain mutant is unable to interact with and inhibit PKA function in vivo lends credence to the need for two domains in Cav-1's ability to negatively regulate certain signaling cascades.

The dominant-negative property of this dual CSD/COOH-terminal domain mutant in Cav-1/PKA signaling is rather intriguing. Only a few caveolin mutants with proposed dominant-negative function have been reported (all for the closely related Cav-3 protein) (17, 40). Roy et al. (40) showed that the Cav-3 mutant exhibited dominant negativity by sequestering the wild-type Cav-3 protein to intracellular vesicles. Galbiati et al. (17) demonstrated that their mutant sequestered the wild-type protein to the Golgi compartment and directed it for proteosomal degradation. The dual CSD/COOH-terminal construct we describe here is unique in its apparent function. Our characterization of its biochemical behavior showed that, similar to its single-domain mutant counterparts, it retained the ability to interact with and be recruited by wild-type Cav-1 to caveolae microdomains. Although the exact membrane topology of Cav-1 is not known, protease protection studies and other deletion analyses have determined a hairpin structure with intracellularly protruding NH2 and COOH termini (28). Additionally, Cav-1 is known to form high-mass (14-16 unit) homooligomers (28, 52). In this conformation, the CSD and COOH-terminal domain are located intracellularly and are able to functionally interact with signaling molecules. It is plausible that coexpression of wild-type and mutant Cav-1 creates a chimeric high-mass heterooligomeric complex. Depending on the relative contribution of the mutant Cav-1 to the oligomer, this chimeric complex will retain only a partial ability to inhibit PKA signaling (Fig. 7). Our result that a gradual increase in expression levels of the mutant has a dose-dependent effect on the inhibitory capacity of wild-type Cav-1 seems to corroborate this idea.

What might be some of the physiological consequences of a Cav-1-PKA interaction? The discovery of the functionally related family of PKA scaffolding proteins known as AKAPs has reformulated the way we view PKA function. It is becoming readily apparent that there are distinct pools of PKA holoenzyme bound in an inactive state to different subcellularly targeted AKAPs. For example, PKA is targeted to the outer mitochondrial membrane by S-AKAP84/D-AKAP, to the plasma membrane by AKAP79 and AKAP18, and to the nuclear matrix by AKAP95 (2, 5, 6, 15, 21). Because the caveolins are a family of membrane-associated, cholesterol-binding proteins (29, 31), their functional interaction with PKA can occur only in distinct cellular compartments, namely, endoplasmic reticulum/Golgi compartments and plasma membrane caveolae. This localization in effect limits the pools of PKA that can physiologically interact with Cav-1 and implicates only a limited number of AKAPs.

The anchoring of PKA to the plasma membrane by AKAP79 and AKAP18 has implications in the functioning of certain ion channels, namely, L-type Ca2+ channels and certain K+ channels (1, 15, 18). Interestingly, L-type Ca2+ channels and some voltage-dependent K+ channels have recently been localized to caveolae (9, 26). It remains to be seen whether Cav-1 can affect the activity of these channels directly or through interactions with channel modulators such as PKA. The recently discovered 18-kDa AKAP18 (15, 55) is also intriguing in other respects. It is the first AKAP shown to be myristoylated and dually palmitoylated, moieties that are essential to its localization to the plasma membrane. For a protein harboring such lipid modifications, a well-known consequence is its targeting to caveolae microdomains. A detailed analysis of this process has been made for several signaling molecules, including the Src family kinases, ras, and endothelial nitric oxide synthase. In future studies, it will be interesting to investigate such compartmentalization of PKA, AKAP18, and associated proteins and to delineate the physiological role the caveolins might play in these PKA-mediated processes.


    ACKNOWLEDGEMENTS

We thank Dr. Roberto Campos-Gonzalez for the Cav-1 MAb clone 2297 and Dr. Bugger Wilson for critical reading of the manuscript.


    FOOTNOTES

This work was supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association, and the Komen Breast Cancer Foundation (to M. P. Lisanti). M. P. Lisanti is the recipient of a Hirschl/Weil-Caulier Career Scientist Award. B. Razani was supported by National Institutes of Health Medical Scientist Training Grant T32-GM-07288.

Address for reprint requests and other correspondence: M. P. Lisanti, Dept. of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (E-mail: lisanti{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 April 2001; accepted in final form 18 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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