Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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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
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INTRODUCTION |
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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 Gs 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
-adrenergic receptors, G
and G
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.
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METHODS |
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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 (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
C constructs then served as templates to further generate scaffolding domain alanine-scan mutants (A-scan and A-scan/
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-PKA-catalytic (PKA
-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.
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RESULTS |
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Cav-1 can interact with PKA-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 PKA
-cat and subjected to
immunoprecipitation using anti-PKA
-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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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
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Functional interaction between Cav-1 and PKA-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
PKA
-cat and with Cav-1 FL or the various mutants and subjected to
immunoprecipitation with anti-PKA
-cat PAb or relevant negative controls (preimmune serum PAb and beads alone). As expected, Cav-1 FL
specifically interacts with PKA
-cat (Fig. 3). Interestingly, Cav-1
A-scan and Cav-1
C also interact with PKA with levels of binding
comparable to that of the full-length protein. However, the A-scan/
C
construct harboring mutations in both putative binding domains is
completely defective in its association with PKA.
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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/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/
C
is not the primary reason for its loss of function.
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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/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).
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DISCUSSION |
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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 PKA-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 PKA-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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Roberto Campos-Gonzalez for the Cav-1 MAb clone 2297 and Dr. Bugger Wilson for critical reading of the manuscript.
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FOOTNOTES |
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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.
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