Role of the membrane interface on the conformation of the caveolin scaffolding domain: a CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V(94)TKYWFYR(101) motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.     

Caveolin scaffolding region and cholesterol-rich domains in membranes

A protein that constitutes a good marker for a type of cholesterol-rich domain in biological membranes is caveolin. A segment of this protein has a sequence that corresponds to a cholesterol recognition/interaction amino acid consensus (CRAC) motif; this motif has been suggested to cause the incorporation of proteins into cholesterol-rich domains. We have studied the interaction of two peptides containing the CRAC motif of caveolin-1 by differential scanning calorimetry, fluorescence, circular dichroism and magic angle spinning NMR. These peptides promote the segregation of cholesterol into domains from mixtures of the sterol with phosphatidylcholine, as shown by depletion of cholesterol from a portion of the membrane and enrichment of cholesterol in another domain. Cholesterol passes its solubility limit in the cholesterol-rich domain, resulting in the formation of cholesterol crystallites, suggesting that not all of the cholesterol recruited to this domain is bound to the peptide. NMR studies show that the peptides insert somewhat more deeply into membranes when cholesterol is present, but their strongest interaction takes place with the interfacial region of the membrane. We conclude that the peptides we studied containing CRAC sequences are more effective in promoting the formation of cholesterol-rich domains than are shorter peptides of this region of caveolin, which although they contain several aromatic amino acids, they have no CRAC motif. The presence or absence of a CRAC motif, however, is not a sufficient criterion to determine the extent to which a protein will promote the segregation of cholesterol in membranes.     

Caveolin-1 is a competitive inhibitor of heme oxygenase-1 (HO-1) with heme: identification of a minimum sequence in caveolin-1 for binding to HO-1

Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 μM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.     

A role for the caveolin scaffolding domain in mediating the membrane attachment of caveolin-1. The caveolin scaffolding domain is both necessary and sufficient for membrane binding in vitro.

Here, we have created a series of caveolin-1 (Cav-1) deletion mutants to examine whether the membrane spanning segment is required for membrane attachment of caveolin-1 in vivo. One mutant, Cav-1-(1-101), contains only the cytoplasmic N-terminal domain and lacks the membrane spanning domain and the C-terminal domain. Interestingly, Cav-1-(1-101) still behaves as an integral membrane protein but lacks any known signals for lipid modification. In striking contrast, another deletion mutant, Cav-1-(1-81), behaved as a soluble protein. These results implicate caveolin-1 residues 82-101 (also known as the caveolin scaffolding domain) in membrane attachment. In accordance with the postulated role of the caveolin-1 scaffolding domain as an inhibitor of signal transduction, Cav-1-(1-101) retained the ability to functionally inhibit signaling along the p42/44 mitogen-activated protein kinase cascade, whereas Cav-1-(1-81) was completely ineffective. To rule out the possibility that membrane attachment mediated by the caveolin scaffolding domain was indirect, we reconstituted the membrane binding of caveolin-1 in vitro. By using purified glutathione S-transferase-caveolin-1 fusion proteins and reconstituted lipid vesicles, we show that the caveolin-1 scaffolding domain and the C-terminal domain (residues 135-178) are both sufficient for membrane attachment in vitro. However, the putative membrane spanning domain (residues 102-134) did not show any physical association with membranes in this in vitro system. Taken together, our results provide strong evidence that the caveolin scaffolding domain contributes to the membrane attachment of caveolin-1.     

In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.     

Regulation of G protein-coupled receptor kinases by caveolin

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

Identification of caveolin-1-interacting sites in neuronal nitric-oxide synthase. Molecular mechanism for inhibition of NO formation

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC(50) value of 1.8 microM. Mutations of Phe(584) and Trp(587) within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (Delta40) and a C-terminal truncation mutant (DeltaC33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of both Delta40 and DeltaC33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed to maintain eNOS in an inactive state; however, increased association of eNOS to heat shock protein 90 (hsp90) is observed following activation. In this study, we investigate the relationship between caveolin and hsp90 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82-101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82-101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90.     

Caveolin scaffolding region and the membrane binding region of SRC form lateral membrane domains

Formation of domains by the membrane binding motifs of caveolin and src were studied in large unilamellar vesicles using fluorescence digital imaging microscopy. Caveolin, a major structural protein of caveolae, contains a scaffolding region (residues 82-101) that contributes to the binding of the protein to the plasma membrane. A caveolin peptide (82-101) corresponding to this scaffolding region induced the formation of membrane domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Cholesterol, another predominant component of caveolae, was also enriched in these domains. Caveolae also contain many different signaling molecules including src family tyrosine kinases. Src proteins bind to the plasma membrane via a N-terminal myristate chain and a cluster of basic residues that can interact electrostatically with negatively charged lipids. A peptide corresponding to the src membrane binding motifs (residues myr-2-19) sequestered acidic lipids into lateral membrane domains. Both the src and the caveolin peptides colocalized together with acidic lipids in the domains. Control experiments show the domains are not the result of vesicle aggregation. Two-photon fluorescence correlation spectroscopy experiments suggest diffusion in the domains was slower, but the domains were dynamic. Protein kinase C phosphorylated src in its N-terminal membrane binding region; however, the caveolin scaffolding peptide inhibited this activity. Consequently, protein-induced membrane domains may affect cell signaling by organizing signal transduction components within the membrane and changing reaction rates.     

Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1

The human immunodeficiency virus, type 1 (HIV-1) gp41 core plays an important role in fusion between viral and target cell membranes. A single chain polypeptide, N36(L8)C34, which forms a six-helix bundle in physiological solution, can be used as a model of gp41 core. Here we identified from a 12-mer phage peptide library a positive phage clone displaying a peptide sequence with high binding activity to the HIV-1 gp41 core. The peptide sequence contains a putative gp41-binding motif, PhiXXXXPhiXPhi (X is any amino acid residue, and Phi is any one of the aromatic amino acid residues Trp, Phe, or Tyr). This motif also exists in the scaffolding domain of caveolin-1 (Cav-1), a known gp41-binding protein. Cav-1-(61-101) and Cav-1-(82-101), two recombinant fusion proteins containing the Cav-1 scaffolding domain, bound significantly to the gp41 expressed in mammalian cells and interacted with the polypeptide N36(L8)C34. These results suggest that the scaffolding domain of Cav-1 may bind to the gp41 core via the motif. This interaction may be essential for formation of fusion pore or endocytosis of HIV-1 and affect the pathogenesis of HIV-1 infection. Further characterization of the gp41 core-binding motifs may shed light on the alternative mechanism by which HIV-1 enters into the target cell.     

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.     

Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCalpha and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain-an essential region for caveolin interaction with signaling molecules--significantly inhibited agonist-induced translocation of both PKCalpha and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCalpha and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.     

Low molecular weight protein tyrosine phosphatase and caveolin-1: interaction and isoenzyme-dependent regulation

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are small enzymes that are ubiquitous in many organisms. They are important in biological processes such as cell proliferation, adhesion, migration, and invasiveness. LMW-PTP is expressed in mammalian cells as two isoforms (IF1 and IF2) originating through alternative splicing. We have previously shown that IF2 targets lipid rafts called caveolae and interacts with caveolin-1, their major structural protein. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that have been implicated in a variety of cellular functions, including signal transduction events. Caveolin-1 contains a scaffolding region that contributes to the binding of the protein to the plasma membrane and mediates protein omo- and etero-oligomerization. Interaction of many signaling molecules with the scaffolding domain sequesters them into caveolae and inhibits or suppresses their activities. Caveolin-interacting proteins usually have a typical sequence motif, also present in all the LMW-PTPs, which is characterized by aromatic or large hydrophobic residues in specific positions. We have examined here the interaction of the LMW-PTP isoforms with caveolin-1 and its molecular mechanism, together with the consequences for their tyrosine phosphatase activities. We found that IF1 and IF2 are both capable of interacting with defined regions of caveolin-1 and that their putative caveolin binding sequence motif is not responsible for the association. The formation of LMW-PTP/caveolin-1 complexes is accompanied by modulation of the enzyme activities, and the inhibitory effect elicited against IF1 is stronger than that against IF2. The caveolin scaffolding domain is directly involved in the observed phenomena.     

The subcellular localization control of integrin linked kinase 1 through its protein-protein interaction with caveolin-1

Integrin linked kinase 1 (ILK1), a member of the serine/threonine kinases, has been shown to be crucial for the cell survival, differentiation, and Wnt signaling. Firstly, by using a confocal microscopy and a transfection approach, we obtained the evidence that ILK1 interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. By ILK1 deletion mutant analysis, we characterized the caveolin-1-binding domain in the kinase domain of ILK1. In addition, we found that native ILK1 is associated with endogenous caveolin-1 in COS-1 cells. Secondly, transient transfection assays showed that a reduction in caveolin-1 binding leads to a substantial increase in the serine/threonine phosphorylation of ILK1. Thirdly, caveolin-1 and its scaffolding peptide (amino acids 82-101) functionally suppressed the auto-kinase activity of purified recombinant ILK1 protein. Fourthly, the association of ILK1 with caveolin-1 regulated its cytoplasmic retention; if it was not associated with caveolin-1, it was transported to the nucleus. Fifthly, we also noticed the putative nuclear localization sequences (nls) in ILK1 near the caveolin-1-binding domain. Thus, our data indicate that caveolin-1 regulates ILK1 auto-phosphorylation activity and its subcellular localization via a specific protein-protein interaction through blocking the exposure of its putative nls motif.     

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.     

Caveolin-1 regulates contractility in differentiated vascular smooth muscle

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.     

A molecular dissection of caveolin-1 membrane attachment and oligomerization. Two separate regions of the caveolin-1 C-terminal domain mediate membrane binding and oligomer/oligomer interactions in vivo

Caveolins form interlocking networks on the cytoplasmic face of caveolae. The cytoplasmically directed N and C termini of caveolins are separated by a central hydrophobic segment, which is believed to form a hairpin within the membrane. Here, we report that the caveolin scaffolding domain (CSD, residues 82-101), and the C terminus (residues 135-178) of caveolin-1 are each sufficient to anchor green fluorescent protein (GFP) to membranes in vivo. We also show that the first 16 residues of the C terminus (i.e. residues 135-150) are necessary and sufficient to attach GFP to membranes. When fused to the caveolin-1 C terminus, GFP co-localizes with two trans-Golgi markers and is excluded from caveolae. In contrast, the CSD targets GFP to caveolae, albeit less efficiently than full-length caveolin-1. Thus, caveolin-1 contains at least two membrane attachment signals: the CSD, dictating caveolar localization, and the C terminus, driving trans-Golgi localization. Additionally, we find that caveolin-1 oligomer/oligomer interactions require the distal third of the caveolin-1 C terminus. Thus, the caveolin-1 C-terminal domain has two separate functions: (i) membrane attachment (proximal third) and (ii) protein/protein interactions (distal third).