May act as a scaffolding protein within caveolar membranes. Interacts directly with G-protein alpha subunits and can functionally regulate their activity (By similarity). Involved in the costimulatory signal essential for T-cell receptor (TCR)-mediated T-cell activation. Its binding to DPP4 induces T-cell proliferation and NF-kappa-B activation in a T-cell receptor/CD3-dependent manner. Recruits CTNNB1 to caveolar membranes and may regulate CTNNB1-mediated signaling through the Wnt pathway.
CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.
Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.
Interacting selectively and non-covalently with cholesterol (cholest-5-en-3-beta-ol); the principal sterol of vertebrates and the precursor of many steroids, including bile acids and steroid hormones.
The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.
Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI(2)/TXA(2) ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer.
Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types, particularly endothelium. A major structural component is the membrane protein caveolin-1 which associates with numerous signalling molecules, including endothelial nitric oxide (eNOS). Caveolin-1, which co-immunoprecipitates with eNOS in preparations from endothelial cells, regulates eNOS activity, holding it inactive. Controversy now exists regarding the presence of caveolae and caveolin-1 in trophoblasts, hence this study was carried out to examine whether the high levels of eNOS expressed in human syncytiotrophoblast are associated with caveolin-1, and to find out if caveolae are present in villous cytotrophoblasts and syncytiotrophoblast. Immunohistochemistry of term placentae revealed only weak labelling for caveolin-1 in the syncytiotrophoblast although the endothelium of the terminal villus vessels stained strongly. By electron microscopy, numerous caveolae were identified in the villus capillary endothelium but were extremely rare in the syncytium. Caveolin-1 staining was extensive in purified, isolated term villous cytotrophoblasts, with the purity of these cytokeratin positive cells confirmed by cytospin analysis and flow cytometry. Caveolae were clearly demonstrated in ultrastructural sections of the purified cytotrophoblasts. The time course of expression of caveolin-1 and eNOS during differentiation of villous cytotrophoblast into syncytiotrophoblast in culture was studied. Western analysis showed that caveolin-1 expression evident in day 1 whole cell lysates decreased at day 3 when the cells had syncytialized and declined further by day 6, while the levels of actin (control) remained high. eNOS expression in the same samples followed a different pattern, with the low levels in day 1 cells increasing substantially by 3 days in culture, subsiding again by day 6. eNOS association with caveolin-1 in day 1 and day 3 trophoblast cultures was evidenced by the demonstration that eNOS co-immunoprecipitates with caveolin-1 and vice versa. We conclude that human villous cytotrophoblasts express caveolin-1, which assembles into caveolae. Differentiation into syncytium results in a decrease, but not disappearance, of expression of caveolin-1 and a marked reduction of the caveolae.
The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.
Evidence
2:
Inferred from Physical InteractionHGNC
The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.
Evidence
3:
Inferred from Physical InteractionIntAct
Recently, ID-1 (inhibitor of differentiation/DNA binding) is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways. However, how Id-1 interacts with these pathways and the immediate downstream effectors of the Id-1 protein are not known. In this study, using a yeast two-hybrid screening technique, we identified a novel Id-1-interacting protein, caveolin-1 (Cav-1), a cell membrane protein, and a positive regulator of cell survival and metastasis in prostate cancer. Using an immunoprecipitation method, we found that the helix-loop-helix domain of the Id-1 protein was essential for the physical interaction between Id-1 and Cav-1. In addition, we also demonstrated that the physical interaction between Id-1 and Cav-1 played a key role in the epithelial-mesenchymal transition and increased cell migration rate as well as resistance to taxol-induced apoptosis in prostate cancer cells. Furthermore, our results revealed that this effect was regulated by Id-1-induced Akt activation through promoting the binding activity between Cav-1 and protein phosphatase 2A. Our study demonstrates a novel Id-1 binding partner and suggests a molecular mechanism that mediates the function of Id-1 in promoting prostate cancer progression through activation of the Akt pathway leading to cancer cell invasion and resistance to anticancer drug-induced apoptosis.
Evidence
4:
Inferred from Physical InteractionBHF-UCL
Multiple cell-signaling pathways converge to modulate large-conductance, voltage- and Ca2+-sensitive K+ channel (maxi-K channel) activity and buffer cell excitability in human myometrial smooth muscle cells (hMSMCs). Recent evidence indicates that maxi-K channel proteins can target to membrane microdomains; however, their association with other proteins within these macromolecular complexes has not been elucidated. Biochemical isolation of detergent-resistant membrane fractions from human myometrium demonstrates the presence of maxi-K channels in lipid raft microdomains, which cofractionate with caveolins. In both nonpregnant and late-pregnant myometrium, maxi-K channels associate and colocalize with caveolar scaffolding proteins caveolin-1 and caveolin-2, but not caveolin-3. Disruption of cultured hMSMC caveolar complexes by cholesterol depletion with cyclodextrin increases an iberiotoxin-sensitive K+ current. Co-immunoprecipitations have indicated that the maxi-K channel also is associated with both alpha- and gamma-actin. Immunocytochemical analysis indicates colocalization of maxi-K channels, actin, and caveolin-1 in primary cultures of hMSMCs. Further experiments using immunoelectron microscopy have shown the proximity of both actin and the maxi-K channel within the same cell surface caveolar structures. Functionally, disruption of the actin cytoskeleton in cultured hMSMCs by cytochalasin D and latrunculin A greatly increased the open-state probability of the channel, while stabilization of actin cytoskeleton with jasplakinolide abolished the effect of latrunculin A. These data indicate that the actin cytoskeleton is involved as part of a caveolar complex in the regulation of myometrial maxi-K channel function.
Evidence
5:
Inferred from Physical InteractionIntAct
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1-/-) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1-Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID-Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
Evidence
6:
Inferred from Physical InteractionBHF-UCL
BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.
Evidence
7:
Inferred from Physical InteractionIntAct
Both caveolin-1 (Cav-1) and Mcl-1 have been implicated in the regulation of cancer cell anoikis, but their relationship and underlying mechanisms of regulation are not known. The present study demonstrated for the first time that Cav-1 regulates Mcl-1 through protein-protein interaction and inhibits its downregulation during cell anoikis in human lung cancer cells. Immunoprecipitation and immunocytochemistry studies showed that Cav-1 interacted with Mcl-1 and prevented it from degradation via the ubiquitin-proteasome pathway. Mcl-1 and Mcl-1-Cav-1 complex were highly elevated in Cav-1-overexpressing cells but were greatly reduced in Cav-1 knockdown cells. Consistent with this finding, we found that Mcl-1 ubiquitination was significantly attenuated by Cav-1 overexpression but increased by Cav-1 knockdown. Together, our results indicate a novel role of Cav-1 in anoikis regulation through Mcl-1 interaction and stabilization, which provides a new insight to the pathogenesis of metastatic lung cancer and its potential treatment.
Evidence
8:
Inferred from Physical InteractionUniProtKB
CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.
Evidence
9:
Inferred from Physical InteractionIntAct
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
Evidence
10:
Inferred from Physical InteractionUniProtKB
Deleted in liver cancer 1 (DLC1) is a recently identified tumor suppressor gene frequently underexpressed in hepatocellular carcinoma (HCC). DLC1 encodes a Rho GTPase-activating protein domain that exhibits growth-suppressive activity in HCC cell lines. Our recent finding has revealed that inhibition of Rho-mediated actin stress fiber formation by DLC1 is associated with its growth inhibitory activity. In the present study, we identified tensin2 as the novel binding partner of DLC1. Tensin2 belongs to a new family of focal adhesion proteins that play key roles in cytoskeleton organization and signal transduction. Dysregulation of tensin proteins has previously been implicated in human cancers. Tensin2 is highly expressed in human liver. Introduction of tensin2 into HCC cell lines with low expression of tensin2 caused significant growth inhibition and induction of apoptosis. Tensin2 directly interacted with DLC1 in vitro and in vivo. Both proteins localized to punctate structures in the cytoplasm. Sequence analysis of DLC1 and tensin2 identified caveolin-1 binding motif in both proteins. In vivo immunoprecipitation study confirmed that both proteins indeed interacted with endogenous caveolin-1, which is the major structural component of caveolae. Our findings presented here suggest a new model for the action of DLC1 in hepatocytes, whereby DLC1-tensin2 complex interacts with Rho GTPases in caveolae to effect cytoskeletal reorganization.
Evidence
11:
Inferred from Physical InteractionBHF-UCL
Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms alpha and beta on the cell surface. BRIa colocalizes predominantly with the caveolin-1 alpha isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 beta. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 alpha. BRII is expressed in regions enriched in caveolin-1 alpha and beta. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 alpha. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 alpha and beta. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 beta isoform inhibits BMP signaling whereas the alpha isoform does not.
Evidence
12:
Inferred from Physical InteractionIntAct
HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.
Evidence
13:
Inferred from Physical InteractionBHF-UCL
Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
Interacting selectively and non-covalently with one or more specific sites on a receptor molecule, a macromolecule that undergoes combination with a hormone, neurotransmitter, drug or intracellular messenger to initiate a change in cell function.
Evidence
1:
Inferred from Physical InteractionBHF-UCL
beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.
J. Cell. Sci. 115, 827-838 (2002)[PubMed:11865038]
Group 1 of plant pathogenesis-related proteins (PR-1) and a variety of related mammalian proteins constitute a superfamily of proteins that share structural similarities. Little is known about their function, but all the family members identified to date are co-translationally translocated to the lumen of the endoplasmic reticulum and are secreted as soluble proteins or are targeted to vacuoles. Here we report the identification of a novel family member that localizes to the cytosolic site of the endomembrane system in mammalian cells. After detergent solubilization of isolated Golgi membranes, a 17 kDa protein was found associated with a low-density detergent-insoluble fraction. The amino-acid sequence, determined by microsequencing and molecular cloning, revealed a significant homology with the superfamily of PR-1 proteins. Golgi-associated PR-1 protein (GAPR-1) showed a brefeldin-A-sensitive Golgi localization in immunofluorescence. Interestingly, the protein remained associated with the microdomain fraction in the presence of Brefeldin A. By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1. Tissue screening revealed that GAPR-1 is not detectably expressed in liver, heart or adrenal glands. High expression was found in monocytes, leukocytes, lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.
A series of molecular signals which triggers the apoptotic death of a cell. The pathway starts with reception of a signal, and ends when the execution phase of apoptosis is triggered.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1-/-) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1-Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID-Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
The aggregation, arrangement and bonding together of a set of components to form a caveola. A caveola is a plasma membrane raft that forms a small pit, depression, or invagination that communicates with the outside of a cell and extends inward, indenting the cytoplasm and the cell membrane.
Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating increased oxygen tension.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1-/-) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1-Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID-Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of deprivation of nourishment.
Evidence
1:
Inferred from Expression PatternBHF-UCL
Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms alpha and beta on the cell surface. BRIa colocalizes predominantly with the caveolin-1 alpha isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 beta. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 alpha. BRII is expressed in regions enriched in caveolin-1 alpha and beta. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 alpha. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 alpha and beta. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 beta isoform inhibits BMP signaling whereas the alpha isoform does not.
The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.
The directed movement of cholesterol, cholest-5-en-3-beta-ol, into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.
Any process involved in the maintenance of an internal steady state of calcium ions within the cytosol of a cell or between the cytosol and its surroundings.
Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
The accumulation and maintenance in cells or tissues of lipids, compounds soluble in organic solvents but insoluble or sparingly soluble in aqueous solvents. Lipid reserves can be accumulated during early developmental stages for mobilization and utilization at later stages of development.
The process whose specific outcome is the progression of the mammary gland over time, from its formation to the mature structure. The mammary gland is a large compound sebaceous gland that in female mammals is modified to secrete milk. Its development starts with the formation of the mammary line and ends as the mature gland cycles between nursing and weaning stages.
An intracellular protein kinase cascade containing at least a MAPK, a MAPKK and a MAP3K. The cascade can also contain two additional tiers: the upstream MAP4K and the downstream MAP Kinase-activated kinase (MAPKAPK). The kinases in each tier phosphorylate and activate the kinases in the downstream tier to transmit a signal within a cell.
The process in which membrane potential changes in the depolarizing direction from the resting potential, usually from negative to positive. For example, the initial depolarization during the rising phase of an action potential is in the direction from the negative resting potential towards the positive membrane potential that will be the peak of the action potential.
Both caveolin-1 (Cav-1) and Mcl-1 have been implicated in the regulation of cancer cell anoikis, but their relationship and underlying mechanisms of regulation are not known. The present study demonstrated for the first time that Cav-1 regulates Mcl-1 through protein-protein interaction and inhibits its downregulation during cell anoikis in human lung cancer cells. Immunoprecipitation and immunocytochemistry studies showed that Cav-1 interacted with Mcl-1 and prevented it from degradation via the ubiquitin-proteasome pathway. Mcl-1 and Mcl-1-Cav-1 complex were highly elevated in Cav-1-overexpressing cells but were greatly reduced in Cav-1 knockdown cells. Consistent with this finding, we found that Mcl-1 ubiquitination was significantly attenuated by Cav-1 overexpression but increased by Cav-1 knockdown. Together, our results indicate a novel role of Cav-1 in anoikis regulation through Mcl-1 interaction and stabilization, which provides a new insight to the pathogenesis of metastatic lung cancer and its potential treatment.
Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms alpha and beta on the cell surface. BRIa colocalizes predominantly with the caveolin-1 alpha isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 beta. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 alpha. BRII is expressed in regions enriched in caveolin-1 alpha and beta. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 alpha. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 alpha and beta. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 beta isoform inhibits BMP signaling whereas the alpha isoform does not.
Any process that decreases the rate, frequency, or extent of the Wnt receptor signaling pathway through beta-catenin, the series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell, followed by propagation of the signal via beta-catenin, and ending with a change in transcription of target genes.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of nitric oxide.
In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.
beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.
Both caveolin-1 (Cav-1) and Mcl-1 have been implicated in the regulation of cancer cell anoikis, but their relationship and underlying mechanisms of regulation are not known. The present study demonstrated for the first time that Cav-1 regulates Mcl-1 through protein-protein interaction and inhibits its downregulation during cell anoikis in human lung cancer cells. Immunoprecipitation and immunocytochemistry studies showed that Cav-1 interacted with Mcl-1 and prevented it from degradation via the ubiquitin-proteasome pathway. Mcl-1 and Mcl-1-Cav-1 complex were highly elevated in Cav-1-overexpressing cells but were greatly reduced in Cav-1 knockdown cells. Consistent with this finding, we found that Mcl-1 ubiquitination was significantly attenuated by Cav-1 overexpression but increased by Cav-1 knockdown. Together, our results indicate a novel role of Cav-1 in anoikis regulation through Mcl-1 interaction and stabilization, which provides a new insight to the pathogenesis of metastatic lung cancer and its potential treatment.
Any process that stops, prevents, or reduces the frequency, rate or extent of the introduction of a phosphate group to a tyrosine residue of a Stat5 protein.
Any process that increases the rate of the directed movement of calcium ions into the cytosol of a cell. The cytosol is that part of the cytoplasm that does not contain membranous or particulate subcellular components.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
Any process that increases the rate, frequency, or extent of the Wnt receptor signaling pathway through beta-catenin, the series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell, followed by propagation of the signal via beta-catenin, and ending with a change in transcription of target genes.
beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1-/-) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1-Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID-Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1-/-) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1-Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID-Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.
The process of creating protein oligomers, compounds composed of a small number, usually between three and ten, of identical component monomers. Oligomers may be formed by the polymerization of a number of monomers or the depolymerization of a large protein polymer.
beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.
BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a calcium ion stimulus.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of stimulus by an estrogen, C18 steroid hormones that can stimulate the development of female sexual characteristics.
It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level.
Any process that results in a change in state or activity of an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a inadequate blood supply.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a progesterone stimulus.
It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.
The developmental sequence of events leading to the formation of adult muscle that occurs in the anima. In vertebrate skeletal muscle the main events are: the fusion of myoblasts to form myotubes that increase in size by further fusion to them of myoblasts, the formation of myofibrils within their cytoplasm and the establishment of functional neuromuscular junctions with motor neurons. At this stage they can be regarded as mature muscle fibers.
The process of providing, via surface-bound receptor-ligand pairs, a second, antigen-independent, signal in addition to that provided by the T cell receptor to augment T cell activation.
CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.
The chemical reactions and pathways involving triglyceride, any triester of glycerol. The three fatty acid residues may all be the same or differ in any permutation. Triglycerides are important components of plant oils, animal fats and animal plasma lipoproteins.
Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.
Viral protein involved in a direct and specific interaction with a host macromolecule. Viruses interact with many cellular pathways to achieve their replication cycle. Entry into the host cell, transport to the viral replication sites or viral budding are all steps that require interaction between the host and the virus. Additionally, the evasion from the host immune response requires a lot of viral proteins to associate with and inhibit cellular proteins with antiviral functions.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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