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• W1965779516 abstract "The transforming growth factor β (TGF-β) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and another endothelial-specific TGF-β receptor, endoglin, both bind TGF-β with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-β, and the mechanism by which these receptors signal in endothelial cells remains unknown. Here we report the identification of the nuclear receptor liver X receptor β (LXRβ) as a modulator/mediator of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to LXRβ in vitro andin vivo. Expression of activated ALK-1 results in translocation of LXRβ from the nuclear compartment to the cytoplasmic compartment. The interaction of activated ALK-1 with LXRβ in the cytoplasmic compartment results in the specific phosphorylation of LXRβ by ALK-1, primarily on serine residues. LXRβ subsequently modulates signaling by ALK-1 and the closely related TGF-β receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRβ as a potential modulator/mediator of ALK-1/ALK-2 signaling. The transforming growth factor β (TGF-β) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and another endothelial-specific TGF-β receptor, endoglin, both bind TGF-β with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-β, and the mechanism by which these receptors signal in endothelial cells remains unknown. Here we report the identification of the nuclear receptor liver X receptor β (LXRβ) as a modulator/mediator of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to LXRβ in vitro andin vivo. Expression of activated ALK-1 results in translocation of LXRβ from the nuclear compartment to the cytoplasmic compartment. The interaction of activated ALK-1 with LXRβ in the cytoplasmic compartment results in the specific phosphorylation of LXRβ by ALK-1, primarily on serine residues. LXRβ subsequently modulates signaling by ALK-1 and the closely related TGF-β receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRβ as a potential modulator/mediator of ALK-1/ALK-2 signaling. transforming growth factor hereditary hemorrhagic telangiectasia hemagglutinin green fluorescence protein activation factor liver X receptor Transforming growth factor β (TGF-β)1 is a member of a family of dimeric polypeptide growth factors that regulate cellular proliferation and differentiation as well as the processes of embryonic development, wound healing, and angiogenesis in a cell- and context-specific manner (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Mutations in TGF-β receptors or their intracellular signaling molecules have been described in association with tumorigenesis and the human disease hereditary hemorrhagic telangiectasia (HHT). HHT is an autosomal dominant disease in which the primary defect is vascular dysplasia resulting in telangiectasia and arteriovenous malformations. Clinically this disorder results in telangiectases of the skin, recurrent epistaxis, and gastrointestinal bleeding as well as shunting phenomena and neurological sequela because of arteriovenous malformations in the pulmonary and central nervous system (2Guttmacher A.E. Marchuk D.A. White R.I., Jr. New Engl. J. Med. 1995; 333: 918-924Crossref PubMed Scopus (907) Google Scholar). The pathological lesions of HHT consist of dilated vessels, lined by a single layer of endothelium attached to a continuous basement membrane, that are thought to form from dilation of postcapillary venules. Genetic linkage studies of families with HHT identified the genes for two receptors in the TGF-β family, endoglin and ALK-1 (a type I receptor in the TGF-β family) as the genes responsible for hereditary hemorrhagic telangiectasia-1 and -2, respectively (3McAllister K.A. Grogg K.M. Johnson D.W. Gallione C.J. Baldwin M.A. Jackson C.E. Helmbold E.A. Markel D.S. McKinnon W.C. Murrell J. et al.Nat. Genet. 1994; 8: 345-351Crossref PubMed Scopus (1268) Google Scholar, 4Johnson D.W. Berg J.N. Baldwin M.A. Gallione C.J. Marondel I. Yoon S.J. Stenzel T.T. Speer M. Pericak-Vance M.A. Diamond A. Guttmacher A.E. Jackson C.E. Attisano L. Kucherlapati R. Porteous M.E. Marchuk D.A. Nat. Genet. 1996; 13: 189-195Crossref PubMed Scopus (884) Google Scholar). Endoglin and ALK-1 are expressed specifically on endothelial cells, and endoglin production is increased in proliferating endothelium (5Burrows F.J. Derbyshire E.J. Tazzari P.L. Amlot P. Gazdar A.F. King S.W. Letarte M. Vitetta E.S. Thorpe P.E. Clin. Cancer Res. 1995; 1: 1623-1634PubMed Google Scholar). Mice lacking endoglin or ALK-1 died at embryonic day 10.5–11.5 because of defects in angiogenesis (6Li D.Y. Sorensen L.K. Brooke B.S. Urness L.D. Davis E.C. Taylor D.G. Boak B.B. Wendel D.P. Science. 1999; 284: 1534-1537Crossref PubMed Scopus (723) Google Scholar, 7Bourdeau A. Dumont D.J. Letarte M. J. Clin. Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (390) Google Scholar, 8Arthur H.M. Ure J. Smith A.J. Renforth G. Wilson D.I. Torsney E. Charlton R. Parums D.V. Jowett T. Marchuk D.A. Burn J. Diamond A.G. Dev. Biol. 2000; 217: 42-53Crossref PubMed Scopus (383) Google Scholar, 9Oh S.P. Seki T. Goss K.A. Imamura T., Yi, Y. Donahoe P.K., Li, L. Miyazono K. ten Dijke P. Kim S. Li E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2626-2631Crossref PubMed Scopus (735) Google Scholar, 10Urness L.D. Sorensen L.K. Li D.Y. Nat. Genet. 2000; 26: 328-331Crossref PubMed Scopus (375) Google Scholar). Histochemical studies of the embryos revealed primary defects in the endothelial and smooth muscle cells of the developing blood vessels, confirming an essential role for these TGF-β receptors in angiogenesis. ALK-1 and endoglin mutations in HHT result in similar phenotypes: both have similar affinity and specificity for TGF-β (with preference for the TGF-β1 and -3 isoforms) and have been shown to associate with one another, suggesting that both receptors transduce similar signals essential for endothelial cell function (11Lux A. Attisano L. Marchuk D.A. J. Biol. Chem. 1999; 274: 9984-9992Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). However, the nature of the signaling pathway downstream from these receptors is unknown. To establish the signaling pathway downstream of ALK-1 and endoglin, yeast two-hybrid screens utilizing the cytoplasmic domains of these receptors were performed. Here we report the identification of the nuclear receptor LXRβ as a protein that specifically binds to the cytoplasmic domain of activated ALK-1. ALK-1 alters LXRβ cellular localization and phosphorylates LXRβ, primarily on serine residues, and LXRβ is able to modulate ALK-1 signaling, establishing LXRβ as a potential member of the ALK-1 signaling pathway. The mating system of James, Halladay, and Craig was utilized (12James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Briefly, a human lung library (Clontech) was screened with baits composed of the cytoplasmic domain of ALK-1 and mutants of the cytoplasmic domain of ALK-1 (ALK-1-Q200D and ALK-K229R) cloned into pGBD, in-frame with the Gal4 DNA binding domain. Yeast containing bait and the prey library cloned in pGAD, in-frame with the Gal4 activation domain, were mated overnight in YPAD (yeast extract, peptone, adenine, and dextrose) media at 30 °C, plated on Trp−, Leu−plates and incubated at 30 °C for 3–5 days to select for yeast containing both bait and prey vectors. Colonies were replica-plated on His− or His−, Ade− plates to assay for interaction as assessed by growth under these conditions. Interacting clones were tested for ability to repeat, bait, and prey dependence and specificity with other baits (lamin, myc, endoglin, ALK-2) to screen out false positives. Clones that passed these screens were sequenced and further analyzed. Appropriate strains of yeast (a strain for bait, α strain for library) were transformed with pGBD control vector or pGBD-ALK-1, -ALK-1Q-D, -ALK-1K-R, -ALK-2, or -END (containing the cytoplasmic domain of these respective proteins) and pGAD-LXRβ (encoding LXRβ lacking the first 14 amino acids). These yeast were mated overnight in YPAD media at 30 °C, plated on Trp−, Leu− plates, and incubated at 30 °C for 3–5 days to allow diploid cells to form visible colonies. Colonies were replica-plated on His−, Ade− plates to assay for interaction as indicated by colony growth. COS-7 or 293T cells expressing HA- or FLAG-tagged LXRβ or HA-tagged LXRα were lysed with 1% Triton X-100 lysis buffer and precleared with glutathione-agarose beads. Equal amounts of cell lysate were incubated with GST fusion proteins of the cytoplasmic domain of ALK-1 (GST-ALK-1), activated ALK-1 (GST-ALK-1Q-D), or GST alone complexed with glutathione-agarose beads. The beads were harvested by centrifugation and washed three times with lysis buffer. Binding proteins were analyzed by SDS-PAGE and Western blot analysis with αHA or αFLAG antibody as appropriate. THP-1 macrophage cells were lysed in RIPA lysis buffer with 1 mm phenylmethylsulfonyl fluoride and 0.02% leupeptin. The lysates was precleared by centrifugation, and 500-μl aliquots were incubated with polyclonal preimmune serum, polyclonal αALK-1 antibody (made to the cytoplasmic domain of ALK-1, peptide KREPVVAVAAPASSESSST), or αLXR antibody (Affinity BioReagents) for 2 h at 4 °C. The immune complexes were harvested by addition of Protein A beads. The pellets were washed three times with ice-cold lysis buffer. 50 μl of 2× sample buffer was added, and samples were boiled and loaded on a 12.5% SDS-PAGE, transferred to nitrocellulose, and visualized by Western blot analysis with αALK-1 antibody. Transfected HEK-293 cells were split onto collagen-coated 35-mm plastic dishes with glass bottoms (MatTek, Ashland, MA) and cultured overnight. GFP images were generated and collected using single line excitation (488 nm) and a 505-nm emission filter with a Zeiss laser scanning confocal microscope (LSM-510). COS-7 cells were transfected with various TGF-β receptors and HA-tagged LXRβ or -α. After 48 h, the cells were washed in phosphate-free medium and labeled with [32P]orthophosphate (1.0 mCi/ml) for 4 h. Cells were washed with phosphate-buffered saline, lysed with RIPA lysis buffer, immunoprecipitated with αHA antibody and Protein G-Sepharose, and analyzed on 10% SDS-PAGE gels; phosphorylation was detected by phosphoimager analysis of the dried gels. In vivo 32P-labeled LXRβ was immunoprecipitated with αHA antibody, resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was washed, and the 32P-labeled LXRβ was detected by autoradiography and excised from the membrane. The protein was hydrolyzed to completion by placing in 6 m HCl at 110 °C for 1 h, dried down, resuspended, and spotted on a TLC plate along with unlabeled phosphoamino acid standards. The TLC was wet with 66% (pH 1.9, electrophoresis buffer (0.58 m formic acid, 1.36m acetic acid)): 34% (pH 3.5, electrophoresis buffer (0.5% (v/v) pyridine, 0.87 m acetic acid, 0.5 mm EDTA)) and electrophoresed for 1 h at 1.5 kV. The plate was dried at 50 °C, sprayed with 0.25% ninhydrin in acetone, reheated to visualize the standards, and then analyzed by phosphoimager analysis. In vivo 32P-labeled LXRβ was resolved by SDS-PAGE, detected by autoradiography, and excised from the dried gel. The gel was rehydrated in 50 mm ammonium bicarbonate, pH 7.3, and the proteins were serially extracted by addition of 1% 2-mercaptoethanol and 0.1% SDS until over 60% of labeled protein was extracted by Cerenkov counting. 20 μg of Rnase (carrier protein) was added; the mixture was trichloroacetic acid-precipitated, oxidized in cold performic acid, dried, and digested overnight in 50 mm ammonium bicarbonate, pH 8.0. with 10 μg of trypsin. The digested peptides were lyophilized, resuspended in pH 1.9 electrophoresis buffer: pH 3.5 electrophoresis buffer (2:1), and spotted on a TLC plate. The TLC was electrophoresed in the first dimension for 20 min at 1.0 kV, then air dried, rotated 90° counterclockwise, and separated in the second dimension by chromatography in standard chromatography buffer until the buffer was 2 cm from the top of plate. The plate was dried at 65 °C and analyzed by phosphoimager analysis. P19 or L6 cells were plated onto 24-well plates at 20,000 cells/ml 1 day prior to transfection. The cells were transiently transfected using FuGene 6 reagent (Roche) with 300 ng/well of total DNA (100 ng of XVent-Lux or pE2.1-Lux, 100 ng of LXRβ or -α expression vectors, and 100 ng of ALK-1 Q-D or -2 Q-D expression vectors). The following morning the cells were washed twice in phosphate-buffered saline and were immediately incubated in α-MEM (minimum essential medium) with 0.2% fetal bovine serum for 18–24 h at 37 °C. Afterward, cells were lysed in 100 μl of passive lysis buffer (Promega) and 25 μl used for luciferase assays (Promega). Data are from experiments done in triplicate and are presented as the mean -fold stimulations (± S.E.) of the luciferase induced by ALK-1 Q-D or -2 Q-D relative to samples transfected with control vector. To elucidate potential downstream signaling molecules for ALK-1, the cytoplasmic domain of ALK-1 was utilized as a bait to screen for interacting proteins with the yeast two-hybrid system of James, Halladay, and Craig (12James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Initial screens of a murine embryonic library (day 10.5) and a human lung library with the entire cytoplasmic domain of ALK-1 did not yield any specific clones, whereas screens with the entire cytoplasmic domain of ALK-2 done in parallel revealed numerous clones of FKBP12 as has been previously reported (13Wang T. Donahoe P.K. Zervos A.S. Science. 1994; 265: 674-676Crossref PubMed Scopus (312) Google Scholar) (data not shown). Because the cytoplasmic domain of ALK-1 contains a serine/threonine protein kinase and constitutively active ALK-1 has been reported to activate BMP-responsive promoters (14Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 15Chen Y.G. Massague J. J. Biol. Chem. 1999; 274: 3672-3677Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and phosphorylate Smad1 and Smad5 (14Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 15Chen Y.G. Massague J. J. Biol. Chem. 1999; 274: 3672-3677Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), baits of the cytoplasmic domain of constitutively active ALK-1 (ALK-1 Q200D, mutating glutamine 200 to aspartic acid in the GS domain) and kinase-dead ALK-1 (ALK-1 K229R, mutating lysine 229 to arginine in the ATP binding site) were utilized to rescreen the human lung library. The ALK-1 K229R bait again did not yield any specific clones; however, the screen with the ALK-1 Q200D bait yielded 17 specific clones. Three of these isolates encoded two separate clones, both encompassing nearly the entire open reading frame of the nuclear receptor LXRβ (Fig. 1 A). LXRβ has been previously identified as an orphan nuclear receptor that forms obligate heterodimers with the retinoid X receptor to bind and regulate promoters with a unique hormone response element (LXRE) (16Shinar D.M. Endo N. Rutledge S.J. Vogel R. Rodan G.A. Schmidt A. Gene. 1994; 147: 273-276Crossref PubMed Scopus (120) Google Scholar, 17Apfel R. Benbrook D. Lernhardt E. Ortiz M.A. Salbert G. Pfahl M. Mol. Cell. Biol. 1994; 14: 7025-7035Crossref PubMed Scopus (296) Google Scholar, 18Song C. Kokontis J.M. Hiipakka R.A. Liao S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10809-10813Crossref PubMed Scopus (210) Google Scholar, 19Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar, 20Teboul M. Enmark E., Li, Q. Wikstrom A.C. Pelto-Huikko M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2096-2100Crossref PubMed Scopus (200) Google Scholar). LXRβ contains an amino-terminal PEST domain followed by a DNA binding domain and then a ligand binding domain containing an activation function (AF-2) (Fig. 1 A). More recently the oxysterols 24(S), 25-epoxycholesterol and 24(S) hydroxycholesterol, have been identified as potential endogenous ligands for LXRβ and the closely related nuclear receptor LXRα (21Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1477) Google Scholar, 22Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B., Su, J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). In addition, LXRα has been established as a sensor of dietary cholesterol, consistent with its restricted expression in tissues rich in lipid metabolism (23Peet D.J. Turley S.D., Ma, W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1252) Google Scholar). In contrast, the role of LXRβ, which is ubiquitously expressed, remains to be established. Significantly, unlike LXRα knockout mice, LXRβ knockout mice lack a metabolic/lipid metabolism phenotype (24Alberti S. Schuster G. Parini P. Feltkamp D. Diczfalusy U. Rudling M. Angelin B. Bjorkhem I. Pettersson S. Gustafsson J.A. J. Clin. Invest. 2001; 107: 565-573Crossref PubMed Scopus (320) Google Scholar). Association of ALK-1 Q200D with LXRβ was specific because LXRβ was not isolated in screens with ALK-1, -1 K229R, or -2 (data not shown). In addition, the cytoplasmic domain of ALK-1 Q200D could interact with LXRβ in the yeast two-hybrid interaction assay, whereas the cytoplasmic domains of ALK-1, -1 K229R, -2, or endoglin could not (Fig. 1 B). To confirm the interaction of ALK-1 with LXRβ, GST fusion proteins of the cytoplasmic domain of ALK-1 and -1 Q200D and an HA-tagged LXRβ lacking the first 14 amino acids of the coding sequence (corresponding to the longer yeast two-hybrid clone isolated, Fig. 1 A) were made. HA-tagged LXRβ was efficiently expressed in COS-7 cells and was specifically pulled down by GST-ALK-1 Q200D as well as by GST-ALK-1 (Fig. 1 C, top panel, lanes 7 and8) but not by GST alone (Fig. 1 C, lane 6). The interaction between ALK-1 and LXRβ in the GST affinity binding assay was unexpected because this interaction could not be detected in the yeast two-hybrid system (Fig. 1 B), suggesting that ALK-1 may be activated in mammalian cell extracts. To further investigate the interaction of ALK-1 and LXRβ, a FLAG-tagged (amino-terminal) full-length LXRβ was made and utilized in the GST affinity binding assay. FLAG-tagged LXRβ was efficiently expressed in 293T cells and specifically pulled down by GST-ALK-1 Q200D (Fig. 1 C, second panel, lane 8) but not by GST-ALK-1 or GST alone (Fig. 1 C, lanes 6 and7), suggesting that the first 14 amino acids of LXRβ may regulate the interaction of ALK-1 and LXRβ. Next, the interaction of ALK-1 with the closely related nuclear receptor, LXRα, was assessed. Neither GST-ALK-1 Q200D nor -ALK-1 was able to pull down LXRα (Fig. 1 C, top panel, lanes 3 and4). Taken together these results confirm a specific interaction between ALK-1 and LXRβ in vitro. To determine the regions important for the interaction between ALK-1 and LXRβ, we examined the sequences of each protein for potential interaction motifs. This examination revealed an LXXLL motif in the cytoplasmic domain of ALK-1 (amino acids 174–178) known to mediate interaction of proteins with the AF-2 domain of nuclear receptors (25Chang C. Norris J.D. Gron H. Paige L.A. Hamilton P.T. Kenan D.J. Fowlkes D. McDonnell D.P. Mol. Cell. Biol. 1999; 19: 8226-8239Crossref PubMed Google Scholar). To establish whether this LXXLL motif was responsible for mediating interaction of ALK-1 with the AF-2 of LXRβ, the LXXLL motif was mutated in GST-ALK-1 Q200D and the mutant assessed for interaction with full-length LXRβ. Mutation of LXXLL (LGDLL to LAAAA) of ALK-1 did not alter the ability of ALK-1 Q200D to bind LXRβ (data not shown). In addition, mutation or deletion of the AF-2 of LXRβ did not alter the ability of LXRβ to bind ALK-1 Q200D (Fig. 1 C,third panel, lanes 7 and 8, data not shown), confirming that these were not the regions responsible for interaction of ALK-1 and LXRβ. We then analyzed the ability of specific regions of LXRβ to bind to ALK-1 Q200D in vitro. These studies revealed that the ligand binding domain of LXRβ (amino acids 286–461) was unable to bind ALK-1 Q200D in vitro(data not shown), suggesting that the binding motif resides in the first 286 amino acids of LXRβ. Attempts to directly confirm binding of amino acids 1–286 of LXRβ containing the DNA binding domain were unsuccessful because constructs of LXRβ (amino acids 1–286) could not be stably expressed (data not shown). Finally, to determine whether this interaction occurred in vivo and without overexpression of ALK-1 and LXRβ (i.e. with physiological levels of expression of ALK-1 and LXRβ), we screened cells known to express LXRβ for expression of ALK-1. This survey revealed that the THP-1 macrophage cell line known to express LXRβ (26Whitney K.D. Watson M.A. Goodwin B. Galardi C.M. Maglich J.M. Wilson J.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2001; 276: 43509-43515Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) also expressed ALK-1 (Fig. 1 D,lane 2). Therefore, we used the THP-1 cell line to perform co-immunoprecipitation studies with antibodies to LXRβ and ALK-1. An antibody to LXRβ was able to co-immunoprecipitate ALK-1 in THP-1 cell lysates (Fig. 1 D, lane 3), whereas preimmune serum was not (Fig. 1 D, lane 1). Conversely, an antibody to ALK-1 co-immunoprecipitated LXRβ in THP-1 cell lysates (data not shown). Taken together, these findings strongly suggest a physiological interaction between ALK-1 and LXRβ. Because the nuclear receptor LXRβ is thought to reside primarily in the nucleus and ALK-1 is a membrane-bound receptor, we explored how these receptors might interact in vivo. To address this issue, we examined the localization of LXRβ in vivo. We constructed and expressed a GFP fusion protein of LXRβ in HEK-293 cells with or without ALK-1 Q-D and examined localization of GFP-LXRβ in live cells by confocal microscopy. In the absence of ALK-1, GFP-LXRβ was predominately, but not exclusively, expressed in the nucleus in most cells (Fig. 2, A andB), whereas GFP alone was uniformly distributed between the nucleus and cytoplasm as previously reported (27Xiao Z. Liu X. Henis Y.I. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7853-7858Crossref PubMed Scopus (107) Google Scholar) (data not shown), and β−arrestin 2-GFP was expressed exclusively in the cytoplasm (Fig. 2 G). However, in some cells in the absence of ALK-1 (∼5%), GFP-LXRβ was more uniformly distributed between the nucleus and cytoplasm (Fig. 2 C). In the presence of ALK-1 Q-D, localization was altered so that GFP-LXRβ was predominately expressed in the nucleus in only a minority of cells (∼20%), (Fig. 2 D), whereas in the majority of cells, GFP-LXRβ was uniformly distributed between the nucleus and cytoplasm (Fig. 2 E). In addition, in some cells (∼5%), a predominant membrane localization of GFP-LXRβ was observed (Fig. 2 F,arrow), suggesting that ALK-1 Q-D results in a nuclear to cytoplasmic translocation of LXRβ and that ALK-1 has access to LXRβ in the cytoplasm. To investigate the potential physiological relevance of the interaction of ALK-1 and LXRβ, we surveyed the expression patterns of ALK-1 and LXRβ by performing virtual Northern blot analysis of SAGE and EST databases and by examining published Northern blot analysis of ALK-1 (28Attisano L. Carcamo J. Ventura F. Weis F.M. Massague J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 29ten Dijke P. Ichijo H. Franzen P. Schulz P. Saras J. Toyoshima H. Heldin C.H. Miyazono K. Oncogene. 1993; 8: 2879-2887PubMed Google Scholar, 30Panchenko M.P. Williams M.C. Brody J.S. Yu Q. Am. J. Physiol. 1996; 270: L547-L558PubMed Google Scholar) and LXRβ (31Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). These surveys confirmed that LXRβ is widely expressed, whereas ALK-1 has a more limited pattern of expression, primarily in vascular tissues (Table I). Significantly, ALK-1 and LXRβ were co-expressed in 94% of tissues, including in endothelial cells, where ALK-1 is specifically expressed.Table ICorrelation of mRNA expression for ALK-1 and LXRβTissueALK-1 mRNA expressionLXRβ mRNA expressionAdipose+++++Brain++++Breast++Colon++++Germ cell++Heart++++Kidney+++++Liver++++Lung++++++Muscle+++++Nerve++Ovary++++Pancreas+++Placenta++++++Prostate++Spleen++++Testis−+++Cell LineHMVEC++Expression of mRNA in various tissues and cell lines for ALK-1 and LXRβ as demonstrated by Northern blot analysis, the presence of expressed sequence tag (ESTs), or a serial analysis of gene expression (SAGE) tag in a library made from the source tissue. + indicates the presence of mRNA; − indicates the absence of mRNA. When relative levels are known through Northern blot analysis, these levels are shown with +++ indicating high levels of expression and ++ indicating lower levels of expression. Data were obtained from Refs.28Attisano L. Carcamo J. Ventura F. Weis F.M. Massague J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 29ten Dijke P. Ichijo H. Franzen P. Schulz P. Saras J. Toyoshima H. Heldin C.H. Miyazono K. Oncogene. 1993; 8: 2879-2887PubMed Google Scholar, 30Panchenko M.P. Williams M.C. Brody J.S. Yu Q. Am. J. Physiol. 1996; 270: L547-L558PubMed Google Scholar, 31Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar. Open table in a new tab Expression of mRNA in various tissues and cell lines for ALK-1 and LXRβ as demonstrated by Northern blot analysis, the presence of expressed sequence tag (ESTs), or a serial analysis of gene expression (SAGE) tag in a library made from the source tissue. + indicates the presence of mRNA; − indicates the absence of mRNA. When relative levels are known through Northern blot analysis, these levels are shown with +++ indicating high levels of expression and ++ indicating lower levels of expression. Data were obtained from Refs.28Attisano L. Carcamo J. Ventura F. Weis F.M. Massague J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 29ten Dijke P. Ichijo H. Franzen P. Schulz P. Saras J. Toyoshima H. Heldin C.H. Miyazono K. Oncogene. 1993; 8: 2879-2887PubMed Google Scholar, 30Panchenko M.P. Williams M.C. Brody J.S. Yu Q. Am. J. Physiol. 1996; 270: L547-L558PubMed Google Scholar, 31Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar. Because LXRβ specifically interacts with activated ALK-1 (ALK-1 Q-D), we explored whether LXRβ was a substrate for the ALK-1 serine/threonine kinase. HA-tagged LXRβ was expressed in COS-7 cells in the presence and absence of either wild type, activated, or kinase-dead ALK-1. The cells were labeled with orthophosphate, and LXRβ was immunoprecipitated with αHA antibody. Activated ALK-1 was able to specifically phosphorylate LXRβ (Fig. 3 A, lane 4), whereas ALK-1 (Fig. 3 A, lane 3) and ALK-1 K-R (Fig. 3 A, lane 5) were not. To characterize the phosphorylation of LXRβ by activated ALK-1, LXRβ phosphorylatedin vivo by activated ALK-1 was subjected to phosphoamino acid analysis. In vivo phosphorylated LXRβ was primarily phosphorylated on serine residues, with a trace amount of threonine phosphorylation and no detectable tyrosine phosphorylation (ratio of pSer:pThr:pTyr of 12:1:0), (Fig. 3 B). To identify the number of phosphorylation sites, phosphopeptide mapping was performed. LXRβ was phosphorylated in vivo on at least two distinct sites, with one major phosphopeptide (Fig. 3 C). ALK-1 has been proposed to form a signaling complex with the type II TGF-β receptor (TβRII) and endoglin in endothelial cells (11Lux A. Attisano L. Marchuk D.A. J. Biol. Chem. 1999; 274: 9984-9992Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To investigate the specificity of phosphorylation of LXRβ by activated ALK-1, we investigated the ability of wild type ALK-1 to phosphorylate LXRβ in the presence of TβRII or TβRII and endoglin in the presence and absence of TGF-β1. Although activated ALK-1 phosphorylated LXRβ (Fig. 4 A, lane 2), wild type ALK-1 could not (Fig. 4 A, lane 1) even in the presence of TβRII (Fig. 4 A, lane 3), TβRII and TGF-β1 (Fig. 4 A, lane 4), or TβRII, endoglin, and TGF-β1 (Fig. 4 A, lane 5), suggesting that an uncharacterized ligand stimulates ALK-1 to phosphorylate LXRβ in vivo. Because TβRII was also HA-tagged (like LXRβ), autophosphorylated TβRII was immunoprecipitated in these experi" @default.
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• W1965779516 title "Regulation of ALK-1 Signaling by the Nuclear Receptor LXRβ" @default.
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• W1965779516 cites W1848404107 @default.
• W1965779516 cites W1862564406 @default.
• W1965779516 cites W1903792504 @default.
• W1965779516 cites W1915833704 @default.
• W1965779516 cites W1968370020 @default.
• W1965779516 cites W1975070287 @default.
• W1965779516 cites W1975522586 @default.
• W1965779516 cites W1976454956 @default.
• W1965779516 cites W1978996505 @default.
• W1965779516 cites W1984789406 @default.
• W1965779516 cites W1988145354 @default.
• W1965779516 cites W1996168740 @default.
• W1965779516 cites W2001332055 @default.
• W1965779516 cites W2004188451 @default.
• W1965779516 cites W2013958991 @default.
• W1965779516 cites W2020475120 @default.
• W1965779516 cites W2028854652 @default.
• W1965779516 cites W2029889395 @default.
• W1965779516 cites W2030662016 @default.
• W1965779516 cites W2040417680 @default.
• W1965779516 cites W2042480227 @default.
• W1965779516 cites W2050902851 @default.
• W1965779516 cites W2060372234 @default.
• W1965779516 cites W2075998623 @default.
• W1965779516 cites W2078107700 @default.
• W1965779516 cites W2081732968 @default.
• W1965779516 cites W2084866479 @default.
• W1965779516 cites W2092099867 @default.
• W1965779516 cites W2099765837 @default.
• W1965779516 cites W2102051477 @default.
• W1965779516 cites W2103031848 @default.
• W1965779516 cites W2106441070 @default.
• W1965779516 cites W2112632255 @default.
• W1965779516 cites W2113574245 @default.
• W1965779516 cites W2119529702 @default.
• W1965779516 cites W2135007868 @default.
• W1965779516 cites W2144599584 @default.
• W1965779516 cites W2149457192 @default.
• W1965779516 cites W2159744955 @default.
• W1965779516 cites W2163504684 @default.
• W1965779516 cites W2168558463 @default.
• W1965779516 cites W2188836137 @default.
• W1965779516 cites W2316067769 @default.
• W1965779516 cites W2770216137 @default.
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• W1965779516 doi "https://doi.org/10.1074/jbc.m210376200" @default.
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