FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2149417967> ?p ?o ?g. }

• W2149417967 endingPage "5760" @default.
• W2149417967 startingPage "5749" @default.
• W2149417967 abstract "CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe454) and lysine (Lys456) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe454 at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr457 and/or Ser459 are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling. CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe454) and lysine (Lys456) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe454 at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr457 and/or Ser459 are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling. CEACAM1 4The abbreviations used are: CEACAM1-4S, carcinoembryonic antigen cell adhesion molecule 1, 4 ecto-domains, short cytoplasmic domain; CEACAM1-4L, carcinoembryonic antigen cell adhesion molecule 1, 4 ectodomains, long cytoplasmic domain; CEA, carcinoembryonic antigen; STD, saturation transfer difference; EDC, N-ethyl-N′-(dimethylaminopropyl)-carbodimide hydrochloride; NHS, N-hydroxysuccinimide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HSQC, heteronuclear single quantum coherence; FBS, fetal bovine serum; eGFP, enhanced green fluorescent protein; MUA, mercaptoundecanoic acid; DBP, vitamin D-binding protein; TOCSY, total correlation spectroscopy. 4The abbreviations used are: CEACAM1-4S, carcinoembryonic antigen cell adhesion molecule 1, 4 ecto-domains, short cytoplasmic domain; CEACAM1-4L, carcinoembryonic antigen cell adhesion molecule 1, 4 ectodomains, long cytoplasmic domain; CEA, carcinoembryonic antigen; STD, saturation transfer difference; EDC, N-ethyl-N′-(dimethylaminopropyl)-carbodimide hydrochloride; NHS, N-hydroxysuccinimide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HSQC, heteronuclear single quantum coherence; FBS, fetal bovine serum; eGFP, enhanced green fluorescent protein; MUA, mercaptoundecanoic acid; DBP, vitamin D-binding protein; TOCSY, total correlation spectroscopy. (carcinoembryonic antigen cell adhesion molecule 1) is a type I membrane glycoprotein that mediates homotypic cell adhesion and belongs to the CEA gene family (1Obrink B. Curr. Opin. Cell Biol. 1997; 9: 616-626Crossref PubMed Scopus (232) Google Scholar). It has been shown to play a role in bacterial and viral uptake (2Blau D.M. Turbide C. Tremblay M. Olson M. Letourneau S. Michaliszyn E. Jothy S. Holmes K.V. Beauchemin N. J. Virol. 2001; 75: 8173-8186Crossref PubMed Scopus (37) Google Scholar, 3Gray-Owen S.D. Dehio C. Haude A. Grunert F. Meyer T.F. EMBO J. 1997; 16: 3435-3445Crossref PubMed Scopus (189) Google Scholar, 4Virji M. Evans D. Griffith J. Hill D. Serino L. Hadfield A. Watt S.M. Mol. Microbiol. 2000; 36: 784-795Crossref PubMed Scopus (94) Google Scholar, 5Boulton I.C. Gray-Owen S.D. Nat. Immunol. 2002; 3: 229-236Crossref PubMed Scopus (238) Google Scholar), in insulin clearance (6Najjar S.M. Trends Endocrinol. Metab. 2002; 13: 240-245Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), angiogenesis (7Ergun S. Kilik N. Ziegeler G. Hansen A. Nollau P. Gotze J. Wurmbach J.H. Horst A. Weil J. Fernando M. Wagener C. Mol. Cell. 2000; 5: 311-320Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), NK and T-cell regulation (8Markel G. Wolf D. Hanna J. Gazit R. Goldman-Wohl D. Lavy Y. Yagel S. Mandelboim O. J. Clin. Investig. 2002; 110: 943-953Crossref PubMed Scopus (110) Google Scholar, 9Kammerer R. Stober D. Singer B.B. Obrink B. Reimann J. J. Immunol. 2001; 166: 6537-6544Crossref PubMed Scopus (73) Google Scholar, 10Iijima H. Neurath M.F. Nagaishi T. Glickman J.N. Nieuwenhuis E.E. Nakajima A. Chen D. Fuss I.J. Utku N. Lewicki D.N. Becker C. Gallagher T.M. Holmes K.V. Blumberg R.S. J. Exp. Med. 2004; 199: 471-482Crossref PubMed Scopus (94) Google Scholar, 11Chen C.J. Shively J.E. J. Immunol. 2004; 172: 3544-3552Crossref PubMed Scopus (57) Google Scholar), and tumor suppression (12Neumaier M. Paululat S. Chan A. Matthaes P. Wagener C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10744-10748Crossref PubMed Scopus (229) Google Scholar, 13Nittka S. Gunther J. Ebisch C. Erbersdobler A. Neumaier M. Oncogene. 2004; 23: 9306-9313Crossref PubMed Scopus (69) Google Scholar). Despite its proposed function as a cell-cell adhesion molecule, CEACAM1 is often found on the lumenal surface of epithelial cells, suggesting the possibility that it also plays a role in cell polarization and lumen formation. In this respect, we have recently shown that CEACAM1 is expressed on the lumenal surface of normal mammary epithelial cells (14Huang J. Simpson J.F. Glackin C. Riethorf L. Wagener C. Shively J.E. Anticancer Res. 1998; 18: 3203-3212PubMed Google Scholar), and that in a three-dimensional model of mammary morphogenesis, blocking its expression with either an antisense gene or anti-CEACAM1 antibodies blocks lumen formation (15Huang J. Hardy J.D. Sun Y. Shively J.E. J. Cell Sci. 1999; 112: 4193-4205Crossref PubMed Google Scholar, 16Kirshner J. Chen C.J. Liu P. Huang J. Shively J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 521-526Crossref PubMed Scopus (147) Google Scholar), while breast cancer cells, which lack CEACAM1 and fail to form lumena in a three-dimensional culture, are restored to normal when transfected with the epithelial specific CEACAM1-4S isoform (16Kirshner J. Chen C.J. Liu P. Huang J. Shively J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 521-526Crossref PubMed Scopus (147) Google Scholar). CEACAM1 gene expression has been shown to be down-regulated early in colon cancer (12Neumaier M. Paululat S. Chan A. Matthaes P. Wagener C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10744-10748Crossref PubMed Scopus (229) Google Scholar, 17Nollau P. Scheller H. Kona-Horstmann M. Rohde S. Hagenmuller F. Wagener C. Neumaier M. Cancer Res. 1997; 57: 2354-2357PubMed Google Scholar), and in a variety of cancer models, re-introduction of the CEACAM1 gene causes reversion of the malignant phenotype (18Luo W. Tapolsky M. Earley K. Wood C.G. Wilson D.R. Logothetis C.J. Lin S.H. Cancer Gene Ther. 1999; 6: 313-321Crossref PubMed Scopus (75) Google Scholar, 19Luo W. Wood C.G. Earley K. Hung M.C. Lin S.H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar, 20Kleinerman D.I. Dinney C.P. Zhang W.W. Lin S.H. Van N.T. Hsieh J.T. Cancer Res. 1996; 56: 3431-3435PubMed Google Scholar). Although CEACAM1 is expressed in multiple isoforms with 1-4 Ig-like ectodomains and either long (72 amino acids) or short (12-14 amino acids) cytoplasmic domains, the short cytoplasmic domain isoforms predominate in epithelial cells. Because the cytoplasmic domain of this isoform is only 12-14 amino acids in length (the exact start of the domain is in doubt) it was originally thought not to play a role in signal transduction, but perhaps acted in a dominant negative fashion in the presence of the long cytoplasmic domain. However, we have shown that peptides or glutathione S-transferase fusion proteins from the short cytoplasmic domain bind directly to actin, tropomyosin, calmodulin, and more recently, to the annexin-2 p11 tetramer AIIt (21Schumann D. Chen C-J. Kaplan B. Shively J.E. J. Biol. Chem. 2001; 276: 47421-47433Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 22Edlund M. Blikstad I. Obrink B. J. Biol. Chem. 1996; 271: 1393-1399Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Kirshner J. Schumann D. Shively J.E. J. Biol. Chem. 2003; 278: 50338-50345Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).In this study, we have used mutational analysis of the short cytoplasmic domain of CEACAM1 to reveal which residues are involved in actin interactions and lumen formation. The results of our studies show that mutants F454A and K456A greatly decrease or increase, respectively, actin interactions, whereas the F454A and the double mutant T457A,S459A greatly reduce lumen formation. Further analysis reveals that either Thr457 or Ser459 are phosphorylated during lumen formation. The studies that identified these key residues are the subject of this report.MATERIALS AND METHODSConstruction of Baits for Yeast Two-hybrid ScreeningThe nucleotide and amino acid sequence for CEACAM1-4S is taken from NCBI entry number NM_001712, gi:4502404, the full-length coding sequence including the N-terminal signal sequence. To construct the bait containing the cytoplasmic domain of CEACAM1-4S (amino acids 451-464), pGKT7/CEACAM1-4S-cyt for yeast two-hybrid screening, an EcoRI site was introduced in front of Phe451 (nucleotide 1352) of CEACAM1-4S by site-directed mutagenesis, using a set of primers, 5′-GCAGTAGCCCTGGAATTCTTTCTGCATTTCG-3′/5′-CGAAATGCAGAAAGAATTCCAGGGCTACTGC-3′, and KS/CEACAM1-4S as template. The plasmid DNA was digested by EcoRI and PstI (located at 3′-untranslated region CEACAM1-4S cDNA). The resulting fragments were then subcloned into the pGKT7 (Clontech) between the EcoRI and PstI sites to create pGKT7/CEACAM1-4S-cyt. All PCR were performed using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA).pGKT7/CEACAM1-4S-cyt MutantsA series of single and dual alanine mutants of CEACAM1-4S-cyt were created by sequence overlap extension PCR. The Matchmaker 5′ DNA-BD vector insert screening sense amplimer was: 5′-TCATCGGAAGAGAGTAGTAAC-3′, and Matchmaker 3′ DNA-BD vector insert screening antisense amplimer was: 3′-GAGTCACTTTAAAATTTGTATAC-5′. For each mutant, a set of inside primers were synthesized (supplementary Table 1S). The pGKT7/CEACAM1-4S plasmid served as the template. In the first PCR run, the inside antisense primer was used with the external sense primer and the inside sense primer was used with external sense primer to create two overlapping fragments. Both of these fragments served as templates to produce the larger fragment using the external primer set. The resulting products were digested with EcoRI and PstI, and ligated into pGKT7 previously cut with the same restriction enzymes. The pGKT7/CEACAM1-4S-cyt was also mutated to create pGKT7/CEACAM1-4S-cyt-DD by site-directed mutagenesis using a set of primers (supplementary Table 1S). The amino acids mutated were T457D,S459D, which mimic phosphorylation at these two sites.Yeast Two-hybrid Screen and AnalysisThe Clontech human cDNA liver library used for screening contains 4 × 106 independent clones. The cytoplasmic domain from CEACAM1-4S (amino acids 451-464) was subcloned into pGKT7, transformed into yeast AH109 cells, and used as bait to screen the library. After mating, cells were plated on plates lacking Leu, Trp, His, and adenine (Ade). Colonies were replica plated onto a plate lacking Leu, Try, His, and Ade and printed onto a filter paper for β-galactosidase analysis using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) as a substrate. Plasmid DNA of positive diploid clones were purified and transformed into Escherichia coli DH5. Transformants carrying the ACTII/prey were selected on LB/ampicillin plates. Plasmid DNA was purified and sequenced. The ACTII/prey plasmid DNA was transformed into yeast AH109 carrying the CEACAM1-4S-cyt bait construct or transformed into yeast Y187 for mating experiments. The co-transformants or mated cells were selected on plates lacking His and Trp. Colonies were picked and the supernatant of the overnight culture was used to assay for α-galactosidase. The cell mass was calculated by measuring the absorbance at A600 nm. The α-galactosidase activity was measured using p-nitrophenyl-α-galactopyranoside as a substrate according to the Clontech manual, and normalized to the cell mass (A410/600 nm) for comparison.Mammalian Expression VectorsTo construct the mammalian expression vector pHβ-CEACAM1-4S-F454A, a single amino acid mutation (nucleotide 1360) was created by PCR site-directed mutagenesis, using KS/CEACAM1-4S as a template. KS/CEACAM1-4S-F454A was digested with SalI and HindIII and ligated into the pHβ-actin vector digested with SalI and HindIII. The β-actin promoter containing expression vector has been previously described (24Gunning P. Leavitt J. Muscat G. Ng S.Y. Kedes L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4831-4835Crossref PubMed Scopus (666) Google Scholar).pCDNA3/CEACAM1-4S-eGFPTo create a XmaI site for the insertion of eGFP into CEACAM1-4S, we first deleted the XmaI site located in the multicloning sites of the KS/CEACAM1-4S vector by PCR site-directed mutagenesis, using a set of primers: 5′-GAATTCCTGCAGCCTGGGGGATCCACTAG-3′/5′-CTAGTGGATCCCCCAGGCTGCAGGAATTC-3′. An XmaI site was introduced between Pro427 (nucleotide 1279) and Gly428 (nucleotide 1281) of CEACAM1-4S by PCR site-directed mutagenesis, using the set of primers: 5′-GAAAATGGCCTCTCACCCGGGGCCATTGCTGGCATTG-3′/5′-CAATGCCAGCAATGGCCCCGGGTGAGAGGCCATTTTC-3′. A full-length eGFP clone containing a XmaI cutting site and a G4 linker on both ends was amplified by PCR using PE-GFP-N2 (Clontech) as the template, a set of primers containing XmaI site and the G4 linker, 5′-TCCCCCCGGGGGAGGAGGAATGGTGAGCAAGGGC-3′/5′-TCCCCCCGGGTCCTCCTCCCTTGTACAGCTCGTC-3′, were used. The PCR product was digested with XmaI, the eGFP fragment was purified and subcloned into KS (XmaI-)/CEACAM1-4S (XmaI+) previously digested with XmaI to create the KS/CEACAM1-4S-eGFP vector. The CEACAM1-4S-eGFP fragment was cut out from KS/CEACAM1-4S-eGFP and subcloned into pCDNA3 previously digested by KpnI and EcoRI. In the resulting vector a G4 linker were introduced after Pro427 of CEACAM1-4S, and a PG3 linker was introduced before Gly428 of CEACAM1-4S.Mutations at Thr457 and Ser459 in the cytoplasmic domain of CEACAM1-4S were constructed according to the QuikChange Site-directed Mutagenesis Kit (Stratagene). In short, mutations were introduced into CEACAM1-4S/pBlue-script II KS by PCR primers shown in supplementary Table 1S. PCR conditions were 95 °C for 1 min, 18 cycles at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 14 min. The parental (unmutated) strand was removed by DpnI digest. Mutants were sequenced and the selected clones were subcloned into the SalI and HindIII restriction sites of the pHβ-actin expression vector. Cell transfections were performed using Lipofectin reagent (Invitrogen) as per the manufacturer’s instructions. Cells were maintained in minimal essential medium with 10% fetal bovine serum (FBS) and selected in 0.1-1.0 mg/ml G418 for 5 weeks to create stable CEACAM1-4S mutant cell lines. Geneticin-resistant cells were sorted for CEACAM1 expression, grown in a Matrigel sandwich assay, and scored for acinus formation. Statistical analysis was performed using Fisher’s Exact test. For the Matrigel sandwich assay, 12-well plates were coated with 150 μl of Matrigel and incubated at 37 °C for 30 min to let the Matrigel solidify. Cells (1 × 105) in 500 μl of mammary epithelial basal medium (MEBM) (Clonetics) plus pituitary gland extract (Cambrex) were added to each well. After 3 h of incubation the floating cells were removed and the bound cells were overlaid with 150 μl of 50% Matrigel (1:1, Matrgiel gel:MEBM plus pituitary gland extract and cholera toxin (100 ng/ml final)). Culture medium was changed every other day. After 6 days the Matrigel cultures were fixed with 2% paraformaldehyde or formaldehyde for histochemistry analysis.Cell Culture and Transfection of Jurkat, HeLa, and MCF7 CellsJurkat cells were grown in RPMI supplemented with 10% FBS. MCF7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Cells (1 × 107/ml) were harvested and washed in Opti-MEM. An aliquot (800 μl) of cell suspension was mixed with 25 μg of DNA (in 20-30 μl) and transferred to an electroporation cuvette with a 0.4-cm electrode gap (Bio-Rad). Electroporation parameters used were 260 V, 900 microfarads, resulting in a pulse time of 20-23 ms. Stable transformants were selected with geneticin and selected clones were shown to have high levels of cell surface expression by fluorescence-activated cell sorter and confocal fluorescence analysis. Cells were grown in Matrigel as described above.Immunohistochemistry and Confocal MicroscopyImmunohistochemistry was performed according to Huang et al. (14Huang J. Simpson J.F. Glackin C. Riethorf L. Wagener C. Shively J.E. Anticancer Res. 1998; 18: 3203-3212PubMed Google Scholar) using anti-CEACAM1 monoclonal antibody 5F4 (kind gift from Dr. Richard Blumberg, Boston, MA). For immunofluorescence, Alexa 546 (Molecular Probes, Eugene, OR) conjugated monoclonal antibody T84.1 (1 μg/ml) was used to detect CEACAM1. Staining was visualized on a Zeiss model 310 confocal microscope. The cellular distribution of CEACAM1-4S in stably transfected Jurkat cells was performed on cells plated on 6-well slides precoated with 20 ng/ml fibronectin. Cells were incubated at 37 °C for 30 min followed by treatment with nocodazole (Sigma) or cytochalasin B (Sigma) at a final concentration of 5 μg/ml. After a 30-min incubation, cells were fixed in 2.5% paraformaldehyde in phosphate-buffered saline for 15 min and incubated for 1 h with anti-CEACAM1 antibody T84.1 (1:500), followed by a 1-h incubation with Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes). After washing three times with cold phosphate-buffered saline, cells were permeabilized in PSG (0.01% saponinin phosphate-buffered saline) containing 0.5% Nonidet P-40 for 20 min at room temperature. Cells were stained for F-actin with Texas Red-conjugated phalloidin (Molecular Probes). For colocalization of CEACAM1-4S-eGFP with F-actin, Jurkat or MCF7 stable transfectants were grown in 6-well chamber slides in Dulbecco’s modified Eagle’s medium with 10% FBS. In some experiments chamber slides were precoated with laminin 10/11 (Sigma), followed by fixation and permeabilization as described above. F-actin was stained with Texas Red-conjugated phalloidin. The slides were mounted in 80% glycerol/phosphate-buffered saline and observed on a Zeiss confocal microscope using fluorescein isothiocyanate and rhodamine filter settings to detect GFP and Texas Red-phalloidin, respectively.Peptide SynthesisSynthetic peptides corresponding to the 12 amino acids adjacent to the transmembrane domain of the CEACAM1 short cytoplasmic domain were synthesized using Fmoc chemistry with an N-terminal mercaptoundecanoic acid (MUA) extension by the City of Hope Peptide Synthesis Core. 15N-labeled Fmoc amino acids were purchased from Cambridge Isotope Labs. MUA was purchased from Aldrich, and the mercapto group was protected by reaction with trityl chloride. The S-trityl derivative was purified by silica gel chromatography. Briefly, 11-mercaptoundecanoic acid (10 mmol, 2.18 g) was dissolved in dry pyridine (20 ml) and trityl chloride (11 mmol, 3.06 g) was added. The reaction mixture was left for 18 h, quenched with 20 ml of ethanol, and concentrated under reduced pressure. The residue was dissolved in 150 ml of dichloromethane, cooled to 0 °C, and washed with ice-cold 1 m citric acid (3 × 50 ml) and saturated brine (50 ml), dried with anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude product was purified on Silica Gel H (120 g) in a gradient of ethanol/dichloromethane (0-3%) with 1% of acetic acid present in both. Chromatography was monitored by TLC (Si60, Merck) in 10% dichloromethane/ethanol/acetic acid (89:10:1). The yield of pure product (RF = 0.65) was 4.0 g (8.75 mmol). Trityl-S-MUA (0.25 mmol) was dissolved in 0.5 ml of 1-methyl-2-pyrrolidinone, 0.5 m HOBt, and reacted with 0.25 ml of 1 m dicyclohexylcarbodiimide in dichloromethane for 30 min, filtered to remove the precipitated urea, and added to the peptide/solid support (0.1 mmol) containing a free amino group. Coupling was performed at 65 °C for 30 min, the resin was washed with N,N-dimethylformamide, dichloromethane, dried, and cleaved with trifluoroacetic acid/water/ethanethiol/triethylsilane (90:5:2.5:2.5) for 30 min at 40 °C. Peptides were purified to >95% purity by reversed-phase high performance liquid chromatography on a Vydac C18 column and their masses confirmed by electrospray ionization mass spectrometry on a Finnigan LCQ ion trap mass spectrometer.BIAcore AnalysisBiomolecular interaction analyses were carried out in HBS buffer (150 mm NaCl, 0.05% (v/v) Surfactant P20, 10 mm HEPES, pH 7.4 or 5.5) using the BIAcore® 2000 (BIAcore, Inc.). Depending on the experiment, 2 mm CaCl2 was added to the HBS. Nonmuscle G-actin (Cytoskeleton, Inc.) was immobilized on a CM5 sensorchip (BIAcore) using the Amine Coupling Kit (BIAcore). The surface of the sensorchip was activated with 30 μl of EDC/NHS (100 mm N-ethyl-N′-(dimethylamino-propyl)-carbodimide hydrochloride, 400 mm N-hydroxysuccinimide) using a flow rate of 5 μl/min. For immobilization of proteins, 1-10 μg of G-actin or vitamin D-binding protein (DBP, Sigma) in 100 μl of 10 mm sodium acetate, pH 4.0, were applied (flow rate: 5 μl/min). Subsequently, the sensorchip was deactivated with 30 μl of 1 m ethanolamine hydrochloride, pH 8.5 (flow rate: 5 μl/min), and conditioned with 10 μl of 25 mm HCl (flow rate: 20 μl/min). Thiol immobilization of MUA peptides was performed according to application note 9 from BIAcore. Briefly, a CM5 chip was activated by NHS/EDC (1:1, 10 μl) followed by pyridine disulfide ethane amine (20 μl of 80 mm in 0.1 m sodium borate, pH 8.5) followed by MUA-peptide (7.5 μl of 2 mg/ml in 10 mm sodium acetate, pH 4.0) followed by cysteine, 1 m NaCl (20 μl of 50 mm in 0.1 m sodium acetate, pH 4.0) to block excess sites. Binding studies and regeneration of the chip surface between injections were carried out at a flow rate of 20 μl/min unless otherwise noted. Samples were diluted in HBS buffer immediately prior to injection. Between sample injections the surface was regenerated with 15 μl of 25 mm HCl. Data were analyzed with BIAevaluation 3.1 software (BIAcore), and curve fitting was done with the assumption of one-to-one (Langmuir) binding.NMR AnalysisHSQC—Peptide solutions were prepared by dissolving lyophilized powders in 5 mm Tris buffer containing 5% D2O, 0.1 mm CaCl2, 0.2 mm ATP, and dithiothreitol (G-buffer, Cytoskeleton, Inc.) and the pH adjusted to 5.4 with 0.1 m HCl. The concentrations of wild-type and F454A mutant peptide were 0.10 and 0.15 mm, respectively. Actin was added to the peptide by dissolving lyophilized actin (Cytoskeleton, Inc.) in the peptide solution and the pH adjusted as necessary. The two-dimensional 1H-15N correlated HSQC experiments were carried out on a Bruker Avance 600 NMR instrument equipped with actively shielded Z-gradient TXI-triple resonance cryoprobe. The Watergate-HSQC with water flip-back pulse was used for water suppression. The experiments were carried out at 15 °C.STD (Saturation Transfer Difference)—Experiments are widely used to identify the interaction between small ligand and large target biomolecules, and to determine the binding epitope of the ligand (25Mayer M. Meyer B. J. Am. Chem. Soc. 2001; 123: 6108-6117Crossref PubMed Scopus (1008) Google Scholar, 26Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D.E. Science. 2003; 300: 976-980Crossref PubMed Scopus (331) Google Scholar). The advantage of the STD approach is that it works on large sized, and low concentrations of biomolecules without a requirement for isotope labeling of biomolecules. The concentration of protein can be as low as in the nanomolar range, and the optimal K ranges from 10-3 to 10-8 d m. STD is composed of two experiments. The first is a saturation experiment with carrier frequency irradiation on protein signals for a period of 2-5 s with soft pulse so that the target signals are saturated and ligand signals are not excited. Next, a reference experiment is carried out with a carrier frequency far away from protein and ligand signals. The two experiments are usually carried out in an interleaved fashion and the subtraction is realized by phase cycling. For a large sized protein, the saturation on any part of the protein (usually the methyl region) is spread out quickly through a spin diffusion mechanism to whole protein as well as the ligand in close contact with the protein. The protein signals from the subtraction of two experiments can be removed by a spin-lock delay (typically 40 ms), and thus the surviving signals are from the ligand that interacts with protein. Furthermore, epitope binding information can be obtained from the intensity of STD signals.STD experiments were performed according to Mayer and Mayer (25Mayer M. Meyer B. J. Am. Chem. Soc. 2001; 123: 6108-6117Crossref PubMed Scopus (1008) Google Scholar). Rabbit muscle actin (1 mg) was dissolved in 2 ml of G buffer, and then dialyzed two times versus 1 liter of G buffer at 4 °C. After dialysis, the G buffer was extensive exchanged to a G buffer prepared with D2O, deuterated dithiothreitol, and deuterated Tris. The final actin concentration was about 0.02 mm. STD experiments were carried out on a 500 MHz Bruker instrument with a TXI probe equipped with triple axis gradients. The irradiation power of the selective Gauss-shaped pulses was set to 86 Hz with duration of 55 ms. Saturation of actin signals was achieved with a train of 70 gauss-shaped pulses separated by a 1-ms delay. The on-resonance irradiation of the actin sample was carried out at -2.4 ppm, and no saturation of CEACAM1 peptide in the absence of actin was observed under these experimental conditions. The off-resonance irradiation was performed at -30 ppm. A 40-ms spinlock pulse with field strength of 4960 Hz was used to suppress the background signals of actin. The saturation time was optimized by arraying the numbers of gauss-shaped pulse, and the highest STD signals were obtained with saturation time of 3.9 s.Molecular Modeling—A molecular model of CEACAM1-4S transmembrane and cytoplasmic domains was built using the HOMOLOGY module within INSIGHT II 2000 software (Accelrys Inc., San Diego, CA). Predicted transmembrane residues 429-452 were modeled using residues 437-460 from the membrane protein AcrB multidrug efflux pump as a template (26Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D.E. Science. 2003; 300: 976-980Crossref PubMed Scopus (331) Google Scholar). Coordinates for the cytoplasmic tail (residues 453-464) were modeled using residues 132-143 of the DBP bound to actin as a template (27Otterbein L.R. Cosio C. Graceffa P. Dominguez R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8003-8008Crossref PubMed Scopus (127) Google Scholar). The DBP-actin complex also served as a template for the initial docking of the cytoplasmic domain of CEACAM1-4S with the terminal unit of F-actin, as modeled by Holmes et al. (28Holmes K.C. Schroder R.R. Sweeney H.L. Houdusse A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 1819-1828Crossref PubMed Scopus (121) Google Scholar), coordinates were courtesy of Ken Holmes, Max Planck Institute for Medical Research, Heidelberg, Germany. To complete the model, the transmembrane helix of CEACAM1-4S was embedded in a standard model of a fluid lipid bilayer. Docking was optimized using the DISCOVER 3.0 module within INSIGHT II. The consistent valence force field was used throughout (29Dauber-Osguthorpe P. Roberts V.A. Osguthorpe D.J. Wolff J. Genest M. Hagler A.T. Proteins. 1988; 4: 31-47Crossref PubMed Scopus (1913) Google Scholar). The conformation of the cytoplasmic domain of CEACAM1-4S and segments of actin within 4.5 Aå were simultaneously optimized using molecular dynamics (50 ps at 300 K) followed by conjugate gradient minimization to a maximum derivative of 0.5 kcal/mol Aå.Phosphorylation of CEACAM1-4S—MCF7 cells (1 × 107) transfected with CEACAM1-4S were grown in a three-dimensional culture for 4 days, incubated with phosphate-free RPMI 1640 plus 1% dialyzed FBS and 11 mCi of [32P]orthophosphate (ICN), harvested with Matrisperse (Collaborative Biomedical Products), and lysed with 2% Nonidet P-40. CEACAM1 was immunoprecipitated with anti-CEACAM1 monoclonal antibody T84.1, and the proteins separated by SDS-gel electrophoresis and Western blot and autoradiography was performed to locate the 32P-labeled CEACAM1 band. The band was excised and counted in a Packard 1600CA scintillation counter. In a second experiment for CEACAM1-4S, transfected and vector control cells were grown for 4 days in Matrigel, harvested with Matrisperse, and lysed with 2% Nonidet P-40. CEACAM1 was immunoprecipit" @default.
• W2149417967 created "2016-06-24" @default.
• W2149417967 creator A5002940955 @default.
• W2149417967 creator A5014673831 @default.
• W2149417967 creator A5014842317 @default.
• W2149417967 creator A5016834515 @default.
• W2149417967 creator A5021017702 @default.
• W2149417967 creator A5056194127 @default.
• W2149417967 date "2007-02-01" @default.
• W2149417967 modified "2023-09-30" @default.
• W2149417967 title "Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation" @default.
• W2149417967 cites W104354955 @default.
• W2149417967 cites W1678855551 @default.
• W2149417967 cites W1719822197 @default.
• W2149417967 cites W1976975154 @default.
• W2149417967 cites W1982635658 @default.
• W2149417967 cites W1985292306 @default.
• W2149417967 cites W1993847375 @default.
• W2149417967 cites W1995036338 @default.
• W2149417967 cites W1999972891 @default.
• W2149417967 cites W2006024947 @default.
• W2149417967 cites W2009060783 @default.
• W2149417967 cites W2021751282 @default.
• W2149417967 cites W2023439329 @default.
• W2149417967 cites W2023802665 @default.
• W2149417967 cites W2033777873 @default.
• W2149417967 cites W2038684541 @default.
• W2149417967 cites W2043664506 @default.
• W2149417967 cites W2044172471 @default.
• W2149417967 cites W2045504138 @default.
• W2149417967 cites W2053190811 @default.
• W2149417967 cites W2061151008 @default.
• W2149417967 cites W2067247128 @default.
• W2149417967 cites W2067874984 @default.
• W2149417967 cites W2071740908 @default.
• W2149417967 cites W2075566899 @default.
• W2149417967 cites W2080510429 @default.
• W2149417967 cites W2089559330 @default.
• W2149417967 cites W2100816704 @default.
• W2149417967 cites W2117159579 @default.
• W2149417967 cites W2117209016 @default.
• W2149417967 cites W2127064732 @default.
• W2149417967 cites W2136156049 @default.
• W2149417967 cites W2144297538 @default.
• W2149417967 cites W2145041874 @default.
• W2149417967 cites W2151079059 @default.
• W2149417967 cites W2160068303 @default.
• W2149417967 cites W4239238212 @default.
• W2149417967 doi "https://doi.org/10.1074/jbc.m610903200" @default.
• W2149417967 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17192268" @default.
• W2149417967 hasPublicationYear "2007" @default.
• W2149417967 type Work @default.
• W2149417967 sameAs 2149417967 @default.
• W2149417967 citedByCount "46" @default.
• W2149417967 countsByYear W21494179672012 @default.
• W2149417967 countsByYear W21494179672013 @default.
• W2149417967 countsByYear W21494179672014 @default.
• W2149417967 countsByYear W21494179672015 @default.
• W2149417967 countsByYear W21494179672016 @default.
• W2149417967 countsByYear W21494179672017 @default.
• W2149417967 countsByYear W21494179672019 @default.
• W2149417967 countsByYear W21494179672021 @default.
• W2149417967 countsByYear W21494179672022 @default.
• W2149417967 countsByYear W21494179672023 @default.
• W2149417967 crossrefType "journal-article" @default.
• W2149417967 hasAuthorship W2149417967A5002940955 @default.
• W2149417967 hasAuthorship W2149417967A5014673831 @default.
• W2149417967 hasAuthorship W2149417967A5014842317 @default.
• W2149417967 hasAuthorship W2149417967A5016834515 @default.
• W2149417967 hasAuthorship W2149417967A5021017702 @default.
• W2149417967 hasAuthorship W2149417967A5056194127 @default.
• W2149417967 hasBestOaLocation W21494179671 @default.
• W2149417967 hasConcept C12554922 @default.
• W2149417967 hasConcept C125705527 @default.
• W2149417967 hasConcept C1491633281 @default.
• W2149417967 hasConcept C16224149 @default.
• W2149417967 hasConcept C178790620 @default.
• W2149417967 hasConcept C185592680 @default.
• W2149417967 hasConcept C190062978 @default.
• W2149417967 hasConcept C55493867 @default.
• W2149417967 hasConcept C84416704 @default.
• W2149417967 hasConcept C85789140 @default.
• W2149417967 hasConcept C86803240 @default.
• W2149417967 hasConcept C95444343 @default.
• W2149417967 hasConceptScore W2149417967C12554922 @default.
• W2149417967 hasConceptScore W2149417967C125705527 @default.
• W2149417967 hasConceptScore W2149417967C1491633281 @default.
• W2149417967 hasConceptScore W2149417967C16224149 @default.
• W2149417967 hasConceptScore W2149417967C178790620 @default.
• W2149417967 hasConceptScore W2149417967C185592680 @default.
• W2149417967 hasConceptScore W2149417967C190062978 @default.
• W2149417967 hasConceptScore W2149417967C55493867 @default.
• W2149417967 hasConceptScore W2149417967C84416704 @default.
• W2149417967 hasConceptScore W2149417967C85789140 @default.
• W2149417967 hasConceptScore W2149417967C86803240 @default.
• W2149417967 hasConceptScore W2149417967C95444343 @default.
• W2149417967 hasIssue "8" @default.
• W2149417967 hasLocation W21494179671 @default.

• next