title: A single amino acid alteration in cytoplasmic domain determines IL-2 promoter activation by ligation of CD28 but not inducible costimulator (ICOS).
abstract: The CD28 family molecules, CD28, and inducible costimulator (ICOS) all provide positive costimulatory signals. However, unlike CD28, ICOS does not costimulate IL-2 secretion. The YMNM motif that exists in the CD28 cytoplasmic domain is a known binding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. ICOS possesses the YMFM motif in the corresponding region of CD28 that binds PI3-K but not Grb2. We postulated that the reason that ICOS does not have the ability to induce IL-2 production is because it fails to recruit Grb2. To verify this hypothesis, we generated a mutant ICOS gene that contains the CD28 YMNM motif and measured IL-2 promoter activation after ICOS ligation. The results indicated that ICOS became competent to activate the IL-2 promoter by this single alteration. Further analysis demonstrated that Grb2 binding to ICOS was sufficient to activate the NFAT/AP-1 site in the IL-2 promoter and that the cytoplasmic domain of CD28 outside of the YMNM motif is required for activation of the CD28RE/AP-1 and NF-kappaB sites. Together, these observations lead us to believe that the difference of a single amino acid, which affects Grb2 binding ability, may define a functional difference between the CD28- and ICOS-mediated costimulatory signals.
authors.affiliation: Research Institute for Biological Sciences, Faculty of Science and Technology, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Yohsuke
authors.initials: Y
authors.lastname: Harada
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ohgai
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Watanabe
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Okano
authors.forename: Osamu
authors.initials: O
authors.lastname: Koiwai
authors.forename: Kazunari
authors.initials: K
authors.lastname: Tanabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Toma
authors.forename: Amnon
authors.initials: A
authors.lastname: Altman
authors.forename: Ryo
authors.initials: R
authors.lastname: Abe
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018106:CD28 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D005786:Gene Expression Regulation; D006801:Humans; D060889:Inducible T-Cell Co-Stimulator Protein; D007376:Interleukin-2 / Q000235:genetics*; D019169:Jurkat Cells; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000945:Antigens, Differentiation, T-Lymphocyte; D018106:CD28 Antigens; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C556692:ICOS protein, human; C556694:Icos protein, mouse; D060889:Inducible T-Cell Co-Stimulator Protein; D007376:Interleukin-2; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D019869:Phosphatidylinositol 3-Kinases