A single amino acid alteration in cytoplasmic domain determines IL-2 promoter activation by ligation of CD28 but not inducible costimulator (ICOS).
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.
Harada
Yohsuke
Y
Research Institute for Biological Sciences, Faculty of Science and Technology, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.
Ohgai
Daisuke
D
Watanabe
Ryosuke
R
Okano
Kazuhiro
K
Koiwai
Osamu
O
Tanabe
Kazunari
K
Toma
Hiroshi
H
Altman
Amnon
A
Abe
Ryo
R
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
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