title: Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation.
abstract: CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y191 mutation (Y191CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M194 mutation (M194CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y191CD28F and M194CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.
authors.affiliation: Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
authors.forename: Y C
authors.initials: YC
authors.lastname: Cai
authors.forename: D
authors.initials: D
authors.lastname: Cefai
authors.forename: H
authors.initials: H
authors.lastname: Schneider
authors.forename: M
authors.initials: M
authors.lastname: Raab
authors.forename: N
authors.initials: N
authors.lastname: Nabavi
authors.forename: C E
authors.initials: CE
authors.lastname: Rudd
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D051940:B7-2 Antigen; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D051940:B7-2 Antigen; D018106:CD28 Antigens; C497964:CD86 protein, human; C497965:Cd86 protein, mouse; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)