Abstract
Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).
Highlights
We show that the last 10 residues of the intracytoplasmic domain of CD28 are required for its costimulatory function
We demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP)
Secretion—We have previously shown that a point mutation of the tyrosine residue at position 173 abolished both PI 3-kinase binding and IL-2 secretion in a murine T-cell hybridoma transfected with human CD28 [12]
Summary
TcR, T-cell receptor; PI 3-kinase, phosphatidylinositol 3-kinase; IL-2, interleukin 2; Th, T helper; SH, Src homology; Ig, immunoglobulin; PLC, phospholipase C; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HPLC, high performance liquid chromatography. We and others have demonstrated previously that ligand stimulation of the human CD28 molecule induces its association with PI 3-kinase activity [11,12,13,14,15] by means of a cytoplasmic YMNM motif at position 173–176 which, when phosphorylated, interacts with the SH2 domains of the p85 adaptor subunit. The T-cell-specific protein-tyrosine kinase ITK has been shown to associate with CD28 and to be phosphorylated on tyrosine residues after CD28 stimulation [19], and the Src-related tyrosine kinases p56lck and p59fyn have been found in CD28 immune complexes from stimulated T cells [20]. We have generated mutants of CD28 containing progressive truncations of its intracytoplasmic tail (10, 21, 30, and 41 residues), as well as a point mutation of the tyrosine residue at position 200 These variants were expressed in a murine T-cell hybri-. We investigated whether these molecules were able to mediate cell adhesion to human CD80-transfected L-cells, to be phosphorylated, bind and activate PI 3-kinase, and to costimulate IL-2 production together with CD31⁄7TcR
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