PHOSPHORYLATION OF EACH OF THE DISTAL 3 TYROSINES OF THE CD28 CYTOPLASMIC TAIL IS REQUIRED FOR CD28-INDUCED T-CELL IL-2 SECRETION

Signaling by the CD28 T cell costimulatory receptor is known to involv e recruitment and activation of phosphatidylinositol 3-kinase (PIS-kin ase) which is dependent upon phosphorylation of tyrosine 173 of the CD 28 cytoplasmic tail, present in a YMNM motif. However, whether this ph osphorylation is required for CD28 costimulation and whether or not ph osphorylation of any of the other three tyrosines of the CD28 cytoplas mic tail (tyrosines 188, 191 and 200) is also important for CD28 induc ed responses is unclear. To address this we examined the ability of ch imeric receptors, consisting of the extracellular plus transmembrane m embrane domain of human CD8 alpha linked to different mutated human CD 28 cytoplasmic tails, to induce IL-2 secretion in Jurkat T leukemia ce lls in the presence of PMA and ionomycin. A receptor in which tyrosine 173 of the CD28 tail was mutated to phenylalanine was able to induce IL-2. By contrast, receptors which contained single tyrosine 188, 191 or 200 to phenylalanine substitutions were unable to induce IL-2. Thes e results imply that in this system phosphorylation of tyrosine 173 an d hence activation of PI3-kinase is not required for CD28 induced IL-2 secretion. Further, they imply that phosphorylation of each of tyrosi nes 188, 191 and 200 is necessary for this response. Despite an appare nt requirement for phosphorylation of all three of these tyrosines, ho wever, receptors which contain tyrosine only at positions 191 or 200 a nd a truncated receptor which does not contain tyrosine 200 induce nor mal IL-2. These last findings, therefore, illustrate the complexity of CD28 mediated activation signals.