ANALYSIS OF CD28 CYTOPLASMIC TAIL TYROSINE RESIDUES AS REGULATORS ANDSUBSTRATES FOR THE PROTEIN-TYROSINE KINASES, EMT AND LCK

The CD28 cell surface receptor provides an important costimulatory sig nal for T cells necessary for their response to Ag. Early events in CD 28 signaling include recruitment and activation of phosphatidylinosito l 3-kinase (PIS-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT, Recruitment and activation of PI3-kinase is know n to be dependent upon phosphorylation of tyrosine 173 of the CD28 cyt oplasmic tail contained within a YMNM motif, By contrast, little is kn own of which residues of the CD28 tail, including tyrosines, are requi red for the activation of PTKs, To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution muta nts of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T l eukemia cells. Our results indicate that 1) activation of EMT is parti ally dependent upon tyrosine 173 of the CD28 fail although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient f or the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for b oth PTKs in in vitro kinase assays, We demonstrate that EMT can phosph orylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo , tyrosines other than tyrosine 173 become phosphorylated following CD 28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.