EFFICIENT CD28 SIGNALING LEADS TO INCREASES IN THE KINASE-ACTIVITIES OF THE TEC FAMILY TYROSINE KINASE EMT ITK/TSK AND THE SRC FAMILY TYROSINE KINASE LCK/

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) productio n, inhibition of anergy and prevention of programmed cell death. Cross linking of CD28 leads to rapid increases in tyrosine phosphorylation o f specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activat e an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 in creases the activity of the intracellular tyrosine kinases EMT/ITK and LCK, The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site i n CD28 is dispensable for optimal IL2 production in Jurkat T cells, We demonstrate herein that murine Y170 (equivalent to human Y173) in CD2 8 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the dist al three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases . The distal three tyrosines of CD28, however, are not required for re cruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in J CaM1 cells which lack LCK and in which EMT/ITK is not activated by lig ation of CD28. Thus optimal activation of LCK or EMT/ITK is not obliga tory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together th e data indicate that the distal three tyrosines in CD28 are integral t o the activation of LCK and EMT/ITK and for subsequent IL2 production.