A DUAL PARTICIPATION OF ZAP-70 AND SCR PROTEIN-TYROSINE KINASES IS REQUIRED FOR TCR-INDUCED TYROSINE PHOSPHORYLATION OF SAM68 IN JURKAT T-CELLS

Sam68 has been initially described as a substrate of src kinases durin g mitosis in fibroblasts. Recent evidence suggests that in T lymphocyt es Sam68 may act as an adaptor protein and participate in the early bi ochemical cascade triggered after CD3 stimulation. A direct interactio n between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase C gamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56 (lck), as well as the role of ZAP-70, the second class of protein tyro sine kinase involved in T cell activation, in Sam68 tyrosine phosphory lation in the human Jurkat T cell line. Using the src inhibitor PP1 [4 -amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a do minant negative form of ZAP-70, we demonstrate that, while both p56(lc k) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kina ses is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p5 6(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 sti mulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p 56(lck) differently participate in regulating the phosphorylation stat e of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule afte r CD3 stimulation.