T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins

Engagement of the T cell antigen receptor initiates signal transduction inv olving tyrosine phosphorylation of multiple effector molecules and the form ation of multimolecular complexes at the receptor site. Adapter proteins th at possess SH2 and SH3 protein-protein interaction domains are implicated i n the assembly of cell activation-induced signaling complexes. We found tha t Crk adapter proteins undergo activation-induced interaction with the zeta -chain associated protein (ZAP-70) tyrosine kinase in the human T cell line , Jurkat, Incubation of various glutathione S-transferase fusion proteins w ith a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyros ine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of a ctivated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between t he two molecules was mediated by direct physical interaction and involved t he Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The as sociation required intact Lck, considered to be an upstream regulator of ZA P-70, because it could not take place in activated JCaM1 cells, which expre ss normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-tr ansferase-Crk fusion proteins were found to interact predominantly with mem brane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphoryl ation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early a ctivation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.