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 involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that 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 with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.
Department of Microbiology and Immunology, Faculty of Health Sciences, Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
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Gelkop
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Isakov
D048868:Adaptor Proteins, Signal Transducing; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology*; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000952:Antigens, Polyomavirus Transforming; C082869:CRKL protein; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human