Inhibition of ZAP-70 kinase activity via an analog-sensitive allele blocks T cell receptor and CD28 superagonist signaling.
ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable, small molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor. We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocked ZAP-70-dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablated CD28 superagonist signaling, thereby demonstrating the utility of this system in dissecting the requirement for ZAP-70 in alternative mechanisms of T cell activation. Thus, we have developed the first specific chemical means of inhibiting ZAP-70 in cells, which serves as a valuable tool for studying the function of ZAP-70 in T cells.
Levin
Susan E
SE
Departments of Medicine and Microbiology & Immunology, Biomedical Sciences Graduate Program, and Howard Hughes Medical Institute, University of California-San Francisco, 5134 Parnassus Avenue, San Francisco, CA 94143, USA.
Zhang
Chao
C
Kadlecek
Theresa A
TA
Shokat
Kevan M
KM
Weiss
Arthur
A
D000483:Alleles; D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011948:Receptors, Antigen, T-Cell / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D018089:Superantigens / Q000494:pharmacology*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D018106:CD28 Antigens; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell; D018089:Superantigens; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human