Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response.
The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (zeta-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca(2+) levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.
Deindl
Sebastian
S
Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
Kadlecek
Theresa A
TA
Cao
Xiaoxian
X
Kuriyan
John
J
Weiss
Arthur
A
D055162:Biocatalysis; D020013:Calcium Signaling; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019169:Jurkat Cells; D020935:MAP Kinase Signaling System; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013329:Structure-Activity Relationship; D013997:Time Factors; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
D050505:Mutant Proteins; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human