Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

Sebastian Deindl, Theresa A. Kadlecek, Xiaoxian Cao, John Kuriyan, and Arthur Weiss

PNAS November 17, 2009, doi:10.1073/pnas.0911512106 (local copy)

Abstract / Figures from the paper


The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (ζ-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 Ca2+ 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.

Figures from the paper.

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Figure 1. Domain architecture (Top) and schematic cartoon representation of the autoinhibited structure of ZAP-70 (Bottom). The kinase domain, SH2-kinase linker, inter-SH2 linker, C-terminal SH2 domain, and N-terminal SH2 domain are shown in blue, green, yellow, red, and orange, respectively. A portion of the docking interface is magnified to show the hydrophobic packing in the linker-kinase sandwich (Center) and the hydrogen-bonding network at the hinge region of the kinase domain. The two regulatory tyrosine residues in the SH2-kinase linker are mutated to phenylalanines in the crystallization construct.
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Figure 2. In vitro catalytic activity of ZAP-70: the rate of peptide substrate phosphorylation by wild-type ZAP-70 and the W131A mutant form of ZAP-70. Rates are relative to the catalytic rate of the wild-type protein. The solid black bars represent catalytic rates in the absence of ITAM peptide, and the light-gray bars depict the rates obtained after incubation with saturating amounts of ITAM peptide.
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Figure 3. Destabilization of the linker-kinase sandwich by mutation affects TCR-induced tyrosine phosphorylation. (A) P116 stable lines were incubated with anti-TCR antibody or left unstimulated for 2 minutes, and lysed in 1% Nonidet P-40 lysis buffer. Phosphorylation levels were assessed by immunoblotting for total tyrosine phosphoproteins (pTyr) (B) or with phosphospecific ZAP-70, PLCγ1, Erk, SLP-76 antibodies (C). Analyses were performed on whole-cell lysates (WCLs) or after immunoprecipitation (IP). The relative expression of the various ZAP-70 mutants are closely matched to the relative expression of the WT protein: YYAA, 0.91; YYFF, 1.44; W131A, 0.97; A141E, 0.87; and KA, 1.26.
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Figure 4. TCR-induced activation of the MAP kinase pathway in P116 cells stably expressing wild-type or W131A mutant ZAP-70. P116 lines stably reconstituted with WT or the W131A mutant of ZAP-70 were incubated with anti-TCR antibody over a 15-minute period. (A) Cells were fixed at various time points, permeabilized, stained intracellularly with phosphospecific Erk antibody, and analyzed by FACS. (B) Dose response to anti-TCR antibody at the 5' time point.
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Figure 5. TCR-induced calcium flux in P116 cells stably reconstituted with wild-type ZAP-70 and the W131A mutant form of ZAP-70. ZAP-70 – deficient P116 Jurkat cells reconstituted with wild-type or the W131A mutant of ZAP-70 were loaded with the calcium-sensitive dye Indo-1 and stimulated with varying doses of anti-TCR Vβ antibody (C305). Calcium changes were monitored over time.
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Figure 6. Time course of TCR-induced tyrosine phosphorylation in wild-type ZAP-70 and W131A-stable transfectants. P116-stable lines were incubated with anti-TCR antibody over a 5-minute period and lysed in 1% Nonidet P-40 lysis buffer. Lysates were separated on SDS-PAGE gels and immunoblotted for total tyrosine phosphoproteins (A) and phosphospecific ZAP-70, PLCγ1, Erk, LAT, and total levels of ZAP-70, PLCγ1, and TCRζ chain (B).
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Figure 7. Binding of the ITAM-ζ1 peptide to ZAP-70. Fluorescence anisotropy increases on binding of fluorescently labeled ITAM-ζ1 peptide to wild-type ZAP-70 (A), ZAP-70 W131A (B), and ZAP-70 YYFF (C). (D) Double-reciprocal plot of ITAM-ζ1 binding to ZAP-70. The data corresponding to the binding of ITAM-ζ1 peptide to ZAP-70 WT, W131A, and YYFF are shown as blue, red, and green lines, respectively. The Kd values depicted here are those obtained from a nonlinear curve fit.
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