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

Abstract

The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (-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.activation ͉ catalytic activity ͉ conformation ͉ ITAM T he protein tyrosine kinase ZAP-70 (-associated protein of 70 kDa) (1) participates in the initial events of T cell antigen receptor (TCR) signaling and thus is of critical importance for the adaptive immune response (2-5). On activation of the TCR, 2 tyrosines in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 or cytoplasmic tails of the TCR are phosphorylated by a Src family kinase member. Through the binding of 2 SH2 (Src homology 2) domains at the N terminus of ZAP-70 to these ITAM phosphotyrosines, ZAP-70 is then specifically recruited to a stimulated TCR. These 2 SH2 domains of ZAP-70 are tightly associated as a tandem unit with a precise structural organization that enables high-affinity ITAM peptide binding (6). In the very closely related Syk (spleen tyrosine) kinase, the destabilization of this structural organization by mutation results in both uncoupling of the 2 SH2 units and a dramatic reduction in ITAM binding affinity (7). Recruitment of ZAP-70 to the TCR results in activation of the catalytic domain located at the C terminus of ZAP-70 and signal propagation by phosphorylation of its downstream target molecules, including the ZAP-70 substrates LAT (linker for the activation of T cells) and SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kDa). Both LAT and SLP-76 are crucial adaptor proteins that, once phosphorylated, provide a platform for the assembly of signaling molecules (8, 9). Ultimately, these signaling events produce effector secretory responses, modulate transcription, and trigger T cell proliferation and differentiation.ZAP-70 plays critical roles in pre-TCR and TCR signaling, thymocyte development, and peripheral T cell function. The loss of ZAP-70 catalytic activity results in profound immunodeficiency, and reduced activity of ZAP-70 may result in autoimmunity (4, 10). Furthermore, the expression of ZAP-70 in B c...

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

“…In the Src family kinases, the side chain of Trp 260 (chicken c-Src numbering) is inserted between the ␣C helix and the main body of the kinase domain, thereby stabilizing the inactive conformation. In ZAP-70, the residue corresponding to Trp 260 in c-Src is Leu 330, but this residue does not appear to be critical for the suppression of kinase activity (18). Indeed, as we now show, it is the side chain of Tyr 319 in the SH2-kinase linker of ZAP-70 that plays a role analogous to that of Trp 260 in c-Src.…”

“…In the first set of experiments, we used a heterologous peptide substrate (poly-Glu/Tyr) and a coupled-enzyme assay that measures ATP consumption (18). The purpose of this set of experiments was to compare the activity of full-length ZAP-70 to that of the isolated kinase domain, because the activity of the latter construct was not measured in our previous studies.…”

“…8, left panel). The W131A mutant was chosen for study because its mutation disrupts the linker-kinase sandwich (18). Each of these constructs was expressed using a recombinant baculovirus system and purified for measurement of kinase activity.…”

“…The conformational change in the tan-dem-SH2 module upon phosphorylated ITAM binding, particularly in interdomain A, is expected to result in the linker-kinase sandwich being dismantled and the tandem-SH2 module being released from the kinase domain. Key aspects of this mechanism have been validated by in vitro and cell-based measurements of the activities of ZAP-70 mutants (18,19). As we explain below, we have now identified a sequence register error in the original model for the SH2-kinase linker, the implications of which are examined in this paper.…”

“…The coupled kinase assay was performed using a continuous spectrophotometric method as described previously (18). Briefly, the final reaction buffer contained 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 150 mM NaCl, 1 mM phosphoenolpyruvate, 0.3 mg/ml of NADH, 75 U/ml of pyruvate kinase, 105 U/ml of lactate dehydrogenase, 2 mg/ml of poly-Glu/Tyr substrate peptide (Glu/Tyr ratio of 4:1; 5 to 20 kDa), 2 M tyrosine kinase, and 1 mM ATP.…”