Intramolecular Regulatory Switch in ZAP-70: Analogy with Receptor Tyrosine Kinases

Abstract

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.Stimulation of the T-cell antigen receptor (TCR) leads to a series of signaling events that result in changes in T-cell function and gene expression. Signaling is initiated by two families of protein tyrosine kinases (PTKs) (17). Src-family kinases first phosphorylate specific immunoreceptor tyrosine-based activation motifs (ITAMs) (4) in the cytoplasmic tails of the CD3 and subunits of TCR. Each ITAM is composed of a pair of tyrosines separated by 9 to 11 amino acids which, when phosphorylated, bind the Syk family PTK ZAP-70. In turn, ZAP-70 becomes activated and subsequently phosphorylates a number of key downstream signaling molecules necessary for further signal propagation.ZAP-70 consists of two N-terminal SH2 domains, responsible for binding to doubly phosphorylated ITAMs, and a Cterminal tyrosine kinase domain (6). The SH2 domains are separated by a linker region, termed interdomain A. The region between the SH2 domains and the kinase domain is known as interdomain B.Upon binding to phosphorylated ITAMs of the activated TCR, ZAP-70 itself becomes phosphorylated on multiple tyrosine residues, both by Src family PTKs and via autophosphorylation. It is well established that tyrosine phosphorylation plays an important role in ZAP-70 activation, but the precise mechanism responsible is still incompletely understood. Among the best-studied phosphorylation sites are tyrosines 492 and 493 (Y492 and Y493) in the activation loop of the kinase (5,33,34). A model has been proposed whereby Y493 becomes phosphorylated first by the Src-family PTK Lck. This phosphorylation is then followed by autophosphorylation at Y492 (5). Phosp...

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“…Radiolabeled tyrosine-phosphorylated proteins were visualized with a Fujifilm FLA 5000 phosphorimaging device (Fujifilm Medical Systems), and the intensity of protein phosphorylation was quantitated with GelPro software. For Zap70, in vitro kinase assays were performed in a similar manner, but reactions were started by the addition of 30 l of kinase buffer containing 50 M ATP, 5 Ci of [␥-32 P]ATP, and 10 g of GST fused to a 14-aa peptide (MEELQDYEDMMEEN) from the cytoplasmic domain of human erythrocyte band 3 (cdb3 peptide) as exogenous substrate (34). The plasmid containing the cdb3 peptide GST fusion protein was a gift from Dr. A. Weiss (University of California, San Francisco, CA).…”

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