No abstract

The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56 lck , in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wildtype ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR chain and was able to bind p56 lck . This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56 lck with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56 lck , which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.Biochemical and genetic studies have indicated that cytoplasmic protein-tyrosine kinases (PTKs) 1 of the Src and Syk families control the early steps of the signaling cascade initiated by T-cell antigen receptor (TCR) triggering (1). The TCR signal-transducing subunits (␥, ␦, and ⑀ chains of the CD3 complex and the homodimer) do not possess intrinsic PTK activity; yet, TCR ligation by antigen/major histocompatibility complex or by anti-TCR mAbs induces rapid tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) present in their cytoplasmic tails (2, 3), an event essential for activation of lymphokine genes (4, 5). Several lines of evidence suggest that the Src family PTKs p56 lck and p59 fyn mediate phosphorylation of the ITAM (6 -9), although the former may have a prominent role (10). ITAM phosphorylation allows recruitment of the Syk-related PTK ZAP-70 to the TCR (11) via binding of the two tandemly arranged ZAP-70 SH2 domains to the two phosphotyrosines of ITAMs (8, 12, 13). As a consequence, ZAP-70 itself is phosphorylated on tyrosine residues, a modification contributing to its catalytic activation (14 -16). The importance of ZAP-70 in TCR signaling has been established by studies of a rare human immunodeficiency caused by the absence of 18). In these individuals, only CD4 ϩ T cells develop but fail to proliferate in response to TCR ligation and show a largely compromised activation-induced tyrosine phosphorylation of intracellular proteins and calcium increase.The mecha...

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are though to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56lck SH2 domain with zeta-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/zeta complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8 alpha alpha and CD8 alpha beta isoforms to the TCR. We demonstrate in vivo in association of CD8 alpha alpha/p56lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56lck specifically impedes the association of ZAP-70 with CD8 alpha alpha/p56lck without affecting the zeta/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8 alpha alpha or CD8 alpha beta isoform with the TCR/CD3/zeta complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated TCR/CD3/zeta complex.

T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.ZAP-70 is a protein tyrosine kinase (PTK) 1 essential for the initiation of the signaling cascade activated by T cell antigen receptor triggering (1). Overall, ZAP-70 displays two structurally and functionally distinct moieties, an N-terminal one composed of two SH2 domains and a C-terminal kinase domain tethered by an ϳ80-amino acid-long linker (2) (also referred to as Interdomain B, (IB); Ref. 3).So far, the only known function of the region comprising the two SH2 domains (hereafter indicated as (SH2) 2 ) is to provide a means to recruit ZAP-70 to the plasma membrane by those TCRs engaged with the ligand (4, 5). This is achieved through the coordinated anchorage of the SH2 domains to di-phosphorylated tyrosine-containing motifs (D/E)XXYXX(I/L)X 6 -8 YXX (I/L) called ITAMs (for Immunoreceptor Tyrosine-based Activation Motifs) present within the cytoplasmic tails of TCR subunits and ⑀ (6 -8). Thereafter, ZAP-70 undergoes tyrosine phosphorylation culminating in the up-regulation of its catalytic activity which in turn is required for phosphorylating cellular substrates (9 -12).The x-ray crystal structure of the (SH2) 2 of human ZAP-70 complexed with a di-phosphorylated ITAM (2pY-ITAM) peptide (3) has revealed an unsuspected structural complementarity and immediate contiguity of the two SH2 domains needed to create a high affinity binding site for the 2pY-ITAM. Thus, while the C-terminal SH2 possesses a binding pocket for the first pY of the ITAM, the corresponding pocket for the second pY in the N-terminal SH2 is contributed, in part, by residues of the C-terminal SH2. Moreover, the 60 amino acids forming the inter-SH2 spacer (hereafter referred to as Interdomain ...

CD45 is a receptor‐like protein tyrosine phosphatase critically involved in the regulation of initial effector functions in B‐ and T‐cells. The protein comprises two phosphatase (PTP) domains in its cytoplasmic region. However, whether each PTP domain has enzyme activity by itself or whether both domains are required to build up a functional enzyme is unclear. We have studied different constructions of human CD45 comprising the two PTP domains, both separately and as a single protein, fused to maltose‐binding protein (MBP). In apparent contrast with previous studies, we show that the first PTP domain of CD45 (when fused to MBP) may be a viable phosphatase in the absence of the second domain. Phosphatase activity resides in the monomeric form of the protein and is lost after proteolytic cleavage of the fusion partner, indicating that MBP specifically activates the first PTP domain. Furthermore, changes in the optimal pH for activity with respect to wild‐type CD45 suggest that protein–protein interactions involving residues in the neighbourhood of the catalytic site mediate enzyme activation.