The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. The tandem SH2 (tSH2) domains of ZAP-70 have been studied by both X-ray and NMR. The crystal structure of the apoprotein is presented, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. The data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation is that the N-terminal SH2 domain can bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data (Folmer, 2002).
Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. A spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA (Sakaguchi, 2003).
One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. Human ZAP-70-deficient Jurkat T cells have been used to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain (Goda, 2004).
Segments of several protein-tyrosine kinase genes from Hydra vulgaris, a member of the ancient metazoan phylum Cnidaria, were amplified using the polymerase chain reaction with primers corresponding to conserved regions in the kinase domain of protein-tyrosine kinases. Characterization of cDNA clones for one of these genes, HTK16, revealed that it encodes a non-receptor protein-tyrosine kinase with two SH2 domains but no SH3 domain. In this regard the predicted HTK16 protein resembles two mammalian non-receptor protein-tyrosine kinases, the products of the ZAP-70 and syk genes. However, the HTK16 protein contains five ankyrin-like repeats, a structural motif that had not previously been found in protein-tyrosine kinases. The HTK16 protein also contains a potential tyrosine phosphorylation site in its carboxyl-terminal tail which resembles the phosphorylation site in members of the src family. RNA hybridization analysis indicates that the HTK16 gene is expressed in epithelial cells, cells which also express the Hydra homolog of the src protein. This finding of the HTK16 gene in Hydra indicates that diversification of genes encoding non-receptor protein-tyrosine kinases was a very early event in metazoan evolution (Chan, 1994).
The beta1 integrins are a family of heterodimeric adhesion receptors involved in cell-to-cell contacts and cell-to-extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, alpha4beta1 and alpha5beta1, can function as costimulatory molecules in T-cell receptor (TCR)-dependent T-cell activation. The Jurkat T-cell line was used as a model system to investigate the TCR-independent role of cell adhesion to fibronectin in the activation of Zap-70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, is induced. Moreover, clustering of fibronectin receptors leads to the detection of a p125FAK/Zap-70 complex. Finally, while the complex between fak-B, another protein kinase localized to focal adhesion sites, and Zap-70 is detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap-70 complex appears specifically induced following fibronectin-mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the beta1 integrin family mediate signalling pathways in T cells (Bearz, 1999).
Syk protein tyrosine kinase is essential for immune system development and function and for the maintenance of vascular integrity. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs). Syk can also be activated by integrin adhesion receptors, but the mechanism of its activation is unknown. A novel mechanism is reported for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. Both Syk and the related kinase ZAP-70 bind the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction is independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreases Syk binding and disruptes its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) fail to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation are unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase (Woodside, 2001).
Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. The interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities [beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)] as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains (Woodside, 2002).
Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. A 70-kDa tyrosine phosphoprotein (ZAP-70) is describes that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro (Chan, 1991).
SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck serves as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (1) inhibits both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (2) reduces the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (3) reduces both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation, and (4) inhibits cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function (Ito, 2003).
Regulation of protein tyrosine kinases (PTKs) by tyrosine phosphorylation is well recognized; in fact, nearly all PTKs require phosphorylation of tyrosine residues in their 'activation loop' for catalytic activity. In contrast, the phosphorylation of PTKs on serine and threonine residues has not been studied nearly as much. The ZAP-70 PTK contains predominately phosphoserine in normal T lymphocytes as well as in Jurkat T leukemia cells. One site of phosphorylation has been identified as Ser-520; this site is important for the recruitment and activation of ZAP-70 in T cells. Mutant ZAP-70-S520A has reduced ability to autophosphorylate and to mediate antigen receptor-induced interleukin 2 gene activation and is not enriched at the plasma membrane. These defects are rescued by addition of a myristylation signal to the N terminus of ZAP-70-S520A to force its plasma membrane and lipid raft localization. It is concluded that phosphorylation of ZAP-70 at Ser-520 plays an important role in the correct localization of ZAP-70 and in priming ZAP-70 for its acute recruitment and activation upon antigen receptor ligation (Yang, 2003).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.