Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

of SH2 domain-containing proteins as a consequence of the ability of SH2 domains to bind specific phospho-Cell Biology and Metabolism Branch National Institute of Child Health tyrosine-containing polypeptides. Additional signaling molecules are subsequently recruited to these newly and Human Development National Institutes of Health TCR-associated proteins via SH2 or other modular interaction domains.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs),

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P 2 and PI-3,4,5-P 3 in the plasma membrane. A major advance in our understanding of signal transduction pathways has come from the realization that many signaling proteins possess one or more self-contained domains that mediate important regulatory interactions with other cellular structures. Many of these domains, such as Src homology domains 2 and 3 (SH2 and SH3 domains), were initially identified as mediators of protein-protein interactions, but it is now apparent that some domains are also involved in high-affinity binding to certain modified phospholipids. Just as SH2 domains have been demonstrated to mediate a regulatable, reversible association between two proteins, depending on the presence or absence of a phosphate group on key tyrosine residues, so it has recently become clear that pleckstrin homology (PH) domains can mediate a similar association with the plasma membrane, depending on the presence or absence of phosphate on the D3 position of the myo-inositol ring of phosphatidylinositides (PI) (6,12,30,44,63). This property of PH domains forms the basis for a regulatable, reversible association of PH domain-containing proteins with the phosphoinositide-rich regions of the plasma membrane, an event that plays an important role in regulating the activities of several enzymes important in signaling pathways.The importance of PH domain-mediated interactions with the plasma membrane is well illustrated by the mechanisms of activation of the ubiquitous serine/threonine kinase Akt (also known as protein kinase B) and the B-cell and mast cell protein tyrosine kinase (PTK) Btk. Both kinases possess PH domains within their amino termini. Akt plays an important role in growth control and protection from apoptosis, and is activated only when sufficient levels of both PI-3,4-P 2 and PI-3,4,5-P 3 are present in the plasma membrane (10,12,16,18). The mechanism of activation involves PH domain-dependent corecruitment of Akt and the Akt kinase PDK1 (which also contains a PH domain) to the plasma membrane by high-affinity interactions with PI-3,4-P 2 and PI-3,4,5-P 3 , respectively. Colocalized PDK1 phosphorylates Akt on Thr-308, catalyzing autophosphorylation on Ser-473, and activating the kinase (2, 71). The Btk kinase plays a vital ...

ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.

No abstract

The Tec family kinase Itk is an important regulator of Ca2+ mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

The adaptive phase of the immune response begins with engagement on CD4ϩ helper T cells of the T cell antigen receptor (TCR) 1 by its ligand, a small foreign peptide bound to a cell surface protein of the class II major histocompatibility complex (peptide-MHC) expressed on an antigen-presenting cell. This engagement initiates a series of biochemical events that can differentially signal the naive T cell to: 1) enter into a pathway leading to generation of effector T cells with the onset of rapid proliferation and production of effector cytokines; 2) enter into a state of antigenic non-responsiveness known as anergy; or 3) die by apoptosis. The type of response elicited depends on multiple factors including the affinity of the interaction, the duration of the interaction, and the presence or absence of various costimulatory signaling inputs such as those provided by the CD4 coreceptor and the CD28 costimulatory receptor. In this review we provide an overview of the signaling events that are associated with the first of these outcomes: T cell activation. To present an overview of sufficiently broad scope, the depth of discussion of each aspect of TCR signaling is by necessity limited, and the reader is referred to the reviews cited throughout this text for consideration of these events in more detail. TCR StructureThe TCR is composed of six different polypeptide chains. The specificity of ligand binding is dictated by the clonotypic TCR␣ and TCR␤ chains, which arise from a process of genetic rearrangement that results in millions of receptor variants. These chains form a heterodimer that binds directly to peptide-MHC. Communication of TCR␣␤ engagement by peptide-MHC to the intracellular signaling machinery occurs via the TCR-associated CD3 chains, which are arranged into three dimers: ␥⑀, ␦⑀, and (1). Each CD3 chain contains immunoreceptor tyrosine-based activation motifs (ITAMs); one each in ␥, ␦ and ⑀ and three in . The eponymous features of these motifs are a pair of tyrosine residues separated by 9 -11 amino acids. These tyrosines become rapidly phosphorylated by the Src-family kinase Lck following TCR stimulation; a required event for initiating TCR signaling (2, 3).