Arthur Weiss

Signal transduction by lymphocyte antigen receptors

Despite the differences in the antigens that they recognize and in the effector functions they carry out, B and T lymphocytes utilize remarkably similar signal transduction components to initiate responses. They both use oligomeric receptors that contain distinct recognition and signal transduction subunits. Antigen receptors on both cells interact with at least two distinct families of PTKs via common sequence motifs, ARAMs, in the cytoplasmic tails of their invariant chains, which have likely evolved from a common evolutionary precursor. Coreceptors appear to serve to increase the sensitivity of both of these receptor systems through events that influence ligand binding and signal transduction. The critical role of tyrosine phosphorylation of downstream signaling components, such as phospholipase C, is the net result of changes in the balance of the action of antigen receptor-regulated PTKs and PTPases. The identification of downstream effectors, including calcineurin and Ras, that regulate cellular responses, such as lymphokine gene expression, promises the future possibility of connecting the complex pathway from the plasma membrane to the nucleus in lymphocytes. Insight gained from studies of the signaling pathways downstream of TCR and BCR stimulation is likely to contribute significantly to future understanding of mechanisms responsible for lymphocyte differentiation and for the discrimination of self from nonself in developing and mature cells.

Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor

Signaling through the T cell antigen receptor (TCR) results both in rapid increases in tyrosine phosphorylation on a number of proteins and in the activation of the phosphatidylinositol pathway. It is not clear how stimulation of the TCR leads to these signaling events. Mutants of the Jurkat T cell line have been previously isolated that fail to show increases in calcium following receptor stimulation. Analysis of one of these mutants, JCaM1, which is defective in the induction of tyrosine phosphorylation, revealed a defect in the expression of functional lck tyrosine kinase. The lack of lck activity was caused in part by a splicing defect. Expression of the lck cDNA in JCaM1 restores the ability of the cell to respond to TCR stimulation. These results indicate that lck is required for normal signal transduction through the TCR.

ZAP-70: A 70 kd protein-tyrosine kinase that associates with the TCR ζ chain

Abstract Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR ζ chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with ζ and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with ζ require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.

Induction of NF-κB by the Akt/PKB kinase

Abstract The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2–4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-κB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-κB inhibitor IκB, and is specific for NF-κB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-κB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-κB induction that could have implications for the control of T-cell growth and survival.

The cytoplasmic domain of the T cell receptor ζ chain is sufficient to couple to receptor-associated signal transduction pathways

The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.

Signal transduction by the TCR for antigen.

The past several years have seen the beginning of a shift in the way that TCR signal transduction is studied. Although many investigators continue to identify new molecules, particularly adaptor proteins, others have attempted to look at signaling events in a larger cellular context. Thus the identification of distinct formations of signaling molecules at junctions between T cells and antigen-presenting cells, the role of the cytoskeleton and the partitioning of molecules into specialized lipid subdomains have been the subjects of many publications. Such concepts are helping to assemble a blueprint of how the myriad adaptors and kinases fit together to effect T cell activation.

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

The function of the Src-family kinases (SFKs) Lck and Fyn in T cells has been intensively studied over the past 15 years. Animal models and cell line studies both indicate a critical role for Lck and Fyn in proximal T-cell antigen receptor (TCR) signal transduction. Recruited SFKs phosphorylate TCR ITAMs (immunoreceptor tyrosine-based activation motifs) in the CD3 and ζ chains, which then serve as docking sites for Syk-family kinases. SFKs then phosphorylate and activate the recruited Syk-family kinase. Lck and Fyn are spatially segregated in cell membranes due to differential lipid raft localization, and may undergo sequential activation. In addition to the CD4 and CD8 coreceptors, a recently described adaptor, Unc119, may link SFKs to the TCR. CD45 and Csk provide positive and negative regulatory control of SFK functions, respectively, and Csk is constitutively bound to the transmembrane adapter protein, PAG/Cbp. TCR-based signaling is required at several stages of T-cell development, including at least pre-TCR signaling, positive selection, peripheral maintenance of naive T cells, and lymphopenia-induced proliferation. SFKs are required for each of these TCR-based signals, and Lck seems to be the major contributor.

T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases.

The mechanisms by which extracellular ligands induce receptor-regulated signal transduction events have been an area of intense investigation. Remarkable progress has been made in understanding howthe binding of extracellular ligands to protein-tyrosine kinase (PTK) growth factor receptors induces the activation of their kinase domains. In contrast, the mechanism whereby receptors without an intrinsic PTK domain activate cytoplasmic PTKs has been enigmatic. However, recent studies of receptors involved in antigen recognition by cells of the hematopoietic lineage provide insight into functionally important cytoplasmic sequence motifs of these receptors and the interaction of these motifs with cytoplasmic PTKs of the Src and non-Src types. This review focuses on mechanisms involved in PTK activation by the T cell antigen receptor (TCR), but similar mechanisms are likely to be relevant for the B cell antigen receptor, the mast cell and basophil high affinity Fc receptorfor immunoglobulin (lg) E (FcsR), and certain Fc receptors expressed on natural killer (NK) cells and myeloid cells. These receptors are involved in the initiation of cellular activation or differentiation but are not directly involved in cell proliferation. All of these receptors, which are responsible for antigen recognition, initiate cellular activation by regulating the function of cytoplasmic PTKs that control the activation of intracellular signaling molecules, such as phospholipase C (reviewed by Samelson and Klausner, 1992). Act/vat/on Motifs in the TCR CD3 and 4 Chains Regulate Cytoplasmic PTKs The TCR is a complex oligomer composed of the products of six genes (Figure l), all of which are required for efficient plasma membrane expression. Together, the a and 8 chains form the ligand-binding subunit responsible for recognition of an antigenic peptide bound to a major histocompatibility complex (MHC) molecule. The CD3 and 5 chains are responsible for signal transduction. Recent studies with chimeric molecules and reconstituted receptors have provided definitive evidence for a signal transduction function for the CD3 and c chains. The transmembrane and membrane-proximal segments of the TCR chains are responsible for the interchain associations and the assembly of the oligomeric receptor. This observation permitted the development of a strategy to study the functions of the individual cytoplasmic domains of CD3 or r;. Chimeric molecules containing the cytoplasmic domain of 6, its alternatively spliced product n, or the related FCER y chains fused to the extracellular and transmembrane domains of another receptor (including CD8, CD4, CD18, and the interleukin-2 [IL-21 receptor a chain) could be expressed independently of theTCR (Irving and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and Seed, 1991). Stimulation of these chimeric receptors with natural ligands or monoclonal antibodies (MAbs) induce all the early and late events characteristically associated with the intact oligomeric TCR. These findings suggest that 1; or the related chains can function to couple the TCR to intracellular signal transduction machinery. Surprisingly, subsequent experiments suggested a similar function for the CD3 chains. Reconstitution of TCR expression on a T cell hybridoma with cDNAs encoding various TCR chains has revealed that a funcfionally active TCR can be restored with a r; chain that is essentially devoid of cytoplasmic sequences (Wegener et al., 1992). A signaling function for the CD3 chains was confirmed when a chimeric molecule incorporating the CD3 E chain cytoplasmic domain was shown to be capable of activating a T cell hybridoma (Letourneur and Klausner, 1992). Thus, the CD3 and r; chains, though structurally distinct, appear to have redundant functions in TCR signaling. Identification of the functional domains of the chimeric receptors (Irving et al., 1993; Letourneur and Klausner, 1992; Romeo et al., 1992) explained the redundancy of function of the CD3 and r; chains. A sequence motif, first noted by Reth (1989), which is triplicated in 5 and present as a single copy in each of the CD3 chains, is responsible for the signal transduction capability of these chains (Figures 1 and 2). This motif, referred to herein as the antigen recognition activation motif (ARAM), is based on tyrosine and leucine (or isoleucine) residues. ARAMs are also present in the cytoplasmic domains of the proteins (Iga and lgp) associated with immunoglobulin on B cells, the 8 and y chains of the FcsR on mast cells and basophils, and, interestingly, the envelope glycoprotein of the bovine leukemia virus that induces polyclonal B cell proliferation. Since the genomic structure of each ARAM within c, CD3, Iga, lgp, and the FcsR j3 and y chains is similar (each motif is

LAT Is Required for TCR-Mediated Activation of PLCγ1 and the Ras Pathway

Abstract In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-ζ chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.

T cell antigen receptor signal transduction

The T cell antigen receptor (TCR) initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs). Considerable progress in the field of TCR signal transduction has been made in three areas recently: first, in understanding the structure and function of the PTK ZAP-70; second, in the elucidation of the function of the substrates and pathways downstream of the PTKs; and third, in the identification of molecules that negatively regulate TCR signalling.