Ronald L. Wange

Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists

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.

Complex Complexes: Signaling at the TCR

The focus of this review has been on the complexity of biochemical events at a very proximal position in the TCR signaling cascade. A number of proteins and protein–protein interactions have been described. It should be noted that the function of many of these proteins (e.g., Vav, Cbl, PI3K, and SLP-76) remains to be defined. Likewise, a full accounting of all of the proteins involved in signaling complexes remains to be performed. The reader should know that extensive analysis of the pathways further downstream in the T cell and in other systems suggests that complexity exists at these levels too (Cantrell 1996xT cell antigen receptor signal transduction pathways. Cantrell, D. Annu. Rev. Immunol. 1996; 14: 259–274Crossref | PubMed | Scopus (551)See all ReferencesCantrell 1996). For example, Ras is likely to be only one of the many small G proteins involved in transduction events. Similarly, the well-known ERK kinase, which is most certainly involved in TCR-coupled events, is only one of several related serine/threonine kinases involved in signaling via TCR and other surface receptors. The complexity of the transcriptional machinery assembled at each lymphokine promoter is also well known. We also acknowledge that we have focused almost entirely on TCR-mediated signaling. Biochemical signals arising from engagement of coreceptors, costimulatory receptors, and adhesion molecules are also integrated to give the complex cellular response. Moreover, complex protein–protein interactions driven by these receptors are likely to be found. T cell activation can no longer be described only in terms of simple linear or even branching biochemical steps, but instead must also be thought of as a complex web of associated proteins.Several observations indicate that careful attention to nuances in the T cell signaling pathways is warranted. Activation of T cells with peptides or MHC molecules bearing subtle changes in sequence, so-called altered peptide ligands, results in dramatic differences in TCRζ and ZAP-70 tyrosine phosphorylation and ZAP-70 activity (64xPartial T cell signaling (altered phospho-ζ and lack of ZAP-70 recruitment in APL-induced T cell anergy) . Sloan-Lancaster, J, Shaw, A.S, Rothbard, J.B, and Allen, P.M. Cell. 1995; 79: 913–922Abstract | Full Text PDF | Scopus (541)See all References, 43xζ phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Madrenas, J, Wange, R.L, Wang, J.L, Isakov, N, Samelson, L.E, and Germain, R.N. Science. 1995; 267: 515–518Crossref | PubMedSee all References). Similarly, T cell activation schemes that result in anergic T cells fail to induce Ras and ERK activation (40xBlocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Li, W, Whaley, C.D, Mondino, A, and Mueller, D.L. Science. 1996; 271: 1272–1276Crossref | PubMedSee all References, 21xBlocked Ras activation in anergic CD4+ T cells. Fields, P.E, Gajewski, T.F, and Fitch, F.W. Science. 1996; 271: 1276–1278Crossref | PubMedSee all References). These examples are likely the first of many instances of variable biochemical responses to receptor engagement. Subtle changes in biochemical signals and in the composition of signaling complexes may have profound effects on cellular physiology. The existence of highly regulated signaling pathways offers the possibility of great flexibility of response to the T cell.

Deficiency of PTEN in Jurkat T Cells Causes Constitutive Localization of Itk to the Plasma Membrane and Hyperresponsiveness to CD3 Stimulation

ABSTRACT 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-P2 and PI-3,4,5-P3 in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-γ1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

ABSTRACT T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70–Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

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.

LAT, the Linker for Activation of T Cells: A Bridge Between T Cell-Specific and General Signaling Pathways

A key event in the regulation of the adaptive immune response is the binding of major histocompatibility complex-bound foreign peptides to T cell antigen receptors (TCRs) that are present on the cell surface of T lymphocytes. Recognition of the presence of cognate antigen in the host animal induces a series of biochemical changes within the T cell; these changes, in the context of additional signals from other surface receptors, ultimately result in massive proliferation of receptor-engaged T cells and the acquisition of effector and memory functions. Early studies established the importance of the activation of the enzymes phospholipase C-γ1 (PLC-γ1) and phosphatidylinositol 3-kinase (PI3K), as well as the small molecular weight heterotrimeric guanine nucleotide binding protein (G protein) Ras, in this process. These biochemical events are dependent on the activity of several protein tyrosine kinases that become activated immediately upon TCR engagement. An unresolved question in the field has been which molecules and what sequence of events tie together the early tyrosine phosphorylation events with the activation of these downstream signaling molecules. A likely candidate for linking the proximal and distal portions of the TCR signaling pathway is the recently described protein, LAT. LAT is a 36-kD transmembrane protein that becomes rapidly tyrosine-phosphorylated after TCR engagement. Phosphorylation of LAT creates binding sites for the Src homology 2 (SH2) domains of other proteins, including PLC-γ1, Grb2, Gads, Grap, 3BP2, and Shb, and indirectly binds SOS, c-Cbl, Vav, SLP-76, and Itk. LAT is localized to the glycolipid-enriched membrane (GEM) subdomains of the plasma membrane by virtue of palmitoylation of two cysteine residues positioned near the endofacial side of the plasma membrane. Notably, in the absence of LAT, TCR engagement does not lead to activation of distal signaling events. This review examines the circumstances surrounding the discovery of LAT and our current understanding of its properties, and discusses current models for how LAT may be functioning to support the transduction of TCR-initiated, T cell-specific signaling events to the distal, general signaling machinery. One of the unresolved questions in T cell receptor (TCR) signaling has been how T cell-specific, TCR-proximal signaling events, such as the activation of the protein tyrosine kinases (PTKs) Lck, Fyn, ZAP-70, Itk, and Tec, lead to the activation of distal, more general signaling elements, such as phospholipase C-γ1 (PLC-γ1), Ras, and phosphatidylinositol 3-kinase (PI3K). With the discovery of LAT, we are much closer to understanding how this occurs. LAT is a 36-kD transmembrane protein that is targeted to lipid rafts and is rapidly tyrosine-phosphorylated after TCR stimulation. LAT appears to serve as a hub between the proximal and distal TCR signaling pathways by facilitating the formation of multimolecular signaling complexes in response to the activation of TCR-associated PTKs. These LAT-assembled complexes are probably involved in ordering interacting proteins into a favorable geometry for productive interaction with one another. In addition to serving as an organizing nucleation scaffold, LAT also serves to localize multiple cytosolic signaling proteins to lipid rafts. Recruitment of signaling proteins to the lipid rafts leads to a very high effective local concentration of these proteins relative to each other, facilitating the formation of additional protein-protein interactions and enhancing enzymatic reaction rates. In addition, LAT-mediated targeting to the lipid rafts may serve to localize enzymes, such as PLC-γ1, PI3K, and SOS, to the vicinity of their substrates. Although it remains to be determined how much of a contribution each of these potential mechanisms makes toward LATs signal transduction capabilities, the discovery and characterization of LAT has considerably advanced our understanding of the ever more complex series of biochemical events that link TCR engagement with T cell activation.

Itk/Emt/Tsk Activation in Response to CD3 Cross-linking in Jurkat T Cells Requires ZAP-70 and Lat and Is Independent of Membrane Recruitment

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivosubstrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.

ZAP-70-dependent and -independent Activation of Erk in Jurkat T Cells DIFFERENCES IN SIGNALING INDUCED BY H2O2 AND CD3 CROSS-LINKING

Oxidative stress in T cells induces signaling events similar to those initiated by T cell antigen receptor engagement, including tyrosine phosphorylation and activation of the critical protein-tyrosine kinase ZAP-70. Distal signaling events such as the activation of mitogen-activated protein kinases and downstream transcription factors are also initiated by oxidative stimuli. In this study P116, a ZAP-70-negative Jurkat T cell line, was used to investigate the role of ZAP-70 in mediating activation of Erk in response to H2O2. Consistent with the hypothesis that ZAP-70 is required for activation of Erk in response to an oxidative stimulus, Erk1 and Erk2 could be rapidly activated in Jurkat cells but not in P116 cells upon addition of H2O2. P116 cells became competent for H2O2-induced Erk activation upon stable transfection with wild-type ZAP-70. An in vivo ZAP-70 substrate, SLP-76, implicated in Erk activation, also became rapidly tyrosine-phosphorylated in Jurkat cells, but not in P116 cells, upon treatment with H2O2. Surprisingly, although ZAP-70 was required for H2O2-mediated Erk activation, Erk activation in response to T cell antigen receptor engagement did not require ZAP-70. In addition to demonstrating a requirement for ZAP-70 in H2O2-stimulated Erk activation, these results provide the first evidence for the existence of a ZAP-70-independent pathway for Erk activation in T cells.

ZAP-70 and SLP-76 regulate protein kinase C-theta and NF-kappa B activation in response to engagement of CD3 and CD28

The transcription factor NF-κB is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-κB activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-κB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-κB activation was examined. NF-κB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-α) activated NF-κB normally. Protein kinase C (PKC) θ activation, which is required for NF-κB activation, also was defective in these cells. Reexpression of ZAP-70 restored PKCθ and NF-κB activation in response to TCR and CD28 coengagement. p95vav (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKCθ and NF-κB activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC θ and NF-κB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.

Membrane Raft-Dependent Regulation of Phospholipase Cγ-1 Activation in T Lymphocytes

ABSTRACT Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cγ-1 (PLCγ1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCγ1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCγ1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCγ1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCγ1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCγ1 over wild-type PLCγ1. These data show that localization of PLCγ1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCγ1 with other signaling components.