scholarly journals The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras.

1994 ◽  
Vol 180 (3) ◽  
pp. 1067-1076 ◽  
Author(s):  
J A Nunès ◽  
Y Collette ◽  
A Truneh ◽  
D Olive ◽  
D A Cantrell
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Signal Transduction Pathway ◽  
Biochemical Basis ◽  
Ras Activation

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.

scholarly journals CD28 and T cell antigen receptor signal transduction coordinately regulate interleukin 2 gene expression in response to superantigen stimulation.

1992 ◽  
Vol 175 (4) ◽  
pp. 1131-1134 ◽  
Author(s):  
J D Fraser ◽  
M E Newton ◽  
A Weiss
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Cell Antigen

Activation of an immune response requires intercellular contact between T lymphocytes and antigen-presenting cells (APC). Interaction of the T cell antigen receptor (TCR) with antigen in the context of major histocompatibility molecules mediates signal transduction, but T cell activation appears to require the induction of a second costimulatory signal transduction pathway. Recent studies suggest that interaction of CD28 with B7 on APC might deliver such a costimulatory signal. To investigate the role of CD28 signal transduction during APC-dependent T cell activation, we have used Staphylococcal enterotoxins (SEs) presented by a B7-positive APC. We used anti-B7 monoclonal antibodies and a mutant interleukin 2 (IL-2) promoter construct, unresponsive to CD28-generated signals, in transient transfection assays to examine the contribution of the CD28-B7 interaction to IL-2 gene activation. These studies indicate that the CD28-regulated signal transduction pathway is activated during SE stimulation of T cells and plays an important role in SE induction of IL-2 gene expression through its influence upon the CD28-responsive element contained within the IL-2 gene promoter. This effect is particularly profound in the activation of the IL-2 gene in peripheral blood T cells.

Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector molecules

1992 ◽  
Vol 263 (6) ◽  
pp. C1119-C1140 ◽  
Author(s):  
B. A. Premack ◽  
P. Gardner
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Phospholipase C ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Binding Proteins ◽  
Cell Receptor ◽  
Binding Domains

There have been major advances over the last several years in understanding the molecular basis of signaling by the T lymphocyte (T-cell) antigen receptor. In this article we discuss the early phases of T-cell activation with an emphasis on receptor-associated signaling molecules, mobilization of Ca, and on the possible roles of Ca in signal transduction. Ligation of the extracellular domains of the T-cell receptor activates receptor-associated tyrosine kinases that can phosphorylate the gamma-isoform of phospholipase C, increasing its catalytic activity. This leads to production of inositol 1,4,5-trisphosphate, release of stored intracellular Ca, and activation of Ca-permeable plasma membrane channels. Many of the critical T-cell signal transducing enzymes such as phospholipase C and protein kinase C contain intrinsic Ca-binding domains, but for the most part the rise in cytoplasmic Ca is transduced by specialized Ca-binding proteins that lack catalytic domains. The Ca-binding proteins found in T-cells include members of both the EF-hand and annexin families, as well as other types of Ca-binding proteins. In T-cells, a number of important kinases, phosphatases, and cytoskeleton-modulating enzymes are functionally Ca dependent but have no Ca-binding domains and therefore must sense changes in the cytoplasmic Ca level through interactions with Ca-binding proteins.

scholarly journals A Role for the Tec Family Tyrosine Kinase Txk in T Cell Activation and Thymocyte Selection

1999 ◽  
Vol 190 (10) ◽  
pp. 1427-1438 ◽  
Author(s):  
Connie L. Sommers ◽  
Ronald L. Rabin ◽  
Alexander Grinberg ◽  
Henry C. Tsay ◽  
Joshua Farber ◽  
...  
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Positive Selection ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Calcium Signal ◽  
Protein Tyrosine

Summary Recent data indicate that several members of the Tec family of protein tyrosine kinases function in antigen receptor signal transduction. Txk, a Tec family protein tyrosine kinase, is expressed in both immature and mature T cells and in mast cells. By overexpressing Txk in T cells throughout development, we found that Txk specifically augments the phospholipase C (PLC)-γ1–mediated calcium signal transduction pathway upon T cell antigen receptor (TCR) engagement. Although Txk is structurally different from inducible T cell kinase (Itk), another Tec family member expressed in T cells, expression of the Txk transgene could partially rescue defects in positive selection and signaling in itk−/− mice. Conversely, in the itk+/+ (wild-type) background, overexpression of Txk inhibited positive selection of TCR transgenic thymocytes, presumably due to induction of cell death. These results identify a role for Txk in TCR signal transduction, T cell development, and selection and suggest that the Tec family kinases Itk and Txk perform analogous functions.

Distinct Roles Of PD-1 Itsm and ITIM In Regulating Interactions With SHP-2, ZAP-70 and Lck, and PD-1-Mediated Inhibitory Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 191-191 ◽  
Author(s):  
Pranam Chatterjee ◽  
Nikolaos Patsoukis ◽  
Gordon J. Freeman ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Fusion Proteins ◽  
Cell Activation ◽  
Cytoplasmic Tail ◽  
Inhibitory Function ◽  
Gst Fusion Proteins

Abstract Programmed death (PD)-1 plays a prominent role in the induction and maintenance of peripheral tolerance. The biochemical mechanisms via which PD-1 mediates its inhibitory function remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with PD-1 ITSM and is involved in PD-1-mediated inhibitory function. We sought to identify the nature of PD-1: SHP-2 interaction and to determine whether other TCR-proximal signaling molecules might interact with PD-1 cytoplasmic tail. SHP-2 has two SH2 domains (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1 we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull down assays with each GST-fusion protein using lysates from naive and activated primary human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after T cell activation along with simultaneous PD-1 ligation. This interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation in order to undergo interaction with SHP-2. To identify the mechanism of PD-1 tyrosine phosphorylation governing PD-1: SHP-2 interaction, we used COS cells to express PD-1 along with either empty vector, the TCR proximal tyrosine kinase Fyn, or a kinase inactive mutant of Fyn, followed by pull down with each SHP-2-GST fusion protein. No interaction between PD-1 and SHP-2-GST fusion proteins was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent tyrosine phosphorylation and was able to interact with GST fusion proteins of SHP-2-FL, SHP-2-N-SH2, SHP-2-C-SH2 and SHP-2-ΔN-SH2 but not SHP-2-PTP, providing evidence that PD-1: SHP-2 interaction requires tyrosine phosphorylation of PD-1 by Src family kinases for subsequent SH2-mediated recruitment of SHP-2. To determine the structural and functional role of each individual tyrosine in the ITIM and the ITSM of PD-1 cytoplasmic tail in PD-1: SHP-2 interaction in vivo, we used Jurkat T cells to express cDNA of either PD-1 wild type, PD-1 with the ITIM tyrosine mutated to phenylalanine (PD-1.Y223F), PD-1 with the ITSM tyrosine mutated to phenylalanine (PD-1.Y248F) or PD-1 with both ITIM and ITSM tyrosines mutated to phenylalanine (PD-1.Y223F/Y248F). After activation, PD-1 wild type underwent tyrosine phosphorylation and developed a robust interaction with SHP-2. PD-1.Y223F retained the ability to undergo interaction with SHP-2 after activation, whereas PD-1.Y248F and PD-1.Y223F/Y248F were unable to interact with SHP-2. We examined whether the PD-1 cytopasmic phosphotyrosines might interact with other SH2 domain containing proteins with critical role in T cell activation. We determined that after T cell activation, PD-1 displayed interaction with ZAP-70 and with activated Lck as determined by PD-1 immunoprecipitation followed by immunoblot with antibodies specific for ZAP-70 and for the activation-specific phospho-LckY394. These interactions remained unaffected in T cells expressing PD-1.Y223F but were abrogated in T cells expressing PD-1.Y248F or PD-1.Y223F/Y248F indicating a mandatory role of phosphorylated ITSM but not ITIM for these associations. However, despite their distinct ability to mediate interactions of PD-1 with SHP-2, Lck and ZAP-70, both phosphorylated ITSM and ITIM had a mandatory role in the inhibitory effect of PD-1 on T cell activation. In T cells expressing either PD-1.Y223F or PD-1.Y248F, PD-1-mediated inhibition of IL-2 production was diminished by 50%, but was almost abrogated in T cells expressing the double mutant PD-1.Y223F/Y248F. Our results indicate that the cytoplasmic tail of PD-1 requires tyrosine phosphorylation in order to mediate phosphorylation-dependent interactions and inhibition on T cell activation. Although phosphorylation-dependent interactions of PD-1 with SHP-2, ZAP-70 and Lck involve Y248 in the ITSM, yet unidentified interactions of Y223 in the ITIM are mandatory for PD-1-mediated inhibitory function on T cell activation. Disclosures: Freeman: Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals PAG-Associated FynT Regulates Calcium Signaling and Promotes Anergy in T Lymphocytes

2007 ◽  
Vol 27 (5) ◽  
pp. 1960-1973 ◽  
Author(s):  
Dominique Davidson ◽  
Burkhart Schraven ◽  
André Veillette
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Anergic T Cells ◽  
Selective Enhancement

ABSTRACT Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3ζ expression

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3851-3859 ◽  
Author(s):  
Sandeep Krishnan ◽  
Vishal G. Warke ◽  
Madhusoodana P. Nambiar ◽  
Henry K. Wong ◽  
George C. Tsokos ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Effector T Cells ◽  
T Cell Differentiation ◽  
Effector T Cell

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon γ (IFN-γ) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3ζ and CD3ε signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3ζ has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3ζ down-regulation were examined. The loss of CD3ζ expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3ζ expression may be a feature of chronic T-cell activation and effector generation in vivo.

scholarly journals How the Discovery of the CD4/CD8-p56lck Complexes Changed Immunology and Immunotherapy

2021 ◽  
Vol 9 ◽  
Author(s):  
Christopher E. Rudd
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Control Cell ◽  
Immune Recognition ◽  
Protein Tyrosine ◽  
Cell Immunity ◽  
Phosphorylation Cascade

The past 25 years have seen enormous progress in uncovering the receptors and signaling mechanisms on T-cells that activate their various effecter functions. Until the late 1980s, most studies on T-cells had focused on the influx of calcium and the levels of cAMP/GMP in T-cells. My laboratory then uncovered the interaction of CD4 and CD8 co-receptors with the protein-tyrosine kinase p56lck which are now widely accepted as the initiators of the tyrosine phosphorylation cascade leading to T-cell activation. The finding explained how immune recognition receptors expressed by many immune cells, which lack intrinsic catalytic activity, can transduce activation signals via non-covalent association with non-receptor tyrosine kinases. The discovery also established the concept that a protein tyrosine phosphorylation cascade operated in T-cells. In this vein, we and others then showed that the CD4- and CD8-p56lck complexes phosphorylate the TCR complexes which led to the identification of other protein-tyrosine kinases such as ZAP-70 and an array of substrates that are now central to studies in T-cell immunity. Other receptors such as B-cell receptor, Fc receptors and others were also subsequently found to use src kinases to control cell growth. In T-cells, p56lck driven phosphorylation targets include co-receptors such as CD28 and CTLA-4 and immune cell-specific adaptor proteins such as LAT and SLP-76 which act to integrate signals proximal to surface receptors. CD4/CD8-p56lck regulated events in T-cells include intracellular calcium mobilization, integrin activation and the induction of transcription factors for gene expression. Lastly, the identification of the targets of p56lck in the TCR and CD28 provided the framework for the development of chimeric antigen receptor (CAR) therapy in the treatment of cancer. In this review, I outline a history of the development of events that led to the development of the “TCR signaling paradigm” and its implications to immunology and immunotherapy.

scholarly journals Positive and negative regulation of T-cell activation through kinases and phosphatases

2003 ◽  
Vol 371 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Tomas MUSTELIN ◽  
Kjetil TASKÉN
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Protein Tyrosine Phosphatases ◽  
Negative Regulation ◽  
Intact Cells ◽  
Protein Tyrosine ◽  
Sequence Of Events

The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components.

scholarly journals Efficient CD28 signalling leads to increases in the kinase activities of the TEC family tyrosine kinase EMT/ITK/TSK and the SRC family tyrosine kinase LCK

1998 ◽  
Vol 330 (3) ◽  
pp. 1123-1128 ◽  
Author(s):  
Spencer GIBSON ◽  
Ken TRUITT ◽  
Yiling LU ◽  
Ruth LAPUSHIN ◽  
Humera KHAN ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

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