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

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

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Phosphorylation-Dependent Regulation of T-Cell Activation by PAG/Cbp, a Lipid Raft-Associated Transmembrane Adaptor

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.2017-2028.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 2017-2028 ◽

Cited By ~ 130

Author(s):

Dominique Davidson ◽

Marcin Bakinowski ◽

Matthew L. Thomas ◽

Vaclav Horejsi ◽

André Veillette

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Protein Tyrosine Phosphatases ◽

Dominant Negative ◽

Negative Regulator ◽

Cell Fractionation ◽

Protein Tyrosine

ABSTRACT PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.

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Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5249-5258.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5249-5258

Author(s):

C Couture ◽

G Baier ◽

C Oetken ◽

S Williams ◽

D Telford ◽

...

Keyword(s):

T Cell ◽

Tyrosine Phosphorylation ◽

T Cell Activation ◽

Cell Activation ◽

Molecular Mechanisms ◽

Sh2 Domain ◽

Lymphoid Cells ◽

Intact Cells ◽

Protein Tyrosine

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

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PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina) PBMC

Clinical and Developmental Immunology ◽

10.1080/17402520500116624 ◽

2005 ◽

Vol 12 (2) ◽

pp. 91-97 ◽

Cited By ~ 15

Author(s):

Jennifer C. C. Neale ◽

Thomas P. Kenny ◽

Ronald S. Tjeerdema ◽

M. Eric Gershwin

Keyword(s):

T Cell ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Critical Role ◽

Harbor Seal ◽

Lymphocyte Function ◽

Altered Expression ◽

Protein Tyrosine

Mechanisms underlyingin vitroimmunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs)FynandItk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP), 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169), a model immunotoxic PCB, or DMSO (vehicle control). Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKsFynandItkwere both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part) by disruption of T cell receptor (TCR) signaling and cytokine production.

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Recombinant glycosyl-phosphatidylinositol-anchored proteins are not associated with protein kinases in transfected thymoma cells

Biochemical Journal ◽

10.1042/bj3040853 ◽

1994 ◽

Vol 304 (3) ◽

pp. 853-859 ◽

Cited By ~ 4

Author(s):

P M Clissold

Keyword(s):

T Cell ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Signal Sequence ◽

Phosphatidyl Inositol ◽

Protein Tyrosine Kinases ◽

Receptor Protein ◽

Protein Tyrosine ◽

Stable Transfectants

The cross-linking by antibody of some glycosyl-phosphatidyl-inositol (GPI)-anchored proteins on the plasma membrane of T cells leads to cell activation. Phosphorylation of proteins on tyrosine residues has a central role in the control of T cell activation, and non-receptor protein tyrosine kinases can be coprecipitated with immune complexes of GPI-anchored proteins in T cell lysates. In order to investigate the nature of this interaction, two recombinant GPI-anchored proteins were constructed (using the GPI signal sequence from Thy-1), and their associations with protein tyrosine kinases in stable transfectants of a mouse thymoma have been investigated. One recombinant GPI protein is the extracellular domain of the human complement receptor-1, normally an integral membrane protein, and the other is the secreted protein, human tissue inhibitor of metalloproteinases. The latter protein should be foreign to the cell surface and yet has been expressed as a GPI-anchored protein at levels equivalent to the highly expressed antigens Thy-1 and Ly6.A2 on mouse thymoma cells. Neither of the two recombinant proteins, when immunoprecipitated from NP40 lysates of transfected cells, was associated with protein tyrosine kinases in contrast with the natural endogenous GPI-anchored proteins Thy-1 and Ly6.A2 in non-transfected parental cells. Moreover, high expression of foreign recombinant GPI protein appears to interfere with the association of the natural GPI proteins with protein tyrosine kinases.

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.626095 ◽

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.

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Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2855 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2855-2866 ◽

Cited By ~ 20

Author(s):

Jane Wong ◽

David Straus ◽

Andrew C. Chan

Keyword(s):

T Cell ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Interleukin 2 ◽

Protein Tyrosine Kinases ◽

Homologous Region ◽

Activation Markers ◽

Protein Tyrosine ◽

Signaling Events

ABSTRACT T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.

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Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.14.6693 ◽

1994 ◽

Vol 91 (14) ◽

pp. 6693-6697 ◽

Cited By ~ 45

Author(s):

S. Koyasu ◽

A. G. Tse ◽

P. Moingeon ◽

R. E. Hussey ◽

A. Mildonian ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Activation Motif

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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

Biochemical Journal ◽

10.1042/bj3301123 ◽

1998 ◽

Vol 330 (3) ◽

pp. 1123-1128 ◽

Cited By ~ 17

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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