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

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5249 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5249-5258 ◽

Cited By ~ 77

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

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

Biochemical Journal ◽

10.1042/bj20021637 ◽

2003 ◽

Vol 371 (1) ◽

pp. 15-27 ◽

Cited By ~ 188

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.

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Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1937 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1937-1943 ◽

Cited By ~ 146

Author(s):

D G Motto ◽

S E Ross ◽

J Wu ◽

L R Hendricks-Taylor ◽

G A Koretzky

Keyword(s):

T Cell ◽

Tyrosine Phosphorylation ◽

T Cell Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Sh2 Domain ◽

Activated T Cells ◽

Amino Terminal

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.

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T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2107 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2107-2113 ◽

Cited By ~ 46

Author(s):

A J da Silva ◽

O Janssen ◽

C E Rudd

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Intracellular Signaling ◽

Cell Activation ◽

Cell Receptor ◽

Sh2 Domain ◽

T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

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CD18 Antibody Application Blocks Unwanted Off-Target T Cell Activation Caused by Bispecific Antibodies

Cancers ◽

10.3390/cancers13184596 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4596

Author(s):

Joseph Kauer ◽

Fabian Vogt ◽

Ilona Hagelstein ◽

Sebastian Hörner ◽

Melanie Märklin ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Lymphoid Cells ◽

Bispecific Antibodies ◽

Target Cells ◽

Cytokine Release ◽

Life Threatening ◽

Blocking Antibodies

T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade—in contrast to CD2—did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

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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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Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

Cell Communication and Signaling ◽

10.1186/s12964-020-00673-z ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Matthias Kästle ◽

Camilla Merten ◽

Roland Hartig ◽

Thilo Kaehne ◽

Ardiyanto Liaunardy-Jopeace ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tyrosine Kinase ◽

Enzymatic Activity ◽

T Cell Activation ◽

Protein Tyrosine Kinase ◽

Cell Activation ◽

Sh2 Domain ◽

Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

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A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

Journal of Experimental Medicine ◽

10.1084/jem.20062066 ◽

2007 ◽

Vol 204 (3) ◽

pp. 681-691 ◽

Cited By ~ 53

Author(s):

Vincenzo Di Bartolo ◽

Benjamin Montagne ◽

Mogjiborahman Salek ◽

Britta Jungwirth ◽

Florent Carrette ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Cell Receptor ◽

Sh2 Domain ◽

Serine Phosphorylation ◽

Binding Partners ◽

Protein Recruitment

The SH2 domain–containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3ε and ζ proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-γ1. Moreover, an SLP-76–S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1–dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

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IL-6 regulates CCR5 expression and immunosuppressive capacity of MDSC in murine melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000949 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000949 ◽

Cited By ~ 4

Author(s):

Rebekka Weber ◽

Zeno Riester ◽

Laura Hüser ◽

Carsten Sticht ◽

Alina Siebenmorgen ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Ccr5 Expression ◽

Myeloid Suppressor Cell ◽

Cell Functions ◽

Arginase 1

BackgroundMyeloid-derived suppressor cells (MDSC) play a major role in the immunosuppressive melanoma microenvironment. They are generated under chronic inflammatory conditions characterized by the constant production of inflammatory cytokines, chemokines and growth factors, including IL-6. Recruitment of MDSC to the tumor is mediated by the interaction between chemokines and chemokine receptors, in particular C–C chemokine receptor (CCR)5. Here, we studied the mechanisms of CCR5 upregulation and increased immunosuppressive function of CCR5+ MDSC.MethodsThe immortalized myeloid suppressor cell line MSC-2, primary immature myeloid cells and in vitro differentiated MDSC were used to determine factors and molecular mechanisms regulating CCR5 expression and immunosuppressive markers at the mRNA and protein levels. The relevance of the identified pathways was validated on the RET transgenic mouse melanoma model, which was also used to target the identified pathways in vivo.ResultsIL-6 upregulated the expression of CCR5 and arginase 1 in MDSC by a STAT3-dependent mechanism. MDSC differentiated in the presence of IL-6 strongly inhibited CD8+ T cell functions compared with MDSC differentiated without IL-6. A correlation between IL-6 levels, phosphorylated STAT3 and CCR5 expression in tumor-infiltrating MDSC was demonstrated in the RET transgenic melanoma mouse model. Surprisingly, IL-6 overexpressing tumors grew significantly slower in mice accompanied by CD8+ T cell activation. Moreover, transgenic melanoma-bearing mice treated with IL-6 blocking antibodies showed significantly accelerated tumor development.ConclusionOur in vitro and ex vivo findings demonstrated that IL-6 induced CCR5 expression and a strong immunosuppressive activity of MDSC, highlighting this cytokine as a promising target for melanoma immunotherapy. However, IL-6 blocking therapy did not prove to be effective in RET transgenic melanoma-bearing mice but rather aggravated tumor progression. Further studies are needed to identify particular combination therapies, cancer entities or patient subsets to benefit from the anti-IL-6 treatment.

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