Lck protein tyrosine kinase is a key regulator of T-cell activation and a target for signal intervention by Herpesvirus saimiri and other viral gene products

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.

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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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ZAP-70 Protein Tyrosine Kinase Is Constitutively Targeted to the T Cell Cortex Independently of its SH2 Domains

The Journal of Cell Biology ◽

10.1083/jcb.137.7.1639 ◽

1997 ◽

Vol 137 (7) ◽

pp. 1639-1649 ◽

Cited By ~ 18

Author(s):

Russell D.J. Huby ◽

Makio Iwashima ◽

Arthur Weiss ◽

Steven C. Ley

Keyword(s):

T Cell ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Domain ◽

Cell Cortex ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Anchoring Protein ◽

High Concentrations ◽

Lck Protein

ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH2-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639–649), ZAP-70 may be localized close to its downstream targets.

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Inhibition of T Cell Receptor Signal Transduction by Tyrosine Kinase–interacting Protein of Herpesvirus saimiri

Journal of Experimental Medicine ◽

10.1084/jem.20040924 ◽

2004 ◽

Vol 200 (5) ◽

pp. 681-687 ◽

Cited By ~ 33

Author(s):

Nam-Hyuk Cho ◽

Pinghui Feng ◽

Sun-Hwa Lee ◽

Bok-Soo Lee ◽

Xiaozhen Liang ◽

...

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

Tyrosine Kinase ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Cell Receptor ◽

Herpesvirus Saimiri ◽

Interacting Protein

T cells play a central role in orchestrating immunity against pathogens, particularly viruses. Thus, impairing T cell activation is an important strategy employed by viruses to escape host immune control. The tyrosine kinase–interacting protein (Tip) of the T lymphotropic Herpesvirus saimiri (HVS) is constitutively present in lipid rafts and interacts with cellular Lck tyrosine kinase and p80 endosomal protein. Here we demonstrate that, due to the sequestration of Lck by HVS Tip, T cell receptor (TCR) stimulation fails to activate ZAP70 tyrosine kinase and to initiate downstream signaling events. TCR ζ chains in Tip-expressing T cells were initially phosphorylated to recruit ZAP70 molecule upon TCR stimulation, but the recruited ZAP70 kinase was not subsequently phosphorylated, resulting in TCR complexes that were stably associated with inactive ZAP70 kinase. Consequently, Tip expression not only markedly inhibited TCR-mediated intracellular signal transduction but also blocked TCR engagement with major histocompatibility complexes on the antigen-presenting cells and immunological synapse formation. These results demonstrate that a lymphotropic herpesvirus has evolved a novel mechanism to deregulate T cell activation to disarm host immune surveillance. This process contributes to the establishment and maintenance of viral latency.

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The role of membrane rafts in Lck transport, regulation and signalling in T-cells

Biochemical Journal ◽

10.1042/bj20130468 ◽

2013 ◽

Vol 454 (2) ◽

pp. 169-179 ◽

Cited By ~ 19

Author(s):

Leandro N. Ventimiglia ◽

Miguel A. Alonso

Keyword(s):

T Cell ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Cell Activation ◽

Protein Tyrosine Kinase ◽

Cell Activation ◽

Protein Transport ◽

Cell Signalling ◽

T Cell Response ◽

Membrane Rafts

Tyrosine phosphorylation is one of the key covalent modifications that occur in multicellular organisms. Since its discovery more than 30 years ago, tyrosine phosphorylation has come to be understood as a fundamentally important mechanism of signal transduction and regulation in all eukaryotic cells. The tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) plays a crucial role in the T-cell response by transducing early activation signals triggered by TCR (T-cell receptor) engagement. These signals result in the phosphorylation of immunoreceptor tyrosine-based activation motifs present within the cytosolic tails of the TCR-associated CD3 subunits that, once phosphorylated, serve as scaffolds for the assembly of a large supramolecular signalling complex responsible for T-cell activation. The existence of membrane nano- or micro-domains or rafts as specialized platforms for protein transport and cell signalling has been proposed. The present review discusses the signals that target Lck to membrane rafts and the importance of these specialized membranes in the transport of Lck to the plasma membrane, the regulation of Lck activity and the phosphorylation of the TCR.

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