The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor ζ chain tyrosine-based activation motif

AbstractEngagement of the T-cell receptor (TCR) results in the activation of Lck/Fyn and ZAP-70/Syk tyrosine kinases. Lck-mediated tyrosine phosphorylation of signaling motifs (ITAMs) in the CD3-ζ subunits of the TCR is an initial step in the transduction of signaling cascades. However, ζ phosphorylation is also promoted by ZAP-70, as TCR-induced ζ phosphorylation is defective in ZAP-70–deficient T cells. We show that this defect is corrected by stable expression of ZAP-70, but not Syk, in primary and transformed T cells. Indeed, these proteins are differentially coupled to the TCR with a 5- to 10-fold higher association of ZAP-70 with ζ as compared to Syk. Low-level Syk-ζ binding is associated with significantly less Lck coupled to the TCR. Moreover, diminished coupling of Lck to ζ correlates with a poor phosphorylation of the positive regulatory tyr352 residue of Syk. Thus, recruitment of Lck into the TCR complex with subsequent ζ chain phosphorylation is promoted by ZAP-70 but not Syk. Importantly, the presence of ZAP-70 positively regulates the TCR-induced tyrosine phosphorylation of Syk. The interplay between Syk and ZAP-70 in thymocytes, certain T cells, and B-chronic lymphocytic leukemia cells, in which they are coexpressed, will therefore modulate the amplitude of antigen-mediated receptor signaling.

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Identification, cloning, and characterization of a novel human T-cell- specific tyrosine kinase located at the hematopoietin complex on chromosome 5q

Blood ◽

10.1182/blood.v82.5.1561.bloodjournal8251561 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1561-1572 ◽

Cited By ~ 3

Author(s):

S Gibson ◽

B Leung ◽

JA Squire ◽

M Hill ◽

N Arima ◽

...

Keyword(s):

Signal Transduction ◽

T Cell ◽

T Lymphocytes ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Cell Receptor ◽

Tyrosine Kinases ◽

Phosphorylation Site ◽

Cell Receptor ◽

Cytokine Receptors

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31–32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31–32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)

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Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes.

Journal of Experimental Medicine ◽

10.1084/jem.183.3.1053 ◽

1996 ◽

Vol 183 (3) ◽

pp. 1053-1062 ◽

Cited By ~ 223

Author(s):

N S van Oers ◽

N Killeen ◽

A Weiss

Keyword(s):

T Cells ◽

T Cell ◽

Tyrosine Phosphorylation ◽

T Cell Receptor ◽

Tyrosine Kinases ◽

Cell Receptor ◽

Protein Tyrosine Kinases ◽

Peripheral T Cells ◽

Tcr Signal Transduction ◽

Deficient Mice

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR-zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.

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The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit.

Journal of Experimental Medicine ◽

10.1084/jem.182.5.1585 ◽

1995 ◽

Vol 182 (5) ◽

pp. 1585-1590 ◽

Cited By ~ 62

Author(s):

N S van Oers ◽

H von Boehmer ◽

A Weiss

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Tyrosine Kinases ◽

Cell Receptor ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Zeta Chain ◽

Proximal Signaling ◽

Zeta Chains

The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR-zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.

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Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation.

Journal of Experimental Medicine ◽

10.1084/jem.184.2.365 ◽

1996 ◽

Vol 184 (2) ◽

pp. 365-376 ◽

Cited By ~ 65

Author(s):

V A Boussiotis ◽

D L Barber ◽

B J Lee ◽

J G Gribben ◽

G J Freeman ◽

...

Keyword(s):

T Cell ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Cell Receptor ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Cell Receptor ◽

Specific Protein ◽

Protein Tyrosine ◽

Potential Applications

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta-associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.

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Identification, cloning, and characterization of a novel human T-cell- specific tyrosine kinase located at the hematopoietin complex on chromosome 5q

Blood ◽

10.1182/blood.v82.5.1561.1561 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1561-1572 ◽

Cited By ~ 51

Author(s):

S Gibson ◽

B Leung ◽

JA Squire ◽

M Hill ◽

N Arima ◽

...

Keyword(s):

Signal Transduction ◽

T Cell ◽

T Lymphocytes ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Cell Receptor ◽

Tyrosine Kinases ◽

Phosphorylation Site ◽

Cell Receptor ◽

Cytokine Receptors

Abstract Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31–32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31–32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)

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