Interaction between the SH2 Domains of ZAP-70 and the Tyrosine-Based Activation Motif 1 Sequence of the ζ Subunit of the T-Cell Receptor

1997 ◽  
Vol 342 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Mark E. Labadia ◽  
Scott Jakes ◽  
Christine A. Grygon ◽  
Daniel J. Greenwood ◽  
Josephine Schembri-King ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Sh2 Domains ◽  
Activation Motif

scholarly journals Dual Fatty Acylation of p59Fyn Is Required for Association with the T Cell Receptor ζ Chain through Phosphotyrosine–Src Homology Domain-2 Interactions

1999 ◽  
Vol 145 (2) ◽  
pp. 377-389 ◽  
Author(s):  
Woutervan't Hof ◽  
Marilyn D. Resh
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Insoluble Fraction ◽  
Sh2 Domain ◽  
Sh2 Domains ◽  
Fatty Acylation ◽  
Src Homology ◽  
Activation Motif

The first 10 residues within the Src homology domain (SH)–4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR ζ chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007–1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR ζ fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with ζ. The Fyn NH2 terminus was necessary but not sufficient for interaction with ζ and both Fyn kinase and SH2 domains were required, directing phosphorylation of ζ ITAM tyrosines and binding to ζ ITAM phosphotyrosines. Fyn/ζ interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with ζ. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR ζ chain ITAMs.

scholarly journals Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

1995 ◽  
Vol 15 (11) ◽  
pp. 5937-5944 ◽  
Author(s):  
J F Cloutier ◽  
L M Chow ◽  
A Veillette
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
T Lymphocytes ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Yeast Cells ◽  
Sh2 Domains ◽  
Tyrosine Protein Kinase ◽  
Carboxy Terminal

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.

scholarly journals Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif.

1994 ◽  
Vol 14 (6) ◽  
pp. 3729-3741 ◽  
Author(s):  
L K Gauen ◽  
Y Zhu ◽  
F Letourneur ◽  
Q Hu ◽  
J B Bolen ◽  
...  
Keyword(s):  
T Cell Receptor ◽  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Kinase Activity ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Single Mutation ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Activation Motif

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.

Phosphorylated T Cell Receptor zeta-chain and ZAP70 Tandem SH2 Domains form a 1:3 Complex in vitro

1996 ◽  
Vol 238 (2) ◽  
pp. 440-445 ◽  
Author(s):  
Winfried Weissenhorn ◽  
Michael J. Eck ◽  
Stephen C. Harrison ◽  
Don C. Wiley
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Sh2 Domains ◽  
Zeta Chain

scholarly journals Characterization of the roles of SH2 domain-containing proteins in T-lymphocyte activation by using dominant negative SH2 domains.

1996 ◽  
Vol 16 (5) ◽  
pp. 2255-2263 ◽  
Author(s):  
J P Northrop ◽  
M J Pustelnik ◽  
A T Lu ◽  
J R Grove
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Signaling Cascade ◽  
Single Point Mutation ◽  
Sh2 Domains

Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.

scholarly journals RAFTK, a Novel Member of the Focal Adhesion Kinase Family, Is Phosphorylated and Associates with Signaling Molecules upon Activation of Mature T Lymphocytes

1997 ◽  
Vol 185 (6) ◽  
pp. 1055-1064 ◽  
Author(s):  
Ramesh K. Ganju ◽  
William C. Hatch ◽  
Hava Avraham ◽  
Mel A. Ona ◽  
Brian Druker ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
T Cell Receptor ◽  
Focal Adhesion ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Sh2 Domains ◽  
Rich Domain ◽  
Kinase Family

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif

1994 ◽  
Vol 14 (6) ◽  
pp. 3729-3741
Author(s):  
L K Gauen ◽  
Y Zhu ◽  
F Letourneur ◽  
Q Hu ◽  
J B Bolen ◽  
...  
Keyword(s):  
T Cell Receptor ◽  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Kinase Activity ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Single Mutation ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Activation Motif

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.

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