scholarly journals An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

2020 ◽  
Vol 477 (7) ◽  
pp. 1287-1308 ◽  
Author(s):  
Kaustav Gangopadhyay ◽  
Bharat Manna ◽  
Swarnendu Roy ◽  
Sunitha Kumari ◽  
Olivia Debnath ◽  
...  
Keyword(s):  
T Cell ◽  
Noncovalent Interactions ◽  
Hot Spot ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Sh2 Domains ◽  
Encounter Complex

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of noncovalent interactions that allosterically couple the two SH2 domains during binding to doubly phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.

scholarly journals An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

2019 ◽  
Author(s):  
Kaustav Gangopadhyay ◽  
Bharat Manna ◽  
Swarnendu Roy ◽  
Sunitha Kumari ◽  
Olivia Debnath ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Signaling ◽  
Structural Transition ◽  
Hot Spot ◽  
Spontaneous Mutation ◽  
Sh2 Domain ◽  
Autoimmune Arthritis ◽  
T Cell Signaling ◽  
Regulatory Module ◽  
Encounter Complex

AbstractT-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.SignificanceT-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand, whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant found in SKG mice.

scholarly journals Loss of tonic T-cell receptor signals alters the generation but not the persistence of CD8+ memory T cells

Blood ◽  
2010 ◽  
Vol 116 (25) ◽  
pp. 5560-5570 ◽  
Author(s):  
Karla R. Wiehagen ◽  
Evann Corbo ◽  
Michelle Schmidt ◽  
Haina Shin ◽  
E. John Wherry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphocytic Choriomeningitis ◽  
Sh2 Domain ◽  
Normal Numbers ◽  
Tcr Signaling ◽  
Tcr Signals

Abstract The requirements for tonic T-cell receptor (TCR) signaling in CD8+ memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain–containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76–dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.

scholarly journals Fyn-Binding Protein (Fyb)/Slp-76–Associated Protein (Slap), Ena/Vasodilator-Stimulated Phosphoprotein (Vasp) Proteins and the Arp2/3 Complex Link T Cell Receptor (Tcr) Signaling to the Actin Cytoskeleton

2000 ◽  
Vol 149 (1) ◽  
pp. 181-194 ◽  
Author(s):  
Matthias Krause ◽  
Antonio S. Sechi ◽  
Marlies Konradt ◽  
David Monner ◽  
Frank B. Gertler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Actin Cytoskeleton ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Antigen Presenting ◽  
Syndrome Protein

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76–associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3–coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

scholarly journals Leukocyte-associated immunoglobulin-like receptor 1 inhibits T-cell signaling by decreasing protein phosphorylation in the T-cell signaling pathway

2020 ◽  
Vol 295 (8) ◽  
pp. 2239-2247 ◽  
Author(s):  
Jeoung-Eun Park ◽  
David D. Brand ◽  
Edward F. Rosloniec ◽  
Ae-Kyung Yi ◽  
John M. Stuart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Signaling ◽  
Signaling Pathway ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Cell Signaling Pathway

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1–sufficient and –deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor–associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal–regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1–induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog–sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

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 Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor SignalingIn Vivo

2016 ◽  
Vol 36 (18) ◽  
pp. 2396-2402 ◽  
Author(s):  
Peter A. Thill ◽  
Arthur Weiss ◽  
Arup K. Chakraborty
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
New Model ◽  
Downstream Signaling ◽  
Conventional Models

The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase Zap70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines,in vivo, Zap70 is bound to phosphorylated ITAMs in resting T cells. However, Zap70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates thesein vivoobservations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed.

scholarly journals Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling

2020 ◽  
Vol 21 (10) ◽  
pp. 3498 ◽  
Author(s):  
Yuanqing Ma ◽  
Yean J. Lim ◽  
Aleš Benda ◽  
Jieqiong Lou ◽  
Jesse Goyette ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spatial Clustering ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
T Cell Receptor Signaling ◽  
Cell Cytosol

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

scholarly journals An allosteric hot spot in the tandem SH2 domain of ZAP‐70 regulates T‐cell signaling

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Kaustav Gangopadhyay ◽  
Swarnendu Roy ◽  
Olivia Debnath ◽  
Sunitha Kumari ◽  
Rahul Das
Keyword(s):  
T Cell ◽  
Cell Signaling ◽  
Hot Spot ◽  
Sh2 Domain ◽  
T Cell Signaling

scholarly journals PLCγ1 promotes phase separation of the T cell signaling clusters

2020 ◽  
Author(s):  
Longhui Zeng ◽  
Ivan Palaia ◽  
Anđela Šarić ◽  
Xiaolei Su
Keyword(s):  
Phase Separation ◽  
T Cell ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Critical Function ◽  
Sh2 Domains ◽  
Patchy Particles ◽  
Tcr Signal Transduction ◽  
Biophysical Principle

SummaryThe T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here we report that phospholipase PLCγ1 promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly crosslinks LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK and SLP76 activation. Intriguingly, a non-monotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.

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