Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

Borroto, Aldo; Arellano, Irene; Dopfer, Elaine P.; Prouza, Marek; Suchánek, Miloslav; Fuentes, Manuel; Órfão, Alberto; Schamel, Wolfgang W. A.; Alarcón, Balbino

doi:10.4049/jimmunol.1202055

Cited by 33 publications

(45 citation statements)

References 30 publications

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“…In the active TCR conformation, the PRS in the cytoplasmic tail of CD3ε is exposed, and only in this conformation can the SH3.1 domain of Nck [SH3.1(Nck)] bind to the CD3ε PRS, as shown in a pull-down assay using a recombinant GST-SH3.1(Nck) fusion protein (13,16,(18)(19)(20)(21)(22). Using biochemical approaches, it has been proposed that the recruitment of Nck to the TCR occurs earlier and independently of tyrosine phosphorylation (13).…”

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confidence: 99%

Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning

Paensuwan

Hartl

Yousefi

et al. 2016

The Journal of Immunology

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Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.

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confidence: 99%

Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning

Paensuwan

Hartl

Yousefi

et al. 2016

The Journal of Immunology

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“…Knock-in mice in which the central prolines of the Nck-binding motif of CD3ε were substituted by alanines displayed defects in thymic development. Functionally, the mutation of the CD3ε proline-rich sequence resulted in a decreased activationdependent recruitment of Nck and impaired CD3ζ phosphorylation and ZAP70 activation [41]. Recent data suggests an important role of the Nck/CD3ε interaction also in mature T cells, since abrogation of Nck recruitment to CD3ε impaired T cell responses to antigen both in vitro and in vivo [42].…”

Section: Discussionmentioning

confidence: 99%

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

2014

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Noncatalytic region of tyrosine kinase (Nck) is an adapter protein that comprises one SH2 (Src homology) domain and three SH3 domains. Nck links receptors and receptorassociated tyrosine kinases or adapter proteins to proteins that regulate the actin cytoskeleton. Whereas the SH2 domain binds to phosphorylated receptors or associated phosphoproteins, individual interactions of the SH3 domains with proline-based recognition motifs result in the formation of larger protein complexes. In T cells, changes in cell polarity and morphology during T-cell activation and effector function require the T-cell receptor-mediated recruitment and activation of actin-regulatory proteins to initiate cytoskeletal reorganization at the immunological synapse. We previously identified the adapter protein HS1 as a putative Nck-interacting protein. We now demonstrate that the SH2 domain of Nck specifically interacts with HS1 upon phosphorylation of its tyrosine residue 378. We report that in human T cells, ligation of the chemokine receptor CXCR4 by stromal cell-derived factor 1α (SDF1α) induces a rapid and transient phosphorylation of tyrosine 378 of HS1 resulting in an increased association with Nck. Consequently, siRNA-mediated downregulation of HS1 and/or Nck impairs SDF1α-induced actin polymerization and T-cell migration.Keywords: CXCR4 r HS1 r Nck r SDF1α r T cells IntroductionT cells govern adaptive immunity by cytokine secretion or cytotoxic effector function. After development in the thymus, naive T lymphocytes constantly recirculate between the blood stream and lymphoid tissues. At the molecular level, this homing and migration processes rely on complex interactions of selected chemokines with their receptors on respective T cells and of T-cell adhesion molecules binding to their ligands on endothelial cells or the extracellular matrix. Individual interactions elicit specific signaling pathways that induce dynamic cytoskeletal rearrangements, which enable lymphocytes to adhere to or pass a certain substrate or to transmigrate through a given endothelium. Morphologically, circular floating T cells adopt a migratory phenotype characterized Correspondence: Dr. Marcus Lettau e-mail: lettau@immunologie.uni-kiel.de by a leading edge at the front and an uropod at the rear. When T cells encounter their antigen on an antigen-presenting cell in the lymph nodes or spleen, migration is halted and the cells rapidly polarize toward the intercellular contact area. The subsequent formation of the so-called immunological synapse (IS) is again accompanied by complex cytoskeletal changes and comprises the structural basis for T-cell activation. This primary antigenic stimulation ultimately results in cell-cycle progression, clonal expansion, and differentiation to T-effector cells. The activated T cells regain migratory potential, leave the lymphoid tissue, and search the periphery for infected or transformed cells displaying their cognate antigen. Upon antigen encounter, the T cells adhere to their target cell and polarize toward the intercell...

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“…To fully prevent the interaction with Nck, whereas minimizing possible interferences with other proteins, we generated a second KI mouse bearing a germline substitution of the two central prolines in the PxxPxxDY motif with alanine. T cell development in these mutant KI (KI-PRS) mice is partly arrested at each step in which pre-TCR or TCR signaling are required (22). Despite this partial defect during development, peripheral lymphoid organs of KI-PRS mice, either non-Tg or Tg for two different TCRs, were normally populated with mature T cells expressing normal levels of TCR and the CD4 and CD8 coreceptors (Supplemental Fig.…”

Section: Inhibition Of T Cell Activation In Mice Bearing a Germline Mmentioning

confidence: 99%

“…Furthermore, in KI mice bearing an 8-aa replacement of the PRS, Nck recruitment to the PRS was shown to regulate TCR levels at the plasma membrane in preselection double-positive CD4 + CD8 + thymocytes, but not at later stages, promoting the degradation of the TCR (15,21). However, a second KI line bearing a more conservative mutation in the PRS did not reveal such TCR degradation defect but rather a partial arrest of thy-mocyte differentiation at a double-positive stage with high TCR expression (22).…”

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Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

Borroto

Arellano

Blanco

et al. 2014

The Journal of Immunology

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On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck–CD3ε interaction may represent a target for pharmacological modulation of the immune response.

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Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

Cited by 33 publications

References 30 publications

Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning

Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

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