GRB2-Mediated Recruitment of THEMIS to LAT Is Essential for Thymocyte Development

Paster, Wolfgang; Brockmeyer, Claudia; Fu, Guo; Simister, Philip C.; Wet, Ben de; Martínez-Riaño, Ana; Hoerter, John A. H.; Feller, Stephan M.; Wülfing, Christoph; Gascoigne, Nicholas R. J.; Acuto, Oreste

doi:10.4049/jimmunol.1203389

Cited by 70 publications

(110 citation statements)

References 30 publications

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“…A group of proteins that selectively, but constitutively, interacted with GRB2 in T cells was reliably identified with high confidence; in contrast, the association between GRB2 and CD28 required CD28 stimulation, consistent with an SH2-pTyr interaction. Among the constitutively GRB2-interacting proteins were CBL, SOS1, and THEMIS (23). Suppressor of T-cell receptor signaling 1 (STS1; gene name UBASH3B), a negative regulator of T-cell activation, is a potential atypical tyrosine phosphatase that contains an SH3 domain that recognizes a proline-rich motif and a UBA domain that binds ubiquitin (24,25).…”

Section: Resultsmentioning

confidence: 99%

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

Tian

Wang²,

Gish

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.uring some forms of intercellular communication, soluble growth factors are secreted by one cell type and bind specific transmembrane receptor tyrosine kinases (RTKs) present on neighboring cells. Activated RTKs then undergo autophosphorylation and also phosphorylate associated scaffold proteins, thereby creating docking sites for effector proteins with phosphotyrosine (pTyr)-recognition modules such as Src homology 2 (SH2) and pTyr-binding (PTB) domains (1). These immediate targets of growth-factor receptors control a variety of intracellular responses, typified by the activation of serine/ threonine protein kinases such as those involved in the ERK MAP kinase pathway. Growth-factor signaling therefore leads to extensive changes in both tyrosine and serine/threonine phosphorylation (2).The alterations in protein-protein interactions and posttranslational modifications elicited by transmembrane receptors can in principle be characterized by mass spectrometry (MS)-based proteomics (3-6). However, under circumstances that involve complex cell-cell interactions, identifying physiologically relevant signals using MS-based proteomics can be challenging. For example, obtaining a sufficiently robust response for MS analysis has often required the use of cells that overexpress a receptor of interest and are subjected to a nonphysiological stimulus. For this reason, experiments have often focused on specific, often stereotypic, aspects of the cellular response, rather than taking a more global approach. Moreover, cell-cell interactions are often typified by the interaction of multiple transmembrane receptor-ligand pairs, frequently involving membrane proteins that are not receptor kinases. Short-peptide motifs from such receptors that serve as potential ligands for downstream targets can be used as affinity probes for binding partners, but these experiments suffer from high fals...

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Section: Resultsmentioning

confidence: 99%

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

Tian

Wang²,

Gish

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The ablation of Themis1 in C57BL/6 mice results in impaired T cell development, particularly of the CD4 + lineage (17)(18)(19)(20)(21). After TCR engagement, Themis1 is phosphorylated by Lck and Zap-70, and recruited to the transmembrane adaptor protein LAT (22,23). Themis1 has been shown to interact with Vav1 and positively regulates its phosphorylation (23).…”

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confidence: 99%

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development

Pedros

Gaud

Bernard

et al. 2015

The Journal of Immunology

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The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown–Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development.

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“…Alternatively, but not mutually exclusively, the role of Grb2 in T cell selection might be explained by its ability to recruit a THEMISGrb2-SHP1/2 complex to LAT in the immunological synapse, where THEMIS and SHP1/2 set the threshold for positive selection (6,7). In the absence of THEMIS or if the THEMIS binding site for Grb2 is mutated, positive selection is drastically impaired (5,7,33,34). The importance of the C-terminal SH3 domain of Grb2 that recruits THEMIS (6) is also highlighted by studies in which mutated forms of Grb2 were transduced into bone marrow cells from Grb2 fl/fl Lckcre tg mice via retroviral transduction.…”

Section: Discussionmentioning

confidence: 99%

“…As a consequence of impaired selection processes, Grb2 fl/fl Lckcre tg mice show reduced T cell numbers in the periphery (2). It was reported recently that Grb2 recruits THEMIS and SHP1 via its SH3 domains to phosphorylated LAT after TCR stimulation (5,6). Thereby, it directs them to the immunological synapse where, in turn, the complex with THEMIS and SHP1 sets the threshold for selection processes (6,7).…”

mentioning

confidence: 99%

“…Thereby, it directs them to the immunological synapse where, in turn, the complex with THEMIS and SHP1 sets the threshold for selection processes (6,7). When the Grb2-dependent recruitment of THEMIS is abrogated, T cell development is impaired; this highlights the importance of Grb2 in thymocyte development (5). However, this model is not mutually exclusive because a complex consisting of two Grb2 molecules and one Sos1 molecule (2:1 complex) is able to cluster LAT proteins, which are phosphorylated after TCR engagement.…”

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confidence: 99%

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Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

Radtke

Lacher

Szumilas

et al. 2016

The Journal of Immunology

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The small adaptor protein growth factor receptor–bound protein 2 (Grb2) modulates and integrates signals from receptors on cellular surfaces in inner signaling pathways. In murine T cells, Grb2 is crucial for amplification of TCR signaling. T cell–specific Grb2fl/fl Lckcretg Grb2-deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study, we found that T cell numbers in Grb2fl/fl CD4cretg mice were normal in the thymus and were only slightly affected in the periphery. Ex vivo analysis of CD4+ Th cell populations revealed an increased amount of Th1 cells within the CD4+ population of Grb2fl/fl CD4cretg mice. Additionally, Grb2-deficient T cells showed a greater potential to differentiate into Th17 cells in vitro. To test whether these changes in Th cell differentiation potential rendered Grb2fl/fl CD4cretg mice more prone to inflammatory diseases, we used the murine Th1 cell– and Th17 cell–driven model of experimental autoimmune encephalomyelitis (EAE). In contrast to our expectations, Grb2fl/fl CD4cretg mice developed a milder form of EAE. The impaired EAE disease can be explained by the reduced proliferation rate of Grb2-deficient CD4+ T cells upon stimulation with IL-2 or upon activation by allogeneic dendritic cells, because the activation of T cells by dendritic cells and the subsequent T cell proliferation are known to be crucial factors for the induction of EAE. In summary, Grb2-deficient T cells show defects in T cell development, increased Th1 and Th17 cell differentiation capacities, and impaired proliferation after activation by dendritic cells, which likely reduce the clinical symptoms of EAE.

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GRB2-Mediated Recruitment of THEMIS to LAT Is Essential for Thymocyte Development

Cited by 70 publications

References 30 publications

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

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