Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination

Lo, Wan‐Lin; Shah, Neel H.; Rubin, Sara A.; Zhang, Weiguo; Horková, Veronika; Fallahee, Ian; Štěpánek, Ondřej; Zon, Leonard I.; Kuriyan, John; Weiss, Arthur

doi:10.1038/s41590-019-0502-2

Cited by 78 publications

(137 citation statements)

References 61 publications

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“…Again, mutation of glycine 131 to aspartate induced increased kinetics and intensity of Erk phosphorylation, with statistical significance at the 3 min time point ( Figure 1C, lower diagram). Altogether, these data confirm that the LAT G131D mutation releases the brake imposed on the TCR/CD3 signaling cassette, as previously observed by Lo et al (2019).…”

Section: Mutation Of Gly 131 To Asp Of Lat Increases Intracellular Sisupporting

confidence: 89%

“…To study the role of the conserved glycine residue preceding tyrosine 132 in LAT we generated lentiviral plasmids to express wild-type LAT or a LAT G131D mutant in J.CaM2 cells, as previously described (Arbulo-Echevarria et al, 2016. This would allow us to verify the effects observed by Lo et al (2019). in Jurkat cells in which CRISPR was performed to eliminate the Lat gene in both chromosomes, and also address other questions of interest.…”

Section: Generation Of Lentiviral Transfectants Of J Cam2 Cells Exprmentioning

confidence: 99%

“…This is bewildering, given the essential role of tyrosine 132 phosphorylation for complete T cells activation (Zhang et al, 2000;Lin and Weiss, 2001;Paz et al, 2001;Aguado et al, 2002;Sommers et al, 2002). Recent work has shown that glycine 131 mutation by an aspartate residue in LAT increases LAT-Y132 phosphorylation, but not the one of tyrosines 171, 191 or 226 (Lo et al, 2019). Moreover, more distal signaling events are also increased in cells expressing the LAT G131D mutant, for example, PLC-γ phosphorylation, Ca 2+ influx, Erk phosphorylation or CD69 expression.…”

Section: Introductionmentioning

confidence: 99%

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Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy

Arbulo-Echevarria

Vico-Barranco

Narbona-Sánchez

et al. 2020

Front. Cell Dev. Biol.

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The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-γ activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LAT G131D) increases phosphorylation of Tyr132, PLC-γ activation and calcium influx generation. Interestingly, the LAT G131D mutant has a slower protein turnover while being equally sensitive to Fasmediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LAT G131D secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LAT G131D mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance.

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Section: Mutation Of Gly 131 To Asp Of Lat Increases Intracellular Sisupporting

confidence: 89%

Section: Generation Of Lentiviral Transfectants Of J Cam2 Cells Exprmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy

Arbulo-Echevarria

Vico-Barranco

Narbona-Sánchez

et al. 2020

Front. Cell Dev. Biol.

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“…In addition to acting between two proteins, regulated unbinding is likely to operate on stable phase-separated signalling assemblies formed by weakly binding multi-domain proteins (55,56). Interestingly, activated ZAP70 catalyses the formation of LAT signalling assemblies (57) and recently, it has been suggested that LAT may be a proofreading step (58,59). Consistent with this, in vitro generated LAT assemblies displayed long half-lives without phosphatases but were disassembled within minutes in their presence (57).…”

Section: Discussionmentioning

confidence: 85%

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Goyette

Depoil

Yang

et al. 2020

Preprint

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Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to the extracellular TCR/antigen half-life and we show that this is the case in both cell lines and primary T cells. The work highlights that binding by kinetic avidity breaks the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discriminated mediated by kinetic proofreading. 1

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“…ZAP-70-targeted sites are characterized by multiple negative residues surrounding the phosphorylated tyrosine; indeed, ZAP-70 is deterred from phosphorylating substrate motifs containing a positive charge anywhere within the surrounding motif [65]. The presence of glycine at the Y-1 position also slows substrate phosphorylation by ZAP-70 [55]. Gads Y45 is preceded by glycine at the Y-1 position, followed by a lysine at the Y+3 position, and has only one negatively charged residue in the surrounding motif, strongly suggesting that it is not a substrate of ZAP-70.…”

Section: P9mentioning

confidence: 99%

Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads

Hallumi

Shalah

Lo³

et al. 2020

Preprint

Self Cite

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The costimulatory receptor, CD28, synergizes with the T cell antigen receptor (TCR) to promote IL-2 production, cell survival and proliferation. Despite their profound synergy, the obligatory interdependence of the signaling pathways initiated by these two receptors is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 at Y173 and PLC-γ1 at Y783. Here we identified Gads Y45 as an additional TCR-inducible, Itk-mediated phosphorylation site. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known binding partners or signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the preferentially-paired binding of Gads to phospho-LAT. Three Itk-related features, Gads Y45, SLP-76 Y173, and a proline-rich Itk SH3-binding motif on SLP-76, were selectively required for activation of the CD28 RE/AP transcriptional element from the IL-2 promoter, but were not required to activate NFAT. This study illuminates a new regulatory module, in which Itk-targeted phosphorylation sites on two adaptor proteins, SLP-76 and Gads, control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

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Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination

Cited by 78 publications

References 61 publications

Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy

Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads

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