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T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein
A Magnan1, V Di Bartolo, A M Mura
1Centre d'Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Univ.Med., Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France.
Abstract:
After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.
Insights
Mutations in ZAP-70
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Tyrosine phosphorylation of ZAP-70 is critical for T cell receptor (TCR) signaling.
- Specific tyrosine residues (Y292, Y315) in ZAP-70 are implicated as docking sites for Cbl and Vav1, influencing TCR signal modulation.
- The precise roles of Y292 and Y315 in T cell development and function remain to be fully elucidated in vivo.
Purpose of the Study:
- To investigate the in vivo functional significance of ZAP-70 tyrosine residues Y292 and Y315 in T cell development and TCR signaling.
- To determine the impact of specific ZAP-70 mutations on T cell selection, cytokine production, and TCR signaling dynamics.
Main Methods:
- Generation of ZAP-70 mutant mice (Y292F, Y315F) in a sensitized TCR transgenic background.
- Analysis of T cell development, including positive and negative selection.
- Assessment of TCR signaling events, such as proximal signaling, cytokine production (IL-2, IFN-gamma), and TCR downmodulation.
- Evaluation of Cbl and Vav1 phosphorylation status.
Main Results:
- The ZAP-70 Y315F mutation impaired positive selection and delayed negative selection, also affecting CD3-zeta phosphoisoform levels.
- The ZAP-70 Y292F mutation enhanced proximal TCR signaling, leading to increased IL-2 and IFN-gamma production and slower TCR downmodulation.
- Neither Y292F nor Y315F mutations prevented TCR-induced phosphorylation of Cbl and Vav1, respectively, suggesting alternative adaptors.
Conclusions:
- ZAP-70 residue Y292 is crucial for attenuating TCR signaling and regulating TCR duration on the cell surface.
- ZAP-70 residue Y315 enhances ZAP-70 function, impacting T cell selection processes.
- Alternative adaptor molecules mediate Cbl and Vav1 recruitment in ZAP-70 signaling.
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