Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Area of Science:

• Immunology
• Molecular Biology
• Cell Signaling

Background:

• Posttranslational modifications, including phosphorylation and ubiquitylation, are critical for regulating protein function.
• T-cell receptor (TCR) signaling relies on proximal events modulated by ubiquitylation.
• Deubiquitylating enzymes (DUBs) play a role in reversing ubiquitylation, but their specific involvement in TCR signaling is not fully understood.

Purpose of the Study:

• To systematically identify deubiquitylating enzymes (DUBs) involved in T-cell receptor (TCR) signaling in primary mouse T lymphocytes.
• To elucidate the function of newly identified DUBs in the TCR signaling pathway.
• To understand the mechanism by which DUBs regulate TCR complex stability and signaling duration.

Main Methods:

• Utilized activity-based probes (HA-Ub-VME) to capture DUBs recruited to the cytosol upon TCR activation in primary T lymphocytes.
• Employed genetic knockout (Usp12-/- Jurkat cells) and reconstitution experiments to assess DUB function.
• Applied proximity-based labeling (BirA-Usp12) to identify interacting TCR signaling proteins.
• Performed Western blotting and flow cytometry to analyze protein expression, phosphorylation, and surface receptor levels.

Main Results:

• Identified Usp12 and Usp46 as novel DUBs participating in TCR signaling.
• Observed translocation of Usp12 from the nucleus to the cytosol upon TCR stimulation, accompanied by its phosphorylation.
• Usp12 deficiency led to impaired NFκB, NFAT, and MAPK signaling due to reduced surface TCR expression.
• Usp12 was found to deubiquitylate and prevent lysosomal degradation of TCR adaptors LAT and Trat1, maintaining proximal TCR complex integrity.

Conclusions:

• Usp12 is essential for sustained TCR signaling by stabilizing key adaptor proteins LAT and Trat1.
• The identification of Usp12 and Usp46 expands our understanding of ubiquitin's role in refining TCR signaling cascades.
• Activity-based probes offer a powerful tool for unbiased DUB discovery in primary cells without prior genetic modification.