Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing spontaneous inflammation

Abstract

ZAP-70 is required for the initiation of T cell receptor (TCR) signaling, and Ssu72 is a phosphatase that regulates RNA polymerase II activity in the nucleus. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that Ssu72 contributed to the fine-tuning of TCR signaling by acting as tyrosine phosphatase for ZAP-70. Affinity purification–mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules and exhibited hyperresponsiveness, which was restored by reducing ZAP-70 phosphorylation. In vitro assay demonstrated that recombinant Ssu72 reduced tyrosine phosphorylation of ZAP-70 via phosphatase activity. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant natural killer T cells and reductions in CD4+ and CD8+ T cell numbers in the periphery but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhi naïve T cells, compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed spontaneous inflammation at 6 mo. In conclusion, Ssu72 phosphatase regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its tyrosine phosphorylation, thereby preventing spontaneous inflammation.

Phosphatase Ssu72 Is Essential for Homeostatic Balance Between CD4+ T Cell Lineages

“…Depletion of Ssu72 in T cells can increase the production of distinct signature cytokines, IFN-γ and IL-4 under Th1- and Th2-polarizing conditions, respectively ( 11 ). Consistently, Ssu72-deleted CD4 + T cells show increased expression of IFN-γ, IL-4, IL-17, GM-CSF, and transcription factors, including Tbx21 , Gata3 and Rorc in response to TCR stimulation both in vivo and in vitro ( 10 ). These results indicate that Ssu72 might contribute to the differentiation of naive T cells to CD4 + Th cells and negatively regulate the polarization of naïve CD4 + T cells to effector CD4 + T cells.…”

“…Recent studies have suggested that appropriate coordination of phosphorylation status of TCR-related target molecules by Ssu72 phosphatase can facilitate balanced differentiation between CD4 + T cell lineages ( 6 10 11 13 ). In conditional knock-out mice, in which the Ssu72 gene was deleted from T cells, Ssu72 deficiency augmented the differentiation into Th1 and Th2 lineages, whereas Treg population was reduced ( 10 11 ). Depletion of Ssu72 in T cells can increase the production of distinct signature cytokines, IFN-γ and IL-4 under Th1- and Th2-polarizing conditions, respectively ( 11 ).…”

“…Interestingly, Ssu72 phosphatase has been newly identified as a fine-tuning regulator of TCR signal by modulating the phosphorylation status of TCR-mediated molecules including ZAP70 in a phosphorylation-dependent manner ( Fig. 2 ) ( 10 ). Ssu72-deleted CD4 + T cells show increased phosphorylation of ZAP70 (Y292, Y319, and Y493) and various downstream molecules including ERK, MEK, and NF-κB after TCR-mediated stimulation ( 10 ).…”

“…Functions of Ssu72 in immune cells have been expanded to include intervening in immune responses by regulating multiple signaling pathways, including TCR engagement and GM-CSF receptor signaling ( 10 11 12 ). Recent studies have provided evidence that Ssu72 contributes to the differentiation, activation, and function of CD4 + T cell lineages by negatively controlling immune receptor signaling pathways ( 6 10 11 13 ).…”

“…Functions of Ssu72 in immune cells have been expanded to include intervening in immune responses by regulating multiple signaling pathways, including TCR engagement and GM-CSF receptor signaling ( 10 11 12 ). Recent studies have provided evidence that Ssu72 contributes to the differentiation, activation, and function of CD4 + T cell lineages by negatively controlling immune receptor signaling pathways ( 6 10 11 13 ). Given that the immune system is largely dependent on the phosphorylation of target proteins for recognition and transduction of extracellular signals, it is not surprising that Ssu72 is involved in regulating signaling cascades in a phosphatase activating-dependent manner.…”