A highly conserved redox-active Mx(2)CWx(6)R motif regulates Zap70 stability and activity

Abstract

ζ-associated protein of 70 kDa (Zap70) is crucial for T-cell receptor (TCR) signaling. Loss of Zap70 in both humans and mice results in severe immunodeficiency. On the other hand, the expression of Zap70 in B-cell malignancies correlates with the severity of the disease. Because of its role in immune-related disorders, Zap70 has become a therapeutic target for the treatment of human diseases. It is well-established that the activity/expression of Zap70 is regulated by post-translational modifications of crucial amino acids including the phosphorylation of tyrosines and the ubiquitination of lysines. Here, we have investigated whether also oxidation of cysteine residues regulates Zap70 functions. We have identified C575 as a major sulfenylation site of Zap70. A C575A substitution results in protein instability, reduced activity, and increased dependency on the Hsp90/Cdc37 chaperone system. Indeed, Cdc37 overexpression reconstituted partially the expression but fully the function of Zap70C575A. C575 lies within a Mx(2)CWx(6)R motif which is highly conserved among almost all human tyrosine kinases. Mutation of any of the conserved amino acids, but not of a non-conserved residue preceding the cysteine, also results in Zap70 instability. Collectively, we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases.

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

“…DNA electroporation of Jurkat T-cell lines was performed using the Gene Pulser II System (BIORAD) as previously described [ 30 ]. For the FLIM/FRET measurements, cells were cultured in RPMI 1640 without phenol red (Gibco).…”

“…For the FLIM/FRET measurements, cells were cultured in RPMI 1640 without phenol red (Gibco). HEK 293T and HEK 293T–TCR/CD3 cells were transfected as described in [ 30 ].…”

The Activity and Stability of p56Lck and TCR Signaling Do Not Depend on the Co-Chaperone Cdc37

“…These data additionally indicate that the overexpression of Cdc37 did not alter the stability of other kinases involved in TCR signaling. Indeed, the expression of Zap-70 ( Figure 1 C and [ 40 ]) as well as of Akt and Raf ( Figure 1 E) were not affected by the overexpression of Cdc37.…”

A comparative view on vitamin C effects on αβ- versus γδ T-cell activation and differentiation

“…The stability of Zap70 is influenced by an intrinsic cysteine‐based redox‐active motif, which stabilizes the protein when oxidized. Vice versa, deletion of this motif or treatment with the anti‐oxidant N‐acetylcysteine (NAC), leads to destabilization of Zap70 . A similar influence on protein stability through cysteine motifs is also suggested for other tyrosine kinases, which can integrate signals into the TCR signaling such as the Src‐kinase Lck .…”

“…By using the anti‐oxidants GSH and NAC, the defective αβ T‐cell development in the Raptor‐deficient mice was partially restored and fewer γδ T cells were generated . High ROS levels are a hallmark of T‐cell activation, and can further enhance the TCR signal strength . A stronger (γδ) TCR signal in turn is known to promote γδ T‐cell over αβ T‐cell development .…”