A Cysteine Residue within the Kinase Domain of Zap70 Regulates Lck Activity and Proximal TCR Signaling

Abstract

Alterations in both the expression and function of the non-receptor tyrosine kinase Zap70 are associated with numerous human diseases including immunodeficiency, autoimmunity, and leukemia. Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLCγ1 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70C564R, is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70C564A displayed the hyperphosphorylation of TCR-ζ and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.

A Story of Kinases and Adaptors: The Role of Lck, ZAP-70 and LAT in Switch Panel Governing T-Cell Development and Activation

“…In addition, it has been proposed that cysteine 564, located in the kinase domain of ZAP70, is palmitoylated, and that a Cys-to-Arg mutation preventing palmitoylation blocks TCR signaling and T-cell activation [ 123 ]. However, it has been recently shown that, although such a mutation prevents palmitoylation, a ZAP70 mutant in which cysteine 564 is replaced by a non-palmitoylable alanine residue is able to transduce TCR signaling, and even enhances the activity of Lck and increases its proximity to the TCR [ 124 ]. Therefore, C564 seems to be another relevant component in the regulation of proximal TCR signaling by ZAP70 kinase.…”

The screening, identification, design and clinical application of tumor-specific neoantigens for TCR-T cells

“…This change enhanced the pairing efficiency and stability of the exogenous α and β chains, reduced the occurrence of mismatch, increased the expression of exogenous TCR on the cell surface, and enhanced the antigen-specific response. Studies have shown that this strategy can reduce the autoimmune pathological reactions caused by TCR mismatch [ 528 ].…”