Functional characterization of cysteine residues in the regulation of Zap-70 activity in physiology and disease

• Applicant: Professor Dr. Luca Simeoni
• Subject Area: Immunology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 416907033

Zap-70 is a key tyrosine kinase orchestrating proximal TCR signaling, which is also involved in signaling downstream of the BCR in leukemic cells. Activation of Zap-70 is regulated via reversible phosphorylation of crucial tyrosine residues and other modifications such as ubiquitination. Recently, we have shown for the first time that also oxidation of cysteine residues (i.e.sulfenylation) is involved in the regulation of Zap-70 function. We have found that C575 of Zap-70 is sulfenylated and that a C575A Zap-70 mutant was unstable and displayed reduced activity. Our data are in line with other studies showing that cysteine residues regulate the activity of tyrosine kinases. Functionally relevant cysteines (such as C797 in the EGFR and C481 in Btk) have also become the target for the development of a new class of therapeutic agents to treat cancer. Thus, the identification of functionally relevant cysteine residues is not only important to unravel how the activity of tyrosine kinases is regulated but it is also of critical importance in the design of novel therapeutic compounds for the treatment of cancer. Based upon these considerations, we have decided to conduct experiments aimed at the identification of additional cysteine residues involved in the regulation of Zap-70 activation. We generated constructs carrying C to A substitutions in Zap-70 and performed functional characterization using Zap-70-deficient Jurkat T cells (P116). Preliminary data indicate that two additional cysteine residues, C39 located in the N-SH2 domain and C564 located in the C-lobe of the kinase domain, are involved in the regulation of Zap-70 activity and TCR signaling. The C39A Zap-70 mutant shows reduced phosphorylation on the activatory residues Y319 and Y493 upon expression in P116 T cells. Accordingly, the phosphorylation of proximal TCR signaling molecules, Ca2+ flux, and the activation of Erk1/2 are significantly reduced in CD3-stimulated P116 cells expressing C39A Zap-70. C39 seems to function as a positive regulator of Zap-70 possibly modulating the recruitment of Zap-70 to the TCR. In contrast to C39A Zap-70, the C564A Zap-70 mutation resulted in an enhanced phosphorylation of Y319 and Y493 and enhanced TCR signaling, thus indicating that the C564A Zap-70 mutant is hyperactive. In this proposal we now aim at: (i) the identification of the molecular mechanisms underlying the regulation of Zap-70 function via C39 and C564 both in vitro and in vivo, (ii) the investigation of the function of Zap-70 cysteines in signaling processes in leukemic cells. We believe that our studies will contribute to the understanding of the mechanisms regulating Zap-70 functions in both physiological conditions and pathological modifications. We hope that our study will also contribute to the development of new pharmacological tools to inhibit Zap-70 functions in human diseases.

• DFG Programme: Research Grants