Functional characterization of cysteines in Lck and Zap-70 and identification of new targets of oxidation in lymphocytes under physiological and pathological conditions.

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Lck and Zap-70 are two key tyrosine kinases that orchestrate proximal TCR signaling. Recent data have shown that they are also involved in signaling downstream of the BCR in leukemic cells. Activation of Lck and Zap-70, as well as of many other kinases, is regulated via reversible phosphorylation of crucial tyrosine residues. The experimental evidence suggests that, in addition to tyrosine phosphorylation, also reversible oxidation (e.g. sulfenylation) of cysteine residues, plays an important role in the regulation of the enzymatic activity of tyrosine kinases. However, whether Lck and Zap-70 are regulated in a cysteine oxidation-dependent manner is not yet completely understood. The aim of this project is to investigate the functional role of cysteine residues within Lck and Zap-70 under both physiological and pathological conditions. To this aim we generated constructs carrying C to A substitutions and performed functional characterization using Lck- or Zap-70-deficient Jurkat T-cell lines. Preliminary data show that cysteines within Lck (C217, C224, C378, and C476) and Zap-70 (C575) are crucial for the function of these kinases, as C to A mutants failed to fully reconstitute TCR-mediated signaling in the Jurkat T-cell lines. The goals of this project are: (i) further biochemical and functional characterization of the cysteine mutants, (ii) generation of mouse models to assess the relevance of the cysteine residues in vivo, and (iii) analysis of the function of Lck and Zap-70 cysteines in leukemic cells (e.g. chronic lymphocytic leukemia, CLL). In addition to Lck and Zap-70, other signaling molecules may be regulated in an oxidation-dependent fashion. Targets of sulfenylation in lymphocytes are still largely unknown. Therefore, using dimedone-based systems, we have explored the sulfenylation pattern in lymphocytes from healthy donors as well as from CLL patients. We have found that both healthy lymphocytes and leukemic cells show several sulfenylated proteins. Interestingly, CLL cells display a specific pattern of protein cysteine sulfenylation, which appears to be different from that of the cells from healthy donors. One additional aim of this project is to identify sulfenylated proteins (redoxome) in lymphocytes from both healthy donors and leukemia patients. We hope that our studies will contribute to the development of new molecular and pharmacological tools to modulate lymphocyte activation and to treat autoimmunity, immunodeficiency, and leukemia.