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

Zap-70是协调近端TCR信号传导的关键酪氨酸激酶，其也参与白血病细胞中BCR下游的信号传导。Zap-70的活化通过关键酪氨酸残基的可逆磷酸化和其他修饰如泛素化来调节。最近，我们首次表明半胱氨酸残基的氧化（即亚磺酰化）也参与Zap-70功能的调节。我们发现Zap-70的C575被亚硫基化，并且C575 A Zap-70突变体不稳定并且活性降低。我们的数据与其他研究一致，表明半胱氨酸残基调节酪氨酸激酶的活性。功能相关的半胱氨酸（例如EGFR中的C797和Btk中的C481）也已成为开发新型癌症治疗剂的目标。因此，功能相关的半胱氨酸残基的鉴定不仅对于揭示酪氨酸激酶的活性如何被调节是重要的，而且在用于治疗癌症的新型治疗化合物的设计中也是至关重要的。基于这些考虑，我们决定进行旨在鉴定参与Zap-70活化调节的其他半胱氨酸残基的实验。我们产生了在Zap-70中携带C至A取代的构建体，并使用Zap-70缺陷型Jurkat T细胞（P116）进行功能表征。初步数据表明，两个额外的半胱氨酸残基，C39位于N-SH 2结构域和C564位于激酶结构域的C端，参与Zap-70活性和TCR信号传导的调节。C39 A Zap-70突变体在P116 T细胞中表达后，在活化残基Y319和Y 493上显示出磷酸化减少。因此，在表达C39 A Zap-70的CD 3刺激的P116细胞中，近端TCR信号分子的磷酸化、Ca 2+通量和Erk 1/2的活化显著降低。C39似乎作为Zap-70的正调节物起作用，可能调节Zap-70向TCR的募集。与C39 A Zap-70相反，C564 A Zap-70突变导致Y319和Y 493的磷酸化增强以及TCR信号传导增强，因此表明C564 A Zap-70突变体是过度活跃的。在这个提议中，我们现在的目标是：（i）通过C39和C564在体外和体内的Zap-70功能的调节的分子机制的鉴定，（ii）在白血病细胞的信号转导过程中Zap-70半胱氨酸的功能的调查。我们相信，我们的研究将有助于了解Zap-70在生理条件和病理变化中的调节机制。我们希望我们的研究也将有助于开发新的药理学工具来抑制Zap-70在人类疾病中的功能。

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.