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

ZAP-70是协调近端TCR信号的关键酪氨酸激酶，它也参与白血病细胞BCR下游的信号传递。ZAP-70的激活是通过关键酪氨酸残基的可逆磷酸化和其他修饰来调节的，例如泛素化。最近，我们首次发现半胱氨酸残基的氧化(即亚硫基化)也参与了ZAP-70功能的调节。我们发现ZAP-70的C575是磺化的，而C575A ZAP-70突变体是不稳定的，活性降低。我们的数据与其他研究表明半胱氨酸残基调节酪氨酸激酶的活性是一致的。功能相关的半胱氨酸(如EGFR中的C797和BTK中的C481)也成为开发一类新的癌症治疗剂的目标。因此，识别功能相关的半胱氨酸残基不仅对揭示酪氨酸激酶的活性是如何调节的很重要，而且在设计新的治疗癌症的化合物时也是至关重要的。基于这些考虑，我们决定进行旨在识别参与ZAP-70激活调控的额外半胱氨酸残基的实验。我们在ZAP-70中产生了带有C到A取代的载体，并使用ZAP-70缺陷的Jurkat T细胞进行了功能鉴定(P116)。初步数据表明，另外两个半胱氨酸残基C39位于N-SH2结构域，C564位于激活域的C叶，参与了ZAP-70活性和TCR信号的调节。C39A ZAP-70突变体在P116 T细胞中表达时，活性残基Y319和Y493的磷酸化水平降低。相应地，在表达C39A Zap-70的CD3刺激的P116细胞中，近端TCR信号分子的磷酸化、Ca~(2+)通量和ERK1/2的激活显著减少。C39似乎是ZAP-70的正向调节因子，可能调节ZAP-70向TCR的募集。与C39A Zap-70相比，C564A Zap-70突变导致Y319和Y493的磷酸化增强，TCR信号增强，从而表明C564A Zap-70突变是过度活跃的。在这项建议中，我们现在的目标是：(I)鉴定体内和体外通过C39和C564调控ZAP-70功能的分子机制，(Ii)研究ZAP-70半胱氨酸在白血病细胞信号转导过程中的功能。我们相信，我们的研究将有助于理解ZAP-70在生理条件和病理修饰下的调节机制。我们希望我们的研究也将有助于开发新的药理工具来抑制人类疾病中的ZAP-70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.