Structure/Function of Protein Tyrosine Phosphatases

描述（由申请人提供）：本研究项目的长期目标是表征蛋白质酪氨酸磷酸酶（PTPs）的结构和功能。PTPs构成了一个大家族的信号传导酶，与蛋白酪氨酸激酶（PTKs）一起调节酪氨酸磷酸化的细胞水平。PTK和PTP活性之间的正常平衡的紊乱导致异常酪氨酸磷酸化，其与包括癌症在内的几种人类疾病的病因学有关。因此，一个完整的了解蛋白质酪氨酸磷酸化的生理作用，以及如何在人类疾病中这一进程是失调必须包括PTPs的表征。这样的理解可能会导致开发新的治疗方法，选择性地靶向治疗人类疾病的信号通路的元素。这种竞争性更新集中在SHP 2（Src同源2（SH 2）-域包含蛋白酪氨酸磷酸酶-2），这是第一个真正的癌蛋白在PTP超家族中确定。SHP 2广泛表达，并通过激活Ras/ERK 1/2级联反应积极调节受体酪氨酸激酶的信号传导。与其致癌作用一致，生殖系常染色体显性SHP 2突变导致临床上相似的LEOPARD综合征（LS）和努南综合征（NS），两者均与恶性肿瘤风险增加相关。此外，体细胞SHP 2突变导致许多形式的白血病和实体瘤。然而，尽管SHP 2突变与许多发育和肿瘤性疾病相关，但仍不清楚SHP 2突变如何改变细胞信号传导以产生疾病表型。例如，NS或瘤形成相关的SHP 2突变体是组成型活性的，导致功能获得效应。相反，与LS相关的突变降低了SHP 2磷酸酶活性。这些发现产生了一个谜：具有相反效应的SHP 2突变如何引起重叠的表型？我们假设致病性SHP 2突变不仅改变了SHP 2磷酸酶活性，还改变了其分子转换机制以驱动疾病结局，因此对SHP 2结构和功能的详细了解将揭示疾病基础的关键信号传导事件。本项目的目标是了解疾病相关的SHP 2突变的分子基础，并确定将SHP 2功能障碍与各种LS异常耦合的分子事件链。将采用涉及X射线晶体学、质谱、组合化学、定点诱变、酶动力学和细胞生物学的创新组合的多学科方法来：1）表征LS突变体的结构和生物化学性质，和2）定义由LS突变体介导的信号传导机制。该项目的成功完成将为理解个体SHP 2突变如何导致疾病创造一个坚实的框架，并为这些疾病的治疗干预提供新的见解。

DESCRIPTION (provided by applicant): The long-term objectives of this research program are to characterize the structure and function of protein tyrosine phosphatases (PTPs). The PTPs constitute a large family of signaling enzymes that together with protein tyrosine kinases (PTKs) modulate the cellular level of tyrosine phosphorylation. Disturbance of the normal balance between PTK and PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. Thus, a complete understanding of the physiological roles of protein tyrosine phosphorylation and how this process is deregulated in human diseases must necessarily encompass the characterization of PTPs. Such understanding may lead to the development of novel therapeutics that selectively target elements of signaling pathways for the treatment of human diseases. This competitive renewal focuses on SHP2 (Src homology 2 (SH2)-domain containing protein tyrosine phosphatase-2), which is the first bona fide oncoprotein identified in the PTP superfamily. SHP2 is ubiquitously expressed and positively regulates signaling from receptor tyrosine kinases through the activation of the Ras/ERK1/2 cascade. Consistent with its oncogenic role, germline autosomal dominant SHP2 mutations cause clinically similar LEOPARD syndrome (LS) and Noonan syndrome (NS), both of which are associated with increased risk of malignancy. In addition, somatic SHP2 mutations contribute to many forms of leukemia and solid tumors. However, although SHP2 mutations are associated with a number of developmental and neoplastic disorders, it remains unclear how SHP2 mutations alter cellular signaling to produce disease phenotypes. For example, NS or neoplasia-associated SHP2 mutants are constitutively active, resulting in gain-of-function effects. In contrast, mutations associated with LS reduce SHP2 phosphatase activity. These findings generated an enigma: how do SHP2 mutations with opposite effects elicit overlapping phenotypes? We hypothesize that pathogenic SHP2 mutations alter not only SHP2 phosphatase activity but also its molecular switching mechanism to drive disease outcomes and thus detailed understanding of the structure and function of SHP2 will reveal critical signaling events that underlie the diseases. The goals of this project ar to understand the molecular basis of disease-associated SHP2 mutations and to define the chain of molecular events coupling SHP2 dysfunction to the various LS abnormalities. A multidisciplinary approach, involving innovative combinations of X-ray crystallography, mass spectrometry, combinatorial chemistry, site-directed mutagenesis, enzyme kinetics, and cell biology will be employed to: 1) characterize the structural and biochemical properties of the LS mutants, and 2) define the signaling mechanisms mediated by the LS mutants. Successful completion of this project will create a solid framework for understanding how individual SHP2 mutations cause diseases and provide insight into novel points of therapeutic intervention for these diseases.