Substrate Profiling of Protein Tyrosine Phosphatases

描述(由申请人提供)：酪氨酸上蛋白质的可逆磷酸化控制许多细胞过程的执行和调节。适当水平的酪氨酸磷酸化对这些过程是至关重要的，并受蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶(PTPs)的相反作用控制。大量的PTP(&gt；100)已经被确定，它们在生理和病理过程中的重要性已经被明确地证明。然而，它们在这些进程中的作用机制仍然不清楚。要了解它们的作用机制，关键的第一步是确定它们参与这些过程的蛋白质底物，这目前是一项非常具有挑战性的任务。该项目描述了一种用于鉴定PTP底物的化学/生物信息学方法的开发和应用。在这种方法中，通过筛选组合多肽文库来系统地确定PTP的底物专一性，并使用共识基序(S)搜索蛋白质和基因组数据库以确定潜在的蛋白质底物。候选蛋白质随后通过常规细胞检测被确认为真正的PTP底物(或被拒绝为假阳性)。在这个项目中，我们将重点研究三个经典的、非受体的PTPs：典型的PTP1B和两个含有Src同源2(SH2)结构域的PTPs，SHP-1和SHP-2。它由三个具体目标组成。具体目的1是进一步发展组合多肽文库方法，并测定PTP1B、SHP-1和SHP-2的底物特异性。具体目的2是鉴定PTP1B、SHP-1和SHP-2的体内蛋白底物。具体目标3是确定与人类疾病有关的SHP-2突变体的特征。将确定SHP-2突变体中SH2和PTP结构域的结合和底物特异性，并将利用所产生的信息来识别SHP-2突变体异常作用的任何蛋白质底物。公共卫生相关性：PTP在生理和病理过程中都发挥着关键作用，因此是化疗干预的重要靶点。PTP1B目前正被用作治疗2型糖尿病的靶点。SHP-2的突变会导致努南综合征、豹子综合征和几种白血病。催化缺陷的SHP-1突变会导致母鼠免疫系统失调，并在出生后2-3周过早死于炎症综合征。本项目将研究这三种PTP在生理和病理过程中发挥作用的分子机制。

DESCRIPTION (provided by applicant): Reversible phosphorylation of proteins on tyrosine controls the execution and regulation of many cellular processes. A proper level of tyrosyl phosphorylation is critical for these processes and is controlled by the opposing actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). A large number of PTPs (>100) have been identified and their importance in physiological and pathological processes has been demonstrated unambiguously. However, their mechanisms of action in these processes remain unclear. To understand their mechanisms of action, a critical first step is to identify their protein substrates involved in these processes, a currently very challenge task. This project describes the development and application of a chemical/bioinformatics approach to the identification of PTP substrates. In this approach, the substrate specificity of PTP is systematically determined by screening a combinatorial peptide library and the consensus motif(s) is used to search protein and genomic databases to identify potential protein substrates. The candidate proteins are subsequently validated as genuine PTP substrates (or rejected as false positives) by conventional cellular assays. In this project, we will focus our studies on three classical, non-receptor PTPs: the prototypical PTP1B and two Src homology 2 (SH2) domain-containing PTPs, SHP-1 and SHP-2. It consists of three specific aims. Specific Aim 1 is to further develop the combinatorial peptide library method and determine the substrate specificity of PTP1B, SHP-1, and SHP-2. Specific Aim 2 is to identify the in vivo protein substrates of PTP1B, SHP-1, and SHP-2. Specific Aim 3 is to characterize SHP-2 mutants that are involved in human diseases. The binding and substrate specificity of the SH2 and PTP domains in SHP-2 mutants will be determined and the resulting information will be utilized to identify any protein substrates abnormally acted upon by SHP-2 mutants. PUBLIC HEALTH RELEVANCE: PTPs play critical roles in both physiological and pathological processes and are therefore an important class of targets for chemotherapeutic intervention. PTP1B is currently being pursued as a target for treatment of type 2 diabetes. Mutations in SHP-2 cause Noonan syndrome, LEOPARD syndrome, and several leukemias. Catalytically defective SHP-1 mutants cause motheaten mice, which have a dysregulated immune system and die prematurely of inflammatory syndrome 2-3 weeks after birth. This project will investigate the molecular mechanisms by which these three PTPs function in physiological and pathological processes.