Substrate Profiling of Protein Tyrosine Phosphatases

项目摘要 酪氨酸上蛋白质的可逆磷酸化控制着许多 细胞过程。适当水平的酪氨酸磷酸化对这些过程是至关重要的，并且 受蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶相反作用的控制 (PTPS)。大量的PTP(&gt；100)已经被发现，它们在生理上的重要性 病理过程已经被明确地证明了。然而，他们的机制是 这些进程中的行动仍不清楚。要了解它们的作用机制，关键的一点是 步骤是确定它们参与这些过程的蛋白质底物，这是目前一项非常具有挑战性的任务。 该项目描述了化学/生物信息学方法的开发和应用 PTP底物的鉴定。在这种方法中，PTP的底物专一性是系统的 通过筛选确定一个组合多肽文库，并使用共识基序(S)进行搜索 蛋白质和基因组数据库，以确定潜在的蛋白质底物。候选蛋白质是 随后通过常规方法验证为真正的PTP底物(或被拒绝为假阳性) 细胞分析。 在这个项目中，我们将集中研究三个经典的、非受体的PTP：原型 PTP1B和两个含有Src同源2(SH2)结构域的PTP，SHP-1和SHP-2。它由以下内容组成 三个具体目标。具体目标1是进一步发展组合多肽文库方法和 确定PTP1B、SHP-1和SHP-2的底物特异性。具体目标2是确定In PTP1B、SHP-1和SHP-2的活体蛋白底物。具体目标3是描述SHP-2的特征 与人类疾病有关的突变体。SH2和SH2的结合和底物特异性 将确定SHP-2突变体中的PTP结构域，并将利用产生的信息来 确定任何由SHP-2突变体异常作用的蛋白质底物。

Project Summary 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.