Chemical Probes of Protein Tyrosine Phosphatase Activity

项目摘要 酪氨酸磷酸化是多种细胞信号通路中的关键调控事件。这个 蛋白酪氨酸激酶催化的酪氨酸磷酸化和去磷酸化 酪氨酸磷酸酶(PTKs)和蛋白酪氨酸磷酸酶(PTPs)分别是一个受到严格调控的过程。 尽管在人类基因组中PTK和PTP的数量大致相同，但类似的 底物选择性和平行生物调节的程度，相对较少知道 关于PTP活性，与大量涉及PTK作用的文献形成对比 在细胞信号方面。特别是，直接报告PTP的基于活性的化学探针 活动非常有趣，因为它们将提供监视PTP活动的方法 同时也有助于研究复杂信号通路中的个体PTP。这个 我们工作的长期目标是开发研究PTP活性的化学方法， 可用于回答有关PTP生物学的关键问题。最近，我们开发了一种PTP- 便于在体外和体内监测单个PTP活性的平台技术 细胞。这项技术涉及将荧光肽底物输送到细胞并监测 当细胞内的PTP作用于底物时，细胞的荧光变化。基于我们的 以前成功地使用多肽底物选择性地监测细胞中的PTP活性，我们 现在，为PTP酶家族的几个成员开发有针对性的探针。具体来说， 我们将开发一系列选择性底物和PTP活性抑制剂，可以 用于监控多个PTP的细胞活动并解决以下关键问题 PTP生物学。我们的方法可以很容易地应用于PTP家族中的几乎任何成员 酶，我们选择了一个具有代表性的基团进行初步调查。此外, 我们的方法应该被证明在开发以PTP为目标的通用PTP方面非常有用， 基于机制的探头也是如此。这里描述的多学科研究计划需要 巴里奥斯实验室在化学合成、生物化学和 将为信号生物学研究提供极具意义的探针。 社区。

Project Summary Tyrosine phosphorylation is a key regulatory event in multiple cellular signaling pathways. The phosphorylation and dephosphorylation of tyrosine, catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively, is a tightly regulated process. Despite the approximately equal numbers of PTKs and PTPs in the human genome and similar degrees of substrate selectivity and parallel biological regulation, comparatively little is known about PTP activity in contrast to the significant body of literature dealing with the roles of PTKs in cellular signaling. In particular, activity-based chemical probes that directly report on PTP activity are of great interest because they would provide the means to monitor PTP activity in living cells and also facilitate the study of individual PTPs in complex signaling pathways. The long-term objective of our work is to develop chemical approaches to studying PTP activity that can be used to answer key questions about PTP biology. Recently, we have developed a PTP- platform technology that facilitates monitoring the activity of individual PTPs both in vitro and in cells. This technology involves delivering fluorogenic peptide substrates to cells and monitoring changes in fluorescence of the cells as intracellular PTPs act on the substrate. Based on our previous success with selectively monitoring PTP activity in cells using peptide substrates, we now develop targeted probes for several members of the PTP family of enzymes. Specifically, we will develop a series of selective substrates and inhibitors for PTP activity that can be used to monitor the cellular activity of multiple PTPs and address key questions about PTP biology. Our approach can readily be applied to almost any member of the PTP family of enzymes and we have selected a representative group for our initial investigation. In addition, our approach should prove very useful in the development of generic PTP-targeted, mechanism-based probes as well. The multidisciplinary research program described here takes advantage of the Barrios laboratory’s expertise in chemical synthesis, biochemistry and enzymology and will provide probes of great interest to the signaling biology research community.