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.