In recent years it has become increasingly clear that protein phosphatases, once thought to simply be housekeeping enzymes, play major roles in cellular signaling. Furthermore, it has been shown that dysregulation of the phosphatome can play a critical role in human disease physiology. Phosphatase activity is regulated in multiple ways (protein-protein interactions, phosphorylation, oxidation, etc.) many of which cannot be reported on by activity proxies such as western blotting. Consequently, methodologies to directly measure alterations in phosphatase catalytic activity in biological samples are currently lacking. It is therefore critical that technologies be developed which allow for the direct monitoring of specific phosphatase activity in order to better understand their individual roles in disease development and progression. Using the fluorescent unnatural amino acid, CSox, we have synthesized a selective, real-time activity probe for the protein tyrosine phosphatase PTP1B, a ubiquitously expressed tyrosine phosphatase that has been identified as a potential target in the treatment of type 2 diabetes. Upon dephosphorylation of our optimized sensor we see a robust 4.6-fold change in signal over background. With recombinant enzyme we are able detect PTP1B activity at concentrations as low as 100 pM. The kinetic parameters of PTP1B with our sensor follow Michaelis-Menten kinetics with KM = 25.0 μM and kcat = 4.8 s-1. Using this assay platform we demonstrate the ability to directly monitor the inhibition of recombinant PTP1B with vanadate, a known tyrosine phosphatase inhibitor, demonstrating the potential of this assay to be used in high-throughput screening efforts for PTP1B inhibitors. Additionally, when assayed against a panel of closely related tyrosine phosphatases (TCPTP, SHP1, SHP2 & PTP-PEST) our sensor is selective for PTP1B. Finally, we demonstrate the ability of our optimized assay platform to quantify the activity of PTP1B isolated from mammalian cell lysates, thus providing a powerful tool to better understand this enzyme in complex systems. In the long term, this general assay format could be readily expanded to monitor the activities of virtually any protein phosphatase, enabling the investigation of phosphatase activity dynamics in human disease development.

General approach to analysis of phosphatase activity in biological samples

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.