Direct Activation of Human Phospholipase C by Its Well Known Inhibitor U73122

John Sondek,Chester L. Costales,Jon D. Williams,Craig D. Wagner,Ryan R. Klein,Stephanie N. Hicks,Dhiren R. Thakker,Wendy L. White,David M. Bourdon

doi:10.1074/jbc.m110.191783

Abstract

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Highlights

U73122, but Not U73343, Increases Human Phospholipase C (PLC)␤3 Activity in a Concentration-dependent Manner—In DDM mixed micelles, U73122 unexpectedly increased the activity of human PLC␤3 (hPLC␤3)
Recent studies have questioned the use of U73122 as a specific inhibitor of PLCs by uncovering a number of “off-target” interactions, likely due to covalent modification of nucleophiles by the maleimide moiety in U73122 [27,28,29]
These findings point to the complication of using U73122 as a tool to study PLC function in complex cellular systems and suggest the need for a clearer understanding of how U73122 interacts with PLCs at the molecular level

Summary

Introduction

13 human PLC isozymes have been identified comprising six distinct and differentially regulated families (␤1– 4, ␥1–2, ␦1,3– 4, ⑀, ␩1–2, and ␨) [1,2,3] They vary in molecular size from the ϳ70-kDa PLC␨ to the much larger ϳ250-kDa PLC⑀, but they share a common core domain structure including the EF domain, the C2 domain, and the highly conserved catalytic core made up of two regions, commonly referred to as X and Y domains [4, 5]. Many studies have since reported that several isozymes of PLC are inhibited by U73122, but not by U73343, a close structural analog of U73122 containing N-alkylsuccinimide moiety in place of N-alkylmaleimide [15,16,17,18,19]. These studies revealed that U73122 activates, not inhibits, the activity of several PLC isozymes, uncovering a novel and potentially general activation mechanism for lipases such as PLCs

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