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

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-((17␤-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 PLC␤3 (hPLC␤3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLC␤3 in a concentrationand time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC 50 ‫؍‬ 13.6 ؎ 5 M. Activation of hPLC␤3 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 M) also activated hPLC␥1 (>10-fold) and hPLC␤2 (ϳ2-fold); PLC␦1 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.

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

“…The precise mechanism by which phospholipase C␤ may regulate this transport event is not clear. Although U73122 is typically used as a phospholipase C␤ inhibitor, the molecule may have a number other actions that contribute to its pharmacological profile (42). Nevertheless, U73122 should be a useful tool to dissect the mechanisms involved in AGS3 translocation going forward.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…It does behave as a PLC inhibitor in many cellular systems with a very narrow concentration range (2-5 M) between effective and toxic doses (1423,1552,1732). However, it either has no effect on purified PLC enzymes or stimulates PLC activity in vitro at somewhat higher concentrations (ϳ10 M) (802). U73122 also inhibits a number of Ca 2ϩ -regulated processes independent of its putative effects on PLC (169,1240,1660,1817).…”

Structural Insights into Phospholipase C-βFunction

“…The maleimide group in U73122, which is required for its inhibitory action, reacts with exposed sulfhydryl groups on the protein surface. For PLCb, several of the modified sulfhydryl groups are on the same face of the catalytic core as the active site, and these hydrophobic adducts are proposed to increase membrane association and activity (Klein et al, 2011).…”