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

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 beta-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 beta 3 (hPLC beta 3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLC beta 3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50 = 13.6 +/- 5 mu M. Activation of hPLC beta 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 mu M) also activated hPLC gamma 1 (> 10-fold) and hPLC beta 2 (similar to 2-fold); PLC delta 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.