Pisiferdiol restores the growth of a mutant yeast suffering from hyper-activated　Ca²⁺-signaling through calcineurin inhibition

Abstract

In the course of our screening program for a new inhibitor of the Ca 2+ signalling pathway using mutant yeast [Saccharomyces cerevisiae (zds1D erg3D pdr1D pdr3D)], a mouse PP2Ca activator, pisiferdiol, isolated from Chamaecyparis pisifera, was found to alleviate the Ca 2+ signal-mediated growth inhibition. Pisiferdiol showed growth inhibition activity against the mpk1D strain compared with the cnb1D strain and induced Li + sensitivity to the wild-type strain, indicating that it suppresses the calcineurin pathway in the yeast. However, the Li + sensitivity to ptc1D strain by pisiferdiol was diminished. Pisiferdiol showed growth restored activity in the zds1D strain without immunophilins Fkb1p or Cph1p, and in the pmc1D strain. It inhibited calcineurin-induced expression in the reporter gene assay and decreased the protein expression (Western blots) of calcineurin (Cnb1p) in addition to a decrease of Swe1p and phosphorylation of Cdc28p in the mutant yeast. These results showed that pisiferdiol could suppress indirectly the action of calcineurin and restored the growth inhibition of the mutant yeast through Ptc1p activation.

A FluoPol-ABPP PAD2 High-Throughput Screen Identifies the First Calcium Site Inhibitor Targeting the PADs

“…Compound 1 (NSC95397) contains a reactive quinone moiety and is known to irreversibly inhibit Cdc25, 28 whereas 2 (ruthenium red) is an inorganic complex that binds specifically to calcium-binding proteins such as calmodulin and has been shown to block calcium flux through calcium ion channels. 29,30 Compound 3 (sanguinarine) is a plant alkaloid isolated from the root of Sanguinaria canadensis 31 and has been demonstrated to target a variety of known cellular proteins including the phosphatases MKP-1 32 and PP2C. 33 Inhibitor Kinetics.…”

Challenges and Opportunities in the Development of Protein Phosphatase-Directed Therapeutics

“…Other structurally unrelated activators of PP2Cα have been identified but they were active only at much higher concentrations. 34 The bioactive sphingolipid ceramide activates not only PP2C but, surprisingly, also the structurally unrelated catalytic subunits of the PPP phosphatases PP1 and PP2A. 35,36 The catalytic subunit of PP1 has a hydrophobic surface groove that is remote from the catalytic site and mediates the binding of regulatory subunits via the so-called RVxF-type (one-letter residue code, x is any residue) docking motif.…”

Temporal Analysis of PP2A Phosphatase Activity During Insulin Stimulation Using a Direct Activity Probe

“…In order to determine the identity of the enzyme(s) responsible for this remaining off-target activity, we performed assays in HeLa cell lysates containing calyculin A (a potent PP1 and PP2A inhibitor) 37 in the presence or absence of the broad spectrum PP2C alpha inhibitor sanguinarine (Figure S5a). 38 In addition, analogous experiments were performed with the dual specificity phosphatase inhibitor orthovanadate (Figure S5b). 39 In either case, we did not observe any significant decrease in activity, thus ruling out these two phosphatase families as the source of off-target activity.…”