Inhibition of Protein Phosphatase-1 by Clavosines A and B

Abstract

Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1c␥) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1c␥ that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1c␥ to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1c␥ Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. Serine/threonine protein phosphatases are highly conserved enzymes, which have been isolated from numerous eukaryotic and prokaryotic organisms (1, 2). Eukaryotic protein phosphatases are inhibited by a structurally diverse array of natural product toxins. These include the polyether okadaic acid from marine dinoflagellates, cyclic peptide microcystins and nodularins from cyanobacteria, and the spiroketal calyculins isolated from marine sponges (3).The first member of the calyculin family of phosphatase inhibitors was identified in 1986 (4). Calyculin A was originally purified from a hydrophobic extract of the marine sponge Discodermia calyx. It was found to be cytotoxic toward P388 and L1210 leukemia cells and to be a strong inhibitor of starfish egg development (4, 5). It was subsequently characterized as a powerful inhibitor of the catalytic subunits of type 1 and 2A protein phosphatases, PP-1c 1 and PP-2Ac, two of the four major eukaryotic serine/threonine protein phosphatases (6). Calyculin A was also shown to have tumor-promoting activity as potent as that of okadaic acid (7). Many additional calyculins (8 -13), and the structurally related calyculinamides (11,14), have since been identified.Recently we reported the isolation of two novel glycosylated members of the calyculin family, clavosines A and B from the marine sponge Myriastra clavosa (15). Clavosines A and B are potent inhibitors of mammalian PP-1c and PP-2Ac and were found to be more cytotoxic overall than the calyculins and the calyculinamides (14, 15) in the National Cancer Institute (NCI) screening panel of tumor cell lines (16). The polyfunctional structure of clavosines A and B and calyculin A is depicted in linear diagrams (Fig. 1a) and space filling models (Fig. 1b). The clavosines (Fig. 1) have many structural features in common with the calyculins but differ markedly from them in having a methyl group at C-32 and an unusual trimethoxy rhamnose group at position C-21. Like the...

Biosynthesis of polyketides by trans-AT polyketide synthases

“…Calyculin A (15) is a highly abundant (0.15% wet weight) and very potent (picomolar range) cytotoxic polyketide isolated from the marine sponge Discodermia calyx. 110 Calyculins [111][112][113] and the related compounds (swinhoeiamide A (16), 114 clavosine A (17) 115,116 and geometricin A (18) 117 ) isolated from geographically and phylogenetically distant marine sponges share a remarkable scaffold containing 5,6-spiroacetal, phosphate, oxazole, and dimethylamino moieties. 118 In addition, 15 contains a terminal nitrile group.…”

Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of the β12-β13 Loop in Inhibitor Binding

“…Purified PP-1c stocks were diluted in phosphatase assay buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 1 mg/ml bovine serum albumin, 0.8 mM MnCl 2 , and 0.2% ␤-mercaptoethanol). Phosphatase was diluted until a 10-l aliquot of diluted enzyme solution caused 15% 32 P release in a 30-l assay (20,21). Duplicate assays were performed for each toxin and inhibitor protein.…”

Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

“…Because our initial goal was to identify the majority of the phosphorylation sites on paxillin, we analyzed protein purified from transiently transfected, but lower-expression paxillin in HEK cells, which were pretreated with the phosphatase inhibitors, peroxovandate and calyculin A. The former is a known inhibitor of protein tyrosine phosphatases, ,, and the latter is specific for serine/threonine phosphatases …”

“…The former is a known inhibitor of protein tyrosine phosphatases, 34,40,41 and the latter is specific for serine/threonine phosphatases. 42 Calyculin A is a potent inhibitor of the catalytic subunits of protein phosphatase (PP)-1c and PP-2Ac. 43 Although other serine/threonine phosphatase inhibitors such as okadaic acid and microcystin are available, the literature suggests they are less potent than calyculin A.…”