Serine−Threonine Protein Phosphatase Inhibitors:  Development of Potential Therapeutic Strategies

McCluskey, Adam; Sim, and Alistair T. R.; Sakoff, Jennette A.

doi:10.1021/jm010066k

Cited by 221 publications

(206 citation statements)

References 297 publications

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“…However, MeOk was a potent disruptor of the actin cytoskeleton of Clone 9 and primary hepatocytes, indicating that this toxin might inhibit another PP, not involved in glucose intake. This is a serious possibility, because OA was reported not only to inhibit PP1 and PP2A, but also PP4, and less efficiently, PP5 (Honkanen et al, 1994;Hastie and Cohen, 1998;Fernandez et al, 2002;McCluskey et al, 2002). This hypothesis agrees with previous reports that MeOk inhibited PP3 more efficiently than PP1 and PP2A (Honkanen et al, 1994).…”

Section: Discussionsupporting

confidence: 91%

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Espiña

Louzao

Cagide

et al. 2010

British J Pharmacology

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The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytesb ph_512 337.. 344 Begoña Espiña Japan Food Research Laboratories, Tama, Tokyo 206-0025, JapanBackground and purpose: Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs. Experimental approach: We tested the effects of MeOK, OA and microcystin-leucine and arginine (MC-LR) on the metabolic rate, the actin cytoskeleton and glucose uptake in a rat hepatocyte cell line (Clone 9) and in primary cultured rat hepatocytes. PP2A was assayed to compare OA and MeOk activity. Key results: MeOk disrupted the actin cytoskeleton and depressed the metabolic rate of both types of rat hepatocytes, being six-fold less potent than OA in Clone 9 cells but nearly six-fold more potent in primary cultured hepatocytes. However, unlike OA, MeOk did not change glucose uptake in these cells, suggesting a weak inhibition of PP2A, as confirmed in direct assays of PP2A activity. Conclusions and implications:Although MeOk was originally described as a weakly bioactive molecule, it clearly depressed the metabolic rate and disrupted the cytoskeleton in primary and immortalized rat hepatocytes. Furthermore, MeOk affected primary hepatocytes at much lower concentrations than those affecting immortalized cells. These effects were unrelated to PP2A inhibition. Our results suggest the risk to public health from MeOk in foodstuffs should be re-evaluated.British Journal of Pharmacology (2010) 159, 337-344; doi:10.1111/j.1476-5381.2009 published online 15 December 2009 Keywords: actin cytoskeleton; glucose uptake kinetics; metabolic rate; methyl okadaate; microcystin; okadaic acid; phycotoxins; protein phosphatase and rat hepatocytes Abbreviations: DSP, diarrheic shellfish poisoning; MC-LR, microcystin-leucine and arginine; MeOk, methyl okadaate; OA, okadaic acid; PP1, protein phosphatase-1; PP2A, protein phosphatase-2A IntroductionMany natural toxins such as okadaic acid (OA) potently inhibit the catalytic activity of serine/threonine protein phosphatases (PPs), of which PP1 and PP2A are the most abundant in cells. Both PPs have a wide spectrum of activity being able to dephosphorylate a large number of substrates in vitro (Fernandez et al., 2002). The list of PPs inhibitors includes a structurally diverse group of compounds: polyether fatty acid substances similar to okadaic acid (OA), cyclic peptides like microcystins (MCs) and nodularins, terpenoids such as cantharidin and thyrsiferyl 23-acetate and polyketides, such as tautomycin and calyculin A (Fernandez et al., 2002). OA is the best representative of the diarrheic shellfish poisoning ...

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Section: Discussionsupporting

confidence: 91%

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Espiña

Louzao

Cagide

et al. 2010

British J Pharmacology

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“…CLA is a member of a family of natural toxins that selectively inhibit the protein phosphatase 1 and 2A classes of serine/threonine phosphatase (10). Immunoblotting cell lysates with an anti-pThr antibody showed that CLA induced a robust increase in threonine phosphorylation in WEHI-231 cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line

Shu

Chen

et al. 2004

Molecular & Cellular Proteomics

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A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine-and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Knowledge about covalent modifications and their regulation is essential for the understanding of protein function. Regulation of protein activity is often modulated by reversible phosphorylation, and information about specific sites of phosphorylation is vital for understanding cellular signaling pathways. Two-dimensional gel electrophoresis is still the most common method used for detecting large-scale changes in phosphorylation (1). However, this method is time consuming and has a number of significant limitations. Although mass spectrometry is a sensitive tool for the identification of phosphopeptides, their detection within a complex peptide mixture can be limited by weak ionization of phosphopeptides (2). Due to the low stoichiometry of phosphorylation and the low abundance of signaling proteins within cells, enrichment of the phosphoproteins or phosphopeptides is often necessary. The ability to isolate phosphopeptides by immobilized metal affinity chromatography (IMAC) 1 was first recognized by Andersson and Porath in 1986 (3). Recently, the use of IMAC in combination with mass spectrometry has allowed the identification of hundreds of phosphorylation sites in yeast (4) and Arabidopsis plasma membrane proteins (5).An important goal of the Alliance for Cellular Signaling (AfCS) is the global analysis of ligand-induced changes in protein phosphorylation (6). Important steps in this process are the identification of phosphoproteins present in the AfCS model cell systems and the determination of their sites of phosphorylation. This information will be used to generate probes to obtain quantitative information on the effects of ligands on phosphorylation of specific sites. Based on recent progress in the application of IMAC and mass spectrometry, we used this approach to identify phosphoproteins and their phosphorylation sites in the murine WEHI-231 B lymphoma cell line. EXPERIMENTAL PROCEDURESProtein Test Samples-One test sample (prepared at a ratio of 1:1:5) contained 1 pmol of a synthetic phosphotyrosine (p-Tyr) phosphopeptide (m/z 1127, DRVpYIHPF), a tryptic digest of 1 pmol of ␣-casein (containing three phosphopeptides: m/z 1467, TV...

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“…Accordingly, we found enhanced cytoplasmic PP1a immunostaining in tumours vs BPH. Further, enhanced cytoplasmic PP1a immunostaining correlated with high Owing to their role as regulators of the cell cycle as well as in apoptosis, protein phosphatases are increasingly discussed as candidates for therapeutic interference (Berndt et al, 1997;McCluskey et al, 2002). Inhibitors of protein phosphatases exert anticancer activity and have already been used in the treatment of primary hepatoma and upper gastrointestinal carcinoma (Berndt et al, 1997;McCluskey et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

et al. 2006

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To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, b-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1a (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1a (P ¼ 0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (Po0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P ¼ 0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

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Serine−Threonine Protein Phosphatase Inhibitors: Development of Potential Therapeutic Strategies

Cited by 221 publications

References 297 publications

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

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