Biochemical characterization and inhibitory effects of dinophysistoxin-1, okadaic acid and microcystine 1-r on protein phosphatase 2a purified from the mussel Mytilus chilensis

Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.     

Apparent "activation" of protein kinases by okadaic acid class tumor promoters

A cytosolic fraction of mouse brain gave two peaks of protein kinase activity on DEAE-cellulose column chromatography. The first peak of protein kinase corresponded to protein kinase C. The second peak contained protein kinases that were "activated" dose-dependently by the okadaic acid class tumor promoters, okadaic acid and dinophysistoxin-1. This "activation" was not achieved by other tumor promoters, such as 12-0-tetradecanoyl-phorbol-13-acetate, teleocidin, aplysiatoxin, or palytoxin. In addition, the second peak contained phosphatases. The phosphate liberation from phosphorylated histone type III-S by incubation with the second peak was inhibited by okadaic acid or dinophysistoxin-1, dose-dependently. The resulting apparent "activation" of protein kinases by okadaic acid is indicated and would imply a new pathway of tumor promotion on mouse skin.     

Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD = 2.3 μg/L) and dinophysistoxin-1 (DTX-1) (LOD = 15.2 μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD = 30.1 μg/L).     

Comparative study of toxicological and cell cycle effects of okadaic acid and dinophysistoxin-2 in primary rat hepatocytes

AimsTo determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures.Main methodsCytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy.Key findingsWe show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm.SignificanceThe results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.     

Inhibition of protein phosphatases-1 and -2A with acanthifolicin: Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition

Acanthifolicin (9,10-epithio-okadaic acid from Pandoras acanthifolium) inhibited protein phosphatase-1 (PP1) similarly to okadaic acid (IC₅₀ = 20 nM and 19 nM, respectively) but was slightly less active against protein phosphatase-2A (PP2A) (IC₅₀ 1 nM and 0.2 nM, respectively). Methyl esterification of acanthifolicin sharply reduced its activity. PP2A was inhibited with an IC₅₀ = 5.0 μM, whilst PP1 was inhibited < 10% at 250 μM toxin. Okadaic acid methyl ester was similarly inactive whereas dinophysistoxin-1 (35-methyl okadaic acid) inhibited PP1/2A almost as potently as okadaic acid. Pure acanthifolicin/okadaic acid methyl ester may be useful as specific inhibitors of PP2A at 1–10 μM concentrations in vitro and perhaps in vivo. The data also indicate that a region on these toxins important for PP1/2A inhibition comprises the single carboxyl group.     

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.     

Synthesis and stereochemistry of the terminal spiroketal domain of the phosphatase inhibitor dinophysistoxin-2

An expedient synthesis of both axially and equatorially C35 methyl substituted spiroketals representing the C28-C38 domain of the potent and selective protein serine/threonine phosphatase inhibitor dinophysistoxin-2 (DTX-2) was developed to enable comparative stereochemical analyses and a stereochemically correct total synthesis of DTX-2. Comparison of proton and carbon NMR data of the synthetic diastereomers with those published for DTX-2 indicates that DTX-2 possesses the (30S *,34R *,35S *)-relative configuration with an axial C35 methyl substituent.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.     

A type 2A protein phosphatase dephosphorylates the elongation factor 2 and is stimulated by the phorbol ester TPA in mouse epidermis in vivo

Mouse epidermal cytosol contains a protein phosphatase with Mr 38,000, which dephosphorylates the elongation factor 2 (EF-2) of protein biosynthesis and is stimulated after topical application of TPA to mouse skin [(1988) Biochem. Biophys. Res. Commun. 153, 1129-1135]. Dephosphorylation of EF-2 by this phosphatase is inhibited by okadaic acid at concentrations as low as 10(-8) M, but not by heparin up to concentrations of 600.micrograms/ml. The catalytic subunit of protein phosphatase 2A (PP2Ac) with EF-2 as a substrate exhibits the same sensitivity towards okadaic acid and insensitivity towards heparin as the EF-2 phosphatase of epidermal cytosol. The catalytic subunit of protein phosphatase 1 (PP1c) is strongly suppressed by heparin and less sensitive towards okadaic acid than PP2Ac. PP2Ac is around 50 times more efficient in dephosphorylating EF-2 than PP1c. These data indicate that the TPA-stimulated EF-2 phosphatase in epidermal cytosol is a type 2A protein phosphatase.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

Protein phosphorylation in rat cardiac microsomes: Effects of inhibitors of protein kinase A and of phosphatases

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 µM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M_r 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 µM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polyp eptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M_r 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates. (Mol Cell Biochem 175: 109–115, 1997     

Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases.

The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Regulation of Repressible Acid Phosphatase by Cyclic Amp in SACCHAROMYCES CEREVISIAE

One of the cyr 1 mutants (cyr 1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25 degrees and was unable to derepress acid phosphatase. Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity. The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity. The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase. The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis. Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase. The cyr1-2 mutant produced significantly low levels of invertase and alpha-D-glucosidase. These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.     

Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.     

Effect of calyculin A on the surface structure of unfertilized sea urchin eggs

Calyculin A, a potent inhibitor of type 1 and type 2A protein phosphatases, induces contractile ring formation when applied to unfertilized sea urchin eggs [Tosuji et al., 1992: Proc. Natl. Acad. Sci. USA 89:10613-10617]. We report here the elongation of microvilli in the unfertilized eggs exposed to calyculin A. The elongated microvilli and associated sperm-egg binding sites (egg receptor for sperm) then became concentrated into a constriction site corresponding to the cleavage furrow. The egg receptor for sperm was also in close connection to the microfilaments. Okadaic acid is another known inhibitor of protein phosphatase type-1 and type-2A. Its effect, however, is about a hundredfold feebler for type-1 phosphatase than type-2A. Even after treatment with okadaic acid, no change was observed, suggesting that these morphological changes were induced by calyculin A solely though its inhibitory effect on the type-1 protein phosphatase.