Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-³H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K_d of 1.30 ± 0.56 nM and 1.54 ± 0.35 nM, respectively. Binding of [24-³H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC₅₀ of 92 ± 8.4 nM and 87 ± 13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.     

Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.     

Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response

Microcystins, potent heptapeptide hepatotoxins produced by certain bloom-forming cyanobacteria, are strong protein phosphatase inhibitors. They covalently bind the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), thereby influencing regulation of cellular protein phosphorylation. The paralytic shellfish poison, okadaic acid, is also a potent inhibitor of these PPs. Inhibition of PP1 and PP2A has a dualistic effect on cells exposed to okadaic acid or microcystin-LR, with both apoptosis and increased cellular proliferation being reported. This review summarises the existing data on the molecular effects of microcystin-LR inhibition of PP1 and PP2A both in vivo and in vitro, and where possible, compares this to the action of okadaic acid.     

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants

The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

Inhibition of antigen and calcium ionophore induced secretion from RBL-2H3 cells by phosphatase inhibitors

The role of serine/threonine protein phosphatases PP1 and PP2A in mast cell secretion was investigated using the phosphatase inhibitors okadaic acid and calyculin A. Calyculin A (5-25 nm) inhibited antigen-induced secretion from a rat mucosal mast cell line (RBL-2H3) when added in conjunction with the activator. Okadaic acid (250-1000 nm) inhibited secretion only when added before activation and did so in a time- and concentration-dependent manner. Both inhibitors caused the cells to become rounder, but only calyculin A induced membrane blebbing and a loss of adherence. Okadaic acid also inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA, indicating that the phosphatase inhibitors act on a component of the secretory pathway downstream of calcium mobilization. Okadaic acid increased the phosphorylation of a number of proteins, as did an analogue methyl okadaate, which also inhibited secretion, but less effectively. Okadaic acid induced the phosphorylation of triton-insoluble proteins of 55, 18 and 16 kDa. The 55 kDa protein was identified as vimentin and okadaic acid induced its partial translocation to the triton-soluble fraction. Our data indicate that full secretory function in mucosal mast cells requires phosphatase activity.     

Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.     

Complementation of the cs dis2-11 cell cycle mutant of Schizosaccharomyces pombe by a protein phosphatase from Arabidopsis thaliana.

The activities of type I protein phosphatases play a central role in eukaryotic cell cycle control. Here, we report the cloning and characterization from the flowering plant Arabidopsis thaliana of a cDNA clone named PP1-At which is highly homologous to protein phosphatase 1. The deduced amino acid sequence of PP1-At shows that the PP1-At protein is 318 amino acid residues long and has a molecular weight of 35,298 Da. The PP1-At protein has strong similarity to all other known protein phosphatase type 1 catalytic subunits. Approximately 62% of the amino acids are identical to type 1 protein phosphatases of rabbit, mouse, Saccharomyces cerevisiae and Schizosaccharomyces pombe. RNA blot analysis revealed a single mRNA species of approximately the same size as the cDNA isolated. The PP1-At-encoded mRNA of 1.3 kb is abundant in most vegetative Arabidopsis tissues, with the lowest level of expression in leaves. When transferred to the fission yeast S.pombe, the PP1-At-encoded protein can rescue a semidominant mutant, cold sensitive (cs) dis2-11, which under nonpermissive conditions is unable to complete chromosome disjunction.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.     

Construction of a metagenomic library for the marine sponge Halichondria okadai

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.     

Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter.

We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest.     

Lectin, a possible basis for symbiosis between bacteria and sponges.

From the marine sponge Halichondria panicea a lectin was isolated and characterized. The homogeneous lectin (composed of protein to 80.7% and of neutral carbohydrates to 14.1%) had a molecular weight of 78,000 (determined by gel filtration) and consisted of four subunits with a molecular weight of 21,000 each (determined by gel electrophoresis in the presence of sodium dodecyl sulfate). The hemagglutinating activity was only slightly dependent upon ionic strength and incubation temperature and did not require divalent cations, but it was inhibited by reagents for thiol groups. The Halichondria lectin was completely inhibited in hemagglutination competition experiments in the presence of fetuin, D-galacturonic acid, D-glucuronic acid, polygalacturonic acid, or L-fucose. The purified Halichondria lectin did not cause reaggregation of dissociated H. panicea cells. From the same sponge species bacteria were isolated and identified as Pseudomonas insolita. These bacteria were cultivated in marine broth 2216. Under these culture conditions the bacteria grew only in the presence of the homologous lectin; the lectin-caused effect was not abolished by D-glucuronic acid or D-galacturonic acid. However, after addition of a polysaccharide-containing fraction isolated from P. insolita, the lectin-caused, growth-promoting effect was abolished. Other lectins were found to exhibit no growth-promoting effect. On the basis of colony counts, P. insolita was the predominant bacterial species in the sponge extract; 1.9 X 10(6) Pseudomonas colonies were measured in extracts isolated from 1 g of sponge. The assumption of an interrelationship between the sponge and the bacterium is supported by the results indicating that the Halichondria lectin has no effect on the growth of such bacteria isolated from six other marine sponge species. Evidence is presented which indicates that the Halichondria lectin is not utilized during growth of the Pseudomonas species. Lectin activity was detected on the surface of mucoid cells from H. panicea. From the data obtained the possibility is discussed that the Halichondria lectin is a basis for a symbiotic relationship between the sponge and the bacterium.     

Protein phosphatase regulation of Na+/H+ exchanger isoform I

We investigated regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) by dephosphorylation. Treatment of primary cultures of cardiomyocytes with the phosphatase inhibitor okadaic acid increased the rate of recovery from an acid load, suggesting that the okadaic acid sensitive PP1 may be involved in NHE1 regulation in vivo. We examined the ability of purified protein phosphatases PP1, PP2A, and PP2B to dephosphorylate the regulatory cytoplasmic tail. NHE1 was completely dephosphorylated by PP1, poorly dephosphorylated by PP2A, and not dephosphorylated by PP2B. Examination of NHE1 binding to PP1 or PP2B revealed that an association occurs between NHE1 and PP1 both in vitro and in vivo, but NHE1 did not associate with full-length PP2B. We expressed PP1 or inhibitor 2, a specific PP1 inhibitor, in cell lines to examine the effect of PP1 on NHE1 activity in vivo. Overexpression of PP1 causes a decrease in NHE1 activity but does not affect stimulation by thrombin. Cell lines expressing the specific PP1 inhibitor, inhibitor 2, had elevated proton efflux rates and could not be further stimulated by the Na(+)/H(+) exchanger agonist thrombin. The results suggest that PP1 is an important regulatory phosphatase of NHE1, that it can bind to and dephosphorylate the protein, and that it regulates NHE1 activity in vivo.     

Importance of the C28-C38 hydrophobic domain of okadaic acid for potent inhibition of protein serine-threonine phosphatases 1 and 2A

Okadaic acid is a potent inhibitor of select serine/threonine protein phosphatases. The importance of the C28-C38 hydrophobic domain of okadaic acid for inhibition of PP1 and PP2A was investigated. The hydrophobic domain is required but not sufficient for potent inhibition, and it also contributes to differential inhibition between PP1 and PP2A.     

Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A, and is a strong tumor promoter that is not an activator of protein kinase C. Treatment of quiescent cultures of rat fibroblastic 3Y1 cells with okadaic acid induced marked activation of a kinase activity that phosphorylated microtubule-associated protein (MAP) 2 and myelin basic protein, but not histone or casein, in vitro. This activated kinase eluted at approximately 0.15 M NaCl on a DEAE-cellulose column and its apparent molecular mass was determined to be approximately 40 kDa by gel filtration. Detection of the kinase activity in polyacrylamide gels containing substrate proteins after sodium dodecyl sulfate gel electrophoresis revealed that the okadaic-acid-activated kinase activity resided mainly in two closely related polypeptides with apparent molecular mass approximately 40 kDa. The characteristics of this kinase were indistinguishable from those of the mitogen-activated MAP kinase in the same cells. The okadaic-acid-activated MAP kinase was deactivated by protein phosphatase 2A treatment in vitro. These results suggest that MAP kinase is negatively regulated by protein phosphatases 1 and/or 2A in quiescent cells and therefore can be activated by inhibiting these protein phosphatases. Interestingly, the okadaic-acid-induced activation of MAP kinase was transient and epidermal-growth-factor-induced activation was also transient, even in the presence of okadaic acid. These data may imply that protein phosphatases 1 and 2A are not involved in the deactivation of MAP kinase in cells.