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.     

Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal

Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.     

Specific binding of okadaic acid, a new tumor promoter in mouse skin

The tumor promoter okadaic acid binds specifically to a particulate as well as a cytosolic fraction of various mouse tissues, e.g., skin, brain, lung and colon. The KD value was 21.7 nM for receptors in the particulate fraction and 1.0 nM for those in the cytosolic fraction of mouse skin. The specific binding of [3H]okadaic acid to the particulate fraction of mouse skin was inhibited dose-dependently by okadaic acid, but not okadaic acid tetramethyl ether, an inactive compound, or by other tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin. The results suggest a new pathway of tumor promotion mediated through the okadaic acid receptor(s).     

Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid.

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.     

Regulation of NDR2 protein kinase by multi-site phosphorylation and the S100B calcium-binding protein

Nuclear Dbf2-related (NDR) protein kinases are a family of AGC group kinases that are involved in the regulation of cell division and cell morphology. We describe the cloning and characterization of the human and mouse NDR2, a second mammalian isoform of NDR protein kinase. NDR1 and NDR2 share 86% amino acid identity and are highly conserved between human and mouse. However, they differ in expression pattern; mouse Ndr1 is expressed mainly in spleen, lung and thymus, whereas mouse Ndr2 shows highest expression in the gastrointestinal tract. NDR2 is potently activated in cells following treatment with the protein phosphatase 2A inhibitor okadaic acid, which also results in phosphorylation on the activation segment residue Ser-282 and the hydrophobic motif residue Thr-442. We show that Ser-282 becomes autophosphorylated in vivo, whereas Thr-442 is targeted by an upstream kinase. This phosphorylation can be mimicked by replacing the hydrophobic motif of NDR2 with a PRK2-derived sequence, resulting in a constitutively active kinase. Similar to NDR1, the autophosphorylation of NDR2 protein kinase was stimulated in vitro by S100B, an EF-hand Ca(2+)-binding protein of the S100 family, suggesting that the two isoforms are regulated by the same mechanisms. Further we show a predominant cytoplasmic localization of ectopically expressed NDR2.     

Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. Comparison with receptor phosphorylation and functions affected by another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate.

Okadaic acid, a potent tumor promoter and inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types. The increases are limited to phosphoserine and phosphothreonine residues. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a distinct tumor promoter and protein kinase C activator, also induces serine/threonine phosphorylation of the EGF receptor and is known to modulate receptor functions. Comparison of okadaic acid and TPA influences on the EGF receptor show significant differences. Okadaic acid did not promote phosphorylation of Thr-654, a major site of TPA-induced phosphorylation. However, other sites of phosphorylation were similar for the two tumor promoters. In vitro experiments with purified protein phosphatase 2A demonstrate the insensitivity of Thr-654 phosphorylation, which regulates EGF receptor function, to dephosphorylation by this okadaic acid-sensitive protein phosphatase. In contrast to TPA, okadaic acid did not attenuate the tyrosine kinase activity or ligand binding capacity of the EGF receptor. However, okadaic acid did produce a decrease in EGF-stimulated inositol phosphate formation in a manner distinct from that of TPA.     

Okadaic Acid Induces Hyperphosphorylation of τ Independently of Mitogen-Activated Protein Kinase Activation

Abstract: Hyperphosphorylation of the microtubule-associated protein τ is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on τ common to Alzheimer's disease. Using an okadaic acid-induced τ hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor {PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]} of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust τ hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in τ phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce τ hyperphosphorylation.     

Identification of gastric cyclic AMP binding proteins

The photoaffinity ligand 8-azidoadenosine 3',5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of 'resting' gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000 and 48 000. When gastric glands were stimulated to produce acid by the addition of 10(-4) M histamine or 1 mM dibutyryl cAMP there was 32-44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine-mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.     

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Author index

The photoaffinity ligand 8-azidoadenosine 3′,5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of ‘resting’ gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000.and 48 000. When gastric glands were stimulated to produce acid by the addition of 10^?4 M histamine or 1 mM dibutyryl cAMP there was 32–44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine- mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.     

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.     

Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain

We have reported previously the cloning and characterization of human and mouse protein kinase B gamma (PKB gamma), the third member of the PKB family of second messenger-regulated serine/threonine kinases (Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133--9136). Here we report the isolation of human and mouse PKB gamma 1, a splice variant lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic C-terminal domain. Expression of PKB gamma 1 is low compared with PKB gamma, and it is regulated in different human tissues. We show that PKB gamma and PKB gamma 1 differ in their response to stimulation by insulin, pervanadate, peroxide, or okadaic acid. Activation of PKB gamma 1 requires phosphorylation at a single regulatory site Thr-305. Interestingly, this site is phosphorylated to a higher extent in PKB gamma compared with PKB gamma 1 upon maximal stimulation by pervanadate, and this is reflected in the respective specific kinase activities. Furthermore, upon insulin stimulation of transfected cells, PKB gamma 1 translocates to the plasma membrane to a lesser extent than PKB gamma. Taken together, these results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of PKB gamma may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1.     

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.     

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets.

The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.     

The non-phorbol ester tumor promoter okadaic acid does not promote morphological transformation or inhibit junctional communication in hamster embryo cells

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.     

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.     

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.     

Ribosomal S6 kinase p90rsk and mRNA cap-binding protein eIF4E phosphorylations correlate with MAP kinase activation during meiotic reinitiation of mouse oocytes

During meiotic reinitiation of the mouse oocyte, entry into M-phase is regulated by changes of protein phosphorylation and by the stimulation of selective mRNA translation following the nuclear membrane dissolution. Our results reveal that M-phase kinases (MAP kinase and histone H1 kinase) are being activated together with S6 kinase and with the phosphorylation of eIF4E, the cap-binding subunit of the initiation factor eIF-4F. In order to test which signaling pathway(s) is(are) involved, okadaic acid and cycloheximide have been used as tools for differentially modulating MAP and histone H1 kinase activities. A role for MAP kinases in the phosphorylation of eIF4E and the activation of S6 kinase is suggested. The possible implication of p90^rsk and/or of p70^s6k in the overall increase in S6 kinase activity has been examined. p70^s6k does not appear to be involved since phosphorylated forms are found in prophase and maturing oocytes. In contrast, p90^rsk is phosphorylated and activated in maturing oocytes. p90^rsk phosphorylation correlates with the activation of S6 kinase. These results suggest that the overall increase of S6 kinase activity is mostly due to p90^rsk activation. The roles of eIF4E phosphorylation and S6 kinase activation in the physiological induction of M-phase and in the okadaic acid-induced premature mitotic events are discussed. Mol. Reprod. Dev. 46:383–391, 1997. © 1997 Wiley-Liss, Inc.