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.     

Enhancement of ganglioside GM3 synthesis in okadaic-acid-treated granulosa cells.

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphorylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer (GM3) and Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer (GM1), demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12-O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.     

Regulation of Ganglioside Metabolism by Phosphorylation and Dephosphorylation

Abstract: Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 α2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t_1/2 = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems.     

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser¹⁴⁴ and at Ser³¹⁵. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser¹⁴⁴ and nearly one-half of them at Ser³¹⁵. Phosphorylation at Ser¹⁴⁴ and at Thr¹⁴⁷ of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.     

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture

Ser55 of neurofilament L (NF-L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Bovine NF-L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin-digested fragments were assigned by MALDI/ TOF (matrix-assisted laser desorption and ionization/ time-of-flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser55 rendered NF-L phosphorylated at Ser55 detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co-treatment with 10 microM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser55 by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.     

Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10–100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca²⁺ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca²⁺-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils. © 1996 Wiley-Liss, Inc.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Use of an activation-specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in rat1 cells

Rap1A and Rap1B are small GTPases of the Ras superfamily whose activation can be measured using a probe that interacts specifically with the GTP-bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyclic AMP-elevating agent forskolin activates both Rap1A and Rap1B in Rat1 cells. Whilst the protein kinase A inhibitor H89 ablated the ability of forskolin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate either Rap1A and Rap1B. Forskolin differentially activated Rap1A and Rap1B isoforms in a time- and dose-dependent manner. The cAMP-specific type 4 family phosphodiesterase inhibitor rolipram potentiated the rate of activation of both Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB but not activation of either Rap1A or Rap1B.     

Okadaic acid,sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase Fa/GSK-3α in A431 cells

Exposure of A431 cells to a rapid and sudden increase from 37°C to 46°C for 30 min could induce an increase in protein level and cellular activity of protein (kinase Fa /GSK-3α) up to ∼200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37°C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37°C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46°C for another 30 min, the heat induction on kinase Fa /GSK-3α was found to be completed blocked. In sharp contrast, when cells were first treated with 1 μM TPA at 37°C for 24 h or with 5 μM sphingosine at 37°C for 30 min to down-regulate cellular PKC, the heat induction on kinase Fa /GSK-3α was found to be reversely promoted up to ∼ 250% of control level, demonstrating that kinase Fa /GSK-3α may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase Fa /GSK-3α in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase Fa /GSK-3α, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process. © 1996 Wiley-Liss, Inc.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

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.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

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     

Increased Production of Paired Helical Filament Epitopes in a Cell Culture System Reduces the Turnover of τ

Abstract: To investigate the regulation of posttranslational modifications of τ that might be pertinent to the production of the paired helical filament (PHF) of Alzheimer's disease, we incubated human neuroblastoma cells with the protein phosphatase inhibitor okadaic acid. This treatment results in increased immunoreactivity of τ with the monoclonal antibodies Alz-50, PHF-1, T3P, and NP8, a reduction in Tau-1 immunoreactivity, and an elevation in apparent molecular weight of τ. Moreover, our data demonstrate that accumulation of phosphates in τ leads to a decrease in the turnover rate of τ in the neuroblastoma cells. It is suggested that similar build-up of hyperphosphorylated τ in the neuronal perikarya may represent an early event in PHF formation. The present system facilitates the investigation of regulatory mechanisms governing the occurrence of PHF epitopes, their effects on neuronal cell metabolism, and possible pharmacological intervention.     

Cytosolic Proteolysis of τ by Cathepsin D in Hippocampus Following Suppression of Cathepsins B and L

Abstract: Incubation of cultured hippocampal slices with an inhibitor [ N -CBZ- l -phenylalanyl- l -alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of τ protein and the production of a 29-kDa τ fragment (τ₂₉). A τ antibody that is sensitive to the phosphorylated state of its epitopes did not recognize τ proteins or the τ₂₉ fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the τ fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. τ₂₉ exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal τ-tubulin interactions. Three lines of evidence indicated that ZPAD-induced τ proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and τ at neutral pH resulted in a loss of intact τ proteins and production of a 29-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced τ proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The τ fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features.     

Okadaic acid-induced actin assembly in neutrophils: Role of protein phosphatases

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of ³²P into cellular proteins using SDS-PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca²⁺] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B₄, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid. © 1993 Wiley-Liss, Inc.