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 novel protein phosphatase inhibitor, tautomycin. Effect on smooth muscle.

The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type-1 and type-2A enzymes. For the catalytic subunits of the type-1 and type-2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca(2+)-independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation.     

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.     

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.     

Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards.

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.     

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.     

Inhibition of protein phosphatase 1 stimulates secretion of Alzheimer amyloid precursor protein.

BACKGROUND: Aberrant metabolism of the Alzheimer amyloid precursor protein (APP) or its amyloidogenic A beta fragment is thought to be centrally involved in Alzheimer's disease. Nonamyloidogenic processing of APP involves its cleavage within the A beta domain by a protease, termed alpha-secretase, and release of the large extracellular domain, termed APPS. Secretion of APPS can be stimulated by phorbol esters, activators of protein kinase C, with concurrent inhibition of A beta production. While the role of protein kinases of APP metabolism has been investigated, considerably less effort has been devoted to elucidating the role played by protein phosphatases. Okadaic acid, a protein phosphatase inhibitor, has been shown to stimulate secretion of APPS, but the identity of the phosphatase involved has not been investigated. MATERIALS AND METHODS: The secretion of APPS from COS-1 cells was measured in the absence or presence of various doses of serine/threonine-specific phosphatase inhibitors. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase involved in the control of APP secretion. RESULTS: The availability of protein phosphatase inhibitors with different relative potencies against the different types of serine/threonine-specific protein phosphatase allowed us to examine which of the four known types of protein phosphatase might be involved in the regulation of APP secretion. Both okadaic acid and calyculin A stimulated the secretion of APP from COS-1 cells in a dose-dependent manner. The half-maximal dose for stimulation of APP secretion was approximately 100-fold higher with okadaic acid than with calyculin A. CONCLUSIONS: The nearly 100-fold difference in the observed IC50 values for okadaic acid and calyculin A implicates a type 1 protein phosphatase in the control of APPS production. Protein phosphatase 1 (PP1) is known to be highly expressed in adult mammalian brain, both in neurons and glia. The identification of a specific phosphatase type in the control of APP secretion opens new avenues to the development of rational therapeutic intervention strategies aimed at the prevention and/or treatment of Alzheimer's Disease.     

Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action.

The pleiotropic nature of insulin action suggests diverse mechanisms of signal transduction for the hormone. The specific protein phosphatase inhibitor, okadaic acid, is utilized to differentiate metabolic pathways that may be regulated by phosphorylation or dephosphorylation of key enzymes. In H-35 hepatoma cells, okadaic acid inhibits insulin-stimulated glycogen synthesis with an IC50 of 400 nM. In contrast, activation of lipogenesis by insulin is inhibited with an IC50 of 50 nM okadaic acid. The toxin also inhibits stimulation of lipogenesis in these cells by the insulin-sensitive inositol glycan enzyme modulator. In isolated rat adipocytes, insulin-stimulated lipogenesis is also inhibited by okadaic acid with an IC50 of approximately 1,700 nM. The antilipolytic effect of insulin in these cells is more sensitive to okadaic acid, exhibiting an IC50 of 150 nM. Maximal activation of lipogenesis by insulin is dramatically reduced by okadaic acid with no effect on the concentration required for half-maximal activation, whereas the sensitivity of insulin-induced antilipolysis is attenuated by okadaic acid, with no apparent reduction in the maximal effect of the hormone. Taken together, these data suggest that specific phosphatases may be differentially involved in some of the metabolic pathways regulated by insulin.     

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

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.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.     

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.     

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

The regulation of carbohydrate metabolism involves changes in the phosphorylation state of enzymes. We used okadaic acid, a potent inhibitor of protein phosphatases type 2A (IC50 0.05-2 nM) and type 1 (IC50 10-20 nM) to determine the role of these phosphatases in the control of carbohydrate metabolism by insulin in rat hepatocytes. In the absence of insulin, okadaic acid caused total inhibition of glycogen synthesis at 100 nM and half-maximal inhibition at 8-9 nM. In the presence of insulin, lower concentrations of okadaic acid (to which type 2A phosphatases are sensitive) were effective at inhibiting glycogen synthesis. 2.5 nM okadaic acid caused total inhibition of the 2-fold stimulation of glycogen synthesis by insulin but had no effect on the basal unstimulated rate of glycogen synthesis. This suggests the involvement of type 2A protein phosphatases in the stimulation of glycogen synthesis by insulin. Okadaic acid (5 nM), partially suppressed but did not abolish the increase in glucokinase mRNA levels caused by insulin, indicating that dephosphorylation mechanisms may be involved in the control of glucokinase mRNA levels by insulin. It is concluded that activation of protein phosphatases type 1 and/or type 2A by insulin may have a widespread role in the control of glucose metabolism at various sites.     

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.     

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.     

Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases

The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. By comparison with protein kinases, however, there have been considerably fewer studies on the functions of serine/threonine protein phosphatases. This is partly due to a lack of specific protein phosphatase inhibitors that can be used as probes. In the present study we characterize the inhibitory effects of microcystin-LR, a hepatotoxic cyclic peptide associated with most strains of the blue-green algae Microcystis aeruginosa found in the Northern hemisphere, that proves to be a potent inhibitor of type 1 (IC50 = 1.7 nM) and type 2A (IC50 = 0.04 nM) protein phosphatases. Microcystin-LR inhibited the activity of both type 1 and type 2A phosphatases greater than 10-fold more potently than okadaic acid under the same conditions. Type 2A protein phosphatases in dilute mammalian cell extracts were found to be completely inhibited by 0.5 nM microcystin-LR while type 1 protein phosphatases were only slightly affected at this concentration. Thus, microcystin-LR may prove to be a useful probe for the study and identification cellular processes which are mediated by protein phosphatases.