Identification and characterization of protein phosphatase 2C activation by ceramide

Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.     

Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells

In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.     

Ceramide stimulates a cytosolic protein phosphatase.

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.     

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.     

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101–11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function

Recently it has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 (Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300). In cells expressing Bcl2, dephosphorylation of Bcl2 appears to be required for ceramide-induced cell death because treatment of cells with low doses of the PP2A inhibitor okadaic acid blocks Bcl2 dephosphorylation and promotes cell survival. Furthermore, the non-phosphorylatable (i.e. PP2A-resistant) gain-of-function S70E mutant Bcl2 can protect cells from ceramide-induced apoptosis. These findings support a model whereby Bcl2 function is regulated by PP2A. PP2A is a heterotrimer that contains a catalytic C-subunit, a structural A-subunit, and a regulatory B-subunit. The A- and C-subunits are fairly conserved and ubiquitously expressed, and they form the catalytic complex of the phosphatase. In contrast, there are at least three families of diverse B-subunit molecules that vary in expression temporally and by tissue type. It is hypothesized that ceramide regulates PP2A via the B-subunit. Thus, understanding the mechanism of how PP2A regulates Bcl2 phosphorylation status and how ceramide might regulate this process requires identification of the regulatory B-subunit of PP2A that comprises the Bcl2 phosphatase. Results indicate that the B56 alpha-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 alpha associates with Bcl2 as evidenced by pull-down experiments, (b) B56 alpha co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 alpha to mitochondrial membranes, and (d) overexpression of B56 alpha promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide. These findings suggest a role for B56 alpha in regulating the Bcl2 phosphatase.     

Ceramide accelerates dephosphorylation of extracellular signal-regulated kinase 1/2 to decrease prostaglandin D(2) production in RBL-2H3 cells.

In the present study, the effect of ceramide on antigen-stimulated phosphorylation of extracellular signal-regulated kinase (ERK) in the mechanism responsible for regulating production of prostaglandin (PG) D(2) was investigated in the mast cell line, RBL-2H3 cells. Cell-permeable C(6)-ceramide (N-hexanoylsphingosine) suppressed antigen-stimulated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase. Ceramide also inhibited production of PGD(2) and an increase in the activity of cytosolic phospholipase A(2) (cPLA(2)), whereas it did not influence the tyrosine phosphorylation of major cellular proteins in response to antigen. The ceramide-induced inhibition of ERK1/2 phosphorylation and of cPLA(2) activation was suppressed by orthovanadate, a tyrosine phosphatase inhibitor, but not by okadaic acid, a serine/threonine phosphatase inhibitor. Addition of ceramide to the lysate prepared from antigen-stimulated cells reduced the phosphorylated ERK1/2, and orthovanadate effectively prevented the reduction. These results suggest that ceramide accelerates the dephosphorylation of phosphorylated ERK1/2 via activation of a protein tyrosine phosphatase, thus preventing activation of cPLA(2) and production of PGD(2).     

Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb,p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2.     

Protein phosphatase 1α mediates ceramide-induced ERM protein dephosphorylation: a novel mechanism independent of phosphatidylinositol 4, 5-biphosphate (PIP2) and myosin/ERM phosphatase

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP(2)) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP(2) hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP(2) and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1α, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1α acted as dominant negative of the endogenous PP1α. Additional results showed that the ceramide mechanism of PP1α activation is largely independent of PIP(2) hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1α and plasma membrane ceramide.     

Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells

Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 (PP-1) and 2A (PP-2A). The phosphorylation and dephosphorylation at the serine/threonine residues on proteins play important roles in regulating gene expression, cell cycle progression, and apoptosis. In this study, phosphatase inhibitor okadaic acid induces apoptosis in U937 cells via a mechanism that appears to involve caspase 3 activation, but not modulation of Bcl-2, Bax, and Bcl-X(L) expression levels. Treatment with 20 or 40 nM okadaic acid for 24 h produced DNA fragmentation in U937 cells. This was associated with caspase 3 activation and PLC-gamma1 degradation. Okadaic acid-induced caspase 3 activation and PLC-gamma1 degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 40 nM. Moreover, PMA (phorbol myristate acetate), PKC (protein kinase C) activator, protected U937 cells from okadaic acid-induced apoptosis, abrogated okadaic acid-induced caspase 3 activation, and specifically inhibited downregulation of XIAP (X-linked inhibitor of apoptosis) by okadaic acid. PMA cotreated U937 cells exhibited less cytochrome c release and sustained expression levels of the IAP (inhibitor of apoptosis) proteins during okadaic acid-induced apoptosis. In addition, these findings indicate that PMA inhibits okadaic acid-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase 3 that is involved in the execution of apoptosis.     

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.     

Protein phosphatase 2A enhances the proapoptotic function of Bax through dephosphorylation

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.     

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.     

Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo (32)P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras.     

Protein phosphatase 2A-linked and -unlinked caspase-dependent pathways for downregulation of Akt kinase triggered by 4-hydroxynonenal

We studied the signal pathways for regulation of serine/threonine protein kinase Akt in Jurkat cells that had been treated with 4-hydroxynonenal (HNE) for caspase-dependent apoptosis induction. Treatment of cells with HNE led to a decrease in the level of Akt activity due to the dephosphorylation at Ser473, a major regulatory phosphorylation site. HNE-mediated dephosphorylation of Akt was prevented by a protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and by a caspase-3 inhibitor, DEVD-CHO. HNE treatment resulted in an increase in the total level of PP2A activity, release of active tyrosine-dephosphorylated PP2A from the cytoskeleton and PP2A-Akt association, which were all dependent on caspase-3 activation. These results suggest that the level of PP2A activity is at least in part determined by its tyrosine phosphorylation, which is dually controlled by okadaic acid-sensitive phosphatases and protein-tyrosine kinases. Possibly underlying the mechanism of caspase-mediated activation of PP2A, HNE treatment resulted in downregulation of the activity of Src kinase, as a representative caspase-sensitive kinase to phosphorylate PP2A at tyrosine. In addition, activated caspase-3 partially cleaved Akt at a late stage of the apoptosis. These results indicate the existence of two distinct caspase-dependent signal pathways for downregulation of Akt that works as a mechanism of positive feedback regulation for HNE-triggered apoptotic signals.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

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

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.     

Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves: Evidence from the effects of okadaic acid and microcystin

Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PP2A). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PP1 and PP2A), while inhibitor-2 (a PP1 inhibitor) or Mg²⁺ (essential for PP2C) were ineffective. In vivo, okadaic add and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO₂ fixation. It is concluded that PP2A is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.