The roles of macromolecular synthesis and phosphorylation in the regulation of a protein kinase activity transiently stimulated by nerve growth factor

Past work identified and characterized an apparently novel protein kinase activity (designated HMK) that is highly and transiently stimulated in PC12 pheochromocytoma cells by nerve growth factor (NGF). In vitro, HMK phosphorylates both high molecular weight microtubule-associated proteins and myelin basic protein. This study investigates the potential mechanisms of HMK regulation in intact PC12 cells and reveals the following. 1) HMK activation is independent of macromolecular synthesis while the subsequent post-induction suppression requires both RNA and protein synthesis. 2) Neither cAMP-dependent nor Ca2+/phospholipid-dependent protein kinases appear to play a role in regulation of HMK activity by NGF. 3) In vitro, HMK activity is inactivated by protein phosphatase 2A. 4) In vivo, HMK activation by NGF is inhibited by the kinase inhibitor, K-252a. (5) Vanadate, a tyrosine phosphatase inhibitor, induces HMK activity in intact cells, while okadaic acid, a serine/threonine phosphatase inhibitor, is much less efficacious. 6) Application of okadaic acid to vanadate-pretreated cells synergistically stimulates HMK activity to a level comparable to that achieved with NGF. (7) Activation of HMK by NGF is not significantly affected when cells are pretreated with okadaic acid. However, the subsequent NGF-promoted deactivation of HMK is greatly accelerated by okadaic acid. (8) NGF down-regulated HMK activity can be heterologously restimulated by exposure to vanadate and okadaic acid. These data suggest that phosphorylation plays a critical role in both the up- and down-regulation of HMK activity in NGF-treated cells. Moreover, suppression of HMK activity requires ongoing macromolecular synthesis and appears to occur by inactivation rather than degradation.     

Increased activity of the eEF-2 specific, Ca2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells.

The activity of the eukaryotic elongation factor (eEF-2) specific, Ca2+ and calmodulin dependent protein kinase III (CaM PK III) was studied in homogenated Ehrlich ascites tumour cells. The eEF-2 kinase activity was determined in the presence of an excess of the substrate, i.e., non-limiting concentrations of eEF-2. The homogenates showed both kinase and phosphatase activity. The latter activity was inhibited by the protein phosphatase 2A inhibitor okadaic acid. Analysis of the kinase using cells from defined stages of the cell cycle showed that the highest activity was found in cells from the early S-phase, whereas the phosphatase activity was most pronounced during the G2+M phase.     

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion.

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.     

Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid.

The involvement of protein phosphatases in regulating platelet activation was studied. The major portion of the phosphorylase phosphatase activity found in platelet lysates appears to be of the type 1 variety. The identification of this enzyme was based on the finding that greater than 80% of protein phosphatase activity was inhibited by the heat-stable inhibitor protein inhibitor 2 and, while only 20% of the phosphorylase phosphatase activity in platelet extracts was inhibited by 2 nM okadaic acid, greater than 95% of the activity was inhibited in the presence of 1 microM okadaic acid. Increases in protein phosphorylations occurred and thrombin-induced release of serotonin was prevented as a result of artificially inhibiting the enzyme with okadaic acid in intact platelets. This implies either that the regulation of okadaic acid sensitive protein phosphatases is necessary for some agonist-induced effects or that okadaic acid sensitive phosphatases are required for maintaining platelets in a responsive state.     

Protein phosphatases regulate DNA-dependent protein kinase activity

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.     

Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1

Cardiomyocyte-derived cell lines deficient in p38alpha are more resistant to apoptosis owing to lower expression of the pro-apoptotic proteins Bax and Fas and upregulation of the ERK survival pathway. Here, we show that increased Akt activity also contributes to the enhanced survival of p38alpha-deficient cardiomyocytes. We found that the serine/threonine phosphatase PP2A can be targeted to caveolae through interaction with caveolin-1 in a p38alpha-dependent manner. In agreement with this, PP2A activity associated with caveolin-1 was higher in wild type than in p38alpha-deficient cells. Akt was also present in caveolae and incubation of wild-type cells with the PP2A inhibitor okadaic acid increases the levels of Akt activity. Thus, p38alpha-induced re-localization of PP2A to caveolae can lead to dephosphorylation and inhibition of Akt, which in turn would contribute to the decreased survival observed in wild type cells. However, cell detachment impairs the formation of the PP2A/caveolin-1 complex and, as a consequence, phospho-Akt levels and survival are no longer regulated by p38alpha in detached wild type cardiomyocytes. Our results suggest that p38alpha can negatively modulate Akt activity, independently of PI3K, by regulating the interaction between caveolin-1 and PP2A through a mechanism dependent on cell attachment.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

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.     

Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2.

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.     

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.     

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro

Interphase Xenopus egg extracts form extensive tubular membrane networks in vitro. These networks are identified here as endoplasmic reticulum by the presence of ER resident proteins, as shown by immunofluorescence, and by the presence of single ribosomes and polysomes, as shown by electron microscopy. The effect of phosphorylation on ER movement in interphase was tested using the phosphatase inhibitor, okadaic acid. Okadaic acid treatment resulted in an increase of up to 27-fold in the number of ER tubules moving and in the extent of ER networks formed compared to control extracts. This activation was blocked by the broad-specificity kinase inhibitor 6- dimethylaminopurine. Okadaic acid had no effect, however, on the direction of ER tubule movement, which occurred towards the minus end of microtubules, and was sensitive to low concentrations of vanadate. Inhibition of phosphatases also had no effect on the speed or duration of ER tubule extensions, and did not stimulate the activity of soluble cytoplasmic dynein. The sensitivity of ER movement to okadaic acid closely matched that of protein phosphatase 1. Although the amount of ER motility was greatly increased by inhibiting protein phosphatase 1 (PP1), the amount of cytoplasmic dynein associated with the membrane was not altered. The data support a model in which phosphorylation regulates ER movement by controlling the activity of cytoplasmic dynein bound to the ER membrane.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

Cell cycle-dependent regulation of telomerase activity by auxin, abscisic acid and protein phosphorylation in tobacco BY-2 suspension culture cells

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats onto the ends of linear eukaryotic chromosomes. It was recently reported that the low, basal level of telomerase activity markedly increased at early S-phase of the cell cycle, and auxin further increased the S-phase-specific telomerase activity in tobacco BY-2 cells. In this study we show that abscisic acid (ABA), a phytohormone known to induce the cyclin-dependent protein kinase inhibitor, effectively abolished both the auxin- and S-phase-specific activation of telomerase in a concentration- and time-dependent fashion in synchronized tobacco BY-2 cells. These results suggest that there exists a hormonal cross-talk between auxin and ABA for the regulation of telomerase activity during the cell cycle of tobacco cells. Treatment of synchronized BY-2 cells with the protein kinase inhibitor staurosporine or H-7 effectively prevented the S-phase-specific activation of telomerase activity. By contrast, when okadaic acid or cantharidin, potent inhibitors of protein phosphatase 2A (PP2A), was applied to the cells, the S-phase-specific high level of telomerase activity was continuously maintained in the cell cycle for at least 14 h after release from M-phase arrest. Incubation of tobacco cell extracts with exogenous PP2A rapidly abrogated in vitro telomerase activity, while okadaic acid and cantharidin blocked the action of PP2A, effectively restoring in vitro telomerase activity. Taken together, these findings are discussed in the light of the suggestion that antagonistic functions of auxin and ABA, and reciprocal phosphorylation and dephosphorylation of telomerase complex, are necessarily involved in the cell cycle-dependent modulation of telomerase activity in tobacco cells.     

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.     

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.     

Okadaic acid suppresses calcium regulation of mitosis onset in sea urchin embryos.

We show that a phosphatase inhibitor, okadaic acid, induces premature and persistent mitosis during the first cell cycle in sea urchin embryos. Okadaic acid-induced mitosis requires protein synthesis, suggesting that it activates the protein synthesis-requiring mitotic H1 kinase. By microinjecting the calcium chelators BAPTA and EGTA and by measuring Cai using fura-2, an indicator dye, we show that okadaic acid-induced mitosis is independent of the calcium signal that usually triggers mitosis onset in sea urchin embryos. Disabling the calmodulin kinase II that is thought to respond to the mitotic Cai signal using a peptide inhibitor fails to prevent mitosis in response to okadaic acid. These data suggest that okadaic acid bypasses calcium regulation of mitosis by inducing constitutive phosphorylation of a site on the H1 kinase that is normally under the control of the calmodulin-regulated kinase.