Okadaic Acid-sensitive Protein Phosphatases Research Articles

Systemic inflammation suppresses spinal respiratory motor plasticity via mechanisms that require serine/threonine protein phosphatase activity

BackgroundInflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system—a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)—a key enzyme necessary for the expression of phrenic motor plasticity.MethodsTo study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons.ResultsWe confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 μg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators.ConclusionsThis study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 μM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca 2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca 2+] cyt) in guard cells. While abscisic acid-induced [Ca 2+] cyt oscillation has been extensively studied, MeJA-induced [Ca 2+] cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca 2+] cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca 2+ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca 2+] cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca 2+] cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.

Glycogen synthase kinase‐3β indirectly facilitates interferon‐γ‐induced nuclear factor‐κB activation and nitric oxide biosynthesis

Either glycogen synthase kinase (GSK)-3β or nuclear factor (NF)-κB regulates interferon (IFN)-γ-induced nitric oxide (NO) biosynthesis; however, the inter-regulation between GSK-3β and NF-κB is unknown. We have previously shown that IFN-γ-activated GSK-3β negatively regulates Src homology-2 domain-containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 1 activation. Because Jaks-IFN-inducible dsRNA-activated serine-threonine protein kinase (PKR) axis signaling is essential for IFN-γ-activation of NF-κB, in this study we investigate the potential mechanism for GSK-3β-facilitated NF-κB activation in IFN-γ-stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK-3β or NF-κB signaling, such as the inhibitor of κB (IκB) kinase β (IKKβ) and IκBα, inhibited IFN-γ-induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK-3β decreased IFN-γ-induced NF-κB phosphorylation (Ser536) and activation. The upstream regulators for GSK-3β activation, including okadaic acid-sensitive protein phosphatase and proline-rich tyrosine kinase 2, were also important for IFN-γ-induced IκBα phosphorylation (Ser32) and degradation. Under IFN-γ stimulation, Jak2-PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK-3β caused SHP2-mediated dephosphorylation of PKR (Thr446), IKKβ (Ser180), and NF-κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK-3β indirectly facilitates IFN-γ-induced NF-κB activation by inhibiting SHP2, in turn activating the PKR-IKKβ-IκBα axis signaling pathway.

Okadaic Acid-Sensitive Protein Phosphatases Constrain Phrenic Long-Term Facilitation after Sustained Hypoxia

Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined.

2-hydroxyestradiol-17β-induced oocyte maturation: involvement of cAMP–protein kinase A and okadaic acid-sensitive protein phosphatases,and their interplay in oocyte maturation in the catfishHeteropneustes fossilis

In Heteropneustes fossilis, in vitro incubation of postvitellogenic follicles with 2-hydroxyestradiol-17beta (2-OHE2, 5 micromol l(-1)) decreased significantly the total cAMP level, concomitant with germinal vesicle breakdown (GVBD). The incubation of the follicles with cAMP or cAMP-elevating drugs [phosphodiesterase (PDE) inhibitors], such as IBMX (3-isobutyl-1-methyl-xanthine), theophylline and caffeine, inhibited the 2-OHE2-induced GVBD in a concentration-dependent manner. The magnitude of the response varied: both cAMP and IBMX were effective at all concentrations (0.1-2.0 mmol l(-1)), followed by theophylline (0.5-2.0 mmol l(-1)) and caffeine (1-2.0 mmol l(-1)). The protein kinase A (PKA) inhibitor H89 stimulated oocyte maturation in a concentration-dependent manner. However, when co-incubated with 2-OHE2 for 24 h it produced a biphasic effect: low concentrations (0.1 and 1.0 micromol l(-1)) did not alter the 2-OHE2-induced GVBD, but high concentrations (5 and 10 micromol l(-1)) inhibited it. The incubation of the follicles with H89 lowered the inhibitory effect of IBMX on the 2-OHE2-induced GVBD. The incubation of the follicles with okadaic acid (OA), a protein phosphatase 1 and 2A inhibitor did not affect GVBD but when co-incubated with 2-OHE2, it enhanced the GVBD response. OA reversed the inhibitory effect of IBMX. The results suggest that OA may overcome the inhibition of 2-OHE2-induced GVBD by IBMX at a step distal to the cAMP-PKA pathway.

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H 2O 2 was determined in Caco-2 cell monolayer. Incubation with H 2O 2 (20 μM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170–250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H 2O 2. However, H 2O 2 reduced 80–85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H 2O 2 reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H 2O 2-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H 2O 2 inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.

Synucleins Are a Novel Class of Substrates for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.

Dexamethasone Differentially Regulates Cytokine Transcription and Translation in Macrophages Responding to Bacteria or Okadaic Acid

Many microorganisms and microbial products induce expression of pro-inflammatory cytokines such as interleukin-1 (IL-1α/β) and tumour necrosis factor-α (TNF-α) in macrophages, primarily by transcriptional activation. We show here, by using mouse macrophages in primary culture, that pre-treatment with dexamethasone inhibits bacteria-induced IL-1β expression as mRNA and cellular pro-IL-1β in parallel, consistent with an effect primarily on transcriptional activation. In contrast, the expression of TNF-α mRNA was only partly inhibited despite virtually complete inhibition of TNF-α protein formation. Furthermore, the selective induction of primarily cell-associated 26,000 M r pro-TNF-α by the protein phosphatase inhibitor okadaic acid also was partly inhibited at the mRNA level by dexamethasone, whereas additional translational inhibition appeared to be lacking. This latter finding is reminiscent of earlier findings regarding signalling to activation of cytosolic phospholipase A 2, which is sensitive to dexamethasone when elicited by bacteria, but not when elicited by okadaic acid. The present results raise the possibility that the inhibitory effect of dexamethasone on TNF-α translation, but not on transcriptional activation, is mediated by one or more okadaic acid-sensitive protein phosphatases.

Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes.

We investigated the effect of carbachol (CCh) on L-type Ca2+ current (ICa(L)) enhanced by dialyzed adenosine 3',5'-cyclic monophosphate (cAMP) and/or bath-applied 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes. At pipette concentrations ([cAMP]pip) from 30 microM to 1 mM, cAMP increased ICa(L) to 25.8 +/- 0.9 microA/cm2 (682 +/- 24.8% increase above control). CCh (100 microM) did not inhibit ICa(L) at any [cAMP]pip. IBMX, a nonselective phosphodiesterase (PDE) inhibitor, increased ICa(L) maximally at 300 microM IBMX (17.9 +/- 0.7 microA/cm2; 449 +/- 20% increase). CCh (100 microM) inhibited ICa(L) by 92 +/- 9.5% at 30 microM IBMX and 78 +/- 4.6% at 100 microM IBMX; this effect was reduced or absent at higher IBMX concentrations (300 and 1,000 microM). Coadministration of cAMP and IBMX also progressively suppressed inhibition by CCh. CCh had a negligible effect on ICa(L) at 750 microM IBMX in the absence of pipette cAMP and at 50 microM IBMX in the presence of 100 microM [cAMP]pip. ACh-activated K+ current (IK(ACh)) was unchanged in atrial myocytes dialyzed with 100 microM cAMP; this excludes a phosphorylation-dependent desensitization of the muscarinic receptor (mAChR) or Gi by cAMP. LY83583 (100 microM), an inhibitor of cyclic guanosine monophosphate (cGMP) production, attenuated inhibition of ICa(L) by CCh in the presence of IBMX. 8-Bromo-cGMP (8-Br-cGMP), an activator of cGMP-dependent protein kinase (PKG), mimicked CCh in its actions on ICa(L) raised by both cAMP (no significant change) and IBMX (49 +/- 5.1% inhibition). Okadaic acid, an inhibitor of type 1 and 2A phosphatases, blocked inhibition of IBMX-stimulated ICa(L) by either CCh or 8-Br-cGMP. Thus the ability of CCh to inhibit ICa(L) appears caused by cGMP/PKG activation of an okadaic acid-sensitive protein phosphatase, and elevated levels of cAMP protect against this action.

Regulatory Mechanisms of Calponin Phosphorylation in Endothelin-1-induced Contraction of Porcine Coronary Artery

Calponin is an actin-associated protein that appears to play an auxiliary regulatory role in the contraction of smooth muscle. We report here on the mechanisms for regulation of calponin phosphorylation in the endothelin-1-induced contraction of porcine coronary artery. Treatment of strips of porcine artery with endothelin-1 increased calponin phosphorylation and contraction in a concentration-dependent manner. The time course of the phosphorylation was biphasic, with the response to endothelin-1. The extent of phosphorylation reached a maximum within 5 min of stimulation with 10−7M endothelin-1 and then it declined rapidly to reach a minimum at 20 min. A potent inhibitor of protein kinase C, GF109203X, inhibited both calponin phosphorylation and contraction that were induced by endothelin-1 at 5 min, without an inhibition for myosin light chain phosphorylation. Protein phosphatase inhibitor, okadaic acid, had no effect on the extent of phosphorylation at 5 min, but it significantly inhibited the subsequent decrease in calponin phosphorylation. In contrast, in PDBu-treated strips of coronary artery, okadaic acid caused a significant steady increase of the extent of calponin phosphorylation. Our results suggest that calponin phosphorylation might be regulated by protein kinase C and okadaic acid sensitive protein phosphatases, in the endothelin-1-induced contraction of porcine coronary artery.

Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin.

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivo showed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

Immunohistochemical identification of Okadaic Acid (OA) sensitive Protein Phosphatases (PP), PPl and PP2A, in murine uterine tissue

Immunohistochemical identification of Okadaic Acid (OA) sensitive Protein Phosphatases (PP), PPl and PP2A, in murine uterine tissue

Inhibition of the Iron-induced ZmFer1 Maize Ferritin Gene Expression by Antioxidants and Serine/Threonine Phosphatase Inhibitors

Two pathways have been implicated in the regulation of maize ferritin synthesis in response to iron. One of them involves the plant hormone abscisic acid (ABA) and controls the expression of ZmFer2 gene(s). Another pathway, ABA-independent, has been characterized in a de-rooted maize plantlet system and involves an oxidative step. The ZmFer1 maize ferritin gene is not regulated by ABA, and it is shown in this paper that the corresponding mRNA accumulates in de-rooted maize plantlets and BMS (Black Mexican Sweet) maize cell suspension cultures in response to iron via the oxidative pathway described previously. To investigate ZmFer1 gene regulation further, the BMS cell system has been used to develop a transient expression assay using a ZmFer1-beta-glucuronidase fusion. Both iron induction and antioxidant inhibition of ZmFer1 gene expression were observed in this system. Using Northern blot analysis and transient expression experiments, it was shown that both okadaic acid and calyculin A, two serine/ threonine phosphatase inhibitors, specifically inhibit ZmFer1 gene expression. These data indicate that an okadaic acid-sensitive protein phosphatase activity is involved in the regulation of the ZmFer1 ferritin gene in maize cells, and this activity is required for iron-induced expression of this gene.

Characterization of ion channel modulator effects on ABA- and malate-induced stomatal movements: strong regulation by kinase and phosphatase inhibitors, and relative insensitivity to mastoparans

In the present study abscisic acid-induced stomatal closing, and malate effects on stomatal apertures were analysed in the presence of guard cell ion channel regulators. A recent study has suggested that abscisic acid (ABA) activation of protein kinases and/or inhibition of protein phosphatases may be central to activation of guard cell slow anion channels and mediation of stomatal closing in Vicia faba (Schmidt et al., 1995). These findings were confirmed and extended in the present study showing that both in Vicia faba and in Commelina communis ABA-induced stomatal closings were abolished by kinase inhibitors and enhanced by the protein phosphatase inhibitor okadaic acid. Further detailed studies demonstrate that very high 40 mM extracellular malate concentrations are required to close stomata only partially and that okadaic acid also enhances malate-induced stomatal closing. In addition, when stomata are widely opened, even at 40 mM malate concentrations, no malate effect on stomatal apertures was observed. This finding may be explained by a complete inactivation of guard cell anion channels when stomatal apertures are opened very widely and suggests that extracellular malate cannot function as a primary CO(2) signal in stomatal regulation. The G-protein regulators mastoparan and mas7 as well as neomycin showed no significant effects on light-induced stomatal opening and ABA-induced stomatal closing. Findings reported here correlate closely to recent findings on slow anion channel regulation in guard cells and support the hypothesis that activation of these anion channels by phosphorylation events and complete inactivation by dephosphorylation events is a rate-limiting component in guard cell signal transduction. Furthermore, the presented data support a model in which ABA-activation of protein kinases and/or inhibition of okadaic acidsensitive protein phosphatases is central to ABA regulation of stomatal movements in Vicia faba and Commelina communis.

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of protein phosphatases, inhibited blue light (BL)-dependent H + pumping in Vicia guard cell protoplasts at half-inhibitory concentrations of 4.5 nM and 400 nM, respectively. Light-induced stomatal opening in Vicia epidermis was completely suppressed by CA at 100 nM and by OA at 1 fiM. These results suggest that CA- and OA-sensitive protein phosphatase is involved in the BL response of stomatal guard cells.

Inhibition of thymidylate synthase and thymidine kinase by okadaic acid in regenerating rat liver after partial hepatectomy

The effects of okadaic acid, a potent and specific inhibitor of protein phosphatases 1 and 2A, on liver regeneration after partial (70%) hepatectomy were investigated. The injection of okadaic acid (25 μg/kg body weight) inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 hr after partial hepatectomy, with a concomitant reduction in DNA content. Northern blot analysis showed that the suppression of thymidylate synthase and thymidine kinase activities was caused by comparable decreases in their mRNA levels. The protein levels of thymidylate synthase and thymidine kinase were confirmed by immunoblotting assay to be proportional to the activity and mRNA levels. These findings suggest that okadaic acid-sensitive protein phosphatases are involved in transcriptional control of the dTMP-synthesizing enzymes during liver regeneration.

Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A.

Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phosphatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases. We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M- to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.