calyculin-a and staurosporine-aglycone

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.

Topics: 14-3-3 Proteins; AMP-Activated Protein Kinase Kinases; Arabidopsis; Arabidopsis Proteins; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Darkness; Deoxyglucose; Glutathione Transferase; Indole Alkaloids; Light; Marine Toxins; Nitrate Reductase; Nitrate Reductases; Oxazoles; Phosphofructokinase-2; Phosphoprotein Phosphatases; Phosphorylation; Plant Leaves; Plant Proteins; Protease Inhibitors; Protein Binding; Protein Kinase Inhibitors; Protein Kinases; Tyrosine 3-Monooxygenase

In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN3, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN3. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells.

Topics: Actins; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cytoplasm; Enzyme Inhibitors; Hydrocharitaceae; Indole Alkaloids; Marine Toxins; Myosins; Oxazoles; Phosphoprotein Phosphatases; Plant Roots; Sodium Nitrite; Thiazoles; Thiazolidines

Three genes (i.e., a zinc finger protein, a lectin-like protein, and AtMPK3), previously shown to respond to chitin elicitation in microarray experiments, were used to examine the response of Arabidopsis spp. to chitin addition. Maximum induction for all three genes was found upon addition of crab-shell chitin at 100 mg per liter. Threefold induction was found with a chitin concentration as low as 10(-4) mg per liter. The specificity of this response was examined using purified chitin oligomers (degree of polymerization = 2 to 8). The larger chitin oligomers (hexamer to octamer), were most effective in inducing expression of the three genes assayed. Gene induction was observed after the addition of 1 nM chitin octamer. The protein kinase inhibitors staurosporine and K252a effectively suppressed chitin-induced gene expression, while the protein phosphatase inhibitors calyculin A and okadaic acid induced the accumulation of mRNA in the absence of chitin. The phosphorylation event necessary for transmission of the chitin signal was completed within the first 20 min of chitin addition. The level of chitin-induced gene expression of the lectin-like protein and AtMPK3 was not significantly changed in mutants blocked in the jasmonic acid (JA, jar1)-, ethylene (ein2)-, or salicylic acid (SA, pad4, npr1, and eds5)-dependent pathway. In contrast, expression of mRNA for the zinc finger protein was reduced in the mutants affected in the JA- or SA-dependent pathway.

Topics: Arabidopsis; Arabidopsis Proteins; Carbazoles; Chitin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Plant; Indole Alkaloids; Lectins; Marine Toxins; Mitogen-Activated Protein Kinase Kinases; Okadaic Acid; Oligosaccharides; Oxazoles; Phosphorylation; Plant Proteins; RNA, Messenger; Signal Transduction; Staurosporine; Transcriptional Activation; Zinc Fingers

We studied the phosphorylation of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to beta-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by beta-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Indole Alkaloids; Indoles; Isoproterenol; Isoquinolines; Maleimides; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Parotid Gland; Peptides, Cyclic; Phenols; Phosphorus Radioisotopes; Phosphorylation; Rats; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Staurosporine; Sulfonamides; Sympathomimetics; Vanadates

Cyclic AMP down-regulates vesicular monoamine transport in PC12 cells and thereby decreased catecholamine reuptake from the extracellular fluid. We examined the effects of protein kinase inhibitors and protein phosphatase inhibitors on this cAMP action. Treatment of cells with a protein kinase inhibitor, K252a, increased vesicular amine transport and cellular amine uptake, thereby antagonizing the regulatory action of cAMP. In contrast, a protein phosphatase inhibitor, okadaic acid, had the opposite effect on the amine transport, i.e. it enhanced the cAMP action. These results suggest the involvement of a protein phosphorylation process in the cAMP-dependent modulation of vesicular monoamine transport.

Topics: Animals; Biogenic Monoamines; Biological Transport; Bucladesine; Carbazoles; Cyclic AMP; Down-Regulation; Ethers, Cyclic; Glycoproteins; Indole Alkaloids; Marine Toxins; Membrane Glycoproteins; Membrane Transport Proteins; Neuropeptides; Norepinephrine; Okadaic Acid; Oxazoles; PC12 Cells; Pheochromocytoma; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Rats; Serotonin; Vesicular Biogenic Amine Transport Proteins

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.

Topics: Actins; Animals; Blotting, Western; Carbazoles; Cell Nucleus; Cells, Cultured; Cytoskeleton; Endothelium, Vascular; Enzyme Inhibitors; Humans; Indole Alkaloids; Marine Toxins; Mice; Microtubules; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinase Inhibitors; Protein Phosphatase 1; Umbilical Veins

The specific phosphatase inhibitor, Calyculin-A (CL-A), decreases high-K stimulated catecholamine secretion in bovine chromaffin cells. This effect can be split into two components: one needs long exposures to the drug to be elicited, and is sensitive to the protein kinase-inhibitor K252a; the other is observed after short incubations of CL-A, and is insensitive to K252a. Here we report that the latter component is due to an external block, by CL-A, of chromaffin cell calcium channels in a voltage-dependent, reversible and phosphorylation-independent manner.

Topics: Adrenal Medulla; Animals; Calcium Channel Blockers; Calcium Channels; Carbazoles; Cattle; Cells, Cultured; Indole Alkaloids; Marine Toxins; Membrane Potentials; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C

Na(+)-K(+)-2Cl- cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na(+)-K(+)-2Cl- cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.

Topics: Animals; Aorta; Carbazoles; Carrier Proteins; Cattle; Cells, Cultured; Endothelium, Vascular; Ethers, Cyclic; Indole Alkaloids; Marine Toxins; Okadaic Acid; Oxazoles; Phosphorylation; Protein Kinase C; Proteins; Sodium-Potassium-Chloride Symporters; Vasoconstrictor Agents