calyculin-a and fostriecin

Topics: Alkenes; Antibiotics, Antineoplastic; Antifungal Agents; Cantharidin; Crystallography, X-Ray; Cyclosporine; Enzyme Inhibitors; Humans; Microcystins; Models, Molecular; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds; Structure-Activity Relationship

Topics: Alkenes; Animals; Antifungal Agents; Cantharidin; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds

We recently reported that p38 MAPK regulates TNF-induced endothelial apoptosis via phosphorylation and downregulation of Bcl-xL. Here, we describe that such apoptosis includes p38 MAPK-mediated, protein phosphatase 2A (PP2A)-dependent, downregulation of the MEK-ERK pathway. Inhibition of PP2A with fostriecin or calyculin A significantly increased MEK phosphorylation, as did exposure to the p38 MAPK inhibitor SB203580. Inhibition of MEK potentiated TNF-induced caspase-3 activity and cell death, and both those events were suppressed by treatment with fostriecin or calyculin A. Immunoprecipitation experiments revealed an association between p38 MAPK, PP2A and MEK, and the results of a phosphatase assay suggested that PP2A is a downstream target of p38 MAPK. Importantly, phosphorylation of Bad at Ser-112 was found to be regulated by p38 MAPK and PP2A. In summary, the present findings indicate a novel p38 MAPK-mediated apoptosis pathway, involving activation of Bad via PP2A-dependent inhibition of the MEK-ERK pathway.

Topics: Alkenes; Apoptosis; bcl-Associated Death Protein; Cell Line; Cell Survival; Cells, Cultured; Down-Regulation; Endothelial Cells; Humans; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Marine Toxins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Phosphatase 2; Pyridines; Pyrones; Signal Transduction; Tumor Necrosis Factor-alpha

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43-46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).

Topics: Alkenes; Animals; Blotting, Western; Cells, Cultured; Connexin 43; Gap Junctions; Gene Expression; Ischemia; Marine Toxins; Myocytes, Cardiac; Okadaic Acid; Oxazoles; Phosphorylation; Polyenes; Pyrones; Rats; Rats, Sprague-Dawley

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

Topics: Alkenes; Animals; Binding Sites; Calcineurin Inhibitors; Calcium; Cholecystokinin; Cyclic AMP; Cyclosporine; DNA-Binding Proteins; Enzyme Inhibitors; Marine Toxins; Okadaic Acid; Oxazoles; Pancreas; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Polyenes; Pyrones; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factors

A wide range of serine/threonine protein phosphatase (PP) inhibitors were studied for effects on the immunoglobulin E (IgE)-mediated release of histamine from human lung mast cells, human skin mast cells and basophils. Okadaic acid (OA) inhibited the release of histamine from all three cell types in a concentration-dependent manner. Two structural analogues of okadaic acid, okadaol and okadaone, known to be less active than the parent molecule as inhibitors of PP, were less active than okadaic acid as inhibitors of histamine release in these three cell types. A number of PP inhibitors, showing differences in selectivity for PP1 and PP2A, were also evaluated. Calyculin, which is roughly equipotent as a PP1 and PP2A inhibitor, attenuated the release of histamine from all three cell types. Similarly, tautomycin (TAU), which shows greater selectivity for PP1 over PP2A, was also effective at inhibiting histamine release in all three cell types. In contrast, fostriecin, which is very much more potent as an inhibitor of PP2A over PP1, was ineffective as an inhibitor in all three cell types. These data indicate that the regulation of mediator release by PPs is similar in lung mast cells, skin mast cells and basophils. Moreover, the data suggest that PP1 is important in the control of cellular activity.

Topics: Alkenes; Antifungal Agents; Basophils; Enzyme Inhibitors; Histamine Release; Humans; Immunoglobulin E; Lung; Marine Toxins; Mast Cells; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Skin; Spiro Compounds

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.

Topics: Adenosine Triphosphate; Alkenes; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ischemic Preconditioning, Myocardial; Marine Toxins; Mitogen-Activated Protein Kinases; Myocardial Ischemia; Myocardium; Naphthalenes; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Kinase C; Pyrones; Rabbits