calyculin-a and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Sphingosine (SPH) is an important bioactive lipid involved in mediating a variety of cell functions including apoptosis. However, the signaling mechanism of SPH-induced apoptosis remains unclear. We have investigated whether SPH inhibits survival signaling in cells by inhibiting Akt kinase activity. This study demonstrates that treatment of Jurkat cells with SPH leads to Akt dephosphorylation as early as 15  min, and the cells undergo apoptosis after 6  h. This Akt dephosphorylation is not mediated through deactivation of upstream kinases, since SPH does not inhibit the upstream phosphoinositide-dependent kinase 1 (PDK1) phosphorylation. Rather, sensitivity to the Ser/Thr protein phosphatase inhibitors (calyculin A, phosphatidic acid, tautomycin, and okadaic acid) indicates an important role for protein phosphatase 1 (PP1) in this process. In vitro phosphatase assay, using Akt immunoprecipitate following treatment with SPH, reveals an increase in Akt-PP1 association as determined by immunoprecipitation analysis. Moreover, SPH-induced dephosphorylation of Akt at Ser(473) subsequently leads to the activation of GSK-3β, caspase 3, PARP cleavage, and ultimately apoptosis. Pre-treatment with caspase 3 inhibitor z-VAD-fmk and Ser/Thr phosphatase inhibitor abrogates the effect of SPH on facilitating apoptosis. Altogether, these results demonstrate that PP1-mediated inhibition of the key anti-apoptotic protein, Akt, plays an important role in SPH-mediated apoptosis in Jurkat cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoprecipitation; Jurkat Cells; Marine Toxins; Okadaic Acid; Oxazoles; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Protein Phosphatase 1; Proto-Oncogene Proteins c-akt; Pyrans; Serine; Signal Transduction; Sphingosine; Spiro Compounds

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V-positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro-apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl-1. zVAD-fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase-3 activity, procaspase-3 processing, and the apoptotic rate of neutrophils. However, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA-induced mobility shift in procaspase-3, caspase-3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase-3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD-fmk. These results suggest that OA induces procaspase-3 processing but that OA-induced apoptosis is caspase-independent and serine protease-dependent.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Caspase 3; Cells, Cultured; Cyclosporine; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Marine Toxins; Myeloblastin; Neutrophils; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase C-alpha; Protein Kinase Inhibitors; Protein Phosphatase 1; Serine Endopeptidases; Serine Proteinase Inhibitors; Sulfones; Time Factors

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.

Topics: Amino Acid Chloromethyl Ketones; Anisomycin; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Biological Transport; Carrier Proteins; Caspase 8; Caspase 9; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Cytosol; HL-60 Cells; Humans; Marine Toxins; Mitochondria; Oxazoles; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Drug Synergism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Fetus; Imidazoles; Marine Toxins; Mice; Mitogen-Activated Protein Kinases; N-Methylaspartate; Necrosis; Neurons; Neurotoxins; Oxazoles; p38 Mitogen-Activated Protein Kinases; Receptors, N-Methyl-D-Aspartate; tau Proteins