okadaic-acid and 4-(2-aminoethyl)benzenesulfonylfluoride

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

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.

Topics: Alleles; alpha-Amylases; Aprotinin; Cysteine Endopeptidases; Enzyme Induction; Enzyme Inhibitors; Gibberellins; Hordeum; Multienzyme Complexes; Mutation; Okadaic Acid; Phenylmethylsulfonyl Fluoride; Plant Leaves; Plant Proteins; Proteasome Endopeptidase Complex; Seeds; Signal Transduction; Sulfones; Vanadates