okadaic-acid and calpeptin

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.

Topics: Amino Acid Sequence; Animals; Autoantigens; Calpain; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Dystrophin-Associated Proteins; Enzyme Activation; Enzyme Inhibitors; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Membrane Proteins; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Binding; Protein Precursors; Protein Tyrosine Phosphatases; Rats; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Receptor-Like Protein Tyrosine Phosphatases, Class 8; Secretory Vesicles; Tumor Cells, Cultured; Utrophin

Heme oxygenase-1 (HO-1), an enzyme important in protection against oxidant stress, is induced in human vascular endothelial cells by the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). However, the signaling mediators that regulate the induction are not known. This study examined the involvement of protein kinase C (PKC), phospholipase A2 (PLA2), calcium, and oxidants in cytokine induction of HO-1. Acute exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated HO-1 mRNA. However, prolonged exposure, which downregulates most PKC isoforms, blocked induction of HO-1 mRNA by IL-1alpha and TNF-alpha. Additionally, the phosphatase inhibitors okadaic acid and calyculin enhanced cytokine induction of HO-1. Mepacrine, a PLA2 inhibitor, prevented HO-1 induction by cytokine, suggesting a role for arachidonate, the product of PLA2 hydrolysis of phospholipids, in HO-1 expression. The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) blocked cytokine induction of HO-1. Paradoxically, the calcium ionophore A-23187 prevented HO-1 induction by cytokine but not by PMA. Finally, the oxidant scavenger N-acetylcysteine inhibited HO-1 induction by cytokines. These results demonstrate that TNF-alpha and IL-1alpha induction of HO-1 requires PKC-mediated phosphorylation and PLA2 activation as well as oxidant generation.

Topics: Acetylcysteine; Blotting, Northern; Calcimycin; Calcium; Carcinogens; Cells, Cultured; Chelating Agents; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Endothelium, Vascular; Enzyme Inhibitors; Free Radical Scavengers; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Interleukin-1; Ionophores; Marine Toxins; Membrane Proteins; Okadaic Acid; Oxazoles; Phospholipases A; Phospholipases A2; Protein Kinase C; Reactive Oxygen Species; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Umbilical Veins