okadaic-acid and Pheochromocytoma

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Serl99/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.

Topics: Animals; Calcineurin; Cell Line; Enzyme Inhibitors; Humans; Neuroblastoma; Okadaic Acid; PC12 Cells; Pheochromocytoma; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Subunits; Rats; Recombinant Proteins; tau Proteins; Transfection; Tumor Cells, Cultured

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

Extracellular deposition of the beta/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. beta/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP751, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the beta/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP695, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-beta/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.

Topics: Adrenal Gland Neoplasms; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Ethers, Cyclic; In Vitro Techniques; Membrane Glycoproteins; Okadaic Acid; Pheochromocytoma; Phorbol Esters; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Secretory Rate; Tumor Cells, Cultured

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.

Topics: Adrenal Gland Neoplasms; Animals; Cell Adhesion; Diglycerides; Ethers, Cyclic; Glioma; Laminin; Neoplasm Proteins; Neuroblastoma; Neurons; Okadaic Acid; Pheochromocytoma; Phorbol Esters; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Tumor Cells, Cultured; Vanadates

The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stimulates protein kinase C, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Topics: Adrenal Gland Neoplasms; Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Animals; Cell Line; Ethers, Cyclic; Humans; Kinetics; Methionine; Molecular Weight; Okadaic Acid; Pheochromocytoma; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Biosynthesis; Protein Kinase C; Protein Precursors; Protein Processing, Post-Translational; Tetradecanoylphorbol Acetate; Transcription, Genetic

PC-12 pheochromocytoma cells contain a growth factor-sensitive protein kinase that phosphorylates microtubule associated protein 2 (MAP-2). This MAP kinase is also activated by the protein phosphatase inhibitor okadaic acid (OA). Additionally, OA potentiates the NGF-dependent activation of MAP kinase, but causes only a modest potentiation (20%) of the maximal activation observed with EGF. Since OA is a specific serine/threonine phosphatase inhibitor, these results suggest that serine/threonine phosphorylation may be involved in the hormonal regulation of MAP kinase.

Topics: Adrenal Gland Neoplasms; Animals; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Epidermal Growth Factor; Ethers, Cyclic; Nerve Growth Factors; Okadaic Acid; Pheochromocytoma; Phosphorylation; Protein Kinases; Serine; Swine; Threonine; Tumor Cells, Cultured

Protein phosphatases and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized PC12 cells. The addition of an exogenous type 2A protein phosphatase caused as much as a 70% decrease in Ca2(+)-dependent norepinephrine secretion. In the presence of okadaic acid, a potent inhibitor of type 2A protein phosphatases, phosphatase 2A had no effect on secretion. The addition of exogenous calcineurin, a Ca2(+)-calmodulin-stimulated phosphatase, also caused decrease in Ca2(+)-dependent secretion, but on a molar basis it was less effective than phosphatase 2A. Two phosphatase inhibitors, 1-naphthylphosphate and sodium pyrophosphate, caused 75-100% increases in the amount of norepinephrine secreted in the absence of Ca2+ without affecting the amount of norepinephrine secreted in the presence of Ca2+. This stimulation of Ca2(+)-independent secretion by 1-naphthylphosphate and pyrophosphate suggests that there is a slow rate of Ca2(+)-independent phosphorylation and that phosphorylation triggers secretion. Unlike the results obtained in the presence of ATP, secretion in the presence of adenosine-5'-O-(3-thiotriphosphate), ATP gamma S, was not affected by the addition of type 2A protein phosphatase or by the addition of phosphatase inhibitors. These results are consistent with secretion in these permeabilized cells being regulated by a Ca2(+)-stimulated phosphorylation.

Topics: Adenosine Triphosphate; Adrenal Gland Neoplasms; Animals; Calcium; Cell Line; Cell Membrane Permeability; Diphosphates; Ethers, Cyclic; Kinetics; Macromolecular Substances; Naphthalenes; Norepinephrine; Okadaic Acid; Organophosphorus Compounds; Pheochromocytoma; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Rats; Tumor Cells, Cultured