Protein phosphatase in neuroblastoma cells: [3H]cantharidin binding site in relation to cytotoxicity.
Protein phosphatase 2A (PP2A) plays a central role in essential phosphorylation-dependent signal transduction pathways. It is also a principal target for many natural toxicants (cantharidin, microcystins, diarrhetic shellfish poisons) and a synthetic herbicide (endothall). This study develops a cellular model to explore the toxicology of PP2A inhibitors by use of a [3H]cantharidic acid ([3H]CA) ligand binding assay to quantify interactions at the toxicant site and cell viability to evaluate in vivo toxicity. Mouse neuroblastoma (N1E-115) cells are similar to mouse brain with respect to the affinity (12-15 nM), number (B(max), 9-22 pmol/mg protein) and ligand specificity of this binding site. In addition, the competitive potency of ten analogs of CA (including endothall) and two potent diarrhetic shellfish poisons (okadaic acid and calyculin A) is correlated (r2 = .9) with and therefore predictive of their cytotoxicity. The only exception is microcystin LR which is a potent inhibitor at the binding site but is not cytotoxic, possibly reflecting a lack of cellular uptake. ATP and several other phosphorus-containing bifunctional acids inhibit [3H]CA binding by phosphorylation-independent pathways; pyrophosphate apparently acts as a competitive inhibitor. Mn++ and five other divalent cations are also inhibitors with a unique action of Mn++ at 25 to 50 microM in increasing [3H]CA binding, which suggests a specific role in PP2A function. Neuroblastoma cells are therefore suitable to study the mechanisms by which the toxicant, ATP and Mn++ binding sites regulate PP2A activity and cell physiology.
Laidley
C W
CW
Department of Environmental Science, Policy, and Management, University of California, Berkeley 94720-3112, USA.
Cohen
E
E
Casida
J E
JE
D000818:Animals; D001665:Binding Sites; D002193:Cantharidin / Q000378:metabolism* / Q000633:toxicity; D002413:Cations, Divalent; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000633:toxicity; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054648:Protein Phosphatase 2; D013329:Structure-Activity Relationship; D014316:Tritium; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D014316:Tritium; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D002193:Cantharidin