Cantharidin-binding protein: identification as protein phosphatase 2A.
The toxic effects of cantharidin from blister beetles and its analogs, including the herbicide endothall, are attributable to their high affinity and specificity for a cantharidin-binding protein (CBP). An ammonium sulfate precipitate of mouse liver cytosol was purified by five chromatographic steps to isolate CBP in 14% yield and > 99% purity as monitored by [3H]cantharidin-binding activity. The purification factor of 2230-fold corresponds to a CBP content of 0.045% of the liver cytosolic protein. CBP is a heterodimer consisting of a 61-kDa alpha subunit and a 39-kDa beta subunit. Amino acid sequences of four peptides from CBP-alpha and three peptides from CBP-beta are identical with deduced amino acid sequences for the A alpha regulatory and C beta catalytic subunits, respectively, of protein phosphatase 2A (PP2A). This assignment of CBP as PP2A-AC from structural evidence is supported by biochemical studies with selective substrates and inhibitors. CBP dephosphorylation of phosphorylase alpha is sensitive not only to okadaic acid, as with PP2A, but also to cantharidin and its analogs, consistent with their potency in blocking the radioligand binding site of CBP. Okadaic acid is a potent inhibitor of [3H]cantharidin binding to CBP. PP2A is present in many mammalian tissues and in plants and is involved in regulatory phosphorylation-dephosphorylation events which modulate multiple cellular functions. Inhibition of PP2A activity may account for the diverse effects and toxicity of cantharidin and its analogs, including the herbicide endothall, in mammals and possibly plants.
Li
Y M
YM
Department of Entomological Sciences, University of California, Berkeley 94720.
Casida
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002193:Cantharidin / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003600:Cytosol / Q000201:enzymology; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D054648:Protein Phosphatase 2; D016415:Sequence Alignment
D002352:Carrier Proteins; D003998:Dicarboxylic Acids; D004988:Ethers, Cyclic; C100030:endothall; D019319:Okadaic Acid; C578496:PPP2CA protein, mouse; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D002193:Cantharidin