Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Dec 15;89(24):11867–11870. doi: 10.1073/pnas.89.24.11867

Cantharidin-binding protein: identification as protein phosphatase 2A.

Y M Li 1, J E Casida 1
PMCID: PMC50658  PMID: 1334551

Abstract

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.

Full text

PDF
11867

Images in this article

11868Image
on p.11868

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagatell F. K., Dugan K., Wilgram G. F. Structural and biochemical changes in tissues isolated from the cantharidin-poisoned rat with special emphasis upon hepatic subcellular particles. Toxicol Appl Pharmacol. 1969 Sep;15(2):249–261. doi: 10.1016/0041-008x(69)90024-6. [DOI] [PubMed] [Google Scholar]
  2. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  3. Cohen P. Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts. Methods Enzymol. 1991;201:389–398. doi: 10.1016/0076-6879(91)01035-z. [DOI] [PubMed] [Google Scholar]
  4. Cohen P., Cohen P. T. Protein phosphatases come of age. J Biol Chem. 1989 Dec 25;264(36):21435–21438. [PubMed] [Google Scholar]
  5. Cohen P., Holmes C. F., Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci. 1990 Mar;15(3):98–102. doi: 10.1016/0968-0004(90)90192-e. [DOI] [PubMed] [Google Scholar]
  6. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  7. Decker R. H. The identification of a phosphoprotein in acantholytic epidermis. J Invest Dermatol. 1968 Aug;51(2):141–146. doi: 10.1038/jid.1968.105. [DOI] [PubMed] [Google Scholar]
  8. Fullmer C. S. Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography. Anal Biochem. 1984 Nov 1;142(2):336–339. doi: 10.1016/0003-2697(84)90473-1. [DOI] [PubMed] [Google Scholar]
  9. Graziano M. J., Casida J. E. Comparison of the acute toxicity of endothal and cantharidic acid on mouse liver in vivo. Toxicol Lett. 1987 Jul;37(2):143–148. doi: 10.1016/0378-4274(87)90150-0. [DOI] [PubMed] [Google Scholar]
  10. Graziano M. J., Pessah I. N., Matsuzawa M., Casida J. E. Partial characterization of specific cantharidin binding sites in mouse tissues. Mol Pharmacol. 1988 Jun;33(6):706–712. [PubMed] [Google Scholar]
  11. Graziano M. J., Waterhouse A. L., Casida J. E. Cantharidin poisoning associated with specific binding site in liver. Biochem Biophys Res Commun. 1987 Nov 30;149(1):79–85. doi: 10.1016/0006-291x(87)91607-x. [DOI] [PubMed] [Google Scholar]
  12. Hemmings B. A., Adams-Pearson C., Maurer F., Müller P., Goris J., Merlevede W., Hofsteenge J., Stone S. R. alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry. 1990 Apr 3;29(13):3166–3173. doi: 10.1021/bi00465a002. [DOI] [PubMed] [Google Scholar]
  13. Ingebritsen T. S., Stewart A. A., Cohen P. The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur J Biochem. 1983 May 2;132(2):297–307. doi: 10.1111/j.1432-1033.1983.tb07362.x. [DOI] [PubMed] [Google Scholar]
  14. Kawamura N., Li Y. M., Engel J. L., Dauben W. G., Casida J. E. Endothall thioanhydride: structural aspects of unusually high mouse toxicity and specific binding site in liver. Chem Res Toxicol. 1990 Jul-Aug;3(4):318–324. doi: 10.1021/tx00016a008. [DOI] [PubMed] [Google Scholar]
  15. Khew-Goodall Y., Mayer R. E., Maurer F., Stone S. R., Hemmings B. A. Structure and transcriptional regulation of protein phosphatase 2A catalytic subunit genes. Biochemistry. 1991 Jan 8;30(1):89–97. doi: 10.1021/bi00215a014. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lee T. H., Solomon M. J., Mumby M. C., Kirschner M. W. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell. 1991 Jan 25;64(2):415–423. doi: 10.1016/0092-8674(91)90649-j. [DOI] [PubMed] [Google Scholar]
  18. MacKintosh C., Coggins J., Cohen P. Plant protein phosphatases. Subcellular distribution, detection of protein phosphatase 2C and identification of protein phosphatase 2A as the major quinate dehydrogenase phosphatase. Biochem J. 1991 Feb 1;273(Pt 3):733–738. doi: 10.1042/bj2730733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MacKintosh R. W., Haycox G., Hardie D. G., Cohen P. T. Identification by molecular cloning of two cDNA sequences from the plant Brassica napus which are very similar to mammalian protein phosphatases-1 and -2A. FEBS Lett. 1990 Dec 10;276(1-2):156–160. doi: 10.1016/0014-5793(90)80531-m. [DOI] [PubMed] [Google Scholar]
  20. Pallas D. C., Shahrik L. K., Martin B. L., Jaspers S., Miller T. B., Brautigan D. L., Roberts T. M. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell. 1990 Jan 12;60(1):167–176. doi: 10.1016/0092-8674(90)90726-u. [DOI] [PubMed] [Google Scholar]
  21. Shenolikar S., Nairn A. C. Protein phosphatases: recent progress. Adv Second Messenger Phosphoprotein Res. 1991;23:1–121. [PubMed] [Google Scholar]
  22. Takai A., Mieskes G. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases. Biochem J. 1991 Apr 1;275(Pt 1):233–239. doi: 10.1042/bj2750233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Usui H., Imazu M., Maeta K., Tsukamoto H., Azuma K., Takeda M. Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. J Biol Chem. 1988 Mar 15;263(8):3752–3761. [PubMed] [Google Scholar]
  24. Walter G., Ferre F., Espiritu O., Carbone-Wiley A. Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8669–8672. doi: 10.1073/pnas.86.22.8669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Walter G., Ruediger R., Slaughter C., Mumby M. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2521–2525. doi: 10.1073/pnas.87.7.2521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Walter W. G. Antitumor imide derivatives of 7-oxabicyclo[2.2.1]heptane-2,3-dimethyl-2,3-dicarboxylic acid. J Pharm Sci. 1989 Jan;78(1):66–67. doi: 10.1002/jps.2600780117. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES