Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11867-11870, December 1992 Pharmacology

Cantharidin-binding protein: Identification as protein phosphatase 2A (mechanism of toxic actlon/endothafl/oxabicydoheptanedicarboxylic acids)

YUE-MING LI* AND JOHN E. CASIDAt Pesticide Chemistry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, CA 94720

Contributed by John E. Casida, September 11, 1992

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 [3Hlcantharidin-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 a subunit and a 39-kDa .8 subunit. Amino acid sequences of four peptides from CBP-a and three peptides from CBP-fl are identical with deduced amino acid sequences for the Aa regulatory and Cfl 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 a 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.

Three 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives are of special toxicological and pharmacological interest (1-6) (Fig. 1). Cantharidin is the toxic constituent of blister beetles and the active ingredient of the purported aphrodisiac Spanish fly. Endothall is a commercial herbicide. Endothall thioanhydride is the most toxic compound of this type. Cantharidin and endothall and many of their dicarboxylic acid and anhydride analogs bind to the same site in mouse liver cytosol, referred to as cantharidin-binding protein (CBP). The binding affinity of cantharidin and its analogs at this site is an excellent predictor of their mammalian toxicity, indicating the toxicological relevance of CBP (7, 8). [3H]Endothall thioanhydride also binds at the same site (unpublished work). Despite many studies on the physiological and biochemical effects of cantharidin its mechanism of action remains unknown (2, 4, 7, 9, 10). This study describes a different approach to determining the mode of action of cantharidin, involving isolation of CBP and identification of its structure and function by comparison with known proteins, leading to the conclusion that CBP is phosphoprotein phosphatase 2A (PP2A), AC form (EC 3.1.3.16).

0

cantharidin 1.0 mg/kg

0

~~OH

6

5

°

0

3

endothall 14 mg/kg

O

endothall thioanhydride 0.31 mg/kg

FIG. 1. Structures of cantharidin, endothall, and endothall thioanhydride showing stars for positions of 3H labels, numbering scheme for substituents, and mouse intraperitoneal LD50 values.

MATERIALS AND METHODS Radioligand Binding Assays. The standard incubation mixture consisted of cytosol or a fraction thereof in 1 ml of "6standard buffer" [50 mM imidazole/HCl (pH 7.4) containing 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 10 juM 2-phenyl4H-1,3,2-benzodioxaphosphorin 2-oxide (a potent protease inhibitor)] to which was added [3H]cantharidin at a final concentration of 2.5 nM. Following incubation for 90 min at 370C with gentle shaking, the samples were filtered under vacuum through polyethylenimine (0.03%)-pretreated Whatman GF/B glass fiber filters and rapidly rinsed with cold buffer (4, 7, 8). Isolation of CBP. Fresh mouse liver was homogenized at 20% (wt/vol) in cold 50 mM Mes buffer (pH 5.7) containing 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 10 ,M 2-phenyl-4H-1,3,2-benzodioxaphosphorin 2-oxide. The PHIcantharidin-binding fraction from precipitation with 60%o saturated ammonium sulfate was purified by five chromatographic steps using standard buffer with different salt gradients (Table 1). The purity of the CBP obtained was evaluated by both 4-15% gradient PAGE under nondenaturing conditions and SDS/12% PAGE (11). Isolation, Digestion, and Sequencing of Two CBP Subunits. Cysteine residues were pyridylethylated with 4-vinylpyridine (12), and the sample was then applied to an ABI C8 column (300 A, 2.1 x 30 mm). Elution with a gradient of 25-75% solvent A (0.085% trifluoroacetic acid in 95% acetonitrile) in solvent B (0.1% trifluoroacetic acid in water) gave two peaks of UV-absorbing material, which were collected for confirmation of purity and subunit structure by SDS/PAGE. Each subunit was digested with endoproteinase Lys-C (Boehringer Mannheim) (13) and applied to a Vydac C8 column (300 A, 2.1 x 250 mm) and then chromatographed as above, but with a gradient of 8-98% solvent A in solvent B. Several peaks were sequenced, and the peptides compared to the Protein Identification Resource data bank (Release 29) by using the IntelliGenetics software for molecular biology. Abbreviation: CBP, cantharidin-binding protein. *Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NaM Acad Sci. USA 89 (1992)

Table 1. Isolation of CBP from mouse liver cytosol Conditions for development with standard buffer Chromatographic Purification column Gradient Salt factor* Phenyl-Sepharose Step 0.57, 0.17, 0 M 26 (NH4)2SO4 Q-Sepharose Linear 0-0.7 M NaCl 322 None None Sephacryl S-300 750 Linear 0.57-0 M Phenyl-Superose 1488 (NH4)2SO4 Mono Q Linear 0-0.7 M NaCl 2230 *Specific binding of [3H]cantharidin as dpm/,ug protein for purified material relative to original cytosol. A portion of the purification in the first step is from precipitation with 60% saturated ammonium sulfate.

Phosphorylase a Phosphatase Activity of CBP. Assays were carried out with CBP (10 ng), candidate inhibitors (described below), and [32P]phosphorylase a (added last) in 10 ,ul of assay buffer [50 mM Tris/HCI (pH 7.0) containing 15 mM caffeine, 0.1 mM EGTA, and 0.1% (vol/vol) 2-mercaptoethanol] (14,15). Okadaic acid (Boehringer Mannheim; 25 ,g, on the basis of supplier's specifications without confirmatory analysis) and cantharidin were dissolved in ethanol and acetone, respectively, and diluted in assay buffer, whereas all other candidate inhibitors were dissolved directly in this buffer. After incubation for 8 min at 25°C, each reaction mixture was treated with 20 ,ul of 50%o trichloroacetic acid and

-

I, CE

'

v----*

_

a-I ....

h-

,

n

t, 41

FiG. 2. Electrophoretic analysis of pure CBP. under nondenaturing conditions with

LAne

1, PAGE

migration frm the cathode

at

the top; lanes 2 and 3, SDS/PAGE for molecular weight determinations of CBP-a and CBP-,8 subunits compared with marker proteins (kDa).

20 ,ul of bovine serum albumin solution (6 mg/ml assay buffer) and cooled on ice for 5 min. Trichioracetic acidinsoluble materials were precipitated by spinning in a microcentrifuge for 3 min; then 45 ,ul of the supernatant was added to 10 ml of scintillation fluid for determination of radioactivity.

CBP-a peptides (italics) compared with PP2A-Aa

151

MAAADGDDSL YPIAVLIDEL LLPFLTDTIY DEDEVLLALA ETVVRDKAVE SLRAISHEHS FSVCYPRVSS AVKAFLRQYF

201 251

VKSEIIPMFS NLASDEQDSV RLLAVEACVN IAQLLPQEDL EALVMPTLRQ AAEDKSWAVR YMVADKFTEL QKAVGPZITK TDLVPAFQNL MKDCEAEVRA

301 351

AASHKVKEFC ENLSADCREN VIMSQILPCI GLSPILGKDN TIEHLLPLFL AQLKDZCPZV ZCPZV SQSLLPAIVE LAEDAKWRVR LAIIEYMPLL LVDHVYAIRE AATSNLKKLV EKFGKEWAHA

1

51 101

RNEDVQLRLN EQLGTFTTLV PSDLEAHFVP RNLCSDDTPM

SIKKLSTIAL GGPEYVHCLL LVKRLAGGDW VRRAAASKLG

ALGVERTRSE PPLESLATVE FTSRTSACGL EFAKVLELDN

AZlLRQY RNLCSDDrPM VRRAAAS

AVGPZI

401 451

501 551

KELVSDANQH RLNIISNLDC RLNIISNLDC AGQLGVEFFD TIIPKVLANS VLZS

VKSALASVIM VNZVIGIRQL VNZVIG EKLNSLCMAW GDPNYLIRKT GDPNYLNRMT TLFCINVLSE VCGQDITTKH MLPTVLRMAG DPVANVRFNV AKSLQKIGPI LDNSTLQSEV KPILEKLTQD QDVDVKYFAQ EALTVLSLA

CBP-( peptides (italics) compared with PP2A-Cp 1

51

101 151 201 251 301

MDDKAFTKEL DQWVEQLNEC KQLNZNQVRT LCZKAKEILT QENzMQVRT LCEK PVTVCGDVHG QFHDLMELFR IGGKSPDTNY LFMGDYVDRG PV VALKVRYPER ITILRGNHES RQITQVYGFY DECLRKYGNA DYLPLTALVD GQIFCLHGGL SPSIDTLDHI RALDRLQEVP SDPDDRGGWG ISPRGAGYTF GQDISETFNH ANGLTLVSRA CHDRNVVTIF SAPNYCYRCG NQAAIMELDD TLKYS1LQFD TRRTPDYFL TRRATPD Y

KESNVQZVRC NVQEVRC YYSVETVTLL NVWKYFTDLF HEGPMCDLLW HQLVMEGYNW PAPRRGZPUV

Y$FZQrD PAPRRGZPNV

FIG. 3. Comparison of peptide sequences for CBP-a and CBP-#8 subunits (Lys-C digestion) of mouse liver cytosol with deduced amino acid (Aa) and catalytic (Cf) subunits of human PP2A [Protein Identification Resource accession numbers A 34541 (16) and B 37135 (17), respectively]. sequences for the regulatory

Proc. Natl. Acad. Sci. USA 89 (1992)

Pharmacology: Li and Casida 100 0

1

0

80

okadaic acid IC

60

50

12 nM

0.

I

cantharidic acid

cyooi poen)n 4

-E

il (Table 1.C50Tf5nM

20

0

V) .C

0

a.

100

10

1

Concentration (nM)

FIG.

Comparison of cantharidic acid and okadaic acid as phosphorylase a phosphatase activity of pure CBP.

4.

inhibitors

of

Inhibition

values

are means

±

SE based

on

three

experiments.

RESULTS

Isolation

of

CBP. Purification of

mouse liver cytosol by precipitation and a five-step chromatoof [3H]canprocedure (monitored by specific of gave pure CBP on 2230-fold purification (63 protein) in 14% yield (Table 1). The final CBP

ammonium sulfate

binding graphic tharidin) tg/g cytosolic preparation consisted of a single band in PAGE under denaturing conditions and two bands in SDS/PAGE at 61 kDa (a) and 39 kDa (ac) (Fig. 2). Other studies showed that the mass of pure CBP was s100 kDa as analyzed by dimethyl suberimide crosslinking and analytical centrifugation; i.e., it

non-

heterodimer (unpublished work). Peptide Sequences of CBP Subunits in Comparison with PP2A (Fig. 3). Attempted direct amino-terminal sequence analysis of the two CBP subunits failed to identify any individual amino acids, suggesting that each subunit was blocked at the amino terminus. Fortunately, several peptides from Lys-C digestion of each subunit were suitable to obtain their internal amino acid sequences. Four peptides from CBP-a gave unambiguous sequences involving 6, 15, 21 (first amino acid not identified), and 24 consecutive residues. A was a

search of the Protein Identification Resource data bank (Release 29) revealed that these four peptides were identical with deduced amino acid sequences for the 61-kDa human transforming protein (18) and the 65-kDa human PP2A-Aa regulatory subunit (16), which are, in fact, one and the same protein (19, 20). The peptides obtained from CBP-a were also consistent with the Lys-C cleavage sites of PP2A-Aa. In a similar manner, the three sequenced peptides of CBP-P involving 9 (first 2 amino acids not identified), 13, and 24 unambiguous consecutive residues were identical with deduced amino acid sequences of the PP2A-CB catalytic chain from eight sources, including human (17). The peptide sequences obtained establish that CBP is the PP2A AC form. Phosphorylase a Phosphatase and p-Nitrophenyl Phosphate Phosphatase Activities of CBP and Their Inhibition by Okadaic Add. Protein phosphatase 1 and PP2A are the only enzymes with significant phosphorylase phosphatase activity in mammalian tissues (21) and are therefore conveniently assayed by measuring the dephosphorylation of phosphorylase a. The expected phosphorylase a phosphatase activity of CBP was readily observed and was also sensitive to okadaic acid and cantharidic acid, with IC50 values of 12 and 50 nM, respectively (Fig. 4). Further, the strong p-nitrophenyl phosphate phosphatase activity ofPP2A (22) was also evident with CBP, and this activity was inhibited by okadaic acid. Cantharidin and Endothall Analogs as Inhibitors of Phosphorylase a Phosphatase Activity of CBP. Nine analogs, including three each with high, intermediate, and low potency as judged from their radioligand binding IC50 values and their LD50 values, were compared as inhibitors of phosphorylase a phosphatase activity of CBP (Table 2). Their potency as phosphatase inhibitors parallels their affinity at the ligand binding site and their toxicity. Okadaic Acid Inhibition of [3H]Cantharidin Binding. Okadaic acid inhibited binding of [3H]cantharidin to pure CBP with an IC50 value of 0.6 nM. Scatchard analysis established that, relative to the control, 0.5 nM okadaic acid reduced the Bmax without changing the Kd (Fig. 5).

DISCUSSION liver cytosol is the established target

CBP of mouse or a suitable model thereof for the toxic effects of cantharidin, endothall, and related oxabicycloheptanedicarboxylic acids (4, 7, 8). This binding site is also present in brain, stomach,

Table 2. Relationship for cantharidin and endothall analogs between inhibition of CBP protein phosphatase activity, inhibition of liver cytosol radioligand binding site, and toxicity to mice Inhibition of IC50 for radioligand phosphatase LD5o,§ Endothall analog* mg/kg bindingt nM activity,t % High potency 2,3-Dimethyl anhydride¶

2,3-Dimethylll 2,3-Trimethylene anhydride Intermediate potency 2-Methyl Unsubstituted (endothall) endo-5-Carboxy

92-95

4-8

1-2

22-51

19-50

5-50

0

200 to >100,000

105 to >400

Low potency

endo-5-Carboethoxy endo-5-Cyanomethyl

1,4-Dimethyl *See Fig. 1 for structure of endothall and numbering scheme.

tPure CBP with [32P]phosphorylase a as the substrate and inhibitors at 250 nM. tBinding assay with [3H]endothall thioanhydride and mouse liver cytosol.

§Mouse intraperitoneal. 1Cantharidin. IICantharidic acid.

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Proc. Nad. Acad. Sci. USA 89 (1992) proteins and sequencing the peptides; Tina H. Lee and Marc W. Kirschner (Department of Biochemistry and Biophysics, University of California at San Francisco) for providing p2P]phosphorylase a; and Philip Cohen (Department of Biochemistry, University of Dundee, Dundee, Scotland), Tina H. Lee, and Marc W. Kirschner for reviewing the manuscript. This work was supported by Grant ES00049 from the National Institute of Environmental Health Sciences.

0.09

*\

control Kd=6.6nM

Bmax U.

0.62

=

pmol

0.06 t\

C0

0

0.03

\

okadaic acid

KKd=6.2nM A,ABmaX =0.15 pmol 0.00 0.0

a

0.2

0.4

0.6

Bound (pmol) FIG. 5. Scatchard plots showing effect of 0.5 nM okadaic acid on binding of [3H]cantharidin to pure CBP (100 ng). Data are based on two experiments.

skin, heart, blood, and many other organs (8). Following isolation, CBP was identified as the PP2A AC form based on seven criteria: (i) the molecular weights for the individual and combined a and subunits, (ii) the amino acid composition of CBP (unpublished work), (iii) the amino acid sequences of several peptides of each subunit, (iv) the presence of protein phosphatase activity, (v) inhibition of this phosphatase activity by oxabicycloheptanedicarboxylic acids in proportion to their affinity for the binding site and their toxicity, (vi) sensitivity of CBP protein phosphorylase a phosphatase activity and p-nitrophenyl phosphate phosphatase activity to inhibition by okadaic acid, and (vii) inhibition of [3H]cantharidin binding to CBP by okadaic acid. PP2A is one of four major classes of serine/threoninespecific protein phosphatases (types 1, 2A, 2B, and 2C) in the cytoplasm of mammalian cells (23). PP2A consists of a heterotrimeric complex (ABC) (24) or a dimeric form (AC) (25). Their catalytic subunits are highly conserved across species (26). PP2A is involved in signaling pathways that control cell proliferation and the activity of a variety of protein kinases and/or phosphatases that are themselves key regulators of cell function. Cytoplasmic PP2A is also implicated in regulating the activity ofmembrane-associated channels and receptors (24, 27). Cantharidin can now be used as an inexpensive and readily available probe for analysis of regulatory phosphorylationdephosphorylation events mediated by PP2A and other protein phosphatases. The mouse intraperitoneal LD50 of cantharidin (1.0 mg/kg; ref. 3) and lethal dose of okadaic acid (0.2 mg/kg; ref. 28) are consistent with their potencies as inhibitors of PP2A activity, suggesting that with both compounds in vivo inhibition may be the cause of sublethal effects and death. The herbicidal activity of endothall is possibly associated with inhibition of PP2A or other protein phosphatase known to be present in plants (29, 30). We thank James Schilling and Kim Brown (Protein Structure Laboratory, University of California at Davis) for digesting the

1. Cavill, G. W. K. & Clark, D. V. (1971) in Naturally Occurring Insecticides, eds. Jacobson, M. & Crosby, D. G. (Dekker, New York), pp. 271-305. 2. Graziano, M. J. & Casida, J. E. (1987) Toxicol. Lett. 37, 143-148. 3. Matsuzawa, M., Graziano, M. J. & Casida, J. E. (1987) J. Agric. Food Chem. 35, 823-829. 4. Kawamura, N., Li, Y.-M., Engel, J. L., Dauben, W. G. & Casida, J. E. (1990) Chem. Res. Toxicol. 3, 318-324. 5. Walter, W. G. (1989) J. Pharm. Sci. 78, 66-67. 6. Liu, X. H., Bravo-Cuellar, A., Br6ard, J., Metzger, G., Comisso, M., Mathe, G. & Orbach-Arbouys, S. (1991) Int. J. Immunother. 7, 37-42. 7. Graziano, M. J., Waterhouse, A. L. & Casida, J. E. (1987) Biochem. Biophys. Res. Commun. 149, 79-85. 8. Graziano, M. J., Pessah, I. N., Matsuzawa, M. & Casida, J. E. (1988) Mol. Pharmacol. 33, 706-712. 9. Decker, R. H. (1968) J. Invest. Dermatol. 51, 141-146. 10. Bagatell, F. K., Dugan, K. & Wilgram, G. F. (1969) Toxicol. Appl. Pharmacol. 15, 249-261. 11. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 12. Fullmer, C. S. (1984) Anal. Biochem. 142, 336-339. 13. Stone, K. L., LoPresti, M. B., Crawford, J. M., DeAngelis, R. & Williams, K. R. (1989) in A Practical Guide to Protein and Peptide Purification for Microsequencing, ed. Matsudaira, P. T. (Academic, New York), pp. 31-47. 14. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., StrAilfors, P. & Tung, H. V. L. (1988) Methods Enzymol. 159, 390-408. 15. Lee, T. H., Solomon, M. J., Mumby, M. C. & Kirschner, M. W. (1991) Cell 64, 415-423. 16. Hemmings, B. A., Adams-Pearson, C., Maurer, F., Muller, P., Goris, J., Merlevede, W., Hofsteenge, J. & Stone, S. R. (1990) Biochemistry 29, 3166-3173. 17. Khew-Goodall, Y., Mayer, R. E., Maurer, F., Stone, S. R. & Hemmings, B. A. (1991) Biochemistry 30, 89-97. 18. Walter, G., Ferre, F., Espiritu, 0. & Carbone-Wiley, A. (1989) Proc. Natl. Acad. Sci. USA 86, 8669-8672. 19. Walter, G., Ruediger, R., Slaughter, C. & Mumby, M. (1990) Proc. Natl. Acad. Sci. USA 87, 2521-2525. 20. Pallas, D. C., Shahrik, L. K., Martin, B. L., Jaspers, S., Miller, T. B., Brautigan, D. L. & Roberts, T. M. (1990) Cell 60, 167-176. 21. Ingebritsen, T. S., Stewart, A. A. & Cohen, P. (1983) Eur. J. Biochem. 132, 297-307. 22. Takai, A. & Mieskes, G. (1991) Biochem. J. 275, 233-239. 23. Cohen, P. (1991) Methods Enzymol. 201, 389-398. 24. Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508. 25. Usui, H., Imazu, M., Maeta, M., Tsukamoto, H., Azuma, K. & Takeda, M. (1988) J. Biol. Chem. 263, 3752-3761. 26. Cohen, P. & Cohen, P. T. W. (1989) J. Biol. Chem. 264, 21435-21438. 27. Shenolikar, S. & Nairn, A. C. (1991) Adv. Second Messenger Phosphoprotein Res. 23, 1-121. 28. Cohen, P., Holmes, C. F. B. & Tsukitani, Y. (1990) Trends Biochem. Sci. 15, 98-102. 29. MacKintosh, R. W., Haycox, G., Hardie, D. G. & Cohen, P. T. W. (1990) FEBS Lett. 276, 156-160. 30. MacKintosh, C., Coggins, J. & Cohen, P. (1991) Biochem. J. 273, 733-738.

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 ...
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