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• W1968580657 abstract "The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo. The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo. Peptidyl-prolyl isomerases (PPIases) 2The abbreviations used are: PPIase, peptidyl-prolyl isomerase; Rab4a(pS204), phospho-Ser204-Rab4a; MEF, mouse embryo fibroblast; PIPES, 1,4-piperazinediethanesulfonic acid; CHO, Chinese hamster ovary; APC/C, anaphase-promoting complex/cyclosome. 2The abbreviations used are: PPIase, peptidyl-prolyl isomerase; Rab4a(pS204), phospho-Ser204-Rab4a; MEF, mouse embryo fibroblast; PIPES, 1,4-piperazinediethanesulfonic acid; CHO, Chinese hamster ovary; APC/C, anaphase-promoting complex/cyclosome. accelerate the cis-trans conversion of peptide bonds preceding prolyl residues, which can cause alterations in protein conformation (e.g. see Refs. 1Schmid F.X. Adv. Protein Chem. 2001; 59: 243-282Crossref PubMed Scopus (78) Google Scholar and 2Shaw P. EMBO Rep. 2007; 8: 40-45Crossref PubMed Scopus (57) Google Scholar). PPIases have been grouped into cyclophilin, FK506-binding protein, and parvulin subfamilies (see Ref. 3Fanghanel J. Fischer G. Front. Biosci. 2004; 9: 3453-3478Crossref PubMed Scopus (195) Google Scholar), for which distinct pharmacological inhibitors are available. The parvulin group is irreversibly inhibited by juglone (4Hennig L. Christner C. Kipping M. Schelbert B. Rucknagel K.P. Grabley S. Kullertz G. Fischer G. Biochemistry. 1998; 37: 5953-5960Crossref PubMed Scopus (257) Google Scholar). Pin1 comprises an N-terminal type IV WW domain, which determines phosphorylation-specific protein-protein interactions, and a C-terminal PPIase domain that harbors the catalytic center (see Ref. 5Lu K.P. Zhou X.Z. Nat. Rev. Cell Mol. Biol. 2007; 8: 904-916Crossref PubMed Scopus (533) Google Scholar). Pin1 is a unique PPIase, because it preferably binds to side chain-phosphorylated S/T-P moieties in numerous proteins, including crucial cell cycle regulators or proteins that become phosphorylated immediately prior to cell division (6Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J.U. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (670) Google Scholar, 7Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (299) Google Scholar). Isomerization of the p(S/T)-P peptide bond regulates, for instance, localization and phosphorylation status of Pin1 client proteins (see Ref. 5Lu K.P. Zhou X.Z. Nat. Rev. Cell Mol. Biol. 2007; 8: 904-916Crossref PubMed Scopus (533) Google Scholar). Pin1 is therefore a regulator that, in concert with proline-directed kinases, phosphatases, and ubiquitin ligases, controls the cell cycle (see Refs. 5Lu K.P. Zhou X.Z. Nat. Rev. Cell Mol. Biol. 2007; 8: 904-916Crossref PubMed Scopus (533) Google Scholar and 8Yeh E.S. Means A.R. Nat. Rev. Cancer. 2007; 7: 381-388Crossref PubMed Scopus (205) Google Scholar). Pin1 is possibly a cancer target gene, because its overexpression enhances transformed phenotypes induced by oncogenic Ras and Neu (see (5Lu K.P. Zhou X.Z. Nat. Rev. Cell Mol. Biol. 2007; 8: 904-916Crossref PubMed Scopus (533) Google Scholar)). Pin1 is overexpressed in many human cancers, and its overexpression correlates with poor prognosis of patients (see Ref. 5Lu K.P. Zhou X.Z. Nat. Rev. Cell Mol. Biol. 2007; 8: 904-916Crossref PubMed Scopus (533) Google Scholar). For that matter, down-regulation of Pin1 activity by pharmacological agents might present an attractive opportunity for controlling tumor growth. The function of Pin1, however, is more complicated, since loss of Pin1 can cause a selective growth disadvantage, which suggests that Pin1 may have a protective function in oncogenesis of certain cell types (see Ref. 8Yeh E.S. Means A.R. Nat. Rev. Cancer. 2007; 7: 381-388Crossref PubMed Scopus (205) Google Scholar). Juglone (5-hydroxy-1,4-naphthalenedione) is a benzoquinone that covalently modifies thiol groups of cysteine residues in parvulin, one of which is essential for PPIase activity (4Hennig L. Christner C. Kipping M. Schelbert B. Rucknagel K.P. Grabley S. Kullertz G. Fischer G. Biochemistry. 1998; 37: 5953-5960Crossref PubMed Scopus (257) Google Scholar). It is thought that the inhibition of isomerase activity by juglone is caused by partial unfolding of the PPIase active site (4Hennig L. Christner C. Kipping M. Schelbert B. Rucknagel K.P. Grabley S. Kullertz G. Fischer G. Biochemistry. 1998; 37: 5953-5960Crossref PubMed Scopus (257) Google Scholar). Although juglone can inhibit other proteins (9Muto N. Inouye K. Inada A. Nakanishi T. Tan L. Biochem. Biophys. Res. Commun. 1987; 146: 487-494Crossref PubMed Scopus (18) Google Scholar, 10Frew T. Powis G. Berggren M. Gallegos A. Abraham R.T. Ashendel C.L. Zalkow L. Hudson C. Gruszecka-Kowalik E. Burgess E.M. Benedetti-Doctorovich V. Kerrigan J.E. Lambropoulos J. Merriman R. Bonjouklian R. Anticancer Drug Des. 1995; 10: 347-359PubMed Google Scholar, 11Varga Z. Bene L. Pieri C. Damjanovich S. Gaspar Jr, R. Biochem. Biophys. Res. Commun. 1996; 218: 828-832Crossref PubMed Scopus (33) Google Scholar), it is frequently used to explore the relevance of Pin1 function in vivo (12Galas M.C. Dourlen P. Begard S. Ando K. Blum D. Hamdane M. Buee L. J. Biol. Chem. 2006; 281: 19296-19304Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Esnault S. Braun R.K. Shen Z.J. Xiang Z. Heninger E. Love R.B. Sandor M. Malter J.S. PLoS One. 2007; 2: e226Crossref PubMed Scopus (33) Google Scholar, 14Kesavapany S. Patel V. Zheng Y.L. Pareek T.K. Bjelogrlic M. Albers W. Amin N. Jaffe H. Gutkind J.S. Strong M.J. Grant P. Pant H.C. Mol. Biol. Cell. 2007; 18: 3645-3655Crossref PubMed Scopus (46) Google Scholar), especially since it often phenocopies effects of Pin1 dominant negative mutants or Pin1 knockdown. For instance, PP2A-mediated dephosphorylation of the Pin1-interacting proteins, Raf-1, Cdc25c, Pim-1, Myc, and Tau, critically relies on Pin1 (15Dougherty M.K. Muller J. Ritt D.A. Zhou M. Zou X.Z. Copeland T.D. Conrads T.P. Veenstra T.D. Lu K.P. Morrison D.K. Mol. Cell. 2005; 17: 215-224Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 16Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Kullertz G. Stark M.A. Fischer G. Lu K. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 17Ma J. Arnold H.K. Lilly M.B. Sears R.C. Kraft A.S. Oncogene. 2007; 26: 5145-5153Crossref PubMed Scopus (60) Google Scholar, 18Yeh E. Cunningham M. Arnold H. Chasse D. Monteith T. Ivaldi G. Hahn W.C. Stukenberg P.T. Shenolikar S. Uchida T. Counter C.M. Nevins J.R. Means A.R. Sears R. Nature Cell Biol. 2004; 6: 308-318Crossref PubMed Scopus (612) Google Scholar). In line with this, juglone prevents the dephosphorylation of MPM2 antigens (19Albert A.L. Lavoie S.B. Vincent M. BMC Cell Biol. 2004; 5: 22Crossref PubMed Scopus (16) Google Scholar), which constitute a subset of Pin1-interacting mitotic phosphoproteins, as well as of NHERF-1 (20He J. Lau A.G. Yaffe M.B. Hall R.A. J. Biol. Chem. 2001; 276: 41559-41565Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and Disabled-2 (21He J. Xu J. Xu X.X. Hall R.A. Oncogene. 2003; 22: 4524-4530Crossref PubMed Scopus (30) Google Scholar). For that matter, it is thought that cis-trans isomerization of p(S/T)-P bonds by Pin1 regulates dephosphorylation of PP2A targets by facilitating the accessibility of this phosphatase to its substrates (see Ref. 8Yeh E.S. Means A.R. Nat. Rev. Cancer. 2007; 7: 381-388Crossref PubMed Scopus (205) Google Scholar). Several Rab GTPases, including endosomal Rab4a, are phosphorylated by Cdk1 on S/T-P sites within their hypervariable region (22Bailly E. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Crossref PubMed Scopus (130) Google Scholar, 23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Rabs are key regulators of membrane traffic (see Ref. 24Pfeffer S.R. Annu. Rev. Biochem. 2007; 76: 629-645Crossref PubMed Scopus (143) Google Scholar), and their phosphorylation at the onset of mitosis might be important in the concomitant down-regulation of intracellular transport (22Bailly E. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Crossref PubMed Scopus (130) Google Scholar, 23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). We previously found that phosphorylated Rab4a binds Pin1 in mitotic cells (25Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z. Lu K.P. Van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar) and that PP2A dephosphorylates Rab4a when cells exit prometaphase. 3C. Fila and P. van der Sluijs, unpublished results. 3C. Fila and P. van der Sluijs, unpublished results. During the analysis of Pin1 function in dephosphorylation of Rab4a, we also employed juglone and made a number of unanticipated observations on the target of the drug. Here we show that treatment of mitotic cells with juglone prevented postmitotic dephosphorylation via pathways that did not involve Pin1. Juglone appears to cause this effect by alkylating sulfhydryl groups in proteins critical for metaphase-anaphase transition, which precludes mitotic exit. Reagents and Materials—An antibody against phospho-Ser204-Rab4a (Rab4a(pS204)) was raised in rabbits with the keyhole limpet hemocyanin-coupled CRQLRpSPRRTQAPN peptide (where pS represents phosphoserine). Rabbit polyclonal antibodies against human Rab4a and human Pin1 have been described (23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar, 25Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z. Lu K.P. Van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Other antibodies were from the indicated sources: mouse monoclonals MPM2 and BubR1 and rabbit antibodies against the catalytic subunit of PP2A (PP2Ac) and methylated PP2Ac (Upstate Cell Signaling Solutions), mouse monoclonal anti-β-catenin (BD Transduction Laboratories), mouse monoclonal anti-α- and anti-β-tubulin (Sigma), and mouse monoclonal cyclin B1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). HRP-labeled and fluorescently labeled secondary antibodies were purchased from Pierce, Jackson Laboratories, and Molecular Probes. Purified protein phosphatase PP2A1 was from Upstate Cell Signaling Solutions, SP600125 was from Sigma, and Alexa488-Annexin V and propidium iodide were from Molecular Probes. Cell Lines and Synchronization—The CHO-Rab4a cell line (23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar) and mouse embryonic fibroblasts (MEFs) from Pin1-/- mice (26Fujimori F. Takahashi K. Uchida C. Uchida T. Biochem. Biophys. Res. Commun. 1999; 265: 658-663Crossref PubMed Scopus (184) Google Scholar) were described in the references indicated. Spontaneously immortalized Pin1-/- MEFs were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin and transfected with pFRSV-Rab4a (23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar) using calcium phosphate. Clones were selected in the presence of 60 μm methotrexate and were tested for the expression of Rab4a by Western blot. Mitotic CHO and Pin1-/- MEFs were obtained as described (26Fujimori F. Takahashi K. Uchida C. Uchida T. Biochem. Biophys. Res. Commun. 1999; 265: 658-663Crossref PubMed Scopus (184) Google Scholar). In brief, cells were synchronized with 2 mm thymidine. After 15 h, the cells were released from the G1/S block for 2.5 h and then incubated with 40 ng/ml nocodazole. After 5 h, the cells were harvested by shake-off. U2OS cells were arrested in prometaphase by treatment with 250 ng/ml nocodazole for 18 h. In Vitro Phosphatase Assay—An in vitro assay was used to determine PP2A activity and was conducted as per the vendor's instructions (Molecular Probes). Briefly, 0.2 milliunits of purified PP2A1 was incubated with 6,8-difluoro-4-methyl-umbelliferyl phosphate in 100 μl of phosphatase reaction buffer (50 mm Tris-HCl, pH 7.0, 100 μm CaCl2, 1 mm NiCl2, 100 μg/ml bovine serum albumin, 0.05% Tween 20) containing either juglone or PP2A inhibitors. Reactions were incubated in a microtiter plate in the dark for 1 h at room temperature. Fluorescence was measured in triplicate in a standard fluorescence microtiter plate reader using excitation at 355 nm and emission at 460 nm. Postmitotic Dephosphorylation Assay—U2OS cells or Rab4-transfected CHO cells and Pin1-/- MEFs were arrested in prometaphase, released from the mitotic block, and incubated in the presence of various concentrations of juglone (with or without 5 mm l-cysteine) for different periods of time. Cell samples were taken and solubilized in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 50 mm NaF, 25 mm β-glycerophosphate, and Roche Complete protease inhibitors). Lysates were cleared by centrifugation at 13,000 rpm in a table-top centrifuge. Supernatants were collected, and cell pellets were resuspended in an equal volume of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% SDS. Fractions were equalized for protein content, resolved on 12% SDS-polyacrylamide gels, and analyzed by Western blot. In Vitro Tubulin Polymerization Assay—Phosphocellulose-purified bovine brain tubulin (>99% purity) was generously provided by Marileen Dogterom (Institute for Atomic and Molecular Physics, Amsterdam) and diluted to 2.2 mg/ml in 80 mm K-PIPES, pH 6.8, 0.5 mm EGTA, 2 mm MgCl2, and 5% glycerol. Tubulin polymerization reactions of 100 μl were prepared in a microtiter plate, and polymerization was started by the addition of 1 mm GTP at 37 °C and followed by A340 readings for up to 30 min. Immunofluorescence Microscopy—Mitotic Pin1-/- MEFs were seeded on poly-l-lysine-coated coverslips and incubated for up to 60 min in the presence of 1 μm juglone. Cells were fixed in ice-cold methanol and subsequently processed for indirect immunofluorescence microscopy. Cells were labeled for α-tubulin and stained with Alexa488-conjugated anti-mouse IgG. DNA was visualized with 4′,6-diamidino-2-phenylindole. Coverslips were mounted and dried, and cells were viewed with a Zeiss LSM5 confocal microscope. Flow Cytometry—Interphase CHO cells were treated with increasing concentrations of juglone for 180 min. Cells were harvested, washed in phosphate-buffered saline, and resuspended at a density of ∼1 × 106 cells/ml in 10 mm Hepes, pH 7.4, 140 mm NaCl, and 2.5 mm CaCl2. Alexa488-Annexin V and propidium iodide were added for 15 min at room temperature according to the manufacturer's instructions. Samples were immediately analyzed on a FACSVantage SE cell sorter (Becton Dickinson) using the 488-nm laser. Results were quantitated in Cellquest. Pin1-/- MEFs were treated for 15 h with increasing concentrations of juglone. During the last hour, Hoechst 33342 (10 μg/ml) was added to stain DNA. Cells were trypsinized, and cell cycle profiles were obtained by cytometric analysis on a FACSVantage SE cell sorter using the UV laser. The number of cells in different cell cycle phases (M1-M4) was evaluated in Cellquest. Rab4a is phosphorylated during mitosis by Cdk1 on Ser204 (23van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar) and dephosphorylated by PP2A when cells exit prometaphase. 4C. Fila and P. van der Sluijs, manuscript in preparation. We here employed the parvulin PPIase inhibitor juglone initially, to analyze a possible role of Pin1 in the dephosphorylation of Ser204. Juglone Titration on CHO Cells—Because high concentrations of juglone affect cell viability (27Rippmann J.F. Hobbie S. Daiber C. Guilliard B. Bauer M. Birk J. Nar H. Garin-Chesa P. Rettig W.J. Schnapp A. Cell Growth Differ. 2000; 11: 409-416PubMed Google Scholar, 28Paulsen M.T. Ljungman M. Toxicol. Appl. Pharmacol. 2005; 209: 1-9Crossref PubMed Scopus (81) Google Scholar), we performed a titration study to establish an optimal concentration of the inhibitor. CHO cells were incubated with increasing concentrations of juglone and stained with Alexa488-Annexin V to assess the extent of apoptosis and with propidium iodide to distinguish between living and dead cells by fluorescence-activated cell sorting analysis. As shown in the bar diagram in Fig. 1A, less than 10 μm juglone did not cause apoptosis (R2 and R3) or necrosis (R1 and R2), compared with controls. Concentrations above 10 μm increased the number of both apoptotic and necrotic cells. Most of the Annexin V-labeled cells were also positive for propidium iodide, showing that the majority had already entered late stages of apoptosis. In the subsequent in vivo experiments in CHO cells, we used 7.5 μm juglone, since this concentration did not induce apoptosis or cell death and is close to the minimal concentration that inactivates parvulins in vitro (4Hennig L. Christner C. Kipping M. Schelbert B. Rucknagel K.P. Grabley S. Kullertz G. Fischer G. Biochemistry. 1998; 37: 5953-5960Crossref PubMed Scopus (257) Google Scholar). Juglone Prevents Postmitotic Dephosphorylation of Rab4a—Mitotic CHO-Rab4a cells were released from prometaphase arrest and incubated in the presence of 7.5 μm juglone. Nonsynchronized (interphase) cells were treated the same and served as negative control. Timed samples were analyzed by Western blot with an antibody against phosphorylated Ser204 in Rab4a. Within 45 min after release of the mitotic block, more than 90% of Rab4a was dephosphorylated in control cells as shown in Fig. 1B. In contrast, Rab4a remained fully phosphorylated in the presence of juglone. Samples were also probed with the MPM2 antibody, which recognizes a multitude of proteins containing phosphorylated S/T-P sites with a molecular mass between ∼35 and 300 kDa (29Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Crossref PubMed Scopus (237) Google Scholar). The entire set of MPM2 antigens was dephosphorylated after 180 min of nocodazole washout in control cells (Fig. 1C). In the juglone-treated cells, MPM2 staining remained at the initial level. We therefore concluded that juglone prevented dephosphorylation of mitotic phosphoproteins, including Rab4a. Similar results were reported for Disabled-2, NHERF-1, and MPM2 antigens and proposed to be caused by inhibition of PP2A-mediated dephosphorylation due to loss of Pin1 function (19Albert A.L. Lavoie S.B. Vincent M. BMC Cell Biol. 2004; 5: 22Crossref PubMed Scopus (16) Google Scholar, 20He J. Lau A.G. Yaffe M.B. Hall R.A. J. Biol. Chem. 2001; 276: 41559-41565Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21He J. Xu J. Xu X.X. Hall R.A. Oncogene. 2003; 22: 4524-4530Crossref PubMed Scopus (30) Google Scholar). Juglone Does Not Inhibit PP2A—PP2A is a heterotrimer consisting of a scaffolding (A), a regulatory (B), and a catalytic (C) subunit (30Van Hoof C. Goris J. Cancer Cell. 2004; 5: 105-106Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Since juglone is known to inhibit proteins other than just parvulins (9Muto N. Inouye K. Inada A. Nakanishi T. Tan L. Biochem. Biophys. Res. Commun. 1987; 146: 487-494Crossref PubMed Scopus (18) Google Scholar, 10Frew T. Powis G. Berggren M. Gallegos A. Abraham R.T. Ashendel C.L. Zalkow L. Hudson C. Gruszecka-Kowalik E. Burgess E.M. Benedetti-Doctorovich V. Kerrigan J.E. Lambropoulos J. Merriman R. Bonjouklian R. Anticancer Drug Des. 1995; 10: 347-359PubMed Google Scholar, 11Varga Z. Bene L. Pieri C. Damjanovich S. Gaspar Jr, R. Biochem. Biophys. Res. Commun. 1996; 218: 828-832Crossref PubMed Scopus (33) Google Scholar), and since naphtoquinones inactivate protein-tyrosine phosphatases (31Iwamoto N. Sumi D. Ishii T. Uchida K. Cho A.K. Froines J.R. Kumagai Y. J. Biol. Chem. 2007; 282: 33396-33404Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), we first investigated the possibility that juglone might act directly on PP2A. We therefore measured the activity of purified heterotrimeric PP2A1 in the presence of juglone in an in vitro assay. The assay measures dephosphorylation of the Ser/Thr phosphatase substrate 6,8-difluoro-4-methyl-umbelliferyl phosphate, which generates fluorescent 6,8-difluoro-4-methyl-umbelliferyl with excitation/emission maxima at 358/452 nm. Whereas established PP2A inhibitors calyculin A and okadaic acid reduced the fluorescence intensity more than 95% with respect to control levels, juglone did not affect PP2A as shown in Fig. 2A. Since maximal activity of PP2A in vivo requires methylation of the C subunit (30Van Hoof C. Goris J. Cancer Cell. 2004; 5: 105-106Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), we also analyzed in cells whether juglone affected this posttranslational modification. As shown in Fig. 2B, juglone did not inhibit PP2A methylation, as we found by Western blot with a specific antibody against the methylated form of the C subunit of PP2A. Collectively, the assays for PP2A activity and PP2A methylation showed that juglone did not inhibit PP2A directly and that postmitotic dephosphorylation of Rab4a is unlikely to be due to inhibition of the phosphatase. Juglone Blocks Postmitotic Dephosphorylation in Pin1-/- Cells—Having shown that inhibited dephosphorylation of mitotic phosphoproteins (Fig. 1, B and C) is not caused by a direct effect of juglone on PP2A (Fig. 2, A and B), we next investigated whether the block was due to inhibition of Pin1. For that matter, we studied dephosphorylation of mitotically phosphorylated Rab4a in MEFs derived from Pin1-/- mice (26Fujimori F. Takahashi K. Uchida C. Uchida T. Biochem. Biophys. Res. Commun. 1999; 265: 658-663Crossref PubMed Scopus (184) Google Scholar). The cells lacked immunologically detectable Pin1, as evidenced by a Western blot with a polyclonal antibody against Pin1 (Fig. S1). As a consequence of Pin1 deficiency, the cells also contained ∼40% less of the Pin1 client protein β-catenin (Fig. S1), whose stability correlates directly with Pin1 expression levels (32Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (418) Google Scholar). The reduced β-catenin levels in Pin1-/- MEFs also suggested that the cells do not have salvage mechanisms to counteract the loss of Pin1 activity. Pin1-/- MEFs were next released from the mitotic block and incubated in the presence of 7.5 μm juglone for up to 60 min. The cells were then harvested at different periods of time after nocodazole washout, and detergent lysates were analyzed for the amounts of Rab4a(pS204) and total Rab4a. The rate of Rab4a dephosphorylation in the absence of Pin1 was comparable with that in CHO cells (Fig. 3). Even in a genetic model for the loss of Pin1 function, juglone inhibited the dephosphorylation of Rab4a in the Pin1-/- MEFs (Fig. 3). These results showed that the effect of juglone is not caused by a mechanism involving Pin1. Juglone Decreased Tubulin Content in Lysates—Careful analysis of the experiments with the Pin1-/- cells revealed an additional effect of juglone treatment. We found that the amount of α- and β-tubulin decreased during the release of the mitotic block in the presence of juglone (Fig. 3). This observation was also made in interphase cells that were treated for 60 min with juglone, documenting that the cell cycle stage was immaterial to this effect of the drug. The disappearance of α- and β-tubulin was not caused by leakage of cytosolic proteins, because actin levels remained the same throughout the experiment. To account for the loss of α- and β-tubulin from the detergent lysates, we evaluated whether the tubulins were sedimented in the Triton X-100-insoluble pellet that was generated during preparation of cleared lysates. Pellets were solubilized in reducing Laemmli buffer and analyzed by Western blot. As shown in Fig. 3, the amount of Triton X-100-insoluble (from now on called insoluble) α- and β-tubulin increased in a time-dependent manner in the presence of juglone. Insoluble, presumably aggregated α- and β-tubulin was also detected in the pelleted material of juglone-treated interphase cells (Fig. 3). Thus, juglone rendered tubulin partially insoluble in Pin1-/- MEFs. Although 7.5 μm juglone caused aggregation of tubulin in Pin1-/- MEFs, we did not observe this in CHO cells (Fig. 1B). To investigate whether or not the effect on the Triton X-100 solubility of tubulin (from now on called solubility) was restricted to Pin1-/- MEFs, we performed juglone titrations on CHO cells and Pin1-/- MEFs. Nocodazole-arrested mitotic cells were harvested and incubated for 60 min in medium containing increasing juglone concentrations. Cell lysates were then analyzed for Rab4a(pS204), MPM2 antigens, α-tubulin, and actin. As shown in Fig. 4A, juglone concentrations above 2 μm inhibited dephosphorylation of Rab4a and MPM2 antigens in CHO cells. The amount of soluble α-tubulin only started to decrease at juglone concentrations above 10 μm. The initial experiments with CHO cells in which we found inhibited post-mitotic dephosphorylation (Fig. 1B) were done with 7.5 μm, which did not affect tubulin solubility (Fig. 4A). In Pin1-/- MEFs, the dephosphorylation of phospho-Rab4a was already inhibited by as little as 0.1 μm juglone (Fig. 4B), whereas tubulin became insoluble at juglone concentrations above 2 μm (Fig. 4B). Thus, the dose-dependent effects of the Pin1 inhibitor on dephosphorylation of mitotic phosphoproteins and tubulins were not limited to Pin1-/- MEFs but were also recapitulated in another cell line. The experiments in Figs. 2 and 4 also showed that dephosphorylation of mitotic phosphoproteins is a more sensitive read-out than the generation of insoluble tubulin. Juglone Affects Tubulin Function via Alkylation of SH Groups—Juglone can covalently modify free SH groups of cysteine residues, which is the basis for the inactivation of Pin1 (4Hennig L. Christner C. Kipping M. Schelbert B. Rucknagel K.P. Grabley S. Kullertz G. Fischer G. Biochemistry. 1998; 37: 5953-5960Crossref PubMed Scopus (257) Google Scholar) and probably of other proteins (33Chao S.H. Greenleaf A.L. Price D.H. Nucleic Acids Res. 2001; 29: 767-773Crossref PubMed Scopus (143) Google Scholar). To examine if the effects of juglone on" @default.
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• W1968580657 date "2008-08-01" @default.
• W1968580657 modified "2023-10-12" @default.
• W1968580657 title "Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis" @default.
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• W1968580657 doi "https://doi.org/10.1074/jbc.m710264200" @default.
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