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Indirubins Inhibit Glycogen Synthase Kinase-3β and CDK5/P25, Two Protein Kinases Involved in Abnormal Tau Phosphorylation in Alzheimer's Disease

A PROPERTY COMMON TO MOST CYCLIN-DEPENDENT KINASE INHIBITORS?*210
Open AccessPublished:January 05, 2001DOI:https://doi.org/10.1074/jbc.M002466200
      The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC50: 50–100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC50: 5–50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3β (GSK-3β). Testing of a series of indoles and bis-indoles against GSK-3β, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3β's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3β, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3′-monoxime also inhibits the in vivophosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3β. To which extent these GSK-3β effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3β to be described.
      CDK
      cyclin-dependent kinase
      AD
      Alzheimer's disease
      HLB
      hypotonic lysis buffer
      PHF
      paired helical filaments
      PKA
      protein kinase A (cAMP-dependent kinase)
      GSK-3β
      glycogen synthase kinase-3β
      EI
      electron impact
      Mops
      4-morpholinepropanesulfonic acid
      DTT
      dithiothreitol
      BSA
      bovine serum albumin
      PAGE
      polyacrylamide gel electrophoresis
      Indigoı̈ds are bis-indoles derived from various natural sources by fermentation, oxidation, and dimerization in the presence of light. The colorful indirubin (1) and indigo (5) originate from the dimerization of colorless precursors, indoxyl and isatin (4) (see Fig. 1). These indoles are released during the fermentation process from conjugates, the nature of which depends on the plant (indican, isatan B) or mollusc (indoxylsulfate) species from which the dyes are prepared (see Fig.1). The use of indigoı̈ds as textile dyes dates back to the Bronze age (−7000), but indigo (now synthetic) remains the most abundantly produced dye in the world (blue jeans, denims, etc.) (
      • Hurry J.B.
      ,
      • Balfour-Paul J.
      ). Indigo-producing plants have also been used in traditional Chinese medicine (
      • Tang W.
      • Eisenbrand G.
      ,

      Lee, H. (1993) in Human Medicinal Agents from Plants(Kinghorn, A. D., and Balandrin, M. F., eds) Chapter 12, pp. 170–190, ACS Symposium Series 534, American Chemical Society, Washington, DC.

      ,
      • Han R.
      ). A well-studied example is Danggui Longhui Wan, a mixture of 11 herbal medicines traditionally utilized against certain types of leukemias. Only one of these ingredients, Qing Dai (Indigo naturalis), a dark blue powder originating from various indigo-producing plants, was found to carry the antileukemic activity (
      • Chen D.H.
      • Xie J.X.
      ). Although it is mostly constituted of indigo, a minor constituent, indirubin, was identified as the active component by the Chinese Academy of Medicine (
      • Institute of Haematology, Chinese Academy of Medical Sciences
      ,
      • Chen D.W.
      • Li Y.F.
      • Ye H.P.
      ,
      • Wu L.M.
      • Yang Y.P.
      • Zhu Z.H.
      ). Preclinical studies performed with indirubin, and more soluble analogues, confirmed that these compounds exhibit good antitumor activity and only minor toxicity (
      • Ji X.J.
      • Zhang F.R.
      • Lei J.L.
      • Xu Y.T.
      ,
      • Sichuan Institute of Traditional Chinese Medicine
      ,
      • Wang J.H.
      • You Y.C.
      • Mi J.X.
      • Ying H.G.
      ,
      • Ji X.J.
      • Zhang F.R.
      ,
      • Li C.
      • Go Y.
      • Mao Z.
      • Koyano K.
      • Kai Y.
      • Kaneshisa N.
      • Zhu Q.
      • Zhou Z.
      • Wu S.
      ). Clinical trials showed that indirubin has a definite efficiency against chronic myelocytic leukemia (
      • Han R.
      ,
      • Institute of Haematology, Chinese Academy of Medical Sciences
      ,
      • Cooperative Group of Clinical Therapy of Indirubin
      ,
      • Gan W.J.
      • Yang T.Y.
      • Wen S.D.
      • Liu Y.Y.
      • Tan Z.
      • Deng C.A.
      • Wu J.X.
      • Liu M.P.
      ,
      • Zhang Z.N.
      • Liu E.K.
      • Zheng T.L.
      ,
      • Zhang Z.N.
      • Liu E.K.
      • Zheng T.L.
      ,
      • Ma M.
      • Yao B.
      ,
      • Chang C.N.
      ).
      Figure thumbnail gr1
      Figure 1The bis-indoles indigo (
      • Han R.
      ), indirubin (
      • Hurry J.B.
      ), and isoindigo (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • De la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ) are derived from the dimerization of indoxyls and isatins, which are themselves derived from the hydrolysis of either indican and isatans (plants) or indoxyl sulfates (molluscs).
      Several mechanisms of action have been brought forward to explain the antimitotic and antitumoral properties of indirubins (
      • Wu G.Y.
      • Fang F.D.
      • Liu J.Z.
      • Chang A.
      • Ho Y.H.
      ,
      • Wu G.Y.
      • Liu J.Z.
      • Fang F.D.
      • Zuo J.
      ,
      • Zhang L.
      • Wu G.Y.
      • Qiu C.C.
      ). We recently reported that indirubins are potent inhibitors of cyclin-dependent kinases (CDKs)1(
      • Hoessel R.
      • Leclerc S.
      • Endicott J.
      • Noble M.
      • Lawrie A.
      • Tunnah P.
      • Leost M.
      • Damiens E.
      • Marie D.
      • Marko D.
      • Niederberger E.
      • Tang W.
      • Eisenbrand G.
      • Meijer L.
      ), a family of key cell cycle regulators (

      Dunphy, W. G. (ed) (1997) Methods Enzymol. 283, 1-67

      ,
      • Morgan D.
      ,
      • Vogt P.K.
      • Reed S.I.
      ). Indirubins act by competing with ATP for binding to the catalytic site of the kinase. The kinase selectivity study showed that indirubins have a strong affinity for CDKs (IC50 values in the range of 50–100 nm) (
      • Hoessel R.
      • Leclerc S.
      • Endicott J.
      • Noble M.
      • Lawrie A.
      • Tunnah P.
      • Leost M.
      • Damiens E.
      • Marie D.
      • Marko D.
      • Niederberger E.
      • Tang W.
      • Eisenbrand G.
      • Meijer L.
      ). Nevertheless, they are not totally devoid of activity toward a few kinases (IC50 values in the 1–10 μm range) (
      • Hoessel R.
      • Leclerc S.
      • Endicott J.
      • Noble M.
      • Lawrie A.
      • Tunnah P.
      • Leost M.
      • Damiens E.
      • Marie D.
      • Marko D.
      • Niederberger E.
      • Tang W.
      • Eisenbrand G.
      • Meijer L.
      ). This rather loose selectivity, when compared with the high specificity of purine inhibitors of CDKs, led us to continue to investigate the selectivity of indirubins as kinase inhibitors.
      We report here that indirubins are very potent inhibitors (IC50 values in the 5–50 nm range) of glycogen synthase kinase-3β (GSK-3β). This kinase is an essential element of the WNT signaling pathway (
      • Willert K.
      • Nusse R.
      ). It is involved in multiple physiological processes, including cell cycle regulation by controlling the levels of cyclin D1 (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ) and β-catenin (
      • Yost C.
      • Torres M.
      • Miller J.R.
      • Huang E.
      • Kimelman D.
      • Moon R.T.
      ), dorsal-ventral patterning during development (
      • Yost C.
      • Torres M.
      • Miller J.R.
      • Huang E.
      • Kimelman D.
      • Moon R.T.
      ,
      • He X.
      • Saint-Jeannet J.-P.
      • Woodgett H.E.
      • Varmus H.E.
      • Dawid I.B.
      ,
      • Emily-Fenouil F.
      • Ghiglione C.
      • Lhomond G.
      • Lepage T.
      • Gache C.
      ), insulin action on glycogen synthesis (
      • Cohen P.
      ,
      • Summers S.A.
      • Kao A.W.
      • Kohn A.D.
      • Backus G.S.
      • Roth R.A.
      • Pessin J.E.
      • Birnbaum M.J.
      ), axonal outgrowth (
      • Lucas F.R.
      • Goold R.G.
      • Gordon-Weeks P.R.
      • Salinas P.C.
      ), HIV-1 Tat-mediated neurotoxicity (
      • Maggirwar S.B.
      • Tong N.
      • Ramirez S.
      • Gelbard H.A.
      • Dewhurst S.
      ), among others. Furthermore, GSK-3β and CDK5 are responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in the paired helical filaments, which are diagnostic for Alzheimer's disease (AD) (
      • Imahori K.
      • Uchida T.
      ,
      • Mandelkow E.-M.
      • Mandelkow E.
      ). It was recently demonstrated that conversion of p35, the regulatory subunit of CDK5, to a truncated form, p25, deregulates CDK5 activity and promotes neurodegeneration (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • De la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ). We here show that indirubins are very potent inhibitors of CDK5/p25. Furthermore, indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivoat Alzheimer's disease-specific sites. Indirubins may thus constitute a lead compound in the study and treatment of neurodegenerative disorders involving abnormal phosphorylation of tau (“taupathies”). We here also show that indirubin-3′-monoxime inhibits phosphorylation of DARPP-32 by CDK5. DARPP-32 is a striatum protein acting downstream of dopamine action, which is either phosphorylated on Thr-34 by cAMP-dependent protein kinase (PKA) (it then acts as a phosphatase 1 inhibitor) or on Thr-75 by CDK5 (it then becomes a PKA inhibitor) (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.-H.
      • Kwon Y.T.
      • Girault J.-A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ). Finally, we also show that many, but not all, CDK inhibitors are potent GSK-3β inhibitors. Whether the antimitotic/antitumoral properties of indirubins (and other CDK inhibitors) derive from their dual inhibitory effects on GSK-3β and CDKs remains to be determined.

      EXPERIMENTAL PROCEDURES

      Chemistry

      Indigo (2) (Fluka), isatin (3) (Fluka), 5,5′,7,7′-indigotetrasulfonic acid potassium salt (25) (Fluka), 5,5′,7-indigotrisulfonic acid potassium salt (26) (Fluka), indigo carmine (27) (Fluka), 5-chloroisatin (30) (Lancaster), 5-fluoroisatin (29) (Aldrich), 5-bromoisatin (31) (Fluka), 5-methylisatin (32) (Sigma), isatin-5-sulfonic acid sodium salt dihydrate (33) (Fluka), 5-nitroisatin (34) (Acros), 1-methylisatin (35) (Acros), 1-phenylisatin (36) (Lancaster), indoxyl acetate (43) (Fluka), 5-bromoindoxyl acetate (44) (Fluka), and other solvents and reagents were obtained from commercial suppliers. They were at least of reagent grade and were used without further purification. Indirubin (1), 5-iodoindirubin (4), 5-bromoindirubin (5), 5-chloroindirubin (6), 5-fluoroindirubin (7), 5-methylindirubin (8), 5-nitroindirubin (9), indirubin-5-sulfonic acid (sodium salt) (10), 5′-bromoindirubin (11), 5,5′-dibromoindirubin (12), 5′-bromoindirubin-5-sulfonic acid (sodium salt) (13), indirubin-3′-monoxime (14), 5-iodoindirubin-3′-oxime (15), 6-iodoindirubin (16), 1-methylindirubin (17), 1-phenylindirubin (18), 3′-hydroxyiminoindirubin-5-sulfonic acid (sodium salt) (19), indirubin-5-sulfonamide (20), indirubin-5-sulfonic acid dimethylamide (21), indirubin-5-sulfonic acid (2-hydroxyethyl)amide (22), indirubin-5-sulfonic acid bis-(2-hydroxyethyl)amide (23), indirubin-5-sulfonic acid methylamide (24), 5-iodoisatin (28), isatin-5-sulfonic acid dimethylamide (37), isatin-5-sulfonic acid bis-(2-hydroxyethyl)amide (38), 6-iodoisatin (39), isoindigo (40), 2,2′-bi-indole (41), 3,3′-diphenyl-2,2′-bi-indole (42), isatin-5-sulfonamide (45), isatin-5-sulfonic acid (2-hydroxyethyl)amide (46), isatin-5-sulfonic acid methylamide (47), and 2-hydroxyimino-N-(3-iodophenyl)acetamide (48) were synthesized and purified as described in the Supplementary material section. Synthesis reactions involving oxygen or moisture-sensitive compounds were performed under a dry argon atmosphere. All reaction mixtures and column chromatographic fractions were analyzed by thin layer chromatography on plates (Alugram Sil G/UV254, purchased from Macherey & Nagel). Column chromatography was carried out using Silica Gel 60 (0.063–0.2 mm, Macherey & Nagel). Melting points of the non-indigoı̈d compounds were determined on a Büchi 510 melting point apparatus and were uncorrected. Melting points over 260 °C were determined on a Wagner and Munz Kupferblock. Elemental analyses were performed using a 2400 CHN elemental analyzer (PerkinElmer Life Sciences). Unless otherwise indicated, NMR spectra were recorded at room temperature.1H NMR spectra were recorded at 400 MHz, 13C NMR spectra at 100 MHz on a Bruker AMX 400, using tetramethylsilane, or Me2SO (δ = 39.4 ppm) as internal standard.J values are reported in hertz. Apparent multiplicities were designated as s, singlet; d, doublet; dd, double doublet; t, triplet; pt, pseudo-triplet; q, quartet; m, multiplet; b, broad. Mass spectra were taken in the positive ion mode under electron impact (EI 70 eV) using a Finnigan MAT 90 mass spectrometer. Gas chromatography/mass spectrometry was performed using a Hewlett-Packard, 5890 Series II gas chromatograph on a 25-m fused silica column (Hewlett-Packard HP-5, I.D. = 0.25 mm; 0.25 μm) and a Hewlett-Packard, HP 5971A mass-selective detector with the following temperature program: 80 °C (4 min), 25 °/min, 320 °C (16.4 min).
      All compounds were dissolved and stored as 10 mm stock solutions in Me2SO. They were diluted in aqueous buffers just prior use.

      Biochemical Reagents

      Sodium orthovanadate, EGTA, EDTA, RNase A, Mops, β-glycerophosphate, phenylphosphate, sodium fluoride, glutathione-agarose, dithiothreitol (DTT), bovine serum albumin (BSA), nitrophenylphosphate, leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, benzamidine, and histone H1 (type III-S) were obtained from Sigma Chemical Co. [γ-32P]ATP (PB 168) was obtained from Amersham Pharmacia Biotech.
      The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE) was synthesized by the Peptide Synthesis Unit, Institute of Biomolecular Sciences, University of Southampton, UK.
      AT-8, AT-180, and AT-100 antibodies were obtained from Innogenetics, SA, Ghent, Belgium, PHF-1 was a gift from Dr. P. Davies (Bronx, NY), and K9JA was obtained from Dako (Hamburg, Germany).

      Buffers

      Homogenization Buffer

      60 mmβ-glycerophosphate, 15 mm p-nitrophenylphosphate, 25 mm Mops (pH 7.2), 15 mm EGTA, 15 mm MgCl2, 1 mm DTT, 1 mm sodium vanadate, 1 mmNaF, 1 mm phenylphosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, and 100 μm benzamidine.

      Buffer A

      Buffer A consisted of 10 mmMgCl2, 1 mm EGTA, 1 mm DTT, 25 mm Tris-HCl, pH 7.5, 50 μg/ml heparin.

      Buffer C

      Buffer C consisted of homogenization buffer but 5 mm EGTA, no NaF, and no protease inhibitors.

      Tris-buffered Saline-Tween 20 (TBST)

      This buffer consisted of 50 mm Tris, pH 7.4, 150 mm NaCl, and 0.1% Tween 20.

      Hypotonic Lysis Buffer (HLB)

      HLB buffer consisted of 50 mm Tris-HCl, pH 7.4, 120 mm NaCl, 10% glycerol, 1% Nonidet-P40, 5 mm DTT, 1 mm EGTA, 20 mm NaF, 1 mm orthovanadate, 5 μm microcystin, and 100 μg/ml each of leupeptin, aprotinin, and pepstatin.

      Kinase Preparations and Assays

      Kinase activities were assayed in Buffer A or C (unless otherwise stated), at 30 °C, at a final ATP concentration of 15 μm. Blank values were subtracted, and activities were calculated as picomoles of phosphate incorporated for a 10-min incubation. The activities are usually expressed in percentage of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethyl sulfoxide. In a few cases phosphorylation of the substrate was assessed by autoradiography after SDS-PAGE (see below).

      GSK-3β

      GSK-3β was expressed in and purified from insect Sf9 cells (
      • Hughes K.
      • Pulverer B.J.
      • Theocharous P.
      • Woodgett J.R.
      ). It was assayed, following a 1/100 dilution in 1 mg/ml BSA, 10 mm DTT, with 5 μl of 40 μm GS-1 peptide as a substrate, in buffer A, in the presence of 15 μm[γ-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 30-min incubation at 30 °C, 25-μl aliquots of supernatant were spotted onto 2.5- × 3-cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters were washed five times (for at least 5 min each time) in a solution of 10 ml of phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml of ACS (Amersham Pharmacia Biotech) scintillation fluid.

      CDK1/Cyclin B

      CDK1/cyclin B was extracted in homogenization buffer from M phase starfish (Marthasterias glacialis) oocytes and purified by affinity chromatography on p9CKShs1-Sepharose beads, from which it was eluted by free p9CKShs1 as described previously (
      • Meijer L.
      • Borgne A.
      • Mulner O.
      • Chong J.P.J.
      • Blow J.J.
      • Inagaki N.
      • Inagaki M.
      • Delcros J.G.
      • Moulinoux J.P.
      ,
      • Borgne A.
      • Ostvold A.C.
      • Flament S.
      • Meijer L.
      ). The kinase activity was assayed in buffer C, with 1 mg/ml histone H1, in the presence of 15 μm [γ-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 10-min incubation at 30 °C, 25-μl aliquots of supernatant were spotted onto P81 phosphocellulose papers and treated as described above.

      CDK/p25

      CDK5/p25 was reconstituted by mixing equal amounts of recombinant mammalian CDK5 and p25 expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified by affinity chromatography on glutathione-agarose (vectors kindly provided by Dr. J. H. Wang). (p25 is a truncated version of p35, the 35-kDa CDK5 activator.) Its activity was assayed in buffer C as described for CDK1/cyclin B.

      In Vitro and in Vivo Tau Phosphorylation

      Cells and Viruses

      Sf9 cells (Invitrogen, San Diego, CA) were grown at 27 °C in monolayer culture Grace's medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin and 2.5 μg/ml amphotericin. BaculoGold was obtained from PharMingen (San Diego, CA), pVL1392 was obtained from Invitrogen.

      Tau Transfection

      The gene for htau23, the shortest human tau isoform, was excised from the bacterial expression vector pNG2 (
      • Biernat J.
      • Gustke N.
      • Drewes G.
      • Mandelkow E.-M.
      • Mandelkow E.
      ) with XbaI and BamHI and inserted into the baculovirus transfer vector pVL1392 cut with the same restriction endonucleases. The BaculoGold system was used to construct the tau baculovirus-containing vector. The BaculoGold DNA is a modified type of baculovirus containing a lethal deletion. Cotransfection of the BaculoGold DNA with a complementing baculovirus transfer vector rescued the lethal deletion of this virus DNA and reconstituted viable virus particles carrying the htau23 coding sequence. Plasmid DNA used for transfections was purified using Qiagen cartridges (Hilden, Germany). Sf9 cells grown in monolayers (2 × 106 cells in a 60-mm cell culture dish) were cotransfected with baculovirus DNA (0.5 μg of BaculoGold DNA) and with vector derivatives of pVL1392 (2 μg) using a calcium phosphate coprecipitation method. The presence of recombinant protein was examined in the infected cells 5 days post-infection by SDS-PAGE and Western blotting.

      Treatment of Sf9 Cells with Kinase Inhibitors

      To determine the effects of aminopurvalanol and indirubin-3′-monoxime on tau phosphorylation, Sf9 cells infected with baculovirus expressing htau23 protein were treated 36 h post-infection (when cells have already expressed levels of tau sufficient for the outgrowth of cell processes (
      • Biernat J.
      • Mandelkow E.-M.
      )) with 20 μm inhibitors for 3 h before being harvested.

      Tau Western Blotting

      Sf9 cells were infected with recombinant virus at a multiplicity of infection of 1–5. Cell lysates were prepared in hypotonic lysis buffer (HLB). After 15-min centrifugation at 16,000 × g, the supernatant was recovered and its NaCl concentration raised to 500 mm. It was then boiled for 10 min and recentrifuged at 16,000 ×g for 15 min. Proteins (3 μg) were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and Western-blotted with the following antibodies: AT-8 (1:2000), AT-180 (1:1000), AT-100 (1:1000), PHF-1 (1:600), and polyclonal anti-tau antibody K9JA. The immunostaining was visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech, Braunschweig, Germany).

      Tau Phosphorylation

      Tau phosphorylation in vitrowas performed using purified GSK-3β and recombinant human tau-32 (provided by Dr. M. Goedert) as a substrate. After 30-min incubation in the presence of various indirubin-3′-monoxime concentrations, under the GSK-3β assay conditions described above, the kinase reaction was stopped by addition of Laemmli sample buffer. Tau was resolved by 10% SDS-PAGE, and its phosphorylation level was visualized by autoradiography.

      In Situ Inhibition of CDK5 in the Striatum

      Adult mouse brain striatal slices were prepared using standard methodology (
      • Nishi A.
      • Snyder G.L.
      • Greengard P.
      ). Following equilibration in Krebs' bicarbonate buffer oxygenated with continuous aeration (95% O2/5% CO2), slices were treated with various concentrations of indirubin-3′-monoxime or 10 μm roscovitine for 60 min or were left in Krebs' bicarbonate buffer for the same period of time. Slices were homogenized by sonication in boiling 1% SDS and 50 mm NaF. Protein concentrations were determined by the BCA method using a BSA standard curve. Equal amounts of protein (80 μg) were subjected to SDS-PAGE using a 15% acrylamide gel, electrophoretically transferred to nitrocellulose membrane, and immunoblotted with a phosphorylation state-specific antibody that selectively detects DARPP-32 phosphorylated at Thr-75 (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.-H.
      • Kwon Y.T.
      • Girault J.-A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ).

      Acknowledgments

      We thank Dr. Ning Xiuren for his help with Chinese ideograms and the fishermen of the Station Biologique de Roscoff for collecting the starfish. We are grateful to our following colleagues for providing reagents: P. Davies, M. Goedert, and J. H. Wang.

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