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Purification of GSK-3 by Affinity Chromatography on Immobilized Axin
Protein Expression and Purification 20, 394 – 404 (2000) doi:10.1006/prep.2000.1321, available online at http://www.idealibrary.com on
Purification of GSK-3 by Affinity Chromatography on Immobilized Axin Aline Primot,* Blandine Baratte,* Marie Gompel,* Annie Borgne,* Sylvie Liabeuf,† Jean-Louis Romette,† Eek-hoon Jho,‡ Frank Costantini,‡ and Laurent Meijer* ,1 *Station Biologique, CNRS, BP 74, 29682 Roscoff cedex, Bretagne, France; †ESIL-GBMA, Faculte´ des Sciences de Luminy, AFMB-DISP, 163, avenue de Luminy, 13288 Marseille Cedex 8, France; and ‡Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, New York 10032
Received May 4, 2000, and in revised form August 9, 2000
Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of -catenin and cyclin D1), Alzheimer’s disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ß, -catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419 – 672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These “Axin-His6” matrices were found to selectively bind recombinant rat GSK-3 and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes. © 2000 Academic Press Key Words: protein kinase; GSK-3; axin; -catenin; APC; WNT; armadillo; kinase inhibitors; screening; cancer; diabetes; Alzheimer’s disease.
1 To whom correspondence and reprint requests should be addressed. Fax: (33).02.98.29.23.42. E-mail: [email protected].
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The human genome encodes an estimated 1500 – 2000 protein kinase genes. Glycogen synthase kinase-3 is a Ser/Thr kinase, which was initially identified as a kinase phosphorylating and inactivating glycogen synthase (1, 2). In mammals GSK-3 2 is present as two very closely homologous isoforms, GSK-3␣ and GSK-3 (3– 5), which are variably expressed in different tissues (6). Numerous substrates of GSK-3 have been described beside glycogen synthase: -catenin, c-jun, the Adenomatous Polyposis Coli protein (APC), inhibitor-2 of protein phosphatase 1, cyclin D1, insulin receptor substrate 1 (IRS-1), the microtubule-binding proteins tau and MAP-1B, elF2B, . . .etc. GSK-3 acts as a downstream element in the Wnt signaling pathway (7–9) and is therefore involved in multiple cellular and physiological processes: (i) GSK-3 regulates dorsoventral patterning during development (10 –13). (ii) GSK-3 is negatively regulated in response to insulin, and its inhibition leads to stimulation of glycogen, fatty acid, and protein synthesis (14, review in 15). (iii) GSK-3 is also involved in cell cycle control; it phosphorylates cyclin D1 on Thr286, thereby leading to its redistribution from the nucleus to the cytoplasm and to its proteolytic degradation (16). GSK-3 (in complex with the tumor suppressor protein APC and axin) phosphorylates -catenin, leading to its ubiquitin-dependent degradation (17). The failure of this degradation, resulting from mutant APC or -catenin, is common in human colon cancer and melanoma (18 –21). -catenin associates with transcription factors of the LEF-1 family to 2 Abbreviations used: Axin-His6, histidine-tagged axin peptide; APC, adenomatous polyposis coli; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GS-1, glycogen synthase peptide 1; GSK-3, glycogen synthase kinase-3; HF cells, high five cells; IPTG, isopropyl--Dthiogalactopyranoside; LB, Luria Bertani medium; Ni-NTA, nickelnitrilotriacetic acid; p-NPP, p-nitrophenylphosphate; SBTI, soybean trypsin inhibitor.
1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
PURIFICATION OF GSK-3 BY AFFINITY CHROMATOGRAPHY
activate the expression of specific genes, in particular the cyclin D1 gene (7, 21, 22, and references therein). These events are clearly linked to cancerogenesis (7, 8, 21, 23). (iv) GSK-3 is also involved in neuronal functions: it regulates axonal outgrowth by phosphorylating MAP-1B (24) and is responsible for HIV-1 Tatmediated neurotoxicity (25). Along with CDK5/p25 (26), GSK-3 is responsible for the phosphorylation of Alzheimer’s disease specific sites on the tau protein, participating thereby to the formation of paired helical filaments, a diagnostic feature of this neurodegenerative disorder (27–31; review in 32, 33). Axin is the product of a mouse gene originally named “Fused” (34), which was cloned on the basis of an insertional mutation in the gene. Axin was subsequently found to modulate signaling through the Wnt pathway which accounts for its effects on embryonic axial development (35). Axin, and its homolog conductin, binds simultaneously to GSK-3 and -catenin, thereby stimulating -catenin phosphorylation (35– 42). Axin also binds several other proteins including the -catenin homologous protein plakoglobin (43), phosphatase 2A (44 – 45), APC (41), MEKK1 (46), and itself (40, 44). The domains binding these various proteins have been located on the axin protein (Fig. 1). In this article we have taken advantage of the highly selective and potent interaction between axin and GSK-3 to set up a simple affinity chromatography protocol for the rapid purification of active GSK-3 to near homogeneity. This method provides a useful tool to monitor the status of GSK-3 (protein levels, posttranslational modifications, kinase activity) in cells and tissues under various physiological conditions. It could also be useful for the purification of large quantities of native GSK-3 to be used in inhibitors screening or, as a purification step, in crystallography programs. MATERIALS AND METHODS
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and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK). [␥- 32P]ATP (PB168) was obtained from Amersham and benzamidine was from Becham. Buffers Lysis buffer. 50 mM NaH 2PO 4 pH 8.0, 300 mM NaCl, 10 mM imidazole. Lysis buffer for cell culture. 155 mM NaCl, 10 mM tris, 1% Triton X-100, 1% deoxycholic acid, 1 mM EGTA. Wash buffer. 50 mM NaH 2PO 4 pH 8.0, 300 mM NaCl, 10% glycerol. Homogenization buffer. 60 mM -glycerophosphate, 15 mM p-NPP, 25 mM Mops, pH 7.2, 15 mM MgCl 2, 0.5 mM DTT, 1 mM Na Vanadate, 1 mM NaF, 1 mM diNa-phenylphosphate, 10 g leupeptin/ml, 10 g aprotinin/ml, 10 g SBTI/ml, 100 M benzamidine. Bead buffer. 50 mM Tris A, pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, 10 g leupeptin/ml, 10 g aprotinin/ml, 10 g SBTI/ml, 100 M benzamidine. Kinase buffer. 30 mM MgCl 2, 3 mM EGTA, 3 mM DTT, 75 mM Tris–HCl, pH 7.5, 150 g heparin/ml. Tris-buffered saline (TBS). 50 mM Tris, pH 7.4, 150 mM NaCl. Anode 1 buffer. 300 mM Tris, pH 10.4, 20% methanol. Anode 2 buffer. 25 mM Tris, pH 10.4, 20% methanol. Cathode buffer. 25 mM Tris, pH 9.4, 40 mM glycine, 20% methanol. Stop buffer. 150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN 3, pH 8. HB buffer. 25 mM Mops, pH 7.2, 15 mM p-NPP, 15 mM MgCl 2, 60 mM -glycerophosphate, 15 mM EGTA, 1 mM DTT, 0.1 mM sodium vanadate, 1% Triton X-100, 1 mM PMSF, 20 g/ml leupeptin, 40 g/ml aprotinin
Chemicals Sodium fluoride, sodium orthovanadate, isopropyl-D-thiogalactopyranoside (IPTG), dithiothreitol (DTT), -glycerophosphate, MgCl 2, NaCl, LiCl, EGTA, MOPS, ampicillin, Luria Bertani (LB) medium, heparin, leupeptin, aprotinin, soybean trypsin inhibitor (SBTI), NaH 2PO 4, Na 2CO 3, methanol, imidazole, CNBr-activated Sepharose 4B beads, formaldehyde 37%, acetate were obtained from Sigma Chemicals. Ethanolamine was obtained from Prolabo. Tris and Coomassie brilliant blue R-250 were obtained from Bio-Rad. Ni-NTA matrix was purchased from Qiagen, p-nitrophenylphosphate (p-NPP) (disodium salt hexahydrate) was from Acros Organics. The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE, where Sp is a phosphorylated serine) was purchased from the University of Southampton (Department of Biochemistry
Expression of Axin-His6 in Bacteria An Escherichia coli strain (BL21 (DE3)) containing a plasmid encoding axin tagged with six histidines (pET32a-Ax(419-672)) was used. Expression of this plasmid is under control of IPTG. E. coli were first grown overnight at 37°C in the presence of 50 g ampicillin/ml LB medium. Five milliliters of this preculture were inoculated/liter of LB containing 50 g ampicillin/ml. Incubation was continued at 37°C until the culture O.D. at 600 nm had reached a value between 0.6 and 0.8 (about 4 –5 h). At this time, 0.4 mM IPTG was added and the incubation was prolonged for 5 h at 30°C. Cells were then pelleted at 6000 rpm for 10 min and stored frozen at ⫺80°C until extraction. They were then disrupted by sonication in lysis buffer (2 ml lysis buffer/50 ml culture). After 20 min centrifugation at 35,000 rpm, the
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supernatant was passed through a 0.45 m filter and frozen at ⫺80°C in 1-ml aliquots. Preparation of Axin-His6 Agarose Beads Axin-His6-containing lysate (75 l) was diluted in 725 l lysis buffer and mixed with 30 l Ni-NTA beads. The mixture was rotated (200 rpm) at 4°C for 15 min. After a 10-s pulse centrifugation, the supernatant was eliminated. The beads were washed four times with 200 l of wash buffer. Coupling of Axin-His6 to CNBr-Activated Sepharose 4B Axin-His6 purified by the Ni-NTA method was dialysed in 0.2 M borate, 0.2 M NaCl, pH 8.2. The required amount of beads was calculated for a final concentration of 2 mg axin/ml beads. Beads are activated with 1 liter of 1 mM HCl. Axin was coupled to beads overnight under constant rotation at 4°C. After the supernatant had been removed, beads were incubated with 1 M ethanolamine pH 9.0 to saturate residual active sites for 2 hours under constant rotation at 4°C. Beads were washed with 0.1 M acetate, pH 4.0, 0.5 M NaCl, and then with bead buffer. They are brought to a 20% suspension in bead buffer and stored at 4°C until further use. Expression of Recombinant GSK-3 in Insect Cells The insect cell lines Sf9 (Spodoptera frugiperda) and HF (High five) were maintained at 27°C in insect Xpress medium (Biowhittaker) supplemented with 10% (v/v) fetal calf serum (heat-inactivated), 1% (v/v) antibiotic/antimycotic, 0.1% (v/v) pluronic F68 and 5 g glucose/l. Working stocks of cells were kept at approximately 0.5 ⫻ 10 6 cells/ml. Amplification of viral stocks and virus titration were carried out with Sf9 cells. HF cells were used for protein production. The rat GSK-3 cDNA was cloned into the baculovirus transfer vector pAcC5. The recombinant virus was kindly provided by K. Hughes and J. R. Woodgett (47). Approximately 150 ml of cell suspension (containing 2 ⫻ 10 8 exponentially growing HF cells) were infected with recombinant AcNPV-GSK-3. The infection was performed at a multiplicity of infection of 1 pfu/cell. The appropriate virus quantity was added to the spinner containing the cell suspension. Cells were incubated for 2 h at 27°C under gentle shaking. One hundred fifty milliliters of media were then added and protein production was allowed to proceed for 4 days before cells were pelleted for 20 min at 2000g. The pellet was resuspended in homogenization buffer (1 ml/2 ⫻ 10 7 cells) and kept on ice for 45 min. After 45 min centrifugation at 40,000 rpm, the supernatant was frozen at ⫺80°C in 1-ml aliquots.
Affinity Purification of Recombinant GSK-3 The axin-His6-Ni-NTA beads (30 l) were mixed with 100 l of GSK-3 lysate diluted with 1.4 ml of homogenization buffer. The mixture was rotated (200 rpm) at 4°C for 30 min. After a 10-s pulse centrifugation, the supernatant was eliminated. The GSK-3/ axin-His6-Ni-NTA beads were washed four times with 200 l homogenization buffer and four times with 200 l kinase buffer. GSK-3/axin-His6 was then eluted by 100 l of kinase buffer containing 200 mM imidazole. The mixture was rotated (200 rpm) at room temperature for 2 min. After a 10-s pulse centrifugation, the supernatant was recovered and kept on ice for immediate testing or frozen at ⫺80°C in the presence of 20% glycerol. Preparation of Sea Urchin Embryos The sea urchins Sphaerechinus granularis, collected by diving in Brittany, were kept in running seawater. Shedding of gametes was induced by injection of 0.2 ml of 0.2 M acetylcholine. Sperm was collected “dry” and kept undiluted at 4°C. Eggs were collected in Milliporefiltered natural seawater (NSW), washed once with NSW and resuspended as a 10% (v/v) suspension. Just before fertilization, glycine (final concentration 0.1%, w/v) was added to the eggs suspension; to facilitate fertilisation membrane elevation. Sperm was diluted just before insemination (1 drop “dry” sperm/5 ml NSW; 1 drop of this dilution/10 ml egg suspension). One to two min after sperm addition the eggs were checked for successful fertilization (100% in all experiments) and the excess sperm was removed by washing the eggs with NSW. When development reached the blastula stage, the embryos were recovered by a short spin. The blastula pellet was immediately frozen in liquid N 2 and kept at ⫺80°C until processing. Purification of Sea Urchin Embryo GSK-3 on Axin-Sepharose Twenty microliters of axin-sepharose beads are washed with 1 ml of bead buffer. The matrix was incubated with 800 l of sea urchin embryo extracts in 400 l of bead buffer during 30 min under constant rotation at 4°C. Beads are washed three times with bead buffer followed by two washes with kinase buffer. The affinity-purified proteins were either assayed for GSK-3 activity or resolved by SDS–PAGE for microsequencing (see below). Preparation of Yeast Extracts and Purification of Yeast GSK-3 on Axin-Sepharose Wild-type fission yeast was grown in liquid Yeast Extract medium (0.5% (w/v) Oxoid yeast extract, 3.0% (w/v) glucose) at 32°C until mid-log phase. Cells were
PURIFICATION OF GSK-3 BY AFFINITY CHROMATOGRAPHY
then harvested by centrifugation at 3000 rpm for 5 min at 4°C and washed once with ice-cold Stop Buffer. The cells were then resuspended at 1 ⫻ 10 10 cells/ml in ice-cold HB buffer and broken by 2–3 min of strong vortexing in the presence of 2 vol of acid-washed glass beads (0.5 mm diameter, Sigma, G-9268). Purification of GSK-3␣ and GSK-3 from Porcine Brain on Axin-Sepharose Porcine brains were obtained from a local slaughter and directly homogenized and processed for affinity chromatography as described for the sea urchin embryos. MCF-7 Cell Culture The human breast carcinoma MCF-7 cell line, cultured in Dulbecco’s modified essential medium medium (Gibco-BRL) supplemented with 2 mM L-glutamine, 10% fetal calf serum, and 25 g gentamycin (Gibco-BRL) were maintained in a humidified atmosphere containing 5% CO 2 in air. For the starvationsynchronization assays, cells were grown to 80% confluence, rinsed twice with serum-free medium and placed in serum-free medium for 24 h. Cells were then incubated in serum-free medium containing 0.2 UI insulin/ml (Umuline NPH, Lilly) for the indicated times. Cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) and lysed for 2 h at 4°C in lysis buffer supplemented with phosphatase and protease inhibitors (10 mM NaF, 200 M sodium orthovanadate, 200 M aprotinin, 50 g/ml leupeptin, 1 mM phenylmethyl sulfonyl fluoride, 1 mM benzamidine, and 10 g/ml soybean trypsin inhibitor). Cellular debris were removed from soluble extracts by centrifugation at 10,000g for 15 min at 4°C and supernatants were frozen until analysis. Equal amounts of proteins (2 mg), determined by the Bradford method, were processed for affinity chromatography and kinase assays as described for the sea urchin embryos. GSK-3 Kinase Assay Kinase activity was assayed in kinase buffer, at 30°C, in presence of 40 M GS-1 (substrate), 15 M [␥- 32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 l. After 10 min incubation at 30°C, 25-l aliquots of the 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 1% phosphoric acid. The wet filters were transferred into 6-ml scintillation vials, 1 ml BCS (Amersham) scintillation fluid was added and the radioactivity measured in a Packard counter. The kinase activity was expressed in pmoles phosphate
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incorporated in GS-1 per 10-min incubation or in percentage of maximal activity. Electrophoresis, Western Blotting, and Protein Microsequencing Samples were run in 10% SDS/polyacrylamide gels. Proteins were stained with Coomassie blue or transferred to 0.1 m nitrocellulose sheets (Schleicher & Schu¨ll) in a Milliblot/SDE system (Millipore) for 30 min at 1.5 mA/cm 2 in transfer buffer. The filters were subsequently blocked with TBS containing 5% low fat milk (Re´gilait) for 1 h at room temperature. The filters were then incubated for 1 h with anti-hGSK-3 antibodies (1:2,500 dilution) (Transduction Laboratories). After four washes of 15 min each with TBS, filters were incubated for 1 h with anti-mouse Ig, horseradish peroxidase-linked F(ab⬘) 2 fragment (from sheep) (Amersham). After another four washes of 15 min each, filters were incubated 1 min with ECL detection reagents (Amersham). They were then exposed to Hyperfilm MP (Amersham). When samples were prepared for microsequencing, gels (1.5 mm thick) were stained with amidoblack (3 mg/100 ml methanol/acetic acid/water, 25/5/20). The amidoblack-stained band was excised from the gel and dried under vacuum and sent to the Pasteur Institute Protein Sequencing Laboratory (Dr. Jacques D’Alayer). The protein was digested with endolysine C and the generated peptides were separated by HPLC and microsequenced. Sequences were compared to available protein sequences using Blastp (Blast 2.0). RESULTS
Expression, Purification, and Immobilization of Axin Axin has been reported to bind several proteins including GSK-3 (35– 42), -catenin (35– 42) and the homologous protein plakoglobin (43), phosphatase 2A (44, 45), APC (41), MEKK1 (46) and itself (40, 44) (Fig. 1A). The GSK-3-binding domain has been located between residues 477 and 561 (37, 38, 41, 42). A sequence coding for an axin fragment encompassing residues 419 to 672 was introduced in a pET-32 vector (Fig. 1A). This axin-His6 fusion protein carries both GSK-3 and -catenin binding sites (Fig. 1A). The resulting fusion protein, a polyhistidine-tagged axin fragment (“AxinHis6”), was expressed in the E. coli strain BL21 (DE3). Following standard induction of the fusion protein with IPTG, the bacteria were pelleted and proteins extracted with lysis buffer. The extract was centrifuged and the supernatant filtered through a 0.45 m filter and stored in aliquots at ⫺80°C until use. Axin-His6 was next purified batchwise by affinity chromatography on Nickel-NTA agarose as described under Material and Methods section (Fig. 1B). Preliminary exper-
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FIG. 1. Protocol for affinity purification and kinase assay of GSK-3. (A) Schematic representation of the axin protein showing the binding sites for APC, axin, GSK-3, -catenin, and phosphatase 2A. Also shown is the Axin-His6 peptide used to generate the affinity chromatography matrix. (B) Generation of Axin-His6 in E.coli, immobilization on Ni-NTA-agarose, affinity purification of GSK-3 (recombinant in HF cells or native from other sources), elution with imidazole, and kinase assay using the GS-1 peptide as a substrate.
iments showed that a 15-min incubation of the bacterial extract with Ni-NTA agarose was sufficient for maximal recovery of Axin-His6. The Ni-NTA-bound Axin-His6 can then be used as an affinity reagent for GSK-3 purification (see below). Alternatively, AxinHis6 can be eluted from the Ni-NTA beads with imidazole, which is dialyzed away. This purified Axin-His6 is then covalently bound to CNBr-activated sepharose according to standard procedures. This also provides an affinity reagent for GSK-3 purification, although, in this case, the ligand is definitively immobilized. Expression and Affinity Purification of Recombinant GSK-3 As an initial series of experiments to validate our affinity purification approach we used recombinant
rat GSK-3 (kindly provided by Dr. Hughes and Woodgett) (47). This enzyme was produced in insect HF cells as described in the Material and Methods section. Following expression, the insect cells were homogenized and the supernatant was incubated for 30 min under constant rotation with Axin-His6-agarose. The beads were thoroughly washed and the bound GSK-3 was eluted with imidazole (optimal concentration: 200 mM). The purified GSK-3 was easily detected by Western blotting (Fig. 2) and kinase assay (Figs. 1 and 2), confirming the validity of this approach. We next attempted to optimize the binding and assay conditions (Fig. 3). Increasing the amounts of insect cell extracts showed that optimal recovery was reached with a ratio of 100 l extract/30 l Axin-His6-Ni-NTA agarose (Fig. 3A). Un-
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FIG. 2. Purification of recombinant GSK-3 by affinity chromatography on axin-His6-Ni-NTA agarose beads. Axin-His6 was bound on Ni-NTA agarose and the beads (30 l) were loaded with either buffer (control) or an HF cell extract after GSK-3 expression (L, lysate; FT, flow through fraction). After extensive washing (4 ⫻ homogenization buffer and 4 ⫻ kinase buffer) the bound proteins were eluted with 100 l 200 mM imidazole (E, GSK-3; eluate; C, control eluate). 48 l of the L and FT fractions and 17.5 l of E and C fractions were loaded on the gel. The gel was stained with Coomassie blue (A) or analyzed by immunoblotting with anti hGSK-3 antibodies (B). The GSK-3 kinase activity of each fraction, expressed in percentage of total input activity (with S.D.), is presented in C. Assays were performed with 1/200 of the initial HF extract (L), the flow through fraction (FT), and four successive elution fractions with imidazole (E1, E2, E3, E4). In addition, control beads, not loaded with insect cell lysates, were eluted with imidazole and tested for GSK-3 activity (C1, C2).
der these conditions the activity of the eluted GSK-3 was linear with respect to volume (0 –5 l) and duration of assay (0 –20 min) (Fig. 3B). The recombinant enzyme displayed the well-described
sensitivity to lithium (48, 49) (Fig. 3C), showing that it could be used in screening assays. Optimal storage conditions for the purified kinase were found to be ⫺80°C and in the presence of 20% glycerol (data not
FIG. 3. Characterization of recombinant rat GSK-3 purified on Axin-His6-agarose. (A) Increasing volumes of HF extracts (0 –250 l) were loaded on 30 l Axin-His6-Ni-NTA beads. The beads were processed as described under Material and Methods. Increasing volumes (0 –5 l) of eluted GSK-3 were assayed for kinase activity. (B) GSK-3 kinase activity as a function of the duration of assay. Assays were performed using 0.5 l of GSK-3 and 40 mM GS-1 peptide as described under Material and Methods. (C) GSK-3 is inhibited by lithium. 0.5 l of affinity purified native GSK-3 and recombinant GSK-3 were assayed in the presence of increasing concentrations of lithium chloride.
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Affinity Purification of Native GSK-3 from Various Sources
FIG. 4. Purification of GSK-3 homologue from fission yeast by affinity chromatography on Axin-His6-Sepharose. A fission yeast extract was loaded on control ethanolamine-sepharose (Eth.) or Axin-His6 Sepharose (Ax.) beads in the presence of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). A 40-kDa protein was eluted and an internal peptide was microsequenced. The 11 amino acids sequence (*) was 100% identical with a sequence present in Skp1p, the fission yeast GSK-3 homologue (Accession No. CAA22609).
shown). As axin is a substrate of GSK-3 (40, 44), it could act as a competitive inhibitor with respect to GS-1. This was found to be the case when high concentrations of axin were added to GSK-3 eluted from axin-His6-Ni-NTA beads or immobilised on axinsepharose (data not shown).
We next decided to validate our affinity purification approach with native GSK-3, using a variety of sources. We first prepared an extract of the fission yeast Schizosaccharomyces pombe. The extract was loaded on control beads or Axin-His6 beads. Following resolution of the bound proteins by SDS–PAGE and silver staining of the gel, one 40-kDa protein was found to bind specifically to immobilized Axin-His6 (Fig. 4). This protein was absent from control ethanolamine beads loaded with yeast extracts (Fig. 4, Eth) and from unloaded Axin-His6 beads (Fig. 4, Ax -). The band was excised from the gel, digested with endolysine C and the peptides were separated by HPLC. One of them was microsequenced and its sequence was found to be 100% identical with a sequence present in Skp1p the S. pombe GSK-3 homologue (Accession No. CAA22609). As an additional example of the potential of the new affinity reagent for GSK-3 purification, we tested extracts of a classical development model, the sea urchin embryo. Eggs were fertilized, and once the embryos reached the blastula stage, they were pelleted and frozen. Extracts were prepared and loaded on immobilized Axin-His6 or on control ethanolamine-sepharose beads. The bound proteins were detected by silver staining following SDS–PAGE (Fig. 5). One major 42kDa protein was detected on the Axin-His6 beads, but not on control beads. Microsequencing of an internal peptide demonstrated that it was bona fide sea urchin
FIG. 5. Purification of active sea urchin GSK-3 by affinity chromatography on Axin-His6-Sepharose. 800 l of sea urchin blastula extracts were loaded on 20 l of Axin-His6 Sepharose beads in the presence of 400 l of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). A 42-kDa protein was eluted and an internal peptide was microsequenced. The 15-amino acids sequence (*) was 100% identical with a sequence present in GSK-3 of the sea urchin Paracentrotus lividus (12). The kinase activity of the purified GSK-3 was measured as a function of time (right).
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mated by autoradiography. GSK-3 activity was also measured using exogenous GS-1 (Fig. 7E). Results show that, as expected, insulin treatment triggers a drop in GSK-3 activity which is detected by a reduction of both axin and GS-1 phosphorylation (Figs. 7D and 7E). -catenin phosphorylation remains low and constant. Interestingly, the protein level of axin-bound GSK-3 (Fig. 7B) appears to be reduced in response to insulin action. This is consistent with the observation that inactive GSK-3 appears to be unable to bind rat axin (38). In other words, immobilized axin seems to purify only active GSK-3. DISCUSSION
Fast, Straightforward, and Selective Affinity Purification of GSK-3 on Immobilized Axin-His6
FIG. 6. Purification of GSK-3␣ and GSK-3 from porcine brain by affinity chromatography on Axin-His6-Sepharose. A porcine brain extract was loaded on Axin-His6 Sepharose beads in the presence of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). The two bound proteins were eluted and an internal peptide was microsequenced. The 15amino acids sequence from the upper band (*) was 100% identical with a sequence present in rat GSK-3␣ (Accession No. P18265). The 15-amino acids sequence from the lower band (**) was 100% identical with a sequence present in rat GSK-3 (Accession No. P18266).
GSK-3 (12) (Accession No. AJ222641) (Fig. 5). In addition the eluted protein displayed kinase activity towards GS-1, a GSK-3 specific substrate (Fig. 5). Finally we loaded porcine brain extracts on AxinHis6 agarose. The beads were extensively washed and the bound proteins detected by silver staining following SDS–PAGE (Fig. 6). The affinity matrix retained essentially two proteins in equal amounts. They were identified, by microsequencing of an internal peptide, as GSK-3␣ (50) and GSK-3 (3) (Accession Nos. P18265 and P18266, respectively). (Fig. 6). Both cross-reacted with anti-GSK-3 antibodies (data not shown). The eluted GSK-3 isoforms displayed high kinase activity and sensitivity to lithium (Fig. 3C). To assess whether a physiological change of GSK-3 activity could be detected by our affinity purification method, we investigated the effect of insulin on GSK-3 activity in mammalian cells. MCF-7 cells were synchronized in G1 by serum starvation and exposed to insulin. Extracts were prepared at various time-points, and loaded on axin-sepharose (2 mg protein/point) (Fig. 7). The levels of -catenin (Fig. 7A) and GSK-3 (Fig. 7B) were assessed by Western blotting. GSK-3 activity was measured by incubation of the axin-bound material with 32P-labeled ATP. Phosphorylation of endogenous -catenin (Fig. 7C) and axin (Fig. 7D) was esti-
The method described here allows the rapid purification of GSK-3 from virtually any source in less than 2 h. It relies on the use of a specific affinity matrix. The mouse axin peptide immobilized on this matrix contains the complete GSK-3 and -catenin binding do-
FIG. 7. Treatment of MCF-7 cells by insulin induces a reduction of GSK-3 activity and axin-sepharose bound GSK-3 protein level. MCF-7 cells were synchronized by serum-starvation and then exposed to insulin. Cells were collected at different time-points and extracted. Extracts were loaded on axin-sepharose and analysed by Western blotting for -catenin (A) and GSK-3 (B) levels, and by in vitro phosphorylation for GSK-3 activity, using endogenous -catenin (C) and axin (D), or exogenous GS-1 peptide (E) as substrates. (A, B) Western blots; (C, D) autoradiographs.
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mains, but not the domains interacting with other proteins (Fig. 1). It is easily produced in unlimited amounts by cheap and standard fermentation procedures. We estimate that a one liter of bacterial culture produces an amount of Axin-His6 sufficient to purify enough GSK-3 (obtained from a one liter insect cell culture) for over 100,000 kinase assays. After induction of Axin-His6 production, the bacterial pellet or extract can be stored for extended periods at ⫺80°C without apparent degradation. Similarly, affinity-purified GSK-3 can also be stored at ⫺80°C without substantial loss of activity. Axin-His6 bound to Ni-NTA-agarose or to CNBr-activated sepharose is equally efficient at binding GSK-3. Batchwise purification of recombinant GSK-3 can be completed within 90 min, while the previous method required two days (47). The AxinHis6 beads display a unique selectivity for GSK-3, whatever the original source (yeast, sea urchin embryos, mammalian brain). However, the matrix does not allow the distinction between GSK-3␣ and GSK-3, which copurify (Fig. 6). The involvement of GSK-3 in various diseases such as cancer, Alzheimer’s disease, HIV-induced neurotoxicity or diabetes calls for an active search of selective and potent GSK-3 inhibitors. We believe our affinity chromatography purification method could be used in the large scale purification of GSK-3 for such screening purposes. It indeed allows the large-scale purification of highly active recombinant or native kinase. The high level of purification, close to homogeneity, ensures that the proper enzyme is assayed and eliminates interference with irrelevant proteins. Use of Immobilized Axin-His6 as a Tool to Investigate GSK-3 Regulation As described in the introduction, GSK-3 is involved in many cellular processes and therefore probably very tightly regulated. Axin-His6 sepharose beads allow a fast purification and concentration of GSK-3 out of extracts prepared from cells at various physiological stages (cell cycle phases, developmental stages, kinetics of insulin action, . . . etc.). As axin-Sepharose appears to bind only active GSK-3, only the active affinity-purified enzyme can be analyzed for changes in abundance, posttranslational modifications, transient association with other proteins, and kinase activity. The reagent readily detects a drop in GSK-3 activity associated with insulin stimulation (Fig. 7). Axin-His6 sepharose beads could also be used as a diagnostic reagent to monitor the status (level and kinase activity) of GSK-3 in pathological tissue samples. An alternative way of measuring GSK-3 activity in crude cells has been presented recently (51). It is based on the use of a peptide derived from a GSK-3 phosphorylation site of eIF2B. This straightforward and simple method also
allows the detection of alterations of GSK-3 activity under various physiological settings. It is rapid and rather specific, but does not allow the study of GSK-3 associated proteins nor a quantification of GSK-3 protein levels. ACKNOWLEDGMENTS We thank Mr. Matthieu Garnier for his help in testing inhibitors and Eve Damiens for her help in cell culture. We are grateful to Dr. Hughes and and J. R. Woodgett for kindly providing the baculovirus transfer vector containing rat GSK-3 cDNA. We also greatly appreciated the protein microsequencing work of Dr. J. d’Alayer and M. Davi (Pasteur Institute). This work was supported by grants from the “Association pour la Recherche sur le Cancer” (ARC5343) and the “Conseil Re´gional de Bretagne” (to L.M.). A.P. and M.G. are recipients of fellowships from the “Conseil Re´gional de Bretagne.”
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30. Munoz-Montano, J. R., Moreno, F. J., Avila, J., and Diaz-Nido, J. (1997) Lithium inhibits Alzheimer’s disease-like tau protein phosphorylation in neurons. FEBS Lett. 411, 183–188. 31. Pei, J. J., Braak, E., Braak, H., Grundke-Iqbal, I., Iqbal, K., Winblad, B., and Cowburn, R. F. (1999) Distribution of active glycogen synthase kinase 3 (GSK-3) in brains staged for Alzheimer disease neurofibrillary changes. J. Neuropathol. Exp. Neurol. 58, 1010 –1019. 32. Imahori, K., and Uchida, T. (1997) Physiology and pathology of tau protein kinases in relation to Alzheimer’s disease. J. Biochem. 121, 179 – 88. 33. Mandelkow, E.-M., and Mandelkow, E. (1998) Tau in Alzheimer’s disease. Trends Cell Biol. 8, 425– 427. 34. Gluecksohn-Schoenheimer, S. (1949) The effects of a lethal mutation responsible for duplications and twinning in mouse embryos. J. Exp. Zool. 110, 47–76. 35. Hart, M. J., de los Santos, R., Albert, I. N., Rubinfeld, B., and Polakis, P. (1998) Down-regulation of -catenin by human axin and its association with the APC tumor suppressor, -catenin, and GSK-3. Curr. Biol. 8, 573–581. 36. Fagoto, F., Jho, E. H., Zeng, L., Kurth, T., Joos, T., Kaufmann, C., and Costantini, F. (1999) Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization. J. Cell Biol. 145, 741–756. 37. Itoh, K., Krupnik, V. E., and Sokol, S. Y. (1998) Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and -catenin. Curr. Biol. 8, 591–594. 38. Ikeda, S., Kishida, M., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998) Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3 and -catenin and promotes GSK-3-dependent phosphorylation of -catenin. EMBO J. 17, 1371–1384. 39. Sakanaka, C., Weiss, J. B., and Williams, L. T. (1998) Bridging of -catenin and glycogen synthase kinase-3 by axin and inhibition of -catenin-mediated transcription. Proc. Natl. Acad. Sci. USA 95, 3020 –3023. 40. Sakanaka, C., and Williams, L. T. (1999) Functional domains of axin. Importance of the C terminus as an oligomerization domain. J. Biol. Chem. 274, 14090 –14093. 41. Behrens, J., Jerchow, B. A., Wu¨rtele, M., Grimm, J., Asbrand, C., Wirtz, R., Ku¨hl, M., Wedlich, D., and Birchmeier, W. (1998) Functional interaction of an axin homolog conductin, with -catenin, APC, and GSK3. Science 280, 596 –599. 42. Li, L., Yuan, H., Weaver, C. D., Mao, J., Farr, G. H., III, Sussman, D. J., Jonkers, J., Kimelman, D., and Wu, D. (1999) Axin and Frat1 interact with Dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1. EMBO J. 18, 4233– 4240. 43. Kodama, S., Ikeda, S., Asahara, T., Kishida, M., and Kikuchi, A. (1999) Axin directly interacts with plakoglobin and regulates its stability. J. Biol. Chem. 274, 27682–27688. 44. Hsu, W., Zeng, L., and Costantini, F. (1999) Identification of a domain of axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. J. Biol. Chem. 274, 3439 – 3445. 45. Ikeda, S., Kishida, M., Matsuura, Y., Usui, H., and Kikuchi, A. (2000) GSK-3-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by -catenin and protein phosphatase 2A complexed with axin. Oncogene 19, 537– 545. 46. Zhang, Y., Neo, S. Y., Wang, X., Han, J., and Lin, S. C. (1999) Axin forms a complex with MEKK1 and activates c-Jun NH2terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling. J. Biol. Chem. 274, 35247– 35254.
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47. Hughes, K., Pulverer, B. J., Theocharous, P., and Woodgett, J. R. (1991) Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3, the mammalian homologue of the Drosophila melanogaster zeste-white homeotic gene product. Eur. J. Biochem. 203, 305–311. 48. Klein, P. S., and Melton, D. A. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93, 8455– 8459. 49. Stambolic, V., Ruel, L., and Woodgett, R. (1996) Lithium inhibits
glycogen synthase kinase-3 activity and mimics wingless signaling in intact cells. Curr. Biol. 6, 1664 –1668. 50. Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J. 12, 803– 808. 51. Welsh, G. I., Patel, J. C., and Proud, C. G. (1997) Peptide substrates suitable for assaying glycogen synthase kinase-3 in crude cell extracts. Anal. Biochem. 244, 16 –21.
Purification of GSK-3 by Affinity Chromatography on Immobilized Axin Aline Primot,* Blandine Baratte,* Marie Gompel,* Annie Borgne,* Sylvie Liabeuf,† Jean-Louis Romette,† Eek-hoon Jho,‡ Frank Costantini,‡ and Laurent Meijer* ,1 *Station Biologique, CNRS, BP 74, 29682 Roscoff cedex, Bretagne, France; †ESIL-GBMA, Faculte´ des Sciences de Luminy, AFMB-DISP, 163, avenue de Luminy, 13288 Marseille Cedex 8, France; and ‡Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, New York 10032
Received May 4, 2000, and in revised form August 9, 2000
Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of -catenin and cyclin D1), Alzheimer’s disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ß, -catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419 – 672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These “Axin-His6” matrices were found to selectively bind recombinant rat GSK-3 and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes. © 2000 Academic Press Key Words: protein kinase; GSK-3; axin; -catenin; APC; WNT; armadillo; kinase inhibitors; screening; cancer; diabetes; Alzheimer’s disease.
1 To whom correspondence and reprint requests should be addressed. Fax: (33).02.98.29.23.42. E-mail: [email protected].
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The human genome encodes an estimated 1500 – 2000 protein kinase genes. Glycogen synthase kinase-3 is a Ser/Thr kinase, which was initially identified as a kinase phosphorylating and inactivating glycogen synthase (1, 2). In mammals GSK-3 2 is present as two very closely homologous isoforms, GSK-3␣ and GSK-3 (3– 5), which are variably expressed in different tissues (6). Numerous substrates of GSK-3 have been described beside glycogen synthase: -catenin, c-jun, the Adenomatous Polyposis Coli protein (APC), inhibitor-2 of protein phosphatase 1, cyclin D1, insulin receptor substrate 1 (IRS-1), the microtubule-binding proteins tau and MAP-1B, elF2B, . . .etc. GSK-3 acts as a downstream element in the Wnt signaling pathway (7–9) and is therefore involved in multiple cellular and physiological processes: (i) GSK-3 regulates dorsoventral patterning during development (10 –13). (ii) GSK-3 is negatively regulated in response to insulin, and its inhibition leads to stimulation of glycogen, fatty acid, and protein synthesis (14, review in 15). (iii) GSK-3 is also involved in cell cycle control; it phosphorylates cyclin D1 on Thr286, thereby leading to its redistribution from the nucleus to the cytoplasm and to its proteolytic degradation (16). GSK-3 (in complex with the tumor suppressor protein APC and axin) phosphorylates -catenin, leading to its ubiquitin-dependent degradation (17). The failure of this degradation, resulting from mutant APC or -catenin, is common in human colon cancer and melanoma (18 –21). -catenin associates with transcription factors of the LEF-1 family to 2 Abbreviations used: Axin-His6, histidine-tagged axin peptide; APC, adenomatous polyposis coli; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GS-1, glycogen synthase peptide 1; GSK-3, glycogen synthase kinase-3; HF cells, high five cells; IPTG, isopropyl--Dthiogalactopyranoside; LB, Luria Bertani medium; Ni-NTA, nickelnitrilotriacetic acid; p-NPP, p-nitrophenylphosphate; SBTI, soybean trypsin inhibitor.
1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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activate the expression of specific genes, in particular the cyclin D1 gene (7, 21, 22, and references therein). These events are clearly linked to cancerogenesis (7, 8, 21, 23). (iv) GSK-3 is also involved in neuronal functions: it regulates axonal outgrowth by phosphorylating MAP-1B (24) and is responsible for HIV-1 Tatmediated neurotoxicity (25). Along with CDK5/p25 (26), GSK-3 is responsible for the phosphorylation of Alzheimer’s disease specific sites on the tau protein, participating thereby to the formation of paired helical filaments, a diagnostic feature of this neurodegenerative disorder (27–31; review in 32, 33). Axin is the product of a mouse gene originally named “Fused” (34), which was cloned on the basis of an insertional mutation in the gene. Axin was subsequently found to modulate signaling through the Wnt pathway which accounts for its effects on embryonic axial development (35). Axin, and its homolog conductin, binds simultaneously to GSK-3 and -catenin, thereby stimulating -catenin phosphorylation (35– 42). Axin also binds several other proteins including the -catenin homologous protein plakoglobin (43), phosphatase 2A (44 – 45), APC (41), MEKK1 (46), and itself (40, 44). The domains binding these various proteins have been located on the axin protein (Fig. 1). In this article we have taken advantage of the highly selective and potent interaction between axin and GSK-3 to set up a simple affinity chromatography protocol for the rapid purification of active GSK-3 to near homogeneity. This method provides a useful tool to monitor the status of GSK-3 (protein levels, posttranslational modifications, kinase activity) in cells and tissues under various physiological conditions. It could also be useful for the purification of large quantities of native GSK-3 to be used in inhibitors screening or, as a purification step, in crystallography programs. MATERIALS AND METHODS
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and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK). [␥- 32P]ATP (PB168) was obtained from Amersham and benzamidine was from Becham. Buffers Lysis buffer. 50 mM NaH 2PO 4 pH 8.0, 300 mM NaCl, 10 mM imidazole. Lysis buffer for cell culture. 155 mM NaCl, 10 mM tris, 1% Triton X-100, 1% deoxycholic acid, 1 mM EGTA. Wash buffer. 50 mM NaH 2PO 4 pH 8.0, 300 mM NaCl, 10% glycerol. Homogenization buffer. 60 mM -glycerophosphate, 15 mM p-NPP, 25 mM Mops, pH 7.2, 15 mM MgCl 2, 0.5 mM DTT, 1 mM Na Vanadate, 1 mM NaF, 1 mM diNa-phenylphosphate, 10 g leupeptin/ml, 10 g aprotinin/ml, 10 g SBTI/ml, 100 M benzamidine. Bead buffer. 50 mM Tris A, pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, 10 g leupeptin/ml, 10 g aprotinin/ml, 10 g SBTI/ml, 100 M benzamidine. Kinase buffer. 30 mM MgCl 2, 3 mM EGTA, 3 mM DTT, 75 mM Tris–HCl, pH 7.5, 150 g heparin/ml. Tris-buffered saline (TBS). 50 mM Tris, pH 7.4, 150 mM NaCl. Anode 1 buffer. 300 mM Tris, pH 10.4, 20% methanol. Anode 2 buffer. 25 mM Tris, pH 10.4, 20% methanol. Cathode buffer. 25 mM Tris, pH 9.4, 40 mM glycine, 20% methanol. Stop buffer. 150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN 3, pH 8. HB buffer. 25 mM Mops, pH 7.2, 15 mM p-NPP, 15 mM MgCl 2, 60 mM -glycerophosphate, 15 mM EGTA, 1 mM DTT, 0.1 mM sodium vanadate, 1% Triton X-100, 1 mM PMSF, 20 g/ml leupeptin, 40 g/ml aprotinin
Chemicals Sodium fluoride, sodium orthovanadate, isopropyl-D-thiogalactopyranoside (IPTG), dithiothreitol (DTT), -glycerophosphate, MgCl 2, NaCl, LiCl, EGTA, MOPS, ampicillin, Luria Bertani (LB) medium, heparin, leupeptin, aprotinin, soybean trypsin inhibitor (SBTI), NaH 2PO 4, Na 2CO 3, methanol, imidazole, CNBr-activated Sepharose 4B beads, formaldehyde 37%, acetate were obtained from Sigma Chemicals. Ethanolamine was obtained from Prolabo. Tris and Coomassie brilliant blue R-250 were obtained from Bio-Rad. Ni-NTA matrix was purchased from Qiagen, p-nitrophenylphosphate (p-NPP) (disodium salt hexahydrate) was from Acros Organics. The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE, where Sp is a phosphorylated serine) was purchased from the University of Southampton (Department of Biochemistry
Expression of Axin-His6 in Bacteria An Escherichia coli strain (BL21 (DE3)) containing a plasmid encoding axin tagged with six histidines (pET32a-Ax(419-672)) was used. Expression of this plasmid is under control of IPTG. E. coli were first grown overnight at 37°C in the presence of 50 g ampicillin/ml LB medium. Five milliliters of this preculture were inoculated/liter of LB containing 50 g ampicillin/ml. Incubation was continued at 37°C until the culture O.D. at 600 nm had reached a value between 0.6 and 0.8 (about 4 –5 h). At this time, 0.4 mM IPTG was added and the incubation was prolonged for 5 h at 30°C. Cells were then pelleted at 6000 rpm for 10 min and stored frozen at ⫺80°C until extraction. They were then disrupted by sonication in lysis buffer (2 ml lysis buffer/50 ml culture). After 20 min centrifugation at 35,000 rpm, the
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supernatant was passed through a 0.45 m filter and frozen at ⫺80°C in 1-ml aliquots. Preparation of Axin-His6 Agarose Beads Axin-His6-containing lysate (75 l) was diluted in 725 l lysis buffer and mixed with 30 l Ni-NTA beads. The mixture was rotated (200 rpm) at 4°C for 15 min. After a 10-s pulse centrifugation, the supernatant was eliminated. The beads were washed four times with 200 l of wash buffer. Coupling of Axin-His6 to CNBr-Activated Sepharose 4B Axin-His6 purified by the Ni-NTA method was dialysed in 0.2 M borate, 0.2 M NaCl, pH 8.2. The required amount of beads was calculated for a final concentration of 2 mg axin/ml beads. Beads are activated with 1 liter of 1 mM HCl. Axin was coupled to beads overnight under constant rotation at 4°C. After the supernatant had been removed, beads were incubated with 1 M ethanolamine pH 9.0 to saturate residual active sites for 2 hours under constant rotation at 4°C. Beads were washed with 0.1 M acetate, pH 4.0, 0.5 M NaCl, and then with bead buffer. They are brought to a 20% suspension in bead buffer and stored at 4°C until further use. Expression of Recombinant GSK-3 in Insect Cells The insect cell lines Sf9 (Spodoptera frugiperda) and HF (High five) were maintained at 27°C in insect Xpress medium (Biowhittaker) supplemented with 10% (v/v) fetal calf serum (heat-inactivated), 1% (v/v) antibiotic/antimycotic, 0.1% (v/v) pluronic F68 and 5 g glucose/l. Working stocks of cells were kept at approximately 0.5 ⫻ 10 6 cells/ml. Amplification of viral stocks and virus titration were carried out with Sf9 cells. HF cells were used for protein production. The rat GSK-3 cDNA was cloned into the baculovirus transfer vector pAcC5. The recombinant virus was kindly provided by K. Hughes and J. R. Woodgett (47). Approximately 150 ml of cell suspension (containing 2 ⫻ 10 8 exponentially growing HF cells) were infected with recombinant AcNPV-GSK-3. The infection was performed at a multiplicity of infection of 1 pfu/cell. The appropriate virus quantity was added to the spinner containing the cell suspension. Cells were incubated for 2 h at 27°C under gentle shaking. One hundred fifty milliliters of media were then added and protein production was allowed to proceed for 4 days before cells were pelleted for 20 min at 2000g. The pellet was resuspended in homogenization buffer (1 ml/2 ⫻ 10 7 cells) and kept on ice for 45 min. After 45 min centrifugation at 40,000 rpm, the supernatant was frozen at ⫺80°C in 1-ml aliquots.
Affinity Purification of Recombinant GSK-3 The axin-His6-Ni-NTA beads (30 l) were mixed with 100 l of GSK-3 lysate diluted with 1.4 ml of homogenization buffer. The mixture was rotated (200 rpm) at 4°C for 30 min. After a 10-s pulse centrifugation, the supernatant was eliminated. The GSK-3/ axin-His6-Ni-NTA beads were washed four times with 200 l homogenization buffer and four times with 200 l kinase buffer. GSK-3/axin-His6 was then eluted by 100 l of kinase buffer containing 200 mM imidazole. The mixture was rotated (200 rpm) at room temperature for 2 min. After a 10-s pulse centrifugation, the supernatant was recovered and kept on ice for immediate testing or frozen at ⫺80°C in the presence of 20% glycerol. Preparation of Sea Urchin Embryos The sea urchins Sphaerechinus granularis, collected by diving in Brittany, were kept in running seawater. Shedding of gametes was induced by injection of 0.2 ml of 0.2 M acetylcholine. Sperm was collected “dry” and kept undiluted at 4°C. Eggs were collected in Milliporefiltered natural seawater (NSW), washed once with NSW and resuspended as a 10% (v/v) suspension. Just before fertilization, glycine (final concentration 0.1%, w/v) was added to the eggs suspension; to facilitate fertilisation membrane elevation. Sperm was diluted just before insemination (1 drop “dry” sperm/5 ml NSW; 1 drop of this dilution/10 ml egg suspension). One to two min after sperm addition the eggs were checked for successful fertilization (100% in all experiments) and the excess sperm was removed by washing the eggs with NSW. When development reached the blastula stage, the embryos were recovered by a short spin. The blastula pellet was immediately frozen in liquid N 2 and kept at ⫺80°C until processing. Purification of Sea Urchin Embryo GSK-3 on Axin-Sepharose Twenty microliters of axin-sepharose beads are washed with 1 ml of bead buffer. The matrix was incubated with 800 l of sea urchin embryo extracts in 400 l of bead buffer during 30 min under constant rotation at 4°C. Beads are washed three times with bead buffer followed by two washes with kinase buffer. The affinity-purified proteins were either assayed for GSK-3 activity or resolved by SDS–PAGE for microsequencing (see below). Preparation of Yeast Extracts and Purification of Yeast GSK-3 on Axin-Sepharose Wild-type fission yeast was grown in liquid Yeast Extract medium (0.5% (w/v) Oxoid yeast extract, 3.0% (w/v) glucose) at 32°C until mid-log phase. Cells were
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then harvested by centrifugation at 3000 rpm for 5 min at 4°C and washed once with ice-cold Stop Buffer. The cells were then resuspended at 1 ⫻ 10 10 cells/ml in ice-cold HB buffer and broken by 2–3 min of strong vortexing in the presence of 2 vol of acid-washed glass beads (0.5 mm diameter, Sigma, G-9268). Purification of GSK-3␣ and GSK-3 from Porcine Brain on Axin-Sepharose Porcine brains were obtained from a local slaughter and directly homogenized and processed for affinity chromatography as described for the sea urchin embryos. MCF-7 Cell Culture The human breast carcinoma MCF-7 cell line, cultured in Dulbecco’s modified essential medium medium (Gibco-BRL) supplemented with 2 mM L-glutamine, 10% fetal calf serum, and 25 g gentamycin (Gibco-BRL) were maintained in a humidified atmosphere containing 5% CO 2 in air. For the starvationsynchronization assays, cells were grown to 80% confluence, rinsed twice with serum-free medium and placed in serum-free medium for 24 h. Cells were then incubated in serum-free medium containing 0.2 UI insulin/ml (Umuline NPH, Lilly) for the indicated times. Cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) and lysed for 2 h at 4°C in lysis buffer supplemented with phosphatase and protease inhibitors (10 mM NaF, 200 M sodium orthovanadate, 200 M aprotinin, 50 g/ml leupeptin, 1 mM phenylmethyl sulfonyl fluoride, 1 mM benzamidine, and 10 g/ml soybean trypsin inhibitor). Cellular debris were removed from soluble extracts by centrifugation at 10,000g for 15 min at 4°C and supernatants were frozen until analysis. Equal amounts of proteins (2 mg), determined by the Bradford method, were processed for affinity chromatography and kinase assays as described for the sea urchin embryos. GSK-3 Kinase Assay Kinase activity was assayed in kinase buffer, at 30°C, in presence of 40 M GS-1 (substrate), 15 M [␥- 32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 l. After 10 min incubation at 30°C, 25-l aliquots of the 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 1% phosphoric acid. The wet filters were transferred into 6-ml scintillation vials, 1 ml BCS (Amersham) scintillation fluid was added and the radioactivity measured in a Packard counter. The kinase activity was expressed in pmoles phosphate
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incorporated in GS-1 per 10-min incubation or in percentage of maximal activity. Electrophoresis, Western Blotting, and Protein Microsequencing Samples were run in 10% SDS/polyacrylamide gels. Proteins were stained with Coomassie blue or transferred to 0.1 m nitrocellulose sheets (Schleicher & Schu¨ll) in a Milliblot/SDE system (Millipore) for 30 min at 1.5 mA/cm 2 in transfer buffer. The filters were subsequently blocked with TBS containing 5% low fat milk (Re´gilait) for 1 h at room temperature. The filters were then incubated for 1 h with anti-hGSK-3 antibodies (1:2,500 dilution) (Transduction Laboratories). After four washes of 15 min each with TBS, filters were incubated for 1 h with anti-mouse Ig, horseradish peroxidase-linked F(ab⬘) 2 fragment (from sheep) (Amersham). After another four washes of 15 min each, filters were incubated 1 min with ECL detection reagents (Amersham). They were then exposed to Hyperfilm MP (Amersham). When samples were prepared for microsequencing, gels (1.5 mm thick) were stained with amidoblack (3 mg/100 ml methanol/acetic acid/water, 25/5/20). The amidoblack-stained band was excised from the gel and dried under vacuum and sent to the Pasteur Institute Protein Sequencing Laboratory (Dr. Jacques D’Alayer). The protein was digested with endolysine C and the generated peptides were separated by HPLC and microsequenced. Sequences were compared to available protein sequences using Blastp (Blast 2.0). RESULTS
Expression, Purification, and Immobilization of Axin Axin has been reported to bind several proteins including GSK-3 (35– 42), -catenin (35– 42) and the homologous protein plakoglobin (43), phosphatase 2A (44, 45), APC (41), MEKK1 (46) and itself (40, 44) (Fig. 1A). The GSK-3-binding domain has been located between residues 477 and 561 (37, 38, 41, 42). A sequence coding for an axin fragment encompassing residues 419 to 672 was introduced in a pET-32 vector (Fig. 1A). This axin-His6 fusion protein carries both GSK-3 and -catenin binding sites (Fig. 1A). The resulting fusion protein, a polyhistidine-tagged axin fragment (“AxinHis6”), was expressed in the E. coli strain BL21 (DE3). Following standard induction of the fusion protein with IPTG, the bacteria were pelleted and proteins extracted with lysis buffer. The extract was centrifuged and the supernatant filtered through a 0.45 m filter and stored in aliquots at ⫺80°C until use. Axin-His6 was next purified batchwise by affinity chromatography on Nickel-NTA agarose as described under Material and Methods section (Fig. 1B). Preliminary exper-
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FIG. 1. Protocol for affinity purification and kinase assay of GSK-3. (A) Schematic representation of the axin protein showing the binding sites for APC, axin, GSK-3, -catenin, and phosphatase 2A. Also shown is the Axin-His6 peptide used to generate the affinity chromatography matrix. (B) Generation of Axin-His6 in E.coli, immobilization on Ni-NTA-agarose, affinity purification of GSK-3 (recombinant in HF cells or native from other sources), elution with imidazole, and kinase assay using the GS-1 peptide as a substrate.
iments showed that a 15-min incubation of the bacterial extract with Ni-NTA agarose was sufficient for maximal recovery of Axin-His6. The Ni-NTA-bound Axin-His6 can then be used as an affinity reagent for GSK-3 purification (see below). Alternatively, AxinHis6 can be eluted from the Ni-NTA beads with imidazole, which is dialyzed away. This purified Axin-His6 is then covalently bound to CNBr-activated sepharose according to standard procedures. This also provides an affinity reagent for GSK-3 purification, although, in this case, the ligand is definitively immobilized. Expression and Affinity Purification of Recombinant GSK-3 As an initial series of experiments to validate our affinity purification approach we used recombinant
rat GSK-3 (kindly provided by Dr. Hughes and Woodgett) (47). This enzyme was produced in insect HF cells as described in the Material and Methods section. Following expression, the insect cells were homogenized and the supernatant was incubated for 30 min under constant rotation with Axin-His6-agarose. The beads were thoroughly washed and the bound GSK-3 was eluted with imidazole (optimal concentration: 200 mM). The purified GSK-3 was easily detected by Western blotting (Fig. 2) and kinase assay (Figs. 1 and 2), confirming the validity of this approach. We next attempted to optimize the binding and assay conditions (Fig. 3). Increasing the amounts of insect cell extracts showed that optimal recovery was reached with a ratio of 100 l extract/30 l Axin-His6-Ni-NTA agarose (Fig. 3A). Un-
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FIG. 2. Purification of recombinant GSK-3 by affinity chromatography on axin-His6-Ni-NTA agarose beads. Axin-His6 was bound on Ni-NTA agarose and the beads (30 l) were loaded with either buffer (control) or an HF cell extract after GSK-3 expression (L, lysate; FT, flow through fraction). After extensive washing (4 ⫻ homogenization buffer and 4 ⫻ kinase buffer) the bound proteins were eluted with 100 l 200 mM imidazole (E, GSK-3; eluate; C, control eluate). 48 l of the L and FT fractions and 17.5 l of E and C fractions were loaded on the gel. The gel was stained with Coomassie blue (A) or analyzed by immunoblotting with anti hGSK-3 antibodies (B). The GSK-3 kinase activity of each fraction, expressed in percentage of total input activity (with S.D.), is presented in C. Assays were performed with 1/200 of the initial HF extract (L), the flow through fraction (FT), and four successive elution fractions with imidazole (E1, E2, E3, E4). In addition, control beads, not loaded with insect cell lysates, were eluted with imidazole and tested for GSK-3 activity (C1, C2).
der these conditions the activity of the eluted GSK-3 was linear with respect to volume (0 –5 l) and duration of assay (0 –20 min) (Fig. 3B). The recombinant enzyme displayed the well-described
sensitivity to lithium (48, 49) (Fig. 3C), showing that it could be used in screening assays. Optimal storage conditions for the purified kinase were found to be ⫺80°C and in the presence of 20% glycerol (data not
FIG. 3. Characterization of recombinant rat GSK-3 purified on Axin-His6-agarose. (A) Increasing volumes of HF extracts (0 –250 l) were loaded on 30 l Axin-His6-Ni-NTA beads. The beads were processed as described under Material and Methods. Increasing volumes (0 –5 l) of eluted GSK-3 were assayed for kinase activity. (B) GSK-3 kinase activity as a function of the duration of assay. Assays were performed using 0.5 l of GSK-3 and 40 mM GS-1 peptide as described under Material and Methods. (C) GSK-3 is inhibited by lithium. 0.5 l of affinity purified native GSK-3 and recombinant GSK-3 were assayed in the presence of increasing concentrations of lithium chloride.
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Affinity Purification of Native GSK-3 from Various Sources
FIG. 4. Purification of GSK-3 homologue from fission yeast by affinity chromatography on Axin-His6-Sepharose. A fission yeast extract was loaded on control ethanolamine-sepharose (Eth.) or Axin-His6 Sepharose (Ax.) beads in the presence of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). A 40-kDa protein was eluted and an internal peptide was microsequenced. The 11 amino acids sequence (*) was 100% identical with a sequence present in Skp1p, the fission yeast GSK-3 homologue (Accession No. CAA22609).
shown). As axin is a substrate of GSK-3 (40, 44), it could act as a competitive inhibitor with respect to GS-1. This was found to be the case when high concentrations of axin were added to GSK-3 eluted from axin-His6-Ni-NTA beads or immobilised on axinsepharose (data not shown).
We next decided to validate our affinity purification approach with native GSK-3, using a variety of sources. We first prepared an extract of the fission yeast Schizosaccharomyces pombe. The extract was loaded on control beads or Axin-His6 beads. Following resolution of the bound proteins by SDS–PAGE and silver staining of the gel, one 40-kDa protein was found to bind specifically to immobilized Axin-His6 (Fig. 4). This protein was absent from control ethanolamine beads loaded with yeast extracts (Fig. 4, Eth) and from unloaded Axin-His6 beads (Fig. 4, Ax -). The band was excised from the gel, digested with endolysine C and the peptides were separated by HPLC. One of them was microsequenced and its sequence was found to be 100% identical with a sequence present in Skp1p the S. pombe GSK-3 homologue (Accession No. CAA22609). As an additional example of the potential of the new affinity reagent for GSK-3 purification, we tested extracts of a classical development model, the sea urchin embryo. Eggs were fertilized, and once the embryos reached the blastula stage, they were pelleted and frozen. Extracts were prepared and loaded on immobilized Axin-His6 or on control ethanolamine-sepharose beads. The bound proteins were detected by silver staining following SDS–PAGE (Fig. 5). One major 42kDa protein was detected on the Axin-His6 beads, but not on control beads. Microsequencing of an internal peptide demonstrated that it was bona fide sea urchin
FIG. 5. Purification of active sea urchin GSK-3 by affinity chromatography on Axin-His6-Sepharose. 800 l of sea urchin blastula extracts were loaded on 20 l of Axin-His6 Sepharose beads in the presence of 400 l of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). A 42-kDa protein was eluted and an internal peptide was microsequenced. The 15-amino acids sequence (*) was 100% identical with a sequence present in GSK-3 of the sea urchin Paracentrotus lividus (12). The kinase activity of the purified GSK-3 was measured as a function of time (right).
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mated by autoradiography. GSK-3 activity was also measured using exogenous GS-1 (Fig. 7E). Results show that, as expected, insulin treatment triggers a drop in GSK-3 activity which is detected by a reduction of both axin and GS-1 phosphorylation (Figs. 7D and 7E). -catenin phosphorylation remains low and constant. Interestingly, the protein level of axin-bound GSK-3 (Fig. 7B) appears to be reduced in response to insulin action. This is consistent with the observation that inactive GSK-3 appears to be unable to bind rat axin (38). In other words, immobilized axin seems to purify only active GSK-3. DISCUSSION
Fast, Straightforward, and Selective Affinity Purification of GSK-3 on Immobilized Axin-His6
FIG. 6. Purification of GSK-3␣ and GSK-3 from porcine brain by affinity chromatography on Axin-His6-Sepharose. A porcine brain extract was loaded on Axin-His6 Sepharose beads in the presence of bead buffer. Proteins purified from the matrix were analyzed by SDS–PAGE followed by silver staining (left). The two bound proteins were eluted and an internal peptide was microsequenced. The 15amino acids sequence from the upper band (*) was 100% identical with a sequence present in rat GSK-3␣ (Accession No. P18265). The 15-amino acids sequence from the lower band (**) was 100% identical with a sequence present in rat GSK-3 (Accession No. P18266).
GSK-3 (12) (Accession No. AJ222641) (Fig. 5). In addition the eluted protein displayed kinase activity towards GS-1, a GSK-3 specific substrate (Fig. 5). Finally we loaded porcine brain extracts on AxinHis6 agarose. The beads were extensively washed and the bound proteins detected by silver staining following SDS–PAGE (Fig. 6). The affinity matrix retained essentially two proteins in equal amounts. They were identified, by microsequencing of an internal peptide, as GSK-3␣ (50) and GSK-3 (3) (Accession Nos. P18265 and P18266, respectively). (Fig. 6). Both cross-reacted with anti-GSK-3 antibodies (data not shown). The eluted GSK-3 isoforms displayed high kinase activity and sensitivity to lithium (Fig. 3C). To assess whether a physiological change of GSK-3 activity could be detected by our affinity purification method, we investigated the effect of insulin on GSK-3 activity in mammalian cells. MCF-7 cells were synchronized in G1 by serum starvation and exposed to insulin. Extracts were prepared at various time-points, and loaded on axin-sepharose (2 mg protein/point) (Fig. 7). The levels of -catenin (Fig. 7A) and GSK-3 (Fig. 7B) were assessed by Western blotting. GSK-3 activity was measured by incubation of the axin-bound material with 32P-labeled ATP. Phosphorylation of endogenous -catenin (Fig. 7C) and axin (Fig. 7D) was esti-
The method described here allows the rapid purification of GSK-3 from virtually any source in less than 2 h. It relies on the use of a specific affinity matrix. The mouse axin peptide immobilized on this matrix contains the complete GSK-3 and -catenin binding do-
FIG. 7. Treatment of MCF-7 cells by insulin induces a reduction of GSK-3 activity and axin-sepharose bound GSK-3 protein level. MCF-7 cells were synchronized by serum-starvation and then exposed to insulin. Cells were collected at different time-points and extracted. Extracts were loaded on axin-sepharose and analysed by Western blotting for -catenin (A) and GSK-3 (B) levels, and by in vitro phosphorylation for GSK-3 activity, using endogenous -catenin (C) and axin (D), or exogenous GS-1 peptide (E) as substrates. (A, B) Western blots; (C, D) autoradiographs.
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mains, but not the domains interacting with other proteins (Fig. 1). It is easily produced in unlimited amounts by cheap and standard fermentation procedures. We estimate that a one liter of bacterial culture produces an amount of Axin-His6 sufficient to purify enough GSK-3 (obtained from a one liter insect cell culture) for over 100,000 kinase assays. After induction of Axin-His6 production, the bacterial pellet or extract can be stored for extended periods at ⫺80°C without apparent degradation. Similarly, affinity-purified GSK-3 can also be stored at ⫺80°C without substantial loss of activity. Axin-His6 bound to Ni-NTA-agarose or to CNBr-activated sepharose is equally efficient at binding GSK-3. Batchwise purification of recombinant GSK-3 can be completed within 90 min, while the previous method required two days (47). The AxinHis6 beads display a unique selectivity for GSK-3, whatever the original source (yeast, sea urchin embryos, mammalian brain). However, the matrix does not allow the distinction between GSK-3␣ and GSK-3, which copurify (Fig. 6). The involvement of GSK-3 in various diseases such as cancer, Alzheimer’s disease, HIV-induced neurotoxicity or diabetes calls for an active search of selective and potent GSK-3 inhibitors. We believe our affinity chromatography purification method could be used in the large scale purification of GSK-3 for such screening purposes. It indeed allows the large-scale purification of highly active recombinant or native kinase. The high level of purification, close to homogeneity, ensures that the proper enzyme is assayed and eliminates interference with irrelevant proteins. Use of Immobilized Axin-His6 as a Tool to Investigate GSK-3 Regulation As described in the introduction, GSK-3 is involved in many cellular processes and therefore probably very tightly regulated. Axin-His6 sepharose beads allow a fast purification and concentration of GSK-3 out of extracts prepared from cells at various physiological stages (cell cycle phases, developmental stages, kinetics of insulin action, . . . etc.). As axin-Sepharose appears to bind only active GSK-3, only the active affinity-purified enzyme can be analyzed for changes in abundance, posttranslational modifications, transient association with other proteins, and kinase activity. The reagent readily detects a drop in GSK-3 activity associated with insulin stimulation (Fig. 7). Axin-His6 sepharose beads could also be used as a diagnostic reagent to monitor the status (level and kinase activity) of GSK-3 in pathological tissue samples. An alternative way of measuring GSK-3 activity in crude cells has been presented recently (51). It is based on the use of a peptide derived from a GSK-3 phosphorylation site of eIF2B. This straightforward and simple method also
allows the detection of alterations of GSK-3 activity under various physiological settings. It is rapid and rather specific, but does not allow the study of GSK-3 associated proteins nor a quantification of GSK-3 protein levels. ACKNOWLEDGMENTS We thank Mr. Matthieu Garnier for his help in testing inhibitors and Eve Damiens for her help in cell culture. We are grateful to Dr. Hughes and and J. R. Woodgett for kindly providing the baculovirus transfer vector containing rat GSK-3 cDNA. We also greatly appreciated the protein microsequencing work of Dr. J. d’Alayer and M. Davi (Pasteur Institute). This work was supported by grants from the “Association pour la Recherche sur le Cancer” (ARC5343) and the “Conseil Re´gional de Bretagne” (to L.M.). A.P. and M.G. are recipients of fellowships from the “Conseil Re´gional de Bretagne.”
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