Preparation and Characterization of Recombinant Protein Phosphatase 1

Section IV Expression Systems

[24]

PREPARATION AND CHARACTERIZATION OF PP1

321

[24] Preparation and Characterization of Recombinant Protein Phosphatase 1 By TAKUO WATANABE, EDGAR F. DA CRUZ E SILVA, HSIEN-BIN HUANG, NATALIA STARKOVA, YOUNG-GUEN KWON, ATSUKO HORIUCHI, PAUL GREENGARD, and ANGUS C. NAIRN

Protein phosphatase-1 (PP1) is a major eukaryotic serine/threonine protein phosphatase that regulates such diverse cellular processes as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, and neuronal signaling.1–4 It appears that little of the free PP1 catalytic subunit (
37 kDa, PP1C) exists in cells. Rather, the precise role played by PP1 in its diverse functions is attributed to its interaction with a large variety of regulatory subunits that include both inhibitor and targeting proteins. Inhibitors include protein inhibitor-1, its homologue DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), and inhibitor2. Phosphorylation of inhibitor-1 at Thr35, or of DARPP-32 at Thr34, by protein kinase A (PKA), is required for PP1 inhibition. In contrast, unphosphorylated inhibitor-2 interacts with PP1C. A growing number of targeting subunits have also been identified that localize PP1C to specific subcellular compartments, thereby influencing its substrate specificity and local function. These include glycogen-targeting subunits, myofibrillartargeting subunits, several nuclear-targeting proteins, and proteins such as spinophilin and neurabin that target PP1C to dendritic spines of neurons. Many of the proteins that interact with PP1C share a common binding, or docking, site, that comprises a docking motif containing one or more basic amino acid followed by two hydrophobic residues separated by a variable amino acid (the so-called RVXF motif).5 X-ray crystallographic analysis has indicated that the docking motif interacts in an extended manner with a hydrophobic channel in PP1C situated on the side opposite that of the active site. Thus, while more complex oligomeric structures may exist, PP1C interacts with its regulatory subunits in a mutually exclusive manner. 1

S. Shenolikar, Annu. Rev. Cell Biol. 10, 55–86 (1994). P. Greengard, P. B. Allen, and A. C. Nairn, Neuron 23, 435–447 (1999). 3 J. B. Aggen, A. C. Nairn, and R. Chamberlin, Chem. Biol. 7, R13–R23 (2000). 4 M. Bollen, Trends Biochem. Sci. 26, 426–431 (2001). 5 M. P. Egloff et al., EMBO J. 16, 1876–1887 (1997). 2

METHODS IN ENZYMOLOGY, VOL. 366

Copyright ß 2003, Elsevier Inc. All rights reserved. 0076-6879/2003 $35.00

322

EXPRESSION SYSTEMS

[24]

Despite the recent advances that have been made in the biochemical characterization of PP1, these studies have been hampered by a lack of easily prepared recombinant PP1C. It has been impossible to overexpress PP1C in mammalian cells, presumably due to the key role it plays in cellular function. We have therefore analyzed various methods for preparation of PP1C in bacteria, and have developed methods for preparation of PP1C in Sf9 cells using the baculovirus expression system. The preparation and characterization of these different recombinant PP1C preparations is discussed.

Materials

Oligonucleotides were synthesized by Operon Technologies, Inc. (Berkley, CA). The QuickChange site-directed mutagenesis kit was from Stratagene. The Bac-to-Bac Baculovirus Expression System including the vector plasmid pFastBac-HT, DH10BAC competent cells, and recombinant TEV protease were from Life Technologies (Gaitherburg, MD). Sf9 cells were from Novagen (Madison, WI). CompleteTM protease inhibitor cocktail tablets and protease inhibitor E-64 were from Boehringer-Mannheim (Indianapolis, IN). NHS-Hi Trap and glutathione-Sepharose were from Pharmacia Biotech (Uppsala, Sweden). Ni-nitrilotriacetic acid (NTA) agarose was from Qiagen (Valencia, CA). Immobilon-P was from Millipore (Bedford, MA). PNUTS peptide was synthesized and purified by reversedphase column HPLC at W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University. The Protein Tyrosine Phosphatase Assay System was from New England Biolabs (NEB). Monoclonal antibody for PP1 (E-9) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Native PP1C was purified from rabbit skeletal muscle, using sequential chromatography on DEAE-cellulose, heparin-Sepharose, phenyl-Sepharose, Sephacryl S-200 and Mono-Q resins (largely as described below for recombinant PP1C, see also Ref. 6). DARPP-32 and inhibitor-2 were prepared from E. coli as described.7 GST-tagged spinophilin and GSTPNUTS were prepared from E. coli as described.8,9

6

H. B. Huang, A. Horiuchi, J. Goldberg, P. Greengard, and A. C. Nairn, Proc. Natl. Acad. Sci. U.S.A. 94, 3530–3535 (1997). 7 H.-B. Huang et al., J. Biol. Chem. 274, 7870–7878 (1999). 8 P. B. Allen, Y. G. Kwon, A. C. Nairn, and P. Greengard, J. Biol. Chem. 273, 4089–4095 (1998). 9 L. C. Hsieh-Wilson, P. B. Allen, T. Watanabe, A. C. Nairn, and P. Greengard, Biochemistry 38, 4365–4373 (1999).

[24]

PREPARATION AND CHARACTERIZATION OF PP1

323

Preparation of Phosphorylated and Thiophosphorylated DARPP-32

Phosphorylated recombinant DARPP-32 was prepared essentially as described.7 DARPP-32 (2 mg) was dissolved in 1 ml of 50 mM Hepes (pH 7.4), 1 mM EGTA, 10 mM magnesium acetate, and 1 mM ATP. The phosphorylation reaction was started by addition of 2 g of PKA catalytic subunit and the incubation was carried out for 60 min. For preparation of [32P]DARPP-32, [32P]ATP replaced ATP. For preparation of thiophosphorylated DARPP-32, 1 mM thiophospho-ATP (Boehringer Mannheim) replaced ATP, and the reaction was carried out at 30 C for 5 days, with fresh PKA (2 g) and thiophospho-ATP (1 mM final concentration) added every 24 hr. Phosphorylated DARPP-32 and thiophospho-DARPP-32 were purified by HPLC using a C-18 column. Preparation of Phosphorylase a

Phosphorylase b (93 mg of lyophilized powder; Sigma) was phosphorylated with phosphorylase kinase (2 mg, Sigma) in 3 ml of reaction buffer containing 0.2 mM ATP, [ -32P]ATP (3000 Ci/mmol, Amersham), 100 mM Tris–HCl, pH 8.2, 100 mM sodium glycerol-1phosphate, 0.1 mM CaCl2 and 10 mM magnesium acetate. Microcystin (final concentration, 100 nM) was added to inhibit endogenous protein phosphatases. The reaction mixture was incubated for 2 hr at 30 C, then 5.45 ml of 70% ammonium sulfate was added (to 45% of saturation). The reaction mixture was put on ice for 30 min and then centrifuged at 19,000g for 15 min. The pellet was resuspended in 240 l of 10 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 10% glycerol and 436 l of 70% ammonium sulfate was added (to 45% of saturation). The mixture was incubated on ice for another 30 min and centrifuged at 19,000g for 15 min. The pellet was resuspended in 600 l of 10 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 10% glycerol, transferred to a dialysis tube and dialyzed at 4 C against 10 mM Tris–HCl, pH 7.5, 0.1 mM EGTA (at least four changes of 1 liter each). Precipitated phosphorylase a and solution from the dialysis tube were transferred to a microcentrifuge tube and incubated on ice for 2 hr. After centrifugation at 19,000g for 15 min, the pellet was resuspended in 600 l of cold 10 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 15 mM 2-mercaptoethanol and stored at 4 C. Dephosphorylation of 32

32

P-phosphorylase a or

32

P-DARPP-32

P-labeled phosphorylase a (10 M) or phospho-Thr34-DARPP-32 (0.5–20 M) was incubated with PP1 in a reaction mixture (40 l)

324

EXPRESSION SYSTEMS

[24]

containing 50 mM Tris–HCl, pH 7.0, 300 mM NaCl, and 0.3 mg/ml bovine serum albumin (BSA). The detergent Brij 35 (0.01%, w/v) was often added to the incubation mixture. Reactions were performed at 30 C for 10 min and stopped by adding 200 l of 20% (phosphorylase) or 50% (DARPP-32) trichloroacetic acid (TCA). Samples were centrifuged at 19,000g for 10 min (for DARPP-32, samples were incubated on ice for 1 h), and 32P in the supernatant was analyzed by Cerenkov counting in a scintillation counter (Beckman). Unless indicated, assays of E. coli PP1 contained 1 mM MnCl2. For assay of inhibitors, PP1 and inhibitors were preincubated for 15–30 min on ice.

Dephosphorylation of p-nitrophenyl Phosphate ( pNPP) and Tyrosine-phosphorylated Myelin Basic Protein

Phosphatase activity was measured in a reaction mixture (0.1 ml) containing 13 mM pNPP, 10 mM HEPES, pH 7.0, and 300 mM NaCl. Reactions were performed at 30 C for 20 min and stopped by adding 1 ml of 0.2 N NaOH. Phosphate release from the reaction product ( p-nitrophenol) was analyzed by measurement of absorbance at 405 nm using a molar extinction coefficient of 18,000 M1 cm1. The rate of nonenzymatic hydrolysis of the substrate was corrected by measuring the optical density in the absence of PP1. [32P]myelin basic protein was phosphorylated by Abl protein tyrosine kinase and tyrosine phosphatase activity was assayed using the Protein Tyrosine Phosphatase Assay System as described by the manufacturer (NEB).

Expression of PP1 in E. coli

Rabbit PP1 cDNA was a generous gift from N. Berndt. PP1 was expressed in E. coli and purified using two different methods. Method 1

The rabbit PP1C cDNA was contained within a pDR540 plasmid containing a trp-lac fusion promoter that preceded the PP1 DNA. E. coli (DH5), harboring the wild-type rabbit PP1 cDNA, were grown in 5 liters of LB medium containing 1 mM MnCl2 and ampicillin (0.1 mg /ml) at 37 C with shaking at 250 rpm. After 4 hr incubation (an OD600nm of
0.6), 0.3 mM isopropyl- -D-thiogalactopyranoside (IPTG) was added to induce the expression of PP1C. Cells were incubated for up to an additional 13 hr,

[24]

PREPARATION AND CHARACTERIZATION OF PP1

325

and then centrifuged at 4000g for 30 min. The pellet was resuspended in 300 ml of 20 mM Tris–HCl buffer (pH 7.5) containing 1 mM MnCl2, 0.2 mM PMSF, 4 mM benzamidine, 15 mM 2-mercaptoethanol, 0.1 mM EGTA, pepstatin A (10 g/ml), leupeptin (10 g/ml) and chymostatin (10 g/ml). Cells were lysed using a French press (1000–1500 psi). The lysate was centrifuged at 20,000g for 20 min, and the supernatant was loaded onto a heparin-Sepharose column (1.5  20 cm), followed by washing with one column volume of buffer A (20 mM Tris–HCl, 1.0 mM MnCl2, 0.1 mM EGTA, 4.0 mM benzamidine, 0.2 mM PMSF and 15 mM 2-mercaptoethanol, pH 7.5) plus 0.1 M NaCl. Proteins were eluted with a linear gradient (500 ml total volume) from 0.1 to 0.6 M NaCl (in buffer A). Fractions containing PP1 activity were pooled and concentrated to 15 ml by ultrafiltration using an Amicon YM-10 membrane. One volume of buffer A containing 3.4 M NaCl was added to the pooled sample to give a final concentration of 1.7 M NaCl. The sample was loaded onto a phenyl-Sepharose column (1.5  18 cm), the column washed with 40 ml of buffer A containing 1.7 M NaCl, and proteins eluted using a linear gradient (500 ml total volume) from 1.7 to 0.5 M NaCl (in buffer A containing 10% glycerol). Fractions containing PP1 activity were pooled and concentrated to 5 ml by ultrafiltration. The concentrated sample was loaded on a Sephacryl S-200 column (2.5  120 cm) equilibrated with 20 mM triethanolamine, pH 7.0, 0.1 mM EGTA, 1.0 mM MnCl2, 4.0 mM benzamidine, 0.2 mM PMSF, 10% glycerol, 0.3 M NaCl, 15 mM 2mercaptoethanol. Fractions containing PP1 activity were pooled and concentrated to 4 ml by ultrafiltration. The sample was diluted by addition of 10 ml of buffer B (20 mM triethanolamine, 0.1 mM EGTA, 10% glycerol, 1 mM MnCl2, 0.1% 2-mercaptoethanol, pH 7.0) to give a final concentration of NaCl of less than 0.1 M. The diluted sample was passed through a 0.45 m filter, and loaded onto a Mono-Q anion exchange column equilibrated in buffer B. The column was washed and proteins eluted using a linear gradient of 0.1–0.4 M NaCl (in buffer B) (1 ml/min over 50 min). PP1C was eluted at
0.22 M NaCl and was stored in 50% glycerol at 20 C. Method 2

PP1C DNA was subcloned from pDR540 (Pharmacia) into pET28a (Novagen) to produce a recombinant enzyme with a His6-tag at the Nterminus. However, the T7 promoter from pET28 proved to be too strong: large amounts of PP1 were synthesized but most of the protein was found to be insoluble. The His6-PP1 was therefore cloned back into pDR540 under control of the tac promoter. BL21 cells were then transformed with the

326

EXPRESSION SYSTEMS

[24]

pDR540 plasmid harboring His6-PP1C. Transformed cells were grown at room temperature (
22–23 C) in LB medium containing 100 g/ml ampicillin and either 1 mM MnCl2 or 100 M CoCl2. At OD600, protein expression was induced with 0.3 mM IPTG and cells were further incubated overnight at room temperature. The cells were harvested by centrifugation, resuspended in buffer C (10 mM Tris–HCl, pH 8.0, 30 mM imidazole, 300 mM NaCl, 10% glycerol, CompleteTM protease inhibitor cocktail, and either 1 mM MnCl2 or 1 mM CoCl2) and lysed using a French press. Cell debris was removed by centrifugation at 35,000g for 15 min, and the supernatant was clarified by centrifugation at 35,000g for 45 min. The resulting supernatant was loaded on a Ni-NTA-agarose column (volume, 3 ml) at a rate of 1 ml/min; the column was washed with buffer C, and His6-PP1C was eluted with an increasing linear gradient of imidazole (30–500 mM) in buffer C (total gradient volume, 30 ml). The fractions were collected and analyzed by SDS-PAGE. Fractions containing His6-PP1 were pooled and stored in small aliquots at 80 C.

Preparation of PP1 in Sf9 Cells

Two different procedures were used to prepare recombinant PP1 using the baculovirus method.

Method 1

Rat PP1C or human PP1C cDNAs was subcloned into pBlueBac (Invitrogen) and recombinant transfer plasmids were transfected into Sf9 cells with linearized AcMNPV DNA (Invitrogen) using lipofectin (PP1C) or into Sf9 cells with BaculoGold (Pharmingen) viral DNA (PP1C ). The medium containing the recombinant virus was collected after 5–7 days. Individual recombinant viral clones were isolated after plaque purification in the presence of Bluo-Gal. High-titre stocks of virus were generated in spinner cultures of Sf9 cells at a low multiplicity of infection (MOI
0.1 pfu/cell). For large scale purification, Sf9 cells (107 cells/ml) were infected with recombinant virus at a MOI of 1–2 for 1 hr at room temperature, followed by dilution with Grace’s medium (without or with 1 mM MnCl2) to give a final cell density of 106/ml. Infected cells were grown in spinner flasks at 27 C with constant stirring at 50–60 rpm. The cells (
1.5 liter) were incubated for 48 hr, and then were harvested by centrifugation at 1000g for 3 min. Cells were washed with 20 mM Tris–HCl, pH 7.5, 0.1 mM EGTA,

[24]

PREPARATION AND CHARACTERIZATION OF PP1

327

15 mM 2-mercaptoethanol, 0.25 M sucrose, resuspended in 20 mM Tris– HCl, pH 7.5, 2 mM EGTA, 2 mM EDTA, 15 mM 2-mercaptoethanol, 0.2 mM PMSF, and lysed by ultrasonication. The lysate was centrifuged at 20,000g for 20 min, and the supernatant was purified by chromatography using DEAE-cellulose (3  25 cm column, linear gradient of 0.08–0.5 M NaCl), heparin-Sepharose and Sephacryl S-200 (as described above for E. coli, Method 1). In some cases, chromatography on Mono-Q resin was carried out as described above. Sf9 PP1C prepared by Method 1 was stored in 50% glycerol at 20 C. If PP1C was expressed with MnCl2 in the incubation medium, 1 mM MnCl2 was also added to all buffers during purification.

Method 2

EcoRI and XbaI restriction sites were added in the 50 end and 30 end of the PP1C DNA, and the DNA was inserted in-frame into pFastBac-HT. For some constructs, cDNA encoding glutathione S-transferase (GST), followed by a TEV protease cleavage site and a FLAG tag, was introduced in-frame between the His6 tag and PP1C sequences. Thus, proteins had either a His6 tag, with a TEV protease cleavage site, or alternatively a His6 tag, a TEV cleavage site, a GST tag, a second TEV cleavage site and a FLAG tag at the N-terminus of PP1C. Recombinant baculovirus stocks were prepared as described by the manufacturer (Life Technologies). In brief, donor plasmids were used for transformation of competent DH10BAC E. coli cells. Recombinant bacmid DNA was isolated and used for transfection of Sf9 cells. After 48 hr incubation at 28 C, supernatant from the culture medium was saved and used for further amplification. For preparation of PP1C, Sf9 cells were grown in suspension culture (28 C, rotating at 135 rpm) in 100 ml of Grace’s insect medium [supplemented with 0.33% yeastolate, 0.33% lactalbumin hydrolysate and 10% (v/v) fetal calf serum] in a 500 ml flask with a ventilation cap (note that Life Technologies serum-free medium, Sf-900 II SFM, should not be used since this results in a Sf9 PP1C preparation that dephosphorylates to some extent phospho-DARPP-32 and tyrosine-phosphorylated myelin basic protein). Sf9 cells (1.5  106 cells/ml) were infected at a MOI between 1 and 2. Cells were then incubated for 72 hr at 28 C, rotating at 135 rpm. Cells were then centrifuged at 500g for 5 min, and the pellets lyzed with gentle swirling in 5 ml (
2  107 cells/ml) of 50 mM Tris– HCl, pH 7.5, 100 mM NaCl (or 100 mM KCl), 1% NP40, 15 mM 2mercaptoethanol, 1 mM AEBSF, 30 g/ml E-64, 5 g/ml pepstatin A, 5 g/ml leupeptin, 5 g/ml chymostatin. Supernatants were prepared by

328

EXPRESSION SYSTEMS

[24]

centrifugation at 100,000g for 30 min and purified by affinity chromatography as described below.

Affinity Chromatography

Several different affinity chromatography procedures were used that utilized the His6 and GST tags attached to recombinant PP1. In addition, PP1 was also purified by affinity chromatography using a docking motif peptide derived from PNUTS (PNUTS[392-408]). In some cases more than one affinity chromatography step was used in a purification procedure. The supernatant obtained after cell lysis was typically diluted in appropriate binding buffer and filtered through a 0.45 m syringe-driven filter unit. For purification of His6-PP1C, filtered supernatant was incubated with Ni-NTA agarose (1–2 ml, Ni-NTA agarose resin, QIAGEN or equivalent) and the agarose slurry transferred into a column. The resin was washed with buffer containing 20 mM Tris–HCl, pH 8.5, 0.5 M KCl, 0.1 mM EGTA, 10% glycerol, 5 mM 2-mercaptoethanol and 20 mM imidazole, then washed with a buffer containing 20 mM Tris–HCl, pH 8.5, 1 M KCl, 0.1 mM EGTA, 10% glycerol, 5 mM 2-mercaptoethanol. Proteins were eluted with buffer containing 20 mM Tris–HCl, pH 8.5, 0.1 M KCl, 0.1 mM EGTA, 10% glycerol, and 150 mM imidazole. Fractions containing active PP1C were dialyzed against buffer containing 50 mM Tris–HCl, pH 7.0, 50% glycerol, 0.1 mM EGTA, and 15 mM 2mercaptoethanol. For purification of GST-tagged PP1C, cells (
3  107 cells per ml) were lysed with buffer containing 50 mM Tris–HCl, pH 7.5, 0.1 M NaCl and 1% NP-40, 15 mM 2-mercaptoethanol, 1 mM AEBSF, 30 g/ml E-64, for 30 min on ice. [In some cases, CompleteTM (EDTA-free) protease inhibitor cocktail tablets (Boehringer-Mannheim), replaced the protease inhibitors.] The supernatant (centrifugation at 100,000g for 30 min) was incubated with
1/10th volume of glutathione-Sepharose resin for 1 hr at 4 C, and the resin was pelleted by centrifugation and washed three times with phosphate-buffered saline. The resin was then resuspended in 50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1 mM DTT, 10% glycerol, and incubated with 200 units of His6-tagged TEV protease for 2 hr at 4 C. After cleavage, Ni-NTA agarose was added to the suspension for 1 hr at 4 C to remove the His6-tagged TEV protease. The suspension was centrifuged and the supernatant was used for protein phosphatase assay. The synthetic peptide PNUTS [392-408] (KGRKRKTVTWPEEGKLR, 1–2 mg) was coupled to HiTrap-NHS resin (1 ml) (Amersham Biosciences) according to the manufacturer’s instructions. The coupling efficiency

[24]

PREPARATION AND CHARACTERIZATION OF PP1

329

(calculated based on the absorbance at 280 nm of the applied sample and first wash sample) was typically >80%. The PNUTS peptide resin was packed in a Hi-Trap column and attached to a FPLC (Pharmacia or equivalent), and the column equilibrated with 5 ml of buffer containing 20 mM triethanolamine, pH 7.0, 50 mM NaCl, 0.1 mM EGTA, 10% glycerol and 15 mM 2-mercaptoethanol, at a flow rate of 0.5 ml/min. The diluted cell lysate was applied to the column at the same flow rate. (Note that the flowthrough can be collected and reloaded onto the column.) After loading, the column was washed with 10 ml of buffer, then PP1C was eluted with a 19.5 ml linear gradient from 0.05 to 2 M NaCl in column equilibration buffer. Fractions of 1 ml were collected, and analyzed for PP1C activity or protein by enzyme assay or immunoblotting. Active PP1C eluted between 0.6 M and 2 M NaCl. (Note that immunologically cross-reactive PP1C that has low phosphatase activity is eluted in fractions between 0.2 and 0.4 M NaCl.) Fractions containing PP1C were dialyzed against 50 mM triethanolamine, pH 7.0, 0.1 mM EGTA, 50% glycerol, 15 mM 2-mercaptoethanol, 0.1 mM PMSF at 4 C overnight, and stored at 70 C until use. In some cases, the PNUTS affinity step was combined with affinity chromatography of His6-PP1 using Ni-NTA. In this case, the sample was applied first to the Ni-NTA column as described above (except that 50 mM triethanolamine, pH 7.0, replaced 20 mM Tris–HCl, pH 8.5). His6-PP1C was eluted with buffer containing 150 mM imidazole (as described above), and fractions containing PP1C activity were loaded directly onto the PNUTS peptide affinity column. (Note that the PNUTS affinity method must only be used after purification on Ni-NTA or glutathione-Sepaharose to avoid contamination with endogenous Sf9 cell PP1.) Although the His6 tag has no apparent influence on PP1C activity, this can be removed using TEV protease (Invitrogen) that is also polyhistidine tagged. The released tagged fragment and the TEV protease can then be removed from the PP1 by chromatography on Ni-NTA resin.

Immunoblotting of Recombinant PP1C

Proteins were separated by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane using standard procedures. Membranes were incubated in PBS containing 0.1% Tween 20, 0.5% nonfat dry milk and 1 g/ml of anti-PP1C monoclonal antibody (E-9). Rabbit anti-mouse IgG antibody and 125I-labeled Protein A were used for detection of signal. Radioactivity was quantified using a PhosphorImager (Molecular Dynamics) and purified recombinant PP1C of known concentration was used as standard.

330

[24]

EXPRESSION SYSTEMS

Characterization of PP1C Expressed in E. coli using Method 1

PP1C has been expressed in E. coli and purified by several investigators using either conventional or affinity chromatography.6,10–14 This type of preparation has been used in many structural and biochemical studies of PP1C (including most studies of PP1C mutants), and was the preparation used in production of protein for X-ray crystallization studies.15,16 Recombinant E. coli PP1C is available commercially from several sources including NEB and Roche. Approximately 1 mg/liter of purified PP1C can be obtained after
460-fold purification with a yield of
50% from the cytosolic fraction of lysed E. coli (Table I). A major limitation in the yield is related to the amount of insoluble PP1C protein that is expressed. Reduced temperature of incubation, or shorter times of induction with IPTG may help to increase the relative expression level of soluble PP1C. Addition of 1 mM MnCl2 to the culture medium, and to buffers during the purification, is essential for preparation of active PP1C.10,11 As shown in several studies, E. coli PP1C exhibits a high specific activity comparable with native PP1C (usually prepared from rabbit skeletal muscle as a mixture of isoforms) [>35 units/mg under standard assay

PURIFICATION Purification step Crude lysate Heparin-Sepharose Phenyl-Sepharose Sephacryl S-200 Mono-Q

10

OF

TABLE I PP1C FROM E.

COLI

USING METHOD 1

Volume (ml)

Protein (mg)

Total activity (units)

Specific activity (units/mg)

Purification (-fold)

Yield (%)

287 130 107 56 7

3961 83.2 ND 8.4 4.1

312 266 ND 194 152

0.08 3.2 ND 23.1 36.9

1 40 ND 289 461

100 85 ND 62 49

Z. Zhang, G. Bai, S. Deans-Zirattu, M. F. Browner, and E. Y. C. Lee, J. Biol. Chem. 267, 1484–1490 (1992). 11 D. R. Alessi, A. J. Street, P. Cohen, and P. T. W. Cohen, Eur. J. Biochem. 213, 1055–1066 (1993). 12 Z. Zhang, S. Zhao, S. D. Zirattu, G. Bai, and E. Y. C. Lee, Arch. Biochem. Biophys. 308, 37–41 (1994). 13 J. Zhang, Z. J. Zhang, K. Brew, and E. Y. C. Lee, Biochemistry 35, 6276–6282 (1996). 14 J. H. Connor et al., J. Biol. Chem. 273, 27716–27724 (1998). 15 J. Goldberg et al., Nature 376, 745–753 (1995). 16 M. P. Egloff, P. T. W. Cohen, P. Reinemer, and D. Barford, J. Mol. Biol. 254, 942–959 (1995).

[24]

PREPARATION AND CHARACTERIZATION OF PP1

331

conditions (Table I); Vmax of > 60 mol/min/mg and Km
10 M using phosphorylase a as substrate].6 The E. coli enzyme is also normal in terms of inhibition by microcystin, calyculin A and okadaic acid (see Table IV) and has therefore been very useful in structure/function studies related to these inhibitors.6,17 However, E. coli PP1C exhibits several anomalous properties. These include the fact that it is dependent on added Mn2 þ for activity, and is able to dephosphorylate phospho-tyrosine-containing substrates and para-nitrophenyl phosphate.6,11,18,19 E. coli PP1C is also relatively insensitive to inhibition by phospho-inhibitor-1 and phosphoDARPP-32, an effect that can be partly explained by the ability of the preparation to dephosphorylate either phospho-inhibitor protein (see Table IV, Ref. 6 and data not shown). Several of the anomalous properties of E. coli PP1C may be explained by a subtle change in the structure of the metal-binding ligands in the active site of the enzyme.6,16,20,21 Thus active E. coli PP1C contains two Mn2 þ ions in its active site rather than Fe2 þ and Zn2 þ , likely present in native PP1C. Subtle changes in the position of Tyr272 that overhangs the phosphate-binding site in the active site, also is the likely cause of the ability of the enzyme to dephosphorylate phospho-inhibitor-1, phosphoDARPP-32, pNPP, and phospho-tyrosine-containing substrates.21 It is notable that E. coli PP1C also exhibits a marked ability to autodephosphorylate phospho-Thr320 when incubated with cdc2 kinase, in contrast to native or Sf9 PP1C (Fig. 1 and see below). In addition to the abnormal catalytic properties, E. coli PP1C exhibits altered interaction with its regulatory subunits that are also likely to be caused by subtle structural changes, perhaps in the region of the protein that binds the docking motif. For example, E. coli PP1C is still relatively insensitive to inhibition by either thiophospho-DARPP-32 (Table IV) or thiophospho-inhibitor-1 (both of which are resistant to the dephosphorylation by PP1). E. coli PP1C is also relatively insensitive to regulation by spinophilin and PNUTS. For example, GST-PNUTS[382– 486] inhibits Sf9 PP1C with an IC50 value of
0.1 nM, but inhibits E. coli PP1C with an IC50 value of
80 nM (data not shown). E. coli PP1C appears to be inhibited normally by full-length inhibitor-2, but displays anomalous regulation by various fragments of inhibitor-2 (Table II). For example, deletion of up to 105 amino acids at the C-terminus of 17

L. F. Zhang, Z. J. Zhang, F. X. Long, and E. Y. C. Lee, Biochemistry 35, 1606–1611 (1996). C. MacKintosh et al., FEBS Lett. 397, 235–238 (1996). 19 S. Endo et al., Biochemistry 36, 6986–6992 (1997). 20 D. Barford, Trends Biochem. Sci. 21, 407–412 (1996). 21 T. Watanabe et al., Proc. Natl. Acad. Sci. U.S.A. 98, 3080–3085 (2001). 18

332

[24]

EXPRESSION SYSTEMS

FIG. 1. In vitro phosphorylation of PP1C expressed in E. coli or Sf9 insect cells by cdc2/ cyclin B. E. coli PP1C (800 nM, upper panel) and Sf9 PP1C (400 nM, lower panel) were phosphorylated at Thr320 for various times at 30 C by purified cdc2/cyclin B (NEB) with [ -32P]ATP in the absence or presence of 1 M microcystin. Proteins were separated by SDSPAGE (10% acrylamide) and [32P]-labeled PP1C was detected by autoradiography. In the absence of microcystin, phosphorylation of E. coli PP1C rapidly reached a steady-state where phosphorylation by cdc2 was balanced by autodephosphorylation. When autodephosphorylation was blocked by microcystin, phosphorylation by cdc2 kinase reached a maximal level that reflected stoichiometric phosphorylation. In contrast, phosphorylation of Sf9 PP1C by cdc2 kinase was not affected by addition of microcystin.

REGULATION

OF

TABLE II E. COLI PP1C AND NATIVE PP1C MUTANT INHIBITOR-2

BY

WILD-TYPE AND

Relative inhibitory activity Inhibitor Full-length I2[1–204] I2[1–180] I2[1–172] I2[1–164] I2[1–159] I2[1–140] I2[1–120] I2[1–99] I2[1–84] I2[9–204] I2[14–204] I2[19–204] I2[I10G] I2[K11E] I2[I13G]

E. coli PP1C

Native PP1C

100% (IC50 1.1 nM) 31.0 48.0 19.0 6.0 7.0 7.0 8.0 4.0 37.0 0.2 0.4 26.0 3.0 1.0

100% (IC50 1.0 nM) ND ND ND ND 50.0 37.0 38.0 7.1 63.0 0.1 0.1 21.0 2.2 0.9

Results for native PP1C are summarized from Huang et al.7 ND, not determined.

inhibitor-2 had little effect on the IC50 for inhibition of native PP1C. However, removal of 40–50 amino acids at the C-terminus of inhibitor-2 rendered the proteins poor inhibitors of E. coli PP1C. This result presumably highlights, in the altered E. coli enzyme preparation, a specific

[24]

PREPARATION AND CHARACTERIZATION OF PP1

333

deficit in one or more of the multiple binding interactions that normally occur with inhibitor-2 and native PP1C.7,22,23 In summary, E. coli PP1C prepared by this standard method is easily obtained in high quantity, is highly active, and is useful for some structure/ function studies. However, as a result of its anomalous enzymatic properties, this preparation is less useful in studies of physiological substrates and of interactions with targeting and inhibitor proteins.

Characterization of PP1 Expressed in E. coli Using Method 2

Previous studies have included examination of the ability of other divalent cations to substitute for Mn2 þ in supporting the activity of E. coli PP1C preparations initially purified in the presence of Mn2 þ .6,24 These studies are possible since at least one of the two Mn2 þ ions required for PP1C activity can be removed by dialysis in the presence of EDTA. The best substitute was found to be Co2 þ , which could form a stable complex with PP1C and activate the enzyme. Based on these results, it seemed possible that if added to the culture medium in place of Mn2 þ , Co2 þ might form a stable complex with PP1C as it was being expressed and folded, and that this preparation might resemble native PP1C. To test this possibility, His6tagged PP1C was expressed in E. coli in the presence of either 1 mM MnCl2 or 100 M CoCl2. In these studies, cells were grown at room temperature, which was important for increasing the level of soluble PP1C. In addition, it was observed that concentrations of CoCl2 higher than 100 M inhibited cell growth. Using a one-step affinity chromatography step, PP1C of greater than 95% purity could be obtained rapidly in the presence of either Mn2 þ or Co2 þ , and approximately similar amounts of PP1C were obtained (yield of 3–5 mg/liter culture). The activity of the Mn-PP1C preparation could be increased
1.5-fold (added 1 mM Mn2 þ ) or
2-fold (added 1 mM Co2 þ ) when divalent cations were added to the assay. In contrast, the CoPP1C preparation was inhibited 20–30% by addition of 1 mM Mn2 þ or Co2 þ . The maximum specific activities of the two preparations were approximately the same (
3–8 mol/min/mg under standard assay conditions). Both preparations also showed similar sensitivity to inhibition by okadaic acid, while as shown previously6,24 Mn-PP1C but not CoPP1C could be inhibited by addition of 1 mM EDTA. However, 22

I.-K. Park and A. A. DePaoli-Roach, J. Biol. Chem. 269, 28919–28928 (1994). J. H. Connor et al., J. Biol. Chem. 275, 18670–18675 (2000). 24 Y. F. Chu, E. Y. C. Lee, and K. K. Schlender, J. Biol. Chem. 271, 2574–2577 (1996). 23

334

EXPRESSION SYSTEMS

[24]

Co-PP1C still displayed anomalous properties in that it dephosphorylated pNPP (specific activity was
50% of that of Mn-PP1C), and could dephosphorylate phospho-Thr34-DARPP-32 (Vmax 0.24 mol/min/mg and Km of 0.6 M compared to Mn-PP1C which had a Vmax of 1.42 mol/ min/mg and a Km of 3.3 M). Thus, while Co-PP1C exhibits properties that are closer to those of native PP1C, the anomalous activities still make it of limited use in many studies of PP1C function. Characterization of PP1 Expressed in Sf9 Cells Using Method 1

Given the anomalous properties of the E. coli PP1C preparations, an alternative procedure was developed using Sf9 cells and the baculovirus method. Purification procedures were developed for two different isoforms, PP1C and PP1C . The optimal time for maximal expression of soluble, active PP1 was established using small-scale cultures, and analysis of membrane and cytosol fractions using phosphatase assays and immunoblotting (data not shown). For PP1C, significant expression of active soluble phosphatase was observed after 30 hr, expression peaked between 50 and 60 hr, and decreased thereafter. At early time points (up to 36 hr), PP1C was mostly found in the cytosol (>60%), but thereafter PP1C was mostly found as an inactive species in the particulate fraction. For PP1C , the time course of expression of active enzyme was similar to that of PP1C, but >70% of the protein was found in the cytosol at all time points. Notably, the activity of PP1C in the soluble fraction did decrease after
60 hr, but the reason for this loss of activity of soluble enzyme was not apparent. Varying the MOI did not have a significant effect on the relative ratios of soluble and particulate PP1C or . Similar patterns of expression were also observed when Sf21, Hi5, or Mg-1 cells were used in place of Sf9 cells (data not shown, note that the amount of PP1C expressed per cell varied somewhat depending on cell size). The purification procedure for untagged PP1C or was very similar to that used for purification of untagged E. coli PP1C (method 1) (Table III shows typical results for PP1C; very similar results were obtained for PP1C ). Approximately 0.7 mg of PP1C (>90% purity) can be obtained after
200-fold purification with a yield of
25% from the cytosolic fraction of lysed Sf9 cells (from
1 liter of culture medium). Sf9 cell PP1C and exhibited high specific activities [up to 20 units/mg under standard assay conditions (Table III and data not shown)]. The two enzyme preparations were also normal in terms of inhibition by microcystin, calyculin A and okadaic acid (Table IV). The two preparations did not require Mn2 þ for activity, and similar to native PP1C, addition of 1 mM Mn2 þ inhibited

[24]

335

PREPARATION AND CHARACTERIZATION OF PP1

TABLE III PURIFICATION OF PP1C FROM Sf9 CELLS Purification step Crude lysate DEAE-cellulose Heparin-Sepharose Sephacryl S-200

REGULATION

OF

USING

METHOD 1

Volume (ml)

Protein (mg)

Total activity Units

Specific activity (units/mg)

Purification (-fold)

Yield (%)

145 155 111 34

544 85 9.4 0.7

44.9 22.7 14.3 10.8

0.08 0.27 1.5 15.2

1 3 19 190

100 51 32 24

TABLE IV VARIOUS RECOMBINANT PREPARATIONS OF PP1C

BY INHIBITORS

IC50 (nM) Source of PP1C P-DARPP-32 S-DARPP-32 Microcystin-LR Calyculin A Okadaic acid Sf9 PP1C Sf9 PP1CMn2 þ E. coli PP1C Rabbit PP1C

2.2 370 450 2.9

5.4 120 115 1.5

0.1 0.3 0.1 0.1

0.8 1.1 0.6 0.8

17 34 36 22

P-DARPP-32 and S-DARPP-32 are proteins phosphorylated by PKA using ATP or thiopho-ATP, respectively.

enzyme activities
50%. Notably, PP1C and did not dephosphorylate phospho-DARPP-32 or phospho-inhibitor-1. Consistent with this, both PP1C and were inhibited by either phospho- or thiophospho-DARPP-32 with IC50 values in the low nM range (Table IV and data not shown), properties similar to those of native PP1C. In addition, PP1C and were both relatively resistant to autodephosphorylation during phosphorylation by cdc2 kinase (Fig. 1 and data not shown). Given the anomalous properties of E. coli PP1C which is expressed in the presence of Mn2 þ , the effect of addition of Mn2 þ to the Sf9 culture medium was also investigated. Addition of 1 mM MnCl2 during the incubation period and purification of PP1C increased the total level of expression, but a greater proportion of protein was found in the particulate fraction (>70% after 36 hr incubation for either PP1C or ). The specific activity of Sf9 PP1CMn was significantly higher (
2-fold) than that of Sf9 PP1C. Like E. coli PP1C, Sf9 PP1CMn was normal in terms of inhibition by microcystin, calyculin A and okadaic acid (Table IV). However, Sf9 PP1CMn was relatively insensitive to either phosphoor thiophospho-DARPP-32 (Table IV). Furthermore, this was associated

336

EXPRESSION SYSTEMS

[24]

with the ability of Sf9 PP1CMn to rapidly dephosphorylate phosphoDARPP-32 or phospho-inhibitor-1 (Vmax of 58 mol/min/mg and Km of 28 M for phospho-inhibitor-1). Thus the addition of extracellular Mn2 þ to Sf9 cells results in the production of PP1C preparation that is very similar to E. coli PP1C. The ability of extracellular Mn2 þ to influence the properties of PP1C (in a cellular environment where the protein has the ability to fold into a native structure) presumably reflects the nonphysiological levels of this divalent cation. Interestingly, chronic Mn2 þ poisoning has been characterized as an irreversible syndrome that bears a striking similarity to Parkinson’s disease, although the detailed neuropathology appears to be distinct.25,26 Specifically, with chronic Mn2 þ , neurodegeneration occurs downstream of the nigrostriatal dopaminergic projection in the striatum and pallidum, rather than in dopaminergic neurons of the substantia nigra (as occurs in Parkinson’s disease). In the medium spiny neurons of the striatum, PP1C has been found to play a key role in mediating the postsynaptic effects of dopamine as a consequence of its regulation by DARPP-32.2 Thus it is conceivable that some of the effects of chronic Mn2 þ poisoning are mediated via the nonphysiological consequences of Mn2 þ on the activities of PP1C in medium spiny neurons.

Characterization of PP1 Expressed in Sf9 Cells Using Method 2

Wild-type, mutant and chimeric PP1C proteins have all been prepared in Sf9 cells using method 2.21 For these various PP1 preparations, expression reached a maximum between 48 and 72 hr postinfection, and approximately 50% of expressed PP1 was recovered in the soluble fraction. The inclusion of the His6 (or GST) tags made the purification more straightforward. Using the two-step affinity chromatography method (either purification on Ni-NTA or glutathione-Sepharose resins then chromatography on the PNUTS affinity column), up to 10 g of purified protein (usually > 90% pure) was generally recovered from 1  108 cells in 100 ml culture. Alternatively, partially purified recombinant PP1 can be rapidly obtained by the one-step procedure, and this is usually sufficient

25

D. B. Calne, N. S. Chu, C. C. Huang, C. S. Lu, and W. Olanow, Neurology 44, 1583–1586 (1994). 26 D. Centonze, P. Gubellini, G. Bernardi, and P. Calabresi, Exp. Neurol. 172, 469–476 (2001).

[24]

PREPARATION AND CHARACTERIZATION OF PP1

337

for the analysis of the effects of various inhibitors or proteins on PP1 activity (
0.2 ng of PP1 protein is sufficient for a single assay reaction). In general, one-step chromatography using glutathione-Sepharose resulted in a purer preparation compared to that of one-step chromatography using Ni-NTA resin. As discussed above for Sf9 cell method 1, tagged Sf9 PP1C exhibited properties essentially identical to those of native PP1, with respect to its lack of dependence on added Mn2 þ , and sensitivity to inhibition by phospho-DARPP-32 and inhibitor-2. Sf9 PP1C also exhibited an ability to bind tightly to spinophilin and PNUTS, and to be inhibited by these two targeting subunits. Importantly, Sf9 PP1C also failed to dephosphorylate tyrosine-phosphorylated myelin basic protein or phosphoDARPP-32. Given the native-like properties of Sf9 PP1C, it has been possible to carry out detailed structure-function analysis using the wild-type enzyme as a control.21 Thus, studies of the potential role of residues in the surface grooves near the active site have revealed that several acidic amino acids do not appear to contribute to regulation of the enzyme by thiophosphoDARPP-32. Other mutation studies of Sf9 PP1C revealed an important role for Tyr272 in enzyme activity. In contrast, previous studies in which Tyr272 has been mutated in E. coli PP1C had not revealed any effect on enzyme activity. In addition, mutation of Tyr272 resulted in an increase in the relative phosphatase activity towards both tyrosine-phosphorylated myelin basic protein and phospho-DARPP-32, with a much larger effect being observed for PP1[Tyr272Ala] than for PP1[Tyr272Phe]. Tyr272 is found in a loop between 12 and 13 strands, and overhangs the active site of the phosphatase.15,16 Based on comparison of the properties of Sf9 and E. coli PP1C, it seems likely that the precise position of this residue may be responsible for some of the abnormal activities of E. coli PP1C.21 In summary, Sf9 PP1 is an excellent preparation for almost all types of biochemical studies. The yield is lower than for E. coli PP1C and is less easy to scale up. However, it is convenient to use affinity chromatography procedures to rapidly obtain PP1C mutants that are suitable for enzyme assays. Most importantly, these studies indicate that PP1C folds in Sf9 cells in the same way that it does in mammalian cells, and the biochemical properties of Sf9 PP1C are indistinguishable from native PP1C typically purified from rabbit skeletal muscle. However, addition of extracellular Mn2 þ to Sf9 cell cultures is sufficient to perturb this normal process, in which case an enzyme preparation is generated that is much like that formed in E. coli. Presumably, a high intracellular level of Mn2 þ is generated that is sufficient to compete with Fe2 þ and Zn2 þ for binding to PP1C as it folds.

338

[24]

EXPRESSION SYSTEMS

Possibly, this effect of Mn2 þ might contribute to neurodegeneration associated with chronic Mn2 þ poisoning. Acknowledgments This work was supported by U.S. Public Health Grant MH40899 (to A. C. N. and P. G.).

[24] An Inducible System to Study the Growth Arrest Properties of Protein Phosphatase 2C By PAULA OFEK, DANIELLA BEN-MEIR, and SARA LAVI Introduction

The PP2C family of phosphatases is one of four major groups of serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) in eukaryotes, which is distinguished from the other groups by its dependence on magnesium ions and its insensitivity to the tumor promoter okadaic acid. The UniGene database (UniGene, NIH) indicates that the human genome contains at least six PP2C paralogs. Several independent reports suggest that different members of the PP2C family regulate transcription of growth-related pathways in mammals.1–5 This protein is highly conserved in evolution.6 Roles for PP2C in response to stress were identified in Arabidopsis7,8 as well as in yeast and mammalian cells.3,9,10 PP2C (also referred as PPM1A) is the most characterized member of the PP2C group. It is a monomeric enzyme of about 42 kDa that shows

1

M. A. Guthridge, P. Bellosta, N. Tavoloni, and C. Basilico, Mol. Cell Biol. 17, 5485 (1997). M. Fiscella, H. Zhang, S. Fan, K. Sakaguchi, S. Shen, W. E. Mercer, G. F. Vande Woude, P. M. O’Connor, and E. Appella, Proc. Natl. Acad. Sci. U.S.A. 94, 6048 (1997). 3 Y. Tong, R. Quirion, and S. H. Shen, J. Biol. Chem. 273, 35282–35290 (1998). 4 A. Cheng, P. Kaldis, and M. J. Solomon, J. Biol. Chem. 275, 34744 (2000). 5 C. Leung-Hagesteijn, A. Mahendra, I. Naruszewicz, and G. E. Hannigan, EMBO J. 20, 2160 (2001). 6 P. Cohen, D. L. Schelling, and M. J. Stark, FEBS Lett. 250, 601 (1989). 7 J. Sheen, Proc. Natl. Acad. Sci. U.S.A. 95, 975 (1998). 8 S. Tahtiharju and T. Palva, Plant J. 26, 461 (2001). 9 M. Hanada, J. Ninomiya-Tsuji, K. Komaki, M. Ohnishi, K. Katsura, R. Kanamaru, K. Matsumoto, and S. Tamura, J. Biol. Chem. 276, 5753 (2001). 10 M. Hanada, T. Kobayashi, M. Ohnishi, S. Ikeda, H. Wang, K. Katsura, Y. Yanagawa, A. Hiraga, R. Kanamaru, and S. Tamura, FEBS Lett. 437(3), 172 (1998). 2

METHODS IN ENZYMOLOGY, VOL. 366

Copyright ß 2003, Elsevier Inc. All rights reserved. 0076-6879/2003 $35.00