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Charbonneau and Ken Walsh, this manuscript could not have been written. We thank them for many helpful discussions. This work was supported by National Institutes of Health Grant DK07902 from the National Institute of Diabetes and Digestive and Kidney Diseases and by a grant from the Muscular Dystrophy Association of America. We thank Carmen Westwater for typing the manuscript.
[39] Resolution and Characterization of Multiple Protein-Tyrosine Phosphatase Activities By THOMASS. IN~EBRrrSEN Introduction Reversible p h o s p h o r y l a t i o n o f proteins on t y r o s i n e residues is one o f the earliest e v e n t s in signal t r a n s d u c t i o n p a t h w a y s leading to the stimulation o f cell proliferation. This regulatory m e c h a n i s m also a p p e a r s to play a critical role in the t r a n s f o r m a t i o n o f animal cells to a tumor-like p h e n o t y p e 1'2 and it m a y also participate in the c o n t r o l o f o t h e r cell functions (e.g., insulin action, neural function, and platelet activation). 3-5 The extent o f p h o s p h o r y l a t i o n o f these proteins is d e p e n d e n t o n the balance o f the protein kinase and p r o t e i n p h o s p h a t a s e activities t o w a r d a particular substrate protein and there is g r o w i n g e v i d e n c e that protein-tyrosine p h o s p h a tases (PTPs) play a critical role in regulating tyrosine p h o s p h o r y l a t i o n reactions. 6-9 P r o t e i n - t y r o s i n e p h o s p h a t a s e s , like protein-tyrosine kinases, can be g r o u p e d into t w o categories d e p e n d i n g on w h e t h e r t h e y are part o f receptor-like molecules.6-9 T h e s e q u e n c e s o f three n o n r e c e p t o r P T P s are k n o w n and t h e y share e x t e n s i v e h o m o l o g y (30-70%) with the receptor-like P T P s 1 T. Hunter and J. A. Cooper, Annu. Rev. Biochem. 54, 897 (1985). 2 T. Hunter and J. A. Cooper, in "The Enzymes" (P. D. Boyer and E. G. Krebs, eds.), Vol. 17, p. 191. Academic Press, Orlando, Florida, 1986. 30. M. Rosen, Science 237, 1452 (1987). 4 j. Brugge, P. Cotton, A. Lustig, W. Yonemoto, L. Lipsich, P. Coussens, J. N. Barrett, D. Nonner, and R. W. Keane, Genes Dev. 1, 287 (1987). A. Golden, S. P. Nemeth, and J. S. Brugge, Proc. Natl. Acad. Sci. U.S.A. 83, 852 (1986). 6 T. Hunter, Cell (Cambridge, Mass.)58, 1013 (1989). 7 T. S. Ingebritsen, S. K. Lewis, V. M. Ingebritsen, B. P. Jena, K. T. Hiriyanna, S. W. Jones, and R. L. Erikson, Adv. Protein Phosphatases 5, 121 (1989). 8 T. S. Ingebritsen and K. T. Hiriyanna, in "Bioinformatics: Information Transduction and Processing Systems from Cell to Whole Body" (O. Hatase and J. H. Wang, eds.), p. 117. Elsevier, Amsterdam, 1989. 9 N. K. Tonks and H. Charbonneau, Trends Biochem. Sci. 14, 497 (1989).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(CD45 and LAR) within a core (perhaps catalytic) domain. 6'9-13 None of the PTP sequences exhibits homology with those of the type 1 and type 2 protein-Ser/Thr phosphatases, indicating that the PTPs represent a distinct family of protein phosphatases. The properties of receptor-like PTPs are reviewed elsewhere in this volume. 14 Six nonreceptor PTPs have been purified to homogeneity or near homogeneity from the cytoplasmic fraction of various mammalian tissues. The enzymes are the human placental 1A PTP (M r 35,000), 15human placental 1B PTP (M r 37,000),15 rabbit kidney type I PTP (M r 34,000), 16rabbit kidney type II PTP (Mr 37,000), 16 bovine spleen PTP (M r 52,000), 17 and bovine brain PTP5 (Mr 48,000). TM Two additional PTPs have been identified via cDNA cloning, the human T cell PTP (M r 48,000) 11and rat brain PTP (M r 50,000). 12 Although the relationships among these PTPs are not fully defined, the available information indicates that there are a minimum of three distinct nonreceptor PTP catalytic subunits. The complete amino acid sequences of the human placental 1B PTP, 10human T cell PTP,11 and rat brain PTP 12 are known. The rat brain PTP sequence is 97% identical to the human placental PTP over the first 321 amino acids but extends for an additional 111 amino acids at the C terminus. This indicates that the two enzymes correspond to the human and rat forms of the same PTP gene product. The C-terminal domain of the PTP may have been selectively lost via limited proteolysis during purification of the enzyme from human placenta. The human T cell PTP is the product of a gene that is distinct from but related to that encoding the rat brain/human placental 1B PTP. This chapter focuses on methods for the assay and purification of bovine brain PTPs and on methods for the assay and purification of two regulatory proteins, inhibitor H and inhibitor L, that function as PTP inhibitors. Bovine brain PTP5 is distinct from the human placental 1A PTP l0 H. Charbonneau, N. K. Tonks, S. Kumar, C. D. Diltz, M. Harrylock, D. E. Cool, E. G. Krebs, E. H. Fischer, and K. A. Walsh, Proc. Natl. Acad. Sci. U.S.A. 86, 5252 (1989). n D. E. Cool, N. K. Tonks, H. Charbonneau, K. A. Walsh, E. H. Fischer, and E. G. Krebs, Proc. Natl. Acad. Sci. U.S.A. 86, 5257 (1989). 12 K. Guan, R. S. Haun, S. J. Watson, R. L. Geahlen, and J. E. Dixon, Proc. Natl. Acad. Sci. U.S.A. 87, 1501 (1990). 13 M. Streuli, N. X. Krueger, A. Y. M. Tsai, and H. Saito, Proc. Natl. Acad. Sci. U.S.A. 86, 8698 (1989). 14 N. K. Tonks, C. D. Diltz, and E. H. Fischer, this volume [38]. 15 N. K. Tonks, C. D. Diltz, and E. H. Fischer, J. Biol. Chem. 263, 6722 (1988). t6 C. L. Shriner and D. L. Brautigan, J. Biol. Chem. 259, 11383 (1984). 17 H. Y. L. Tung and L. J. Reed, Anal. Biochem. 161, 412 (1987). is S. W. Jones, R. L. Erikson, V. M. Ingebritsen, and T. S. Ingebritsen, J. Biol. Chem. 264, 7747 (1989).
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and rabbit kidney type II PTP. 18The relationship of PTP5 to the rat brain/ human placental 1B PTP, human T cell PTP, rabbit kidney type I PTP, and bovine spleen PTP is unknown. In addition to PTP5, six other PTP activities, termed PTP1A, PTP1B, PTP2, PTP3, PTP4, and PTP6, have been partially purified from the cytosolic fraction from bovine brain. TM Like PTP5 these activities are distinct from protein-Ser/Thr phosphatases but differ from PTP5 in their chromatographic properties on DEAE-cellulose, phosphocellulose, and/or gel filtration. The seven PTPs also differ in their sensitivity to inhibitor proteins (see below) 19 and in their sensitivity to inhibition by macromolecular polyanions [heparin, poly(Glu8°,Tyr2°), and the synthetic polynucleotide, poly(G,I)].7 It is not yet clear whether the seven bovine brain activities correspond to distinct gene products, different forms of the same gene product, or some combination of these two possibilities. The turnover number of bovine brain PTP5 ~8 is at least an order of magnitude greater than those of the known protein-tyrosine kinases. This suggests that the activity of PTP5 may need to be closely regulated in order to allow tyrosine phosphorylation reactions to occur in intact cells. Pursuing this idea we have identified two regulatory proteins, inhibitor H (>500 kDa) and inhibitor L (38 kDa) in bovine brain, that potently and preferentially inhibit PTP5.19 Inhibitor H and inhibitor L also inhibit bovine brain PTP4 but the IC50 values for inhibition of PTP4 are 10- and 2fold greater, respectively, than those for the inhibition of PTP5. The two inhibitor proteins only inhibit the other five bovine brain protein-tyrosine phosphatases at very high concentrations (IC50 - 100-fold higher than those for PTP5). This suggests that PTP4 and PTP5 may be closely related enzymes. While the precise functions of PTPs are poorly understood, the physiological importance of these enzymes is underscored by several types of observations. First, addition of vanadate, a potent PTP inhibitor, to normal cells results in a dramatic rise in tyrosine phosphorylation, 6 although it should be noted that vanadate may also have a stimulatory effect on some protein-tyrosine kinases. Second, the existence of receptor-like PTPs and of protein inhibitors of nonreceptor PTPs suggests that PTPs may be tightly regulated and that this regulation in some cases may initiate signal transduction events. Third, PTP4 and/or PTP5 like activities are present in a wide range of vertebrate cell types and they are particularly enriched in proliferating cell populations. 7 Fourth, the similarity of receptor-like PTP LAR to neural adhesion molecules suggests that it may have a role
J9 T. S. Ingebritsen, J. Biol. Chem. 264, 7754 (1989).
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in cell-cell or cell-matrix interactions. 2° Fifth, the receptor-like PTP, CD45, is required for antigen-induced T lymphocyte proliferation2~ and influences signal transduction events mediated via the CD2, CD3, and CD4 cell surface antigens of T cells.22 Sixth, microinjection of the placental PTP into X e n o p u s oocytes delays the insulin-induced maturation response. 9
Assay Methods Protein-Tyrosine P h o s p h a t a s e A s s a y Principle. Protein-tyrosine phosphatases are assayed by following the time-dependent release of trichloroacetic acid-soluble radioactivity from [32p]casein. Reagents
Bovine serum albumin (BSA): Ultrapure grade from Boehringer-Mannheim (Cat. #238 031) Assay buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-0.05 mM EDTA-1 mg/ ml BSA-0.3% (v/v) 2-mercaptoethanol Tris-Brij buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-0.01% (w/v) Brij 35 [32p]Casein: The stock solution of casein phosphorylated by the insulin receptor kinase (see below) is diluted to 300 nM with assay buffer. Between assays the diluted [32P]casein is stored at 4° Procedure
Mix 20/xl of protein phosphatase diluted in assay buffer with 20/xl of Tris-Brij buffer and preincubate for 5 rain at 30°. Start the assay by adding 20/zl of [32p]casein to the reaction mixture. Continue the incubation for 1-30 min and then stop the reaction with 100/xl of 20% (w/v) trichloroacetic acid (TCA). Vortex the mixture and hold on ice for l0 min. Vortex again, centrifuge at room temperature for 2 min at 12,000 g in a microfuge, and count 120/xl of the supernatant. The protein phosphatase assays are linear with respect to enzyme concentration and time up to 30% release of inorganic phosphate from casein. One unit of protein-tyrosine phosphatase is the amount that re2oM. Streuli, N. X. Krueger, L. R. Hall, S. F. Schlossman,and H. Saito,J. Exp. Med. 168, 1523 (1988). 2J j. T. Pingel and M. L. Thomas, Cell (Cambridge, Mass.) 58, 1055 (1989). 22j. A. Ledbetter, N. K. Tonks, E. H. Fischer, and E. A. Clark, Proc. Natl. Acad. Sci. U.S.A. 85, 8628 (1988).
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leases 1 nmol of inorganic phosphate from casein per minute under the standard assay conditions. When initially characterizing a new proteintyrosine phosphatase using this assay it is important to establish that the trichloroacetic acid-soluble counts represent inorganic phosphate rather than small phosphopeptides that may also be trichloroacetic acid soluble. This can readily be done by treating the TCA supernatant with molybdate and extracting the resulting inorganic phosphate-molybdate complexes into organic solvent. 23 Phosphate esters or anhydrides do not form complexes with molybdate.
Protein-Tyrosine Phosphatase Inhibitor Assay Principle. Inhibitor H and inhibitor L are assayed based on their ability to inhibit the activity of PTP5. Reagents PTP5: The enzyme is partially or highly purified from bovine brain as described below and diluted to a concentration of 10 mU/ml in assay buffer Other reagents: The preparation of these are described above (see Protein-Tyrosine Phosphatase Assay)
Procedure Fractions containing inhibitor activity are incubated at 95 ° for 5 min to inactivate any endogenous protein-tyrosine phosphatase activity. The mixture is cooled on ice, centrifuged for 2 min at 15,000 g to remove denatured protein, and diluted in Tris-Brij buffer. Then, mix 20 /~l of protein phosphatase diluted in assay buffer with 20/zl of diluted inhibitor H or inhibitor L, preincubate for 5 min at 30°, and initiate the proteintyrosine phosphatase assay by adding 20/A of [32p]casein to the reaction mixture. After 10 min the reaction is terminated and further processed as described above (see Protein-Tyrosine Phosphatase Assay). It is important to use high-quality BSA (Boehringer Mannheim, ultrapure grade) in the assay buffers since fraction V BSA contains contaminants that interfere with the inhibition of PTP5 by the two inhibitor proteins. Inhibitor activity is quantitated based on the concentration-dependent decrease in PTP5 compared to a control incubation lacking inhibitor (Fig.
23 S. Shenolikar and T. S. Ingebritsen, this series, Vol. 107, p. 102.
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I00
80
J
80
i 4° 2O
O
0
i
i
i
I
I
I
2
3
4
5
6
Inhibitor Activity (Units/assay) FIG. 1. Standard curve for the inhibition of PTP5. The standard inhibitor assay was used and the amounts of inhibitor (DEAE-cellulose fraction) added are indicated. See text for definition of units. (Reprinted with permission from Ingebritsen. 19)
I). Concentrations producing up to 50-60% PTP5 inhibition can be estimated with little loss of accuracy. The sensitivity of PTP5 to inhibition by inhibitor H and inhibitor L decreases as the concentration of PTP5 in the assay increases) 9 The standard assay contains 0.2 mU PTP5, which is equivalent to a PTP5 concentration of 20 pM based on a molecular weight of 46,000, and Km and Vmaxvalues of 130 nM and 10,000 U/mg, respectively. This concentration of PTP5 gives near-maximal sensitivity of the enzyme to the two inhibitor proteins. One unit of inhibitor is the amount that decreases the activity of 0.2 mU PTP5 by 50% under the standard assay conditions. 24
Phosphorylation of Casein by Insulin Receptor Kinase Principle. [32p]Casein, phosphorylated exclusively on tyrosine residues to a stoichiometry of 1-2 nmol/mg, is prepared by incubation with the insulin receptor kinase and [y-32p]ATP. The definition of inhibitor units given here is equivalent to that given in Ref. 19. The apparent difference in the amount of PTP5 in the standard assay reflects a PTP5 activity unit definition based on a substrate concentration of 100 nM vs 1 nM in Ref. 19. The actual PTP5 concentation (pM) is approximately the same as in Ref. 19.
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Reagents
Buffer A: 50 mM HEPES, pH 7.4-0.1% (v/v) Triton X-100-10 mM MgCI2 Buffer B: 166.7 mM HEPES, pH 7.4-16.7 mM MgC12-1.7 mM MNC12-3.3 mM EDTA Casein: This is a mixture of 70% ~s~- + ~s2-Caseins and 30%/3- + Kcaseins. It is purchased from Sigma (Cat. #C-7891; St. Louis, MO) Insulin receptor kinase: The enzyme is partially purified from NIH 3T3 HIR3.5 cells as described by Treadwell et al. 25 and stored at - 8 0 ° in 50 mM Tris-HCl (pH 8.0 at 25°)-0.3 M N-acetylglucosamine-0.05% (v/v) Triton X-100-10% (v/v) glycerol at a concentration of 0.4-0.8 U/ml. Insulin receptor kinase activity is assayed essentially as described by Tonks et al. 15 using poly(GluS°,Tyr 2°) as substrate (2 mg/ ml) and 0.1 mM [y-3Zp]ATP (1000 cpm/pmol). One unit of insulin receptor kinase is the amount that incorporates 1 nmol of 32p into poly(Glu8°,Tyr 2°) in 1 min under the standard assay conditions [y-3Zp]ATP (5 mM, 10,000-20,000 cpm/pmol) in H20 Phosphorylation Procedure
Mix 320/zl of buffer A containing 12.5 mg/ml casein and 7/zg/ml insulin with 240 tzl buffer B plus 80 tzl insulin receptor. Preincubate the mixture for 15 min at 30°. Add 160/xl [-y-32p]ATP and incubate overnight at 30°. Remove a 5-tzl aliquot and process to determine phosphate incorporation (see below). Terminate incubation by adding 160/zl of 100% TCA. Hold on ice for 60 min and centrifuge for 2 min at 12,000 g in a microfuge. Wash the pellet six times with 0.5 ml ice-cold 20% TCA (centrifuge 1 min after each wash). Add 0.5 ml of 0.5 M Tris, pH 8.5 to the washed pellet and allow the pellet to redissolve overnight in the refrigerator. Gel filter the solution on a Sephadex G-50 column (0.7 x 17 cm) equilibrated with 50 mM Tris (pH 7.0 at 25°)-0.05 mM EDTA. Pool the 32p-labeled protein peak, divide into aliquots containing 300 pmol of [32p]casein, and store at - 2 0 °. Measurement o f Phosphate Incorporation into Casein
Dilute 5/zl of the reaction mixture to 100/xl with H20. Transfer four 5-/xl aliquots of the diluted reaction mixture to 2 x 2 cm squares of Whatman (Clifton, NJ) P-81 phosphocellulose paper. Wash the filter papers three times with 200 ml of 0.5% (w/v) H3PO4 (5 min for each wash). Wash the filter papers once with 200 ml of ethanol, dry, and count in scintillant. 25 j. L. Treadwell, J. Whittaker, and J. E. Pessin, J. Biol. Chem. 264, 15136 (1989).
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Extract Chjrtosol DEAE-CeHulose I 0 - 9 0 m M NaCI
90-500mMNaCI (PTP6, PrP2A)
(PTPIA, PTPIB, PTP2, PTP3, PTP4, PTP5) Phosphocellulose I Breakthrough
tPTPI~ PTPaBI
SephaerylS-300 [
Gradient
~
~
PTP2
PTP3
PTP6
PrP2A
~ PTP4
PTP5
SephacrylS-300 I
l~rPIA
PTP1B
FIG. 2. Outline of the procedure for the separation of the seven bovine brain proteintyrosine phosphatases (PTPs) from each other and from protein-Ser/Thr Phosphatase 2A (~P2A).
Protein-Tyrosine Phosphatase and Phosphatase Inhibitor Preparations
Separation of Bovine Brain Protein-Tyrosine Phosphatases Principle. Most of the protein-tyrosine phosphatase activity in bovine brain extracts (70%) is in the cytosolic fraction. Seven protein-tyrosine phosphatases (PTP1A, PTP1B, PTP2, PTP3, PTP4, PTP5, and PTP6) account for 90-95% of the cytosolic activity. These activities are resolved by successive chromatographies on DEAE-cellulose, phosphocellulose, and Sephacryl S-300. The remaining 5-10% of the activity is due to proteinSer/Thr phosphatase 2A. The separation protocol is shown in schematic form in Fig. 2. Reagents Homogenization solution: 4.0 mM EDTA, pH 7.0-250 mM sucrose0.1 mM phenylmethylsulfonyl fluoride (PMSF)-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol
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459
Buffer C: 20 mM Tris-HC1 (pH 7.0 at 25°)-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol Buffer D: 50 mM Tris-HCl (pH 7.0 at 25°)-50 mM NaC1-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.01% (w/v) Brij 35-0.2% (v/v) 2-mercaptoethanol Buffer E: 50 mM Tris-HC1 (pH 7.0 at 25°)-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-50% (v/v) glycerol-0.2% (v/v) 2-mercaptoethanol Procedure. The entire procedure is performed at 4° . Two fresh bovine brains are deveined, passed through a meat grinder, and homogenized in a Waring blender (2 min at low speed) in 2 vol of homogenization solution. The homogenate is centrifuged at 12,000 g for 20 min and the resulting supernatant (extract) is further centrifuged at 100,000 g for 60 min. DEAE-Cellulose Chromatography Step. The supernatant (cytosol) is applied to a DEAE-cellulose column (7.5 × 14 cm) equilibrated with buffer C at a flow rate of 300-400 ml/hr. The column is washed with buffer C containing 90 mM NaC1 until the A280 of the effluent is PTP4 > PTP3 ~ PTP2 > P T P I > PTP6.19 PTP5 is 2- and 10-fold m o r e sensitive to inhibition b y inhibitor L and inhibitor H, respectively, than PTP4 and
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---50- to lO0-fold more sensitive than the other five protein-tyrosine phosphatases. PTP5 is potently inhibited by three classes of macromolecular polyanions: polynucleotides (e.g., DNA, RNA, and synthetic oligonucleotides), acidic polyamino acids [e.g., poly(GluS°,Tyr2°), poly(Glu)], and the acidic polysaccharide heparin.7 The IC50 values for inhibition of PTP5 by heparin, poly(Glu8°,Tyr2°), and poly(G,I) are -0.2/zg/ml. The three types of polyanions inhibit the bovine brain protein-tyrosine phosphatases with the following order of potencies: PTP5 > PTP4 ~ PTP3 > PTP2 ~ PTP1 > PTP6. The separation procedure can also be applied to other tissues. Using this procedure and the sensitivity to inhibition by inhibitor H, we have identified PTP4- and PTP5-1ike activities in seven rabbit tissues (brain, spleen, kidney, liver, heart, adipose tissue, and skeletal muscle) as well as several vertebrate cell types in culture (chicken embryo fibroblasts, a mouse T cell hybridoma, and a mouse B cell hybridoma). 7
Purification of PTP5 to Near Homogeneity Principle. Highly purified PTP5 is prepared from bovine brain using a seven-step procedure employing successive chromatographies of the cytosolic fraction on DEAE-cellulose, phosphocellulose, Affi-Gel Blue, heparin-agarose, Mono S, and TSK G3000 SW. Early steps in the procedure are similar to those used for the separation of the seven bovine brain PTPs; however, they have been shortened by using a batch adsorption procedure at the DEAE-cellulose step and step NaCI cuts at the phosphocellulose step. It is critical to maintain the protease inhibitor, PMSF, in all buffers used in the purification procedure to avoid limited proteolysis of the enzyme. Starting with 750 g of bovine brain, approximately 6/zg of protein is obtained after the final purification step with the M r 48,000 PTP5 peptide accounting for about 10-25% of the total protein. The overall yield based on total activity in the brain extracts is about 1.5% with a 13,000fold purification. Reagents Homogenization solution: See Separation of Bovine Brain Protein-Tyrosine Phosphatases Buffer F: 10 mM Tris-HCl (pH 7.0 at 25°)-0.1 mM EDTA-0.2 mM PMSF-0.2% (v/v) 2-mercaptoethanol Buffer G: 10 mM potassium phosphate, pH 7.0-0.1 mM EDTA-0.2 mM PMSF-0.2 (v/v) 2-mercaptoethanol Buffer H: 10 mM potassium phosphate, pH 7.0-0.1 mM EDTA-5 mM DTT-0.05% (w/v) Brij 35-10% (v/v) glycerol
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Buffer I: 50 mM potassium phosphate, pH 7.0-150 mM NaCI-0.1 mM EDTA-0.05% (w/v) Brij 35-10% (v/v) glycerol-0.2% (v/v) 2-mercaptoethanol PTP5 storage buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-50 mM NaC1-0.1 mM EDTA-5 mM DTT-50% (v/v) glycerol Purification Procedure. All procedures are carried out at 4° unless otherwise specified. Brain (750 g) is homogenized (see above) in 1.5 liters of homogenization solution. The homogenate is centrifuged at 7700 g, the supernatant (extract) is decanted through glass wool, and the pH adjusted to 7.5 with NaOH. The extract is centrifuged at 53,000 g for 5.5 hr. DEAE-Cellulose Chromatography. The supernatant is added to 500 ml (packed volume) of DEAE-cellulose equilibrated with buffer F. The mixture is stirred for 30 min, allowed to settle for 20 min, and decanted through a sintered glass funnel. The DEAE-cellulose is washed in the funnel with buffer F until the volume of flow through plus wash totals about 2 liters. Solid ammonium sulfate (472 g/liter) is added to the combined flow through and wash fractions to give a 70% saturation solution and the mixture is stirred for 20 min. Precipitated proteins are collected by centrifugation at 7700 g for 20 min, dissolved in buffer F, dialyzed for 8 hr against 20 liters of buffer F with two changes of buffer, and fresh PMSF is added to give a final concentration of 1 mM. Phosphocellulose Chromatography. The resulting solution (DEAEcellulose fraction) is applied at a flow rate of 2.5 ml/min to a phosphocellulose column (50-ml bed volume) equilibrated in buffer F. The column is washed with 250 mM NaC1 in buffer F ( - 4 liters) at a flow rate of 3.6 ml/ min and PTP5 is eluted with 350 mM NaCI in buffer F at a flow rate of 0.8 ml/min. Fractions (15 ml) containing the activity peak are pooled. Affi-Gel Blue Chromatography. The pooled phosphocellulose fraction is applied at a flow rate of 1 ml/min to an Affi-Gel Blue column (10-ml bed volume) equilibrated with buffer F containing 350 mM NaC1. PTP5 is eluted from the column at a flow rate of 0.75 ml/min with a 500-ml linear gradient of 0.5-1.0 M NaC1 in buffer F (adjusted to pH 7.5 at 25°). Fractions (15 ml) containing the activity peak are pooled. Heparin-Agarose Chromatography. The pooled fractions from the Affi-Gel Blue chromatography step are dialyzed for 8 hr against 20 liters of buffer F with two changes of buffer and applied at a flow rate of 1 ml/ min to a heparin-agarose column (5-ml bed volume) equilibrated in buffer F. PTP5 is eluted at a flow rate of 0.5 ml/min with a 250-ml linear gradient of 0-1 M NaC1 in buffer G. Fractions (5 ml) containing the activity peak are pooled. Mono S Chromatography. The pooled fractions from the heparinagarose step are dialyzed overnight against 2 liters of buffer F and applied
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to a Pharmacia (Piscataway, N J) Mono S cation-exchange column (1-ml bed volume) equilibrated with buffer H. PTP5 is eluted with a 28-ml gradient of 0-600 mM NaCI in buffer H using a Pharmacia FPLC (fast protein liquid chromatography) system (flow rate of 0.8 ml/min). Fractions (1 ml) containing the PTP5 activity peak are pooled. Gel-Permeation Chromatography. The pooled fractions from the Mono S chromatography step are dialyzed against 1 liter of buffer I for 4 hr and concentrated to about 0.5 ml by ultrafiltration using an Amicon (Danvers, MA) YM10 membrane. The resulting fraction is applied to a TSK G3000 SW column (0.75 × 60 cm) equilibrated with buffer I. The procedure is carried out at room temperature at a flow rate of 0.5 ml/min. Fractions (0.25 ml) are collected, immediately placed on ice, and assayed for proteintyrosine phosphatase activity. The peak of activity is pooled, dialyzed against PTP5 storage buffer, and stored at - 2 0 °.
Properties of Highly Purified PTP5 PTP5 is a monomeric protein with a rather broad pH optimum of 7.0. The enzyme dephosphorylates four of six substrates that have been tested to date, including casein, RCM lysozyme (phosphorylated by the insulin receptor kinase), pp60 v-~rc(Tyr-416 autophosphorylation site), and insulin receptor kinase (autophosphorylation sites). 7 In the latter case, the cloned cytoplasmic domain of the insulin receptor kinase expressed in insect cells using the baculovirus expression system 26 was used as substrate after autophosphorylation in vitro. Caseins phosphorylated by the insulin receptor kinase, pp60 v~rc, or pp60 c-~rc are equivalent substrates for PTP5. 27 Kinetic parameters (Kin, 100-130 nM; kcat, 6-8 sec-t) for dephosphorylation of casein and lysozyme are similar. At a fixed substrate concentration, pp60 ..... and insulin receptor kinase are dephosphorylated at 10% of the rate with casein or RCM lysozyme as substrates. Calpactin I (phosphorylated by pp60 v-~r~) and mixed histones [phosphorylated by epidermal growth factor (EGF) receptor kinase] are not dephosphorylated at significant rates, p-Nitrophenyl phosphate is also a substrate for PTP5, although the enzyme is not a major alkaline phosphatase activity in bovine brain) 8
Partial Purification of Protein-Tyrosine Phosphatase Inhibitors Principle. Inhibitor H and inhibitor L are partially purified by successive chromatographies of the bovine brain cytosolic fraction on DEAEcellulose and Sephacryl S-300. The DEAE-cellulose chromatography step 26 R. Herrera, D. Lebwohl, A. G. Herreros, R. G. Kallen, and O. M. Rosen, J. Biol. Chem. 263, 5560 (1988). 27 S. K. Jakes, K. L. Hipper., and T. S. Ingebritsen, unpublished observations.
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gives a 40-fold purification with a 300-400% yield and separates the two inhibitor proteins from the seven bovine brain PTP activities. The reason for the increase in activity at this step is not known. The Sephacryl S-300 step separates inhibitor H (Mr > 500,000) from inhibitor L (Mr 38,000).
Reagents Buffers C and D: See Separation of Bovine Brain Protein-Tyrosine Phosphatases Buffer J: 50 mM Tris-HCl (pH 7.0 at 25°)-0.05 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol Procedure. The starting point for the preparation is the unboiled 12,000 g supernatant (extract) obtained from a single bovine brain (see Separation of Bovine Brain Protein-Tyrosine Phosphatases). The extract is centrifuged at 100,000 g for 60 min at 4 ° and the resulting supernatant (cytosol) is applied to a DEAE-cellulose (DE-52) column (5.5 x 16 cm) equilibrated with buffer C at a flow rate of 300 ml/hr. The column is washed with 400 ml buffer C and then further washed with buffer C containing 200 mM NaCI until the A280of the effluent is
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Charbonneau and Ken Walsh, this manuscript could not have been written. We thank them for many helpful discussions. This work was supported by National Institutes of Health Grant DK07902 from the National Institute of Diabetes and Digestive and Kidney Diseases and by a grant from the Muscular Dystrophy Association of America. We thank Carmen Westwater for typing the manuscript.
[39] Resolution and Characterization of Multiple Protein-Tyrosine Phosphatase Activities By THOMASS. IN~EBRrrSEN Introduction Reversible p h o s p h o r y l a t i o n o f proteins on t y r o s i n e residues is one o f the earliest e v e n t s in signal t r a n s d u c t i o n p a t h w a y s leading to the stimulation o f cell proliferation. This regulatory m e c h a n i s m also a p p e a r s to play a critical role in the t r a n s f o r m a t i o n o f animal cells to a tumor-like p h e n o t y p e 1'2 and it m a y also participate in the c o n t r o l o f o t h e r cell functions (e.g., insulin action, neural function, and platelet activation). 3-5 The extent o f p h o s p h o r y l a t i o n o f these proteins is d e p e n d e n t o n the balance o f the protein kinase and p r o t e i n p h o s p h a t a s e activities t o w a r d a particular substrate protein and there is g r o w i n g e v i d e n c e that protein-tyrosine p h o s p h a tases (PTPs) play a critical role in regulating tyrosine p h o s p h o r y l a t i o n reactions. 6-9 P r o t e i n - t y r o s i n e p h o s p h a t a s e s , like protein-tyrosine kinases, can be g r o u p e d into t w o categories d e p e n d i n g on w h e t h e r t h e y are part o f receptor-like molecules.6-9 T h e s e q u e n c e s o f three n o n r e c e p t o r P T P s are k n o w n and t h e y share e x t e n s i v e h o m o l o g y (30-70%) with the receptor-like P T P s 1 T. Hunter and J. A. Cooper, Annu. Rev. Biochem. 54, 897 (1985). 2 T. Hunter and J. A. Cooper, in "The Enzymes" (P. D. Boyer and E. G. Krebs, eds.), Vol. 17, p. 191. Academic Press, Orlando, Florida, 1986. 30. M. Rosen, Science 237, 1452 (1987). 4 j. Brugge, P. Cotton, A. Lustig, W. Yonemoto, L. Lipsich, P. Coussens, J. N. Barrett, D. Nonner, and R. W. Keane, Genes Dev. 1, 287 (1987). A. Golden, S. P. Nemeth, and J. S. Brugge, Proc. Natl. Acad. Sci. U.S.A. 83, 852 (1986). 6 T. Hunter, Cell (Cambridge, Mass.)58, 1013 (1989). 7 T. S. Ingebritsen, S. K. Lewis, V. M. Ingebritsen, B. P. Jena, K. T. Hiriyanna, S. W. Jones, and R. L. Erikson, Adv. Protein Phosphatases 5, 121 (1989). 8 T. S. Ingebritsen and K. T. Hiriyanna, in "Bioinformatics: Information Transduction and Processing Systems from Cell to Whole Body" (O. Hatase and J. H. Wang, eds.), p. 117. Elsevier, Amsterdam, 1989. 9 N. K. Tonks and H. Charbonneau, Trends Biochem. Sci. 14, 497 (1989).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(CD45 and LAR) within a core (perhaps catalytic) domain. 6'9-13 None of the PTP sequences exhibits homology with those of the type 1 and type 2 protein-Ser/Thr phosphatases, indicating that the PTPs represent a distinct family of protein phosphatases. The properties of receptor-like PTPs are reviewed elsewhere in this volume. 14 Six nonreceptor PTPs have been purified to homogeneity or near homogeneity from the cytoplasmic fraction of various mammalian tissues. The enzymes are the human placental 1A PTP (M r 35,000), 15human placental 1B PTP (M r 37,000),15 rabbit kidney type I PTP (M r 34,000), 16rabbit kidney type II PTP (Mr 37,000), 16 bovine spleen PTP (M r 52,000), 17 and bovine brain PTP5 (Mr 48,000). TM Two additional PTPs have been identified via cDNA cloning, the human T cell PTP (M r 48,000) 11and rat brain PTP (M r 50,000). 12 Although the relationships among these PTPs are not fully defined, the available information indicates that there are a minimum of three distinct nonreceptor PTP catalytic subunits. The complete amino acid sequences of the human placental 1B PTP, 10human T cell PTP,11 and rat brain PTP 12 are known. The rat brain PTP sequence is 97% identical to the human placental PTP over the first 321 amino acids but extends for an additional 111 amino acids at the C terminus. This indicates that the two enzymes correspond to the human and rat forms of the same PTP gene product. The C-terminal domain of the PTP may have been selectively lost via limited proteolysis during purification of the enzyme from human placenta. The human T cell PTP is the product of a gene that is distinct from but related to that encoding the rat brain/human placental 1B PTP. This chapter focuses on methods for the assay and purification of bovine brain PTPs and on methods for the assay and purification of two regulatory proteins, inhibitor H and inhibitor L, that function as PTP inhibitors. Bovine brain PTP5 is distinct from the human placental 1A PTP l0 H. Charbonneau, N. K. Tonks, S. Kumar, C. D. Diltz, M. Harrylock, D. E. Cool, E. G. Krebs, E. H. Fischer, and K. A. Walsh, Proc. Natl. Acad. Sci. U.S.A. 86, 5252 (1989). n D. E. Cool, N. K. Tonks, H. Charbonneau, K. A. Walsh, E. H. Fischer, and E. G. Krebs, Proc. Natl. Acad. Sci. U.S.A. 86, 5257 (1989). 12 K. Guan, R. S. Haun, S. J. Watson, R. L. Geahlen, and J. E. Dixon, Proc. Natl. Acad. Sci. U.S.A. 87, 1501 (1990). 13 M. Streuli, N. X. Krueger, A. Y. M. Tsai, and H. Saito, Proc. Natl. Acad. Sci. U.S.A. 86, 8698 (1989). 14 N. K. Tonks, C. D. Diltz, and E. H. Fischer, this volume [38]. 15 N. K. Tonks, C. D. Diltz, and E. H. Fischer, J. Biol. Chem. 263, 6722 (1988). t6 C. L. Shriner and D. L. Brautigan, J. Biol. Chem. 259, 11383 (1984). 17 H. Y. L. Tung and L. J. Reed, Anal. Biochem. 161, 412 (1987). is S. W. Jones, R. L. Erikson, V. M. Ingebritsen, and T. S. Ingebritsen, J. Biol. Chem. 264, 7747 (1989).
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and rabbit kidney type II PTP. 18The relationship of PTP5 to the rat brain/ human placental 1B PTP, human T cell PTP, rabbit kidney type I PTP, and bovine spleen PTP is unknown. In addition to PTP5, six other PTP activities, termed PTP1A, PTP1B, PTP2, PTP3, PTP4, and PTP6, have been partially purified from the cytosolic fraction from bovine brain. TM Like PTP5 these activities are distinct from protein-Ser/Thr phosphatases but differ from PTP5 in their chromatographic properties on DEAE-cellulose, phosphocellulose, and/or gel filtration. The seven PTPs also differ in their sensitivity to inhibitor proteins (see below) 19 and in their sensitivity to inhibition by macromolecular polyanions [heparin, poly(Glu8°,Tyr2°), and the synthetic polynucleotide, poly(G,I)].7 It is not yet clear whether the seven bovine brain activities correspond to distinct gene products, different forms of the same gene product, or some combination of these two possibilities. The turnover number of bovine brain PTP5 ~8 is at least an order of magnitude greater than those of the known protein-tyrosine kinases. This suggests that the activity of PTP5 may need to be closely regulated in order to allow tyrosine phosphorylation reactions to occur in intact cells. Pursuing this idea we have identified two regulatory proteins, inhibitor H (>500 kDa) and inhibitor L (38 kDa) in bovine brain, that potently and preferentially inhibit PTP5.19 Inhibitor H and inhibitor L also inhibit bovine brain PTP4 but the IC50 values for inhibition of PTP4 are 10- and 2fold greater, respectively, than those for the inhibition of PTP5. The two inhibitor proteins only inhibit the other five bovine brain protein-tyrosine phosphatases at very high concentrations (IC50 - 100-fold higher than those for PTP5). This suggests that PTP4 and PTP5 may be closely related enzymes. While the precise functions of PTPs are poorly understood, the physiological importance of these enzymes is underscored by several types of observations. First, addition of vanadate, a potent PTP inhibitor, to normal cells results in a dramatic rise in tyrosine phosphorylation, 6 although it should be noted that vanadate may also have a stimulatory effect on some protein-tyrosine kinases. Second, the existence of receptor-like PTPs and of protein inhibitors of nonreceptor PTPs suggests that PTPs may be tightly regulated and that this regulation in some cases may initiate signal transduction events. Third, PTP4 and/or PTP5 like activities are present in a wide range of vertebrate cell types and they are particularly enriched in proliferating cell populations. 7 Fourth, the similarity of receptor-like PTP LAR to neural adhesion molecules suggests that it may have a role
J9 T. S. Ingebritsen, J. Biol. Chem. 264, 7754 (1989).
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in cell-cell or cell-matrix interactions. 2° Fifth, the receptor-like PTP, CD45, is required for antigen-induced T lymphocyte proliferation2~ and influences signal transduction events mediated via the CD2, CD3, and CD4 cell surface antigens of T cells.22 Sixth, microinjection of the placental PTP into X e n o p u s oocytes delays the insulin-induced maturation response. 9
Assay Methods Protein-Tyrosine P h o s p h a t a s e A s s a y Principle. Protein-tyrosine phosphatases are assayed by following the time-dependent release of trichloroacetic acid-soluble radioactivity from [32p]casein. Reagents
Bovine serum albumin (BSA): Ultrapure grade from Boehringer-Mannheim (Cat. #238 031) Assay buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-0.05 mM EDTA-1 mg/ ml BSA-0.3% (v/v) 2-mercaptoethanol Tris-Brij buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-0.01% (w/v) Brij 35 [32p]Casein: The stock solution of casein phosphorylated by the insulin receptor kinase (see below) is diluted to 300 nM with assay buffer. Between assays the diluted [32P]casein is stored at 4° Procedure
Mix 20/xl of protein phosphatase diluted in assay buffer with 20/xl of Tris-Brij buffer and preincubate for 5 rain at 30°. Start the assay by adding 20/zl of [32p]casein to the reaction mixture. Continue the incubation for 1-30 min and then stop the reaction with 100/xl of 20% (w/v) trichloroacetic acid (TCA). Vortex the mixture and hold on ice for l0 min. Vortex again, centrifuge at room temperature for 2 min at 12,000 g in a microfuge, and count 120/xl of the supernatant. The protein phosphatase assays are linear with respect to enzyme concentration and time up to 30% release of inorganic phosphate from casein. One unit of protein-tyrosine phosphatase is the amount that re2oM. Streuli, N. X. Krueger, L. R. Hall, S. F. Schlossman,and H. Saito,J. Exp. Med. 168, 1523 (1988). 2J j. T. Pingel and M. L. Thomas, Cell (Cambridge, Mass.) 58, 1055 (1989). 22j. A. Ledbetter, N. K. Tonks, E. H. Fischer, and E. A. Clark, Proc. Natl. Acad. Sci. U.S.A. 85, 8628 (1988).
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leases 1 nmol of inorganic phosphate from casein per minute under the standard assay conditions. When initially characterizing a new proteintyrosine phosphatase using this assay it is important to establish that the trichloroacetic acid-soluble counts represent inorganic phosphate rather than small phosphopeptides that may also be trichloroacetic acid soluble. This can readily be done by treating the TCA supernatant with molybdate and extracting the resulting inorganic phosphate-molybdate complexes into organic solvent. 23 Phosphate esters or anhydrides do not form complexes with molybdate.
Protein-Tyrosine Phosphatase Inhibitor Assay Principle. Inhibitor H and inhibitor L are assayed based on their ability to inhibit the activity of PTP5. Reagents PTP5: The enzyme is partially or highly purified from bovine brain as described below and diluted to a concentration of 10 mU/ml in assay buffer Other reagents: The preparation of these are described above (see Protein-Tyrosine Phosphatase Assay)
Procedure Fractions containing inhibitor activity are incubated at 95 ° for 5 min to inactivate any endogenous protein-tyrosine phosphatase activity. The mixture is cooled on ice, centrifuged for 2 min at 15,000 g to remove denatured protein, and diluted in Tris-Brij buffer. Then, mix 20 /~l of protein phosphatase diluted in assay buffer with 20/zl of diluted inhibitor H or inhibitor L, preincubate for 5 min at 30°, and initiate the proteintyrosine phosphatase assay by adding 20/A of [32p]casein to the reaction mixture. After 10 min the reaction is terminated and further processed as described above (see Protein-Tyrosine Phosphatase Assay). It is important to use high-quality BSA (Boehringer Mannheim, ultrapure grade) in the assay buffers since fraction V BSA contains contaminants that interfere with the inhibition of PTP5 by the two inhibitor proteins. Inhibitor activity is quantitated based on the concentration-dependent decrease in PTP5 compared to a control incubation lacking inhibitor (Fig.
23 S. Shenolikar and T. S. Ingebritsen, this series, Vol. 107, p. 102.
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PROTEIN PHOSPHATASES
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I00
80
J
80
i 4° 2O
O
0
i
i
i
I
I
I
2
3
4
5
6
Inhibitor Activity (Units/assay) FIG. 1. Standard curve for the inhibition of PTP5. The standard inhibitor assay was used and the amounts of inhibitor (DEAE-cellulose fraction) added are indicated. See text for definition of units. (Reprinted with permission from Ingebritsen. 19)
I). Concentrations producing up to 50-60% PTP5 inhibition can be estimated with little loss of accuracy. The sensitivity of PTP5 to inhibition by inhibitor H and inhibitor L decreases as the concentration of PTP5 in the assay increases) 9 The standard assay contains 0.2 mU PTP5, which is equivalent to a PTP5 concentration of 20 pM based on a molecular weight of 46,000, and Km and Vmaxvalues of 130 nM and 10,000 U/mg, respectively. This concentration of PTP5 gives near-maximal sensitivity of the enzyme to the two inhibitor proteins. One unit of inhibitor is the amount that decreases the activity of 0.2 mU PTP5 by 50% under the standard assay conditions. 24
Phosphorylation of Casein by Insulin Receptor Kinase Principle. [32p]Casein, phosphorylated exclusively on tyrosine residues to a stoichiometry of 1-2 nmol/mg, is prepared by incubation with the insulin receptor kinase and [y-32p]ATP. The definition of inhibitor units given here is equivalent to that given in Ref. 19. The apparent difference in the amount of PTP5 in the standard assay reflects a PTP5 activity unit definition based on a substrate concentration of 100 nM vs 1 nM in Ref. 19. The actual PTP5 concentation (pM) is approximately the same as in Ref. 19.
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Reagents
Buffer A: 50 mM HEPES, pH 7.4-0.1% (v/v) Triton X-100-10 mM MgCI2 Buffer B: 166.7 mM HEPES, pH 7.4-16.7 mM MgC12-1.7 mM MNC12-3.3 mM EDTA Casein: This is a mixture of 70% ~s~- + ~s2-Caseins and 30%/3- + Kcaseins. It is purchased from Sigma (Cat. #C-7891; St. Louis, MO) Insulin receptor kinase: The enzyme is partially purified from NIH 3T3 HIR3.5 cells as described by Treadwell et al. 25 and stored at - 8 0 ° in 50 mM Tris-HCl (pH 8.0 at 25°)-0.3 M N-acetylglucosamine-0.05% (v/v) Triton X-100-10% (v/v) glycerol at a concentration of 0.4-0.8 U/ml. Insulin receptor kinase activity is assayed essentially as described by Tonks et al. 15 using poly(GluS°,Tyr 2°) as substrate (2 mg/ ml) and 0.1 mM [y-3Zp]ATP (1000 cpm/pmol). One unit of insulin receptor kinase is the amount that incorporates 1 nmol of 32p into poly(Glu8°,Tyr 2°) in 1 min under the standard assay conditions [y-3Zp]ATP (5 mM, 10,000-20,000 cpm/pmol) in H20 Phosphorylation Procedure
Mix 320/zl of buffer A containing 12.5 mg/ml casein and 7/zg/ml insulin with 240 tzl buffer B plus 80 tzl insulin receptor. Preincubate the mixture for 15 min at 30°. Add 160/xl [-y-32p]ATP and incubate overnight at 30°. Remove a 5-tzl aliquot and process to determine phosphate incorporation (see below). Terminate incubation by adding 160/zl of 100% TCA. Hold on ice for 60 min and centrifuge for 2 min at 12,000 g in a microfuge. Wash the pellet six times with 0.5 ml ice-cold 20% TCA (centrifuge 1 min after each wash). Add 0.5 ml of 0.5 M Tris, pH 8.5 to the washed pellet and allow the pellet to redissolve overnight in the refrigerator. Gel filter the solution on a Sephadex G-50 column (0.7 x 17 cm) equilibrated with 50 mM Tris (pH 7.0 at 25°)-0.05 mM EDTA. Pool the 32p-labeled protein peak, divide into aliquots containing 300 pmol of [32p]casein, and store at - 2 0 °. Measurement o f Phosphate Incorporation into Casein
Dilute 5/zl of the reaction mixture to 100/xl with H20. Transfer four 5-/xl aliquots of the diluted reaction mixture to 2 x 2 cm squares of Whatman (Clifton, NJ) P-81 phosphocellulose paper. Wash the filter papers three times with 200 ml of 0.5% (w/v) H3PO4 (5 min for each wash). Wash the filter papers once with 200 ml of ethanol, dry, and count in scintillant. 25 j. L. Treadwell, J. Whittaker, and J. E. Pessin, J. Biol. Chem. 264, 15136 (1989).
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Extract Chjrtosol DEAE-CeHulose I 0 - 9 0 m M NaCI
90-500mMNaCI (PTP6, PrP2A)
(PTPIA, PTPIB, PTP2, PTP3, PTP4, PTP5) Phosphocellulose I Breakthrough
tPTPI~ PTPaBI
SephaerylS-300 [
Gradient
~
~
PTP2
PTP3
PTP6
PrP2A
~ PTP4
PTP5
SephacrylS-300 I
l~rPIA
PTP1B
FIG. 2. Outline of the procedure for the separation of the seven bovine brain proteintyrosine phosphatases (PTPs) from each other and from protein-Ser/Thr Phosphatase 2A (~P2A).
Protein-Tyrosine Phosphatase and Phosphatase Inhibitor Preparations
Separation of Bovine Brain Protein-Tyrosine Phosphatases Principle. Most of the protein-tyrosine phosphatase activity in bovine brain extracts (70%) is in the cytosolic fraction. Seven protein-tyrosine phosphatases (PTP1A, PTP1B, PTP2, PTP3, PTP4, PTP5, and PTP6) account for 90-95% of the cytosolic activity. These activities are resolved by successive chromatographies on DEAE-cellulose, phosphocellulose, and Sephacryl S-300. The remaining 5-10% of the activity is due to proteinSer/Thr phosphatase 2A. The separation protocol is shown in schematic form in Fig. 2. Reagents Homogenization solution: 4.0 mM EDTA, pH 7.0-250 mM sucrose0.1 mM phenylmethylsulfonyl fluoride (PMSF)-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol
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459
Buffer C: 20 mM Tris-HC1 (pH 7.0 at 25°)-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol Buffer D: 50 mM Tris-HCl (pH 7.0 at 25°)-50 mM NaC1-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.01% (w/v) Brij 35-0.2% (v/v) 2-mercaptoethanol Buffer E: 50 mM Tris-HC1 (pH 7.0 at 25°)-0.1 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-50% (v/v) glycerol-0.2% (v/v) 2-mercaptoethanol Procedure. The entire procedure is performed at 4° . Two fresh bovine brains are deveined, passed through a meat grinder, and homogenized in a Waring blender (2 min at low speed) in 2 vol of homogenization solution. The homogenate is centrifuged at 12,000 g for 20 min and the resulting supernatant (extract) is further centrifuged at 100,000 g for 60 min. DEAE-Cellulose Chromatography Step. The supernatant (cytosol) is applied to a DEAE-cellulose column (7.5 × 14 cm) equilibrated with buffer C at a flow rate of 300-400 ml/hr. The column is washed with buffer C containing 90 mM NaC1 until the A280 of the effluent is PTP4 > PTP3 ~ PTP2 > P T P I > PTP6.19 PTP5 is 2- and 10-fold m o r e sensitive to inhibition b y inhibitor L and inhibitor H, respectively, than PTP4 and
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---50- to lO0-fold more sensitive than the other five protein-tyrosine phosphatases. PTP5 is potently inhibited by three classes of macromolecular polyanions: polynucleotides (e.g., DNA, RNA, and synthetic oligonucleotides), acidic polyamino acids [e.g., poly(GluS°,Tyr2°), poly(Glu)], and the acidic polysaccharide heparin.7 The IC50 values for inhibition of PTP5 by heparin, poly(Glu8°,Tyr2°), and poly(G,I) are -0.2/zg/ml. The three types of polyanions inhibit the bovine brain protein-tyrosine phosphatases with the following order of potencies: PTP5 > PTP4 ~ PTP3 > PTP2 ~ PTP1 > PTP6. The separation procedure can also be applied to other tissues. Using this procedure and the sensitivity to inhibition by inhibitor H, we have identified PTP4- and PTP5-1ike activities in seven rabbit tissues (brain, spleen, kidney, liver, heart, adipose tissue, and skeletal muscle) as well as several vertebrate cell types in culture (chicken embryo fibroblasts, a mouse T cell hybridoma, and a mouse B cell hybridoma). 7
Purification of PTP5 to Near Homogeneity Principle. Highly purified PTP5 is prepared from bovine brain using a seven-step procedure employing successive chromatographies of the cytosolic fraction on DEAE-cellulose, phosphocellulose, Affi-Gel Blue, heparin-agarose, Mono S, and TSK G3000 SW. Early steps in the procedure are similar to those used for the separation of the seven bovine brain PTPs; however, they have been shortened by using a batch adsorption procedure at the DEAE-cellulose step and step NaCI cuts at the phosphocellulose step. It is critical to maintain the protease inhibitor, PMSF, in all buffers used in the purification procedure to avoid limited proteolysis of the enzyme. Starting with 750 g of bovine brain, approximately 6/zg of protein is obtained after the final purification step with the M r 48,000 PTP5 peptide accounting for about 10-25% of the total protein. The overall yield based on total activity in the brain extracts is about 1.5% with a 13,000fold purification. Reagents Homogenization solution: See Separation of Bovine Brain Protein-Tyrosine Phosphatases Buffer F: 10 mM Tris-HCl (pH 7.0 at 25°)-0.1 mM EDTA-0.2 mM PMSF-0.2% (v/v) 2-mercaptoethanol Buffer G: 10 mM potassium phosphate, pH 7.0-0.1 mM EDTA-0.2 mM PMSF-0.2 (v/v) 2-mercaptoethanol Buffer H: 10 mM potassium phosphate, pH 7.0-0.1 mM EDTA-5 mM DTT-0.05% (w/v) Brij 35-10% (v/v) glycerol
462
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Buffer I: 50 mM potassium phosphate, pH 7.0-150 mM NaCI-0.1 mM EDTA-0.05% (w/v) Brij 35-10% (v/v) glycerol-0.2% (v/v) 2-mercaptoethanol PTP5 storage buffer: 50 mM Tris-HCl (pH 7.0 at 25°)-50 mM NaC1-0.1 mM EDTA-5 mM DTT-50% (v/v) glycerol Purification Procedure. All procedures are carried out at 4° unless otherwise specified. Brain (750 g) is homogenized (see above) in 1.5 liters of homogenization solution. The homogenate is centrifuged at 7700 g, the supernatant (extract) is decanted through glass wool, and the pH adjusted to 7.5 with NaOH. The extract is centrifuged at 53,000 g for 5.5 hr. DEAE-Cellulose Chromatography. The supernatant is added to 500 ml (packed volume) of DEAE-cellulose equilibrated with buffer F. The mixture is stirred for 30 min, allowed to settle for 20 min, and decanted through a sintered glass funnel. The DEAE-cellulose is washed in the funnel with buffer F until the volume of flow through plus wash totals about 2 liters. Solid ammonium sulfate (472 g/liter) is added to the combined flow through and wash fractions to give a 70% saturation solution and the mixture is stirred for 20 min. Precipitated proteins are collected by centrifugation at 7700 g for 20 min, dissolved in buffer F, dialyzed for 8 hr against 20 liters of buffer F with two changes of buffer, and fresh PMSF is added to give a final concentration of 1 mM. Phosphocellulose Chromatography. The resulting solution (DEAEcellulose fraction) is applied at a flow rate of 2.5 ml/min to a phosphocellulose column (50-ml bed volume) equilibrated in buffer F. The column is washed with 250 mM NaC1 in buffer F ( - 4 liters) at a flow rate of 3.6 ml/ min and PTP5 is eluted with 350 mM NaCI in buffer F at a flow rate of 0.8 ml/min. Fractions (15 ml) containing the activity peak are pooled. Affi-Gel Blue Chromatography. The pooled phosphocellulose fraction is applied at a flow rate of 1 ml/min to an Affi-Gel Blue column (10-ml bed volume) equilibrated with buffer F containing 350 mM NaC1. PTP5 is eluted from the column at a flow rate of 0.75 ml/min with a 500-ml linear gradient of 0.5-1.0 M NaC1 in buffer F (adjusted to pH 7.5 at 25°). Fractions (15 ml) containing the activity peak are pooled. Heparin-Agarose Chromatography. The pooled fractions from the Affi-Gel Blue chromatography step are dialyzed for 8 hr against 20 liters of buffer F with two changes of buffer and applied at a flow rate of 1 ml/ min to a heparin-agarose column (5-ml bed volume) equilibrated in buffer F. PTP5 is eluted at a flow rate of 0.5 ml/min with a 250-ml linear gradient of 0-1 M NaC1 in buffer G. Fractions (5 ml) containing the activity peak are pooled. Mono S Chromatography. The pooled fractions from the heparinagarose step are dialyzed overnight against 2 liters of buffer F and applied
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MULTIPLE PROTEIN-TYROSINE PHOSPHATASES
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to a Pharmacia (Piscataway, N J) Mono S cation-exchange column (1-ml bed volume) equilibrated with buffer H. PTP5 is eluted with a 28-ml gradient of 0-600 mM NaCI in buffer H using a Pharmacia FPLC (fast protein liquid chromatography) system (flow rate of 0.8 ml/min). Fractions (1 ml) containing the PTP5 activity peak are pooled. Gel-Permeation Chromatography. The pooled fractions from the Mono S chromatography step are dialyzed against 1 liter of buffer I for 4 hr and concentrated to about 0.5 ml by ultrafiltration using an Amicon (Danvers, MA) YM10 membrane. The resulting fraction is applied to a TSK G3000 SW column (0.75 × 60 cm) equilibrated with buffer I. The procedure is carried out at room temperature at a flow rate of 0.5 ml/min. Fractions (0.25 ml) are collected, immediately placed on ice, and assayed for proteintyrosine phosphatase activity. The peak of activity is pooled, dialyzed against PTP5 storage buffer, and stored at - 2 0 °.
Properties of Highly Purified PTP5 PTP5 is a monomeric protein with a rather broad pH optimum of 7.0. The enzyme dephosphorylates four of six substrates that have been tested to date, including casein, RCM lysozyme (phosphorylated by the insulin receptor kinase), pp60 v-~rc(Tyr-416 autophosphorylation site), and insulin receptor kinase (autophosphorylation sites). 7 In the latter case, the cloned cytoplasmic domain of the insulin receptor kinase expressed in insect cells using the baculovirus expression system 26 was used as substrate after autophosphorylation in vitro. Caseins phosphorylated by the insulin receptor kinase, pp60 v~rc, or pp60 c-~rc are equivalent substrates for PTP5. 27 Kinetic parameters (Kin, 100-130 nM; kcat, 6-8 sec-t) for dephosphorylation of casein and lysozyme are similar. At a fixed substrate concentration, pp60 ..... and insulin receptor kinase are dephosphorylated at 10% of the rate with casein or RCM lysozyme as substrates. Calpactin I (phosphorylated by pp60 v-~r~) and mixed histones [phosphorylated by epidermal growth factor (EGF) receptor kinase] are not dephosphorylated at significant rates, p-Nitrophenyl phosphate is also a substrate for PTP5, although the enzyme is not a major alkaline phosphatase activity in bovine brain) 8
Partial Purification of Protein-Tyrosine Phosphatase Inhibitors Principle. Inhibitor H and inhibitor L are partially purified by successive chromatographies of the bovine brain cytosolic fraction on DEAEcellulose and Sephacryl S-300. The DEAE-cellulose chromatography step 26 R. Herrera, D. Lebwohl, A. G. Herreros, R. G. Kallen, and O. M. Rosen, J. Biol. Chem. 263, 5560 (1988). 27 S. K. Jakes, K. L. Hipper., and T. S. Ingebritsen, unpublished observations.
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gives a 40-fold purification with a 300-400% yield and separates the two inhibitor proteins from the seven bovine brain PTP activities. The reason for the increase in activity at this step is not known. The Sephacryl S-300 step separates inhibitor H (Mr > 500,000) from inhibitor L (Mr 38,000).
Reagents Buffers C and D: See Separation of Bovine Brain Protein-Tyrosine Phosphatases Buffer J: 50 mM Tris-HCl (pH 7.0 at 25°)-0.05 mM EDTA-0.1 mM PMSF-0.1 mM benzamidine-0.2% (v/v) 2-mercaptoethanol Procedure. The starting point for the preparation is the unboiled 12,000 g supernatant (extract) obtained from a single bovine brain (see Separation of Bovine Brain Protein-Tyrosine Phosphatases). The extract is centrifuged at 100,000 g for 60 min at 4 ° and the resulting supernatant (cytosol) is applied to a DEAE-cellulose (DE-52) column (5.5 x 16 cm) equilibrated with buffer C at a flow rate of 300 ml/hr. The column is washed with 400 ml buffer C and then further washed with buffer C containing 200 mM NaCI until the A280of the effluent is
