414
PROTEIN PHOSPHATASES
[36]
adding the inactive enzyme to the appropriate volume of dilution buffer, leads to precipitation: The method yields 0.1 mg of active PP1 from 10 ml of cultured S. frugiperda cells within a few hours, the cell culture and infection with recombinant virus requiring minimal man hours. The active, expressed enzyme and renatured enzyme are indistinguishable from PP1 isolated from rabbit skeletal muscle by a number of criteria. These include specific activity toward glycogen phosphorylase, and sensitivity to inhibition by okadaic acid and inhibitor 1 and 2. 5 The expression of PPI in this system will be useful for studies of structure/function relationships using sitedirected mutagenesis. Although the method can, in principle, be used to generate the large amounts of enzyme required for crystallization and analysis of the three-dimensional structure of PP1, the volumes involved in scaling up the procedure are still a problem. The procedure should also be useful for the expression of other related protein-serine/threonine phosphatases, such as PP2A, PP2B, and other enzymes of this gene family (e.g., PPX, PPY, and PPZ) that have been identified by cDNA cloning. H Expression of the latter enzymes will be critical to elucidate their substrate specificities and regulatory behavior. It p. T. W. Cohen, this volume [34].
[36] T a r g e t i n g S u b u n i t s for P r o t e i n P h o s p h a t a s e s
By MICHAEL J. HUBBARD and PHILIP COHEN Protein phosphatase 1 (PP1), one of the major protein-serine/threonine phosphatase catalytic subunits in eukaryotic cells, does not exist as a monomer in vivo, but as complexes with other proteins that target it to particular subcellular locations, modify its substrate specificity, and appear to be the key to its regulation (reviewed in Ref. I). The best characterized situation is in rabbit skeletal muscle, where PP1 is found associated with glycogen particles, the sarcoplasmic reticulum (SR), and the myofibrils, as well as in the cytosol. The glycogen-associated enzyme, termed PP1G, is extremely similar, if not identical, to the form that is associated with the SR. 2 PPI~ is a heterodimer composed of the catalytic C subunit and a G subunit, which is responsible for targeting the enzyme to both the J P. Cohen, Annu. Rev. Biochem. 58, 453 (1989). 2 M. J. Hubbard, P. Dent, C. Smythe, and P. Cohen, Eur. J. Biochem. 189, 243 (1990).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
PHOSPHATASE TARGETING SUBUNITS
415
glycogen particles 3-5 and the SR. 2 The G subunit is phosphorylated by cyclic AMP-dependent protein kinase (A-kinase) at two serine residues, site 1 and site 2, 6 and by an insulin-stimulated protein kinase (ISPK) at site 1. 7 Phosphorylation of site 1 increases the rate at which PP1 o dephosphorylates (activates) glycogen synthase and dephosphorylates (inactivates) phosphorylase kinase and appears to underlie the effects of insulin on glycogen metabolism. 7 In contrast, phosphorylation of site 2, which is located 19 residues C terminal to site 1,6 causes dissociation of the C subunit from the G subunit, resulting in translocation of the former from glycogen particles 6 and SR 2to the cytosol. Conversely, dephosphorylation of site 2 permits rebinding of the C subunit to the G subunit. 6 At physiological ionic strength, in the presence of glycogen, and under conditions where both the phosphatase and its substrates are largely bound to glycogen, PP1 C is at least five- to eightfold more active in dephosphorylating glycogen phosphorylase and glycogen synthase than the released C subunit.8 In contrast, PP 1Gand the released C subunit are equally effective in dephosphorylating substrates that do not bind to glycogen, such as myosin. 8 These observations, together with in vivo studies, 9-11 indicate that phosphorylation of the G subunit at site 2 represents a mechanism by which adrenalin (acting via cyclic AMP) overides the effect of insulin and prevents PP1 from dephosphorylating glycogen-bound substrates. The phosphorylation of site 1 by A-kinase may be a device for accelerating the rate of reactivation of glycogen synthesis after adrenergic stimulation has terminated and site 2 has been dephosphorylated. 7 Two further serines, four and eight residues N terminal to site l, are phosphorylated by glycogen synthase kinase-3, but only after prior phosphorylation of site 1 by A-kinase) z'13 The role of these phosphorylations is unknown. In view of the multiple phosphorylation sites and role of A-kinase and ISPK phosphorylation, this important region of the G subunit is termed the phosphoregulatory domain. 6 3 p. Stralfors, A. Hiraga, and P. Cohen, Eur. J. Biochem. 149, 295 (1985). 4 A. Hiraga, B. E. Kemp, and P. Cohen, Eur. J. Biochem. 163, 253 (1987). 5 M. J. Hubbard and P. Cohen, Eur. J. Biochem. 180, 457 (1989). 6 M. J. Hubbard and P. Cohen, Eur. J. Biochem. 186, 701 (1989). 7 p. Dent, A. Lavoinne, S. Nakielny, F. B. Caudwell, P. Watt, and P. Cohen, Nature (London) 348, 302 (1990). s M. J. Hubbard and P. Cohen, Eur. J. Biochem. 186, 711 (1989). 9 A. Hiraga and P. Cohen, Eur. J. Biochem. 161, 763 (1986). 10 C. MacKintosh, D. G. Campbell, A. Hiraga, and P. Cohen, FEBS Lett. 2,34, 189 (1988). IIp. Dent, D. G. Campbell, F. B. Caudwell, and P. Cohen, FEBS Lett. 259, 281 (1990). 12 p. Dent, D. G. Campbell, M. J. Hubbard, and P. Cohen, FEBS Lett. 248, 67 (1989). J3 C. J. Fiol, J. H. Haseman, Y. Wang, P. J. Roach, R. W. Roeske, M. Kawalczuk, and A. A. DePaoli-Roach, Arch. Biochem. Biophys. 267, 797 (1988).
416
PROTEIN PHOSPHATASES
[36]
The form of PP 1 as sociated with myofibrils, termed PP 1M, is composed of the C subunit complexed to a protein, distinct from the G subunit, which appears to enhance the ability of PP1M to dephosphorylate myosin. 14'15 The major form of PP1 in the cytosol is inactive and therefore termed PPll. It is a heterodimer composed of the C subunit and a thermostable protein, inhibitor 2, and can be activated in vitro through a mechanism that involves the reversible phosphorylation of inhibitor-2.1 Here, we detail methods that have been used to characterize the structure, localization, and regulation of PP1 G, several of which may be applicable to the analysis of other targeting subunits of PP 1, as they are identified and purified.
Buffer Solutions Buffer A: 50 mM Tris-HCl, pH 7.5 (22°), 0.1 mM EDTA, 0.1 mM EGTA, 10% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 4/~g ml-1 leupeptin, 0.1 mg ml-1 tosylphenyl chloromethyl ketone (TPCK), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) Buffer B: 25 mM Tris-HC1, 25 mM bis-Tris-HCl, pH 7.4 (4°), 0.2 mM EGTA, 1 mM benzamidine, 2 mg ml-1 bovine serum albumin, with freshly added I mM dithiothreitol Buffer C: 50 mM Tris-HC1, pH 7.5 (22°), 5% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 0.05% (v/v) Brij 35, and the proteinase inhibitors of buffer A Buffer D: 50 mM Tris-HC1, pH 7.5 (22°), 5% (v/v) glycerol, 0.1% 2mercaptoethanol, 0.1 mM EDTA, 0. I mM EGTA, ! mM benzamidine, 0.01% (w/v) Triton X-100 Buffer E: 50 mM Tris-HC1, pH 7.0, 0.1 mM EGTA, 0.1% (v/v) 2mercaptoethanol, 1 mg ml- 1 ovalbumin, and the proteinase inhibitors present in buffer A Buffer F: 10 mM Tris, 75 mM glycine, pH 8.3
Purification of Protein Phosphatase 1o The purification of glycogen-associated PP1G was described earlier in and only minor modifications have been introduced subsequently. 5'6 These procedures result in purification to near homogeneity of PP1c, in which the native 161-kDa G subunit is partially proteolyzed to t h i s s e r i e s 16
14 A. A. K. Chisholm and P. Cohen, Biochim. Biophys. Acta 968, 392 (1988). 15 A. A. K. Chisholm and P. Cohen, Biochirn. Biophys. Acta 971, 163 (1988). 16 p. Cohen, S. Alemany, B. A. Hemmings, T. J. Resink, P. Stralfors, and H. Y. L. Tung, this series, Vol. 159, p. 390.
[36]
PHOSPHATASE TARGETING SUBUNITS
417
the 103-kDa G' fragment (this occurs principally at the chromatographic step on DEAE-celluloseS). Both the G' subunit and activity of purified PP1G are stable at - 8 0 ° in solutions containing 10% (v/v) glycerol for > 1 year. The same procedure has also been used to purify PP1G from the SR following its extraction from the myofibrillar pellet with Triton X-100. 2 For studies of the in vivo phosphorylation state of PP1G , the phosphatase inhibitors NaF and inorganic pyrophosphate are included. ~°'N A more rapid procedure for isolating glycogen-associated PP1G described below takes only 26 hr and yields nearly homogenous enzyme in which some intact (161 kDa) G subunit is still present. 5 1. Isolate glycogen-protein particles from the skeletal muscle of three New Zealand White rabbits as described 16 and resuspend them in 210 ml of 50 mM Tris-HC1, pH 7.5, 0.1 mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 8/zg ml- 1leupeptin, 0.1 mg mlTPCK, 1 mM PMSF. 2. Add a-amylase (5/xg ml-1) to degrade the glycogen. Incubate the suspension for 45 min at 30° (fresh 0.1 mM PMSF being added at 20 rain), chill on ice, and add EGTA (0.1 mM) and fresh PMSF. 3. Centrifuge the suspension and chromatograph the supernatant, containing 75% of the PP1G activity, on a 60-ml column of DEAE-cellulose as described, 16 but with 0.1 mM EDTA and 0.1 mM EGTA in the solutions. 4. Apply the 0.15 M Tris-HCl eluate directly to an 8-ml column of poly(L-lysine)-Sepharose, prepared as described. 16Wash with buffer A + 0.2 M NaCI, and elute PP1G with buffer A + 0,5 M NaCI. 5. Dilute the active fractions with an equal volume of buffer A and apply to a 3-ml column of aminohexyl-Sepharose. Elute with a 36-ml linear gradient of 0.25-1.0 M NaCI in buffer A. 6. Concentrate the active fractions by ultrafiltration (YM30 membrane; Amicon, Danvers, MA), dilute with buffer A to 40 kDa) are recognized as well as the intact G subunit. The sensitivity of detection during immunoblotting is severalfold less than with the antibody preparations described below, consistent with low epitope density resulting from the use of a peptide 'immunogen. In addition, the anti-site 1 antibodies do not recognize the phosphorylated G-subunit nearly as well as the dephosphorylated protein. Preparation of Anti-Native G Subunit Antibodies 2°
1. To hyperimmunize an adult female sheep, absorb 0.16 mg homogeneous PPlc to a piece (3 × 1 cm) of nitrocellulose membrane filter (e.g., BA85; Schleicher and Schull), then quench the filter by incubating with 10 mg ml-1 keyhole limpet hemocyanin for 2 hr at room temperature. Rinse the filter in phosphate-buffered saline and implant it in the peritoneal cavity through a small ( - 1 cm) stab incision in the shaved right side. Close the wound with intramuscular and dermal sutures and spray with plastic skin. The operation is done under extensive local analgesia. 2. Emulsify 0.4 mg homogeneous PP1G in 2 ml Freund's complete 2o M. J. Hubbard, unpublished observations (1990).
[36]
PHOSPHATASE TARGETING SUBUNITS
425
adjuvant and inject at multiple sites in the neck. Give three boost injections of 0.16 mg PP1G in 1 ml Freund's incomplete adjuvant every 2 weeks. 3. After 7 weeks (from the time of implantation), test bleed the animal and verify the presence of immunoreactivity. Obtain the antiserum, isolate the ,/-globulin fraction by (NH4)2SO 4 fractionation, dialyze against Trisbuffered saline, and store at - 2 0 °. Anti-PP1G antibodies obtained by this procedure react strongly with PP1G on dot blots and with the G subunit on immunoblots. Preparation o f Anti-Denatured G' Subunit Antibodies l°
1. Denature 2 mg PP1G by boiling for 2 min in solubilization buffer containing 2% (w/v) SDS, then subject to preparative scale SDS-polyacrylamide gel electrophoresis. 2. Lightly stain the gel with aqueous 0.1% (w/v) Coomassie Blue, locate and excise the band corresponding to the major 103-kDa G' fragment of the G subunit, then lyophilize and powder it. 3. To hyperimmunize a sheep, emulsify the powdered gel with Freund's incomplete adjuvant and inject one-third of the material at multiple subcutaneous sites. Repeat at 2 to 3 week intervals. After 9 weeks, collect the antiserum and store at - 2 0 ° . Anti-G' subunit antibodies obtained by this procedure reacted strongly with G subunit and proteolytic fragments (---30 kDa) on immunoblots (the phospho and dephospho forms stain equally well), 6 and with PP1G on dot blots, 2'8'1° but do not immunoprecipitate significant amounts of PPI~. Immunoprecipitation 4,2°
1. Dilute P P l c , or an unknown phosphatase, to - I mU ml -~ and antibodies (anti-site 1, or anti-native G subunit) to 0.2 mg ml-~ in buffer E. An equivalent amount of nonimmune IgG is used routinely as a control. For anti-site 1 antibodies, specificity is illustrated by preincubating the antibodies with excess synthetic site 1 peptide to prevent immunoprecipitation.4 2. Mix I0/xl of diluted phosphatase with 10/zl antibody or control IgG and incubate for 10 min at room temperature or 30 min on ice. 3. For rabbit antibodies, precipitate immune complexes by adding 5/zl of 10% (v/v) heat-treated, formalinized Staphylococcus aureus suspension (e.g., Pansorbin; Calbiochem, San Diego, CA), and after 3 min at room temperature or 10 min on ice centrifuge for 2 min at 14,000 g. For sheep antibodies, use Pansorbin pretreated with an excess of rabbit anti-sheep IgG, and double the incubation time. In both cases, wash Pansorbin imme-
426
PROTEIN PHOSPHATASES
[36]
diately before use by centrifuging and resuspending three times in buffer E. 4. Assess specific immunoprecipitation by measuring PP1 activity in the supernatant of test control samples, using the standard phosphatase assay. Immunoprecipitation of PP1 c by antibodies to the G subunit was used to establish that the dephosphorylated G subunit is tightly associated with the C subunit 4 and showed that extensive proteolytic fragmentation of the G subunit did not destroy the site(s) of interaction with the C subunit. 4'5 Immunoprecipitation experiments also indicated that dissociation of PP1 from glycogen at high dilution results from release of the PPI~ holoenzyme and not dissociation of the C subunit from the G subunit. 5 Immunoprecipitation of SR-associated PP1 was one of the experiments which indicated that this enzyme was very similar or identical to PP1G ,2 while the failure to immunoprecipitate myofibrillar PP1 suggested that PPI Mwas a distinct species. 14
Immunoblotting the G Subuni: 1. Soak SDS-polyacrylamide gel in electrotransfer buffer F for 30 min at room temperature. 2. Prepare the usual sandwich of gel with 0.45-/.~m nominal pore size nitrocellulose and subject to transverse electrophoresis in a Transblot apparatus (Bio-Rad) configured for high field intensity (4 cm between electrode plates). Transfer for at least 200 V hr cm-~ with stirred water cooling. 3. For immunodetection, quench the nitrocellulose with albumin or skim milk proteins and expose to primary antibodies and an amplified chromogenic detection system in the normal w a y ) The 161-kDa G subunit is slow to move from the gel, being only partially transferred under conditions that give quantitative transfer of the 205kDa myosin heavy chain) The above procedure uses half the normal electrotransfer buffer concentration, and with methanol omitted to reduce heating and facilitate transfer of high molecular weight species. To avoid distortion artifacts, it is essential to equilibrate the gel thoroughly in electrotransfer buffer prior to transfer. Many low-molecular-weight species are poorly retained on the nitrocellulose under these high field transfer conditions) Quantitative comparisons between different molecular weight species must therefore be approached with caution. Immunoblotting experiments are used to demonstrate the intact form (161 kDa) and degradation of the G subunit, 5 to monitor association of G subunit with glycogen under various conditions, 5,6,~° and to identify SR-associated PP1G .2
[37]
PTPases FROMHUMANPLACENTA
427
Concluding Remarks The procedures described here have led to increased understanding of the structure, localization, and regulation of glycogen-associated PPlc. The observed effects of PP1 c activity of ionic strength, pH, G - C subunit interaction, and binding of glycogen to PPI C and substrates emphasizes the importance of using near-physiological conditions for assessment of phosphatase function. Together with associated studies of myofibrillar and SR-associated PP1, this work has led to the concept of PP1 regulation in skeletal muscle by targeting subunits. It remains to be seen whether the control mechanisms now being elucidated for PPlo are general ones, utilized by PP1 in other tissues and perhaps by other phosphatases. Acknowledgment This work was supported by a Programme Grant and Group Support from the Medical Research Council, London, the British Diabetic Association, and the Royal Society. Michael Hubbard was a Postdoctoral Fellow of the Juvenile Diabetes Foundation International. We thank our colleagues for useful suggestions during the development of several of the methods described here.
[37] P u r i f i c a t i o n o f 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 f r o m Human Placenta
By
NICHOLAS
K. TONKS, CURTIS D. DILTZ, and EDMOND H. FISCHER
There is now a substantial body of evidence implicating the phosphorylation of proteins on tyrosyl residues as an essential element in the control of normal and neoplastic cell growth) The phosphorylation state of a protein obviously reflects the relative activities of the kinase that phosphorylates it and the phosphatase that removes the phosphate. Thus, while considerable progress has been made in the characterization of the proteintyrosine kinases, such a focus of attention furnishes an incomplete picture of this dynamic and reversible process. Characterization of the proteintyrosine phosphatases (PTPases) provides a necessary complementary perspective from which to achieve an overall understanding of the control l y . Yarden and A. Ullrich, Annu. Reu. Biochem. 57, 443 (1988).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright 6) 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROTEIN PHOSPHATASES
[36]
adding the inactive enzyme to the appropriate volume of dilution buffer, leads to precipitation: The method yields 0.1 mg of active PP1 from 10 ml of cultured S. frugiperda cells within a few hours, the cell culture and infection with recombinant virus requiring minimal man hours. The active, expressed enzyme and renatured enzyme are indistinguishable from PP1 isolated from rabbit skeletal muscle by a number of criteria. These include specific activity toward glycogen phosphorylase, and sensitivity to inhibition by okadaic acid and inhibitor 1 and 2. 5 The expression of PPI in this system will be useful for studies of structure/function relationships using sitedirected mutagenesis. Although the method can, in principle, be used to generate the large amounts of enzyme required for crystallization and analysis of the three-dimensional structure of PP1, the volumes involved in scaling up the procedure are still a problem. The procedure should also be useful for the expression of other related protein-serine/threonine phosphatases, such as PP2A, PP2B, and other enzymes of this gene family (e.g., PPX, PPY, and PPZ) that have been identified by cDNA cloning. H Expression of the latter enzymes will be critical to elucidate their substrate specificities and regulatory behavior. It p. T. W. Cohen, this volume [34].
[36] T a r g e t i n g S u b u n i t s for P r o t e i n P h o s p h a t a s e s
By MICHAEL J. HUBBARD and PHILIP COHEN Protein phosphatase 1 (PP1), one of the major protein-serine/threonine phosphatase catalytic subunits in eukaryotic cells, does not exist as a monomer in vivo, but as complexes with other proteins that target it to particular subcellular locations, modify its substrate specificity, and appear to be the key to its regulation (reviewed in Ref. I). The best characterized situation is in rabbit skeletal muscle, where PP1 is found associated with glycogen particles, the sarcoplasmic reticulum (SR), and the myofibrils, as well as in the cytosol. The glycogen-associated enzyme, termed PP1G, is extremely similar, if not identical, to the form that is associated with the SR. 2 PPI~ is a heterodimer composed of the catalytic C subunit and a G subunit, which is responsible for targeting the enzyme to both the J P. Cohen, Annu. Rev. Biochem. 58, 453 (1989). 2 M. J. Hubbard, P. Dent, C. Smythe, and P. Cohen, Eur. J. Biochem. 189, 243 (1990).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
PHOSPHATASE TARGETING SUBUNITS
415
glycogen particles 3-5 and the SR. 2 The G subunit is phosphorylated by cyclic AMP-dependent protein kinase (A-kinase) at two serine residues, site 1 and site 2, 6 and by an insulin-stimulated protein kinase (ISPK) at site 1. 7 Phosphorylation of site 1 increases the rate at which PP1 o dephosphorylates (activates) glycogen synthase and dephosphorylates (inactivates) phosphorylase kinase and appears to underlie the effects of insulin on glycogen metabolism. 7 In contrast, phosphorylation of site 2, which is located 19 residues C terminal to site 1,6 causes dissociation of the C subunit from the G subunit, resulting in translocation of the former from glycogen particles 6 and SR 2to the cytosol. Conversely, dephosphorylation of site 2 permits rebinding of the C subunit to the G subunit. 6 At physiological ionic strength, in the presence of glycogen, and under conditions where both the phosphatase and its substrates are largely bound to glycogen, PP1 C is at least five- to eightfold more active in dephosphorylating glycogen phosphorylase and glycogen synthase than the released C subunit.8 In contrast, PP 1Gand the released C subunit are equally effective in dephosphorylating substrates that do not bind to glycogen, such as myosin. 8 These observations, together with in vivo studies, 9-11 indicate that phosphorylation of the G subunit at site 2 represents a mechanism by which adrenalin (acting via cyclic AMP) overides the effect of insulin and prevents PP1 from dephosphorylating glycogen-bound substrates. The phosphorylation of site 1 by A-kinase may be a device for accelerating the rate of reactivation of glycogen synthesis after adrenergic stimulation has terminated and site 2 has been dephosphorylated. 7 Two further serines, four and eight residues N terminal to site l, are phosphorylated by glycogen synthase kinase-3, but only after prior phosphorylation of site 1 by A-kinase) z'13 The role of these phosphorylations is unknown. In view of the multiple phosphorylation sites and role of A-kinase and ISPK phosphorylation, this important region of the G subunit is termed the phosphoregulatory domain. 6 3 p. Stralfors, A. Hiraga, and P. Cohen, Eur. J. Biochem. 149, 295 (1985). 4 A. Hiraga, B. E. Kemp, and P. Cohen, Eur. J. Biochem. 163, 253 (1987). 5 M. J. Hubbard and P. Cohen, Eur. J. Biochem. 180, 457 (1989). 6 M. J. Hubbard and P. Cohen, Eur. J. Biochem. 186, 701 (1989). 7 p. Dent, A. Lavoinne, S. Nakielny, F. B. Caudwell, P. Watt, and P. Cohen, Nature (London) 348, 302 (1990). s M. J. Hubbard and P. Cohen, Eur. J. Biochem. 186, 711 (1989). 9 A. Hiraga and P. Cohen, Eur. J. Biochem. 161, 763 (1986). 10 C. MacKintosh, D. G. Campbell, A. Hiraga, and P. Cohen, FEBS Lett. 2,34, 189 (1988). IIp. Dent, D. G. Campbell, F. B. Caudwell, and P. Cohen, FEBS Lett. 259, 281 (1990). 12 p. Dent, D. G. Campbell, M. J. Hubbard, and P. Cohen, FEBS Lett. 248, 67 (1989). J3 C. J. Fiol, J. H. Haseman, Y. Wang, P. J. Roach, R. W. Roeske, M. Kawalczuk, and A. A. DePaoli-Roach, Arch. Biochem. Biophys. 267, 797 (1988).
416
PROTEIN PHOSPHATASES
[36]
The form of PP 1 as sociated with myofibrils, termed PP 1M, is composed of the C subunit complexed to a protein, distinct from the G subunit, which appears to enhance the ability of PP1M to dephosphorylate myosin. 14'15 The major form of PP1 in the cytosol is inactive and therefore termed PPll. It is a heterodimer composed of the C subunit and a thermostable protein, inhibitor 2, and can be activated in vitro through a mechanism that involves the reversible phosphorylation of inhibitor-2.1 Here, we detail methods that have been used to characterize the structure, localization, and regulation of PP1 G, several of which may be applicable to the analysis of other targeting subunits of PP 1, as they are identified and purified.
Buffer Solutions Buffer A: 50 mM Tris-HCl, pH 7.5 (22°), 0.1 mM EDTA, 0.1 mM EGTA, 10% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 4/~g ml-1 leupeptin, 0.1 mg ml-1 tosylphenyl chloromethyl ketone (TPCK), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) Buffer B: 25 mM Tris-HC1, 25 mM bis-Tris-HCl, pH 7.4 (4°), 0.2 mM EGTA, 1 mM benzamidine, 2 mg ml-1 bovine serum albumin, with freshly added I mM dithiothreitol Buffer C: 50 mM Tris-HC1, pH 7.5 (22°), 5% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 0.05% (v/v) Brij 35, and the proteinase inhibitors of buffer A Buffer D: 50 mM Tris-HC1, pH 7.5 (22°), 5% (v/v) glycerol, 0.1% 2mercaptoethanol, 0.1 mM EDTA, 0. I mM EGTA, ! mM benzamidine, 0.01% (w/v) Triton X-100 Buffer E: 50 mM Tris-HC1, pH 7.0, 0.1 mM EGTA, 0.1% (v/v) 2mercaptoethanol, 1 mg ml- 1 ovalbumin, and the proteinase inhibitors present in buffer A Buffer F: 10 mM Tris, 75 mM glycine, pH 8.3
Purification of Protein Phosphatase 1o The purification of glycogen-associated PP1G was described earlier in and only minor modifications have been introduced subsequently. 5'6 These procedures result in purification to near homogeneity of PP1c, in which the native 161-kDa G subunit is partially proteolyzed to t h i s s e r i e s 16
14 A. A. K. Chisholm and P. Cohen, Biochim. Biophys. Acta 968, 392 (1988). 15 A. A. K. Chisholm and P. Cohen, Biochirn. Biophys. Acta 971, 163 (1988). 16 p. Cohen, S. Alemany, B. A. Hemmings, T. J. Resink, P. Stralfors, and H. Y. L. Tung, this series, Vol. 159, p. 390.
[36]
PHOSPHATASE TARGETING SUBUNITS
417
the 103-kDa G' fragment (this occurs principally at the chromatographic step on DEAE-celluloseS). Both the G' subunit and activity of purified PP1G are stable at - 8 0 ° in solutions containing 10% (v/v) glycerol for > 1 year. The same procedure has also been used to purify PP1G from the SR following its extraction from the myofibrillar pellet with Triton X-100. 2 For studies of the in vivo phosphorylation state of PP1G , the phosphatase inhibitors NaF and inorganic pyrophosphate are included. ~°'N A more rapid procedure for isolating glycogen-associated PP1G described below takes only 26 hr and yields nearly homogenous enzyme in which some intact (161 kDa) G subunit is still present. 5 1. Isolate glycogen-protein particles from the skeletal muscle of three New Zealand White rabbits as described 16 and resuspend them in 210 ml of 50 mM Tris-HC1, pH 7.5, 0.1 mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 8/zg ml- 1leupeptin, 0.1 mg mlTPCK, 1 mM PMSF. 2. Add a-amylase (5/xg ml-1) to degrade the glycogen. Incubate the suspension for 45 min at 30° (fresh 0.1 mM PMSF being added at 20 rain), chill on ice, and add EGTA (0.1 mM) and fresh PMSF. 3. Centrifuge the suspension and chromatograph the supernatant, containing 75% of the PP1G activity, on a 60-ml column of DEAE-cellulose as described, 16 but with 0.1 mM EDTA and 0.1 mM EGTA in the solutions. 4. Apply the 0.15 M Tris-HCl eluate directly to an 8-ml column of poly(L-lysine)-Sepharose, prepared as described. 16Wash with buffer A + 0.2 M NaCI, and elute PP1G with buffer A + 0,5 M NaCI. 5. Dilute the active fractions with an equal volume of buffer A and apply to a 3-ml column of aminohexyl-Sepharose. Elute with a 36-ml linear gradient of 0.25-1.0 M NaCI in buffer A. 6. Concentrate the active fractions by ultrafiltration (YM30 membrane; Amicon, Danvers, MA), dilute with buffer A to 40 kDa) are recognized as well as the intact G subunit. The sensitivity of detection during immunoblotting is severalfold less than with the antibody preparations described below, consistent with low epitope density resulting from the use of a peptide 'immunogen. In addition, the anti-site 1 antibodies do not recognize the phosphorylated G-subunit nearly as well as the dephosphorylated protein. Preparation of Anti-Native G Subunit Antibodies 2°
1. To hyperimmunize an adult female sheep, absorb 0.16 mg homogeneous PPlc to a piece (3 × 1 cm) of nitrocellulose membrane filter (e.g., BA85; Schleicher and Schull), then quench the filter by incubating with 10 mg ml-1 keyhole limpet hemocyanin for 2 hr at room temperature. Rinse the filter in phosphate-buffered saline and implant it in the peritoneal cavity through a small ( - 1 cm) stab incision in the shaved right side. Close the wound with intramuscular and dermal sutures and spray with plastic skin. The operation is done under extensive local analgesia. 2. Emulsify 0.4 mg homogeneous PP1G in 2 ml Freund's complete 2o M. J. Hubbard, unpublished observations (1990).
[36]
PHOSPHATASE TARGETING SUBUNITS
425
adjuvant and inject at multiple sites in the neck. Give three boost injections of 0.16 mg PP1G in 1 ml Freund's incomplete adjuvant every 2 weeks. 3. After 7 weeks (from the time of implantation), test bleed the animal and verify the presence of immunoreactivity. Obtain the antiserum, isolate the ,/-globulin fraction by (NH4)2SO 4 fractionation, dialyze against Trisbuffered saline, and store at - 2 0 °. Anti-PP1G antibodies obtained by this procedure react strongly with PP1G on dot blots and with the G subunit on immunoblots. Preparation o f Anti-Denatured G' Subunit Antibodies l°
1. Denature 2 mg PP1G by boiling for 2 min in solubilization buffer containing 2% (w/v) SDS, then subject to preparative scale SDS-polyacrylamide gel electrophoresis. 2. Lightly stain the gel with aqueous 0.1% (w/v) Coomassie Blue, locate and excise the band corresponding to the major 103-kDa G' fragment of the G subunit, then lyophilize and powder it. 3. To hyperimmunize a sheep, emulsify the powdered gel with Freund's incomplete adjuvant and inject one-third of the material at multiple subcutaneous sites. Repeat at 2 to 3 week intervals. After 9 weeks, collect the antiserum and store at - 2 0 ° . Anti-G' subunit antibodies obtained by this procedure reacted strongly with G subunit and proteolytic fragments (---30 kDa) on immunoblots (the phospho and dephospho forms stain equally well), 6 and with PP1G on dot blots, 2'8'1° but do not immunoprecipitate significant amounts of PPI~. Immunoprecipitation 4,2°
1. Dilute P P l c , or an unknown phosphatase, to - I mU ml -~ and antibodies (anti-site 1, or anti-native G subunit) to 0.2 mg ml-~ in buffer E. An equivalent amount of nonimmune IgG is used routinely as a control. For anti-site 1 antibodies, specificity is illustrated by preincubating the antibodies with excess synthetic site 1 peptide to prevent immunoprecipitation.4 2. Mix I0/xl of diluted phosphatase with 10/zl antibody or control IgG and incubate for 10 min at room temperature or 30 min on ice. 3. For rabbit antibodies, precipitate immune complexes by adding 5/zl of 10% (v/v) heat-treated, formalinized Staphylococcus aureus suspension (e.g., Pansorbin; Calbiochem, San Diego, CA), and after 3 min at room temperature or 10 min on ice centrifuge for 2 min at 14,000 g. For sheep antibodies, use Pansorbin pretreated with an excess of rabbit anti-sheep IgG, and double the incubation time. In both cases, wash Pansorbin imme-
426
PROTEIN PHOSPHATASES
[36]
diately before use by centrifuging and resuspending three times in buffer E. 4. Assess specific immunoprecipitation by measuring PP1 activity in the supernatant of test control samples, using the standard phosphatase assay. Immunoprecipitation of PP1 c by antibodies to the G subunit was used to establish that the dephosphorylated G subunit is tightly associated with the C subunit 4 and showed that extensive proteolytic fragmentation of the G subunit did not destroy the site(s) of interaction with the C subunit. 4'5 Immunoprecipitation experiments also indicated that dissociation of PP1 from glycogen at high dilution results from release of the PPI~ holoenzyme and not dissociation of the C subunit from the G subunit. 5 Immunoprecipitation of SR-associated PP1 was one of the experiments which indicated that this enzyme was very similar or identical to PP1G ,2 while the failure to immunoprecipitate myofibrillar PP1 suggested that PPI Mwas a distinct species. 14
Immunoblotting the G Subuni: 1. Soak SDS-polyacrylamide gel in electrotransfer buffer F for 30 min at room temperature. 2. Prepare the usual sandwich of gel with 0.45-/.~m nominal pore size nitrocellulose and subject to transverse electrophoresis in a Transblot apparatus (Bio-Rad) configured for high field intensity (4 cm between electrode plates). Transfer for at least 200 V hr cm-~ with stirred water cooling. 3. For immunodetection, quench the nitrocellulose with albumin or skim milk proteins and expose to primary antibodies and an amplified chromogenic detection system in the normal w a y ) The 161-kDa G subunit is slow to move from the gel, being only partially transferred under conditions that give quantitative transfer of the 205kDa myosin heavy chain) The above procedure uses half the normal electrotransfer buffer concentration, and with methanol omitted to reduce heating and facilitate transfer of high molecular weight species. To avoid distortion artifacts, it is essential to equilibrate the gel thoroughly in electrotransfer buffer prior to transfer. Many low-molecular-weight species are poorly retained on the nitrocellulose under these high field transfer conditions) Quantitative comparisons between different molecular weight species must therefore be approached with caution. Immunoblotting experiments are used to demonstrate the intact form (161 kDa) and degradation of the G subunit, 5 to monitor association of G subunit with glycogen under various conditions, 5,6,~° and to identify SR-associated PP1G .2
[37]
PTPases FROMHUMANPLACENTA
427
Concluding Remarks The procedures described here have led to increased understanding of the structure, localization, and regulation of glycogen-associated PPlc. The observed effects of PP1 c activity of ionic strength, pH, G - C subunit interaction, and binding of glycogen to PPI C and substrates emphasizes the importance of using near-physiological conditions for assessment of phosphatase function. Together with associated studies of myofibrillar and SR-associated PP1, this work has led to the concept of PP1 regulation in skeletal muscle by targeting subunits. It remains to be seen whether the control mechanisms now being elucidated for PPlo are general ones, utilized by PP1 in other tissues and perhaps by other phosphatases. Acknowledgment This work was supported by a Programme Grant and Group Support from the Medical Research Council, London, the British Diabetic Association, and the Royal Society. Michael Hubbard was a Postdoctoral Fellow of the Juvenile Diabetes Foundation International. We thank our colleagues for useful suggestions during the development of several of the methods described here.
[37] P u r i f i c a t i o n o f 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 f r o m Human Placenta
By
NICHOLAS
K. TONKS, CURTIS D. DILTZ, and EDMOND H. FISCHER
There is now a substantial body of evidence implicating the phosphorylation of proteins on tyrosyl residues as an essential element in the control of normal and neoplastic cell growth) The phosphorylation state of a protein obviously reflects the relative activities of the kinase that phosphorylates it and the phosphatase that removes the phosphate. Thus, while considerable progress has been made in the characterization of the proteintyrosine kinases, such a focus of attention furnishes an incomplete picture of this dynamic and reversible process. Characterization of the proteintyrosine phosphatases (PTPases) provides a necessary complementary perspective from which to achieve an overall understanding of the control l y . Yarden and A. Ullrich, Annu. Reu. Biochem. 57, 443 (1988).
METHODS IN ENZYMOLOGY, VOL. 201
Copyright 6) 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
