- Email: [email protected]
[38] Purification and characterization of protein phosphatase type 2C in photoreceptors
570
PHOTORECEPTOR PROTEIN PHOSPHATASES
[381
suitable basic species, the equilibrium is shifted from a closed structure, of the type shown in Fig. 5C, into an open conformation (Fig. 5B or 5D) in which the phosphate group is exposed for effective interaction with the phosphatase. The fact that the small phosphopeptides are efficiently hydrolyzed without requiring the presence of protamine is explained by invoking that with these substrates the ground state is populated by a large number of rapidly equilibrating structural forms from which the species containing unbridged structures are readily scavenged by the phosphatase. The hypothesis thus envisages that the stimulation of the activity of the two ROS phosphatases (catalytic subunit and holoenzyme) by basic amines may be a substrate-directed phenomenon as illustrated in the model of Fig. 5. For further comments on the model see Ref. 23. Acknowledgments This research was supported by the Wellcome Trust, Medical Research Council, and Ulverscroft Foundation.
[38] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f P r o t e i n Phosphatase
By S U S A N N E
Type 2C in Photoreceptors KLUMPP and DAGMAR SELKE
Introduction Increasing appreciation of the importance of protein phosphatases has generated considerable interest in methods for their separation, detection, and characterization. Phosphatases catalyzing the dephosphorylation of phosphoserine- and phosphothreonine-containing proteins are composed of two structurally distinct families (old nomenclature is given in parentheses)l'2: The PPP family, which includes PPP1 (PP1), PPP2 (PP2A), PPP3 (PP2B or calcineurin), etc.; and the PPM family of Mg2+- or Mn2+-dependent phosphatases including PPM1 (PP2C) enzymes. The presence of five distinct PP2C genes (c~,/3,y = FIN13, and ~-Wipl) has been reported in mammalian c e l l s . 3-7 PP2Ca and PP2C/3 are the major and best characterized isoforms 1 T. S. Ingebritsen and P. Cohen, Science 221, 331 (1983). 2 p. T. W. Cohen, Adv. Prot. Phosphatases 8, 371 (1994). 3 C. H. McGowan, D. G. Campbell, and P. Cohen, Biochim. Biophys. Acta 930, 279 (1987). 4 C. H. M c G o w a n and P. Cohen, Eur. J. Biochern. 166, 713 (1987). 5 S. M. Travis and M. J. Welsh, FEBS Lett. 412, 415 (1997).
METHODS IN ENZYMOLOGY,VOL. 315
Copyright © 2000 by AcademicPress All rights of reproduction in any form reserved. 0076-6879/00$30.00
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
571
with a molecular mass of 43-48 kDa (75% identity in primary structure). ~ Mouse PP2C/3 has been found to have five distinct isoforms, which are splicing variants originating from a single pre-mRNA. 9 The crystal structure of recombinant human 2Cot is known. 1° PP2C isoforms are ubiquitously expressed. The major differences of PP2C compared to the other Ser/Thr protein phosphatases include (1) strict requirement for Mg2+ or Mn 2+ ions for activityll; (2) inhibition by Ca 2+ ions12'13; (3) insensitivity toward okadaic acid, tautomycin, microcystin, etc., and insensitivity toward low molecular weight, acidic, and thermostable inhibitor proteins affecting PP1 and PP2A--thus, compounds to specifically enhance or inhibit PP2C activity are not available; and (4) monomeric structure: regulatory or targeting subunits referring localization and substrate specificity to the catalytic entity are unknown. With regard to regulation of PP2C activity, phosphorylation of the a isozyme by casein kinase II has been reported. 14'15 Both a and/3 isozymes were found to be sensitive to activation by unsaturated fatty acids. 16 Determination of Phosphatase Type 2C Activity PP2C activity is measured in the presence of magnesium ions using 32p-labeled casein as a substrate. Phosphorylation of bovine casein is performed slightly modifying the procedure of McGowan and CohenJ 7 Casein (10 mg) (Sigma, St. Louis, MO) and 2 mg cAMP-dependent protein kinase (Sigma) are incubated in the presence of 50/xM cAMP, I0 mM magnesium
6 M. A. Guthridge, P. Bellosta, N. Tavoloni, and C. Basilico, Mol. Cell, Biol. 17, 5485 (1997). 7 M. Fiscella, H. L. Zhang, S. Fan, K. Sakaguchi, S. Shen, W. E. Mercer, G. F. Van de Woude, P. M. O'Connor, and E. Appella, Proc. Natl. Acad. Sci. U.S.A. 94, 6048 (1997). D. J. Mann, D. G. Campbell, C. H. McGowan, and P. T. W. Cohen, Biochim. Biophys. Acta 1130, 100 (1992). S. Kato, T. Terasawa, T. Kobayashi, M. Ohnishi, Y. Sasahara, K. Kusuda, Y. Yanagawa, A. Hiraga, Y. Matsui, and S. Tamura, Arch. Biochem. Biophys. 318, 387 (1995). 10 A. K. Das, N. R. Helps, P. T. W. Cohen, and D. Barford, EMBO Z 15, 6798 (1996). 11M. D. Pato and R. S. Adelstein, J. Biol. Chem. 258, 7055 (1983). 12M. D. Pato and E. Kerc, Mol. Cell. Biochem. 101, 31 (1991). 13S. Klumpp, D. Selke, D. Fischer, A. Baumann, F. MUller, and S. Thanos, J. Neurosci. Res. 51, 328 (1998). t4 T. Kobayashi, S. Kanno, T. Terasawa, T. Murakami, M. Ohnishi, K. Ohtsuki, A. Hiraga, and S. Tamura, Biochem. Biophys. Res. Commun. 195, 484 (1993). t5 T. Kobayashi, K. Kusuda, M. Ohnishi, H. Wang, S. Ikeda, M. Hanada, Y. Yanagawa, and S. Tamura, FEBS Lett. 430, 222 (1998). 16 S. Klumpp, D. Selke, and J. Hermesmeier, FEBS Lett. 437, 229 (1998). 17 C. H. McGowan and P. Cohen, Methods Enzymol. 159, 416 (1988).
572
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
acetate, 200 ~ M adenosine triphosphate (ATP), and 37 MBq [y-3Zp]ATP (110 TBq/mmol). The phosphorylation reaction (1 ml) is incubated for 6-8 hr at 30° (incorporation rate 5-10%) and terminated by addition of 100/A containing 100 mM Na4P207, 100 mM EDTA, pH 7. The centrifugation supernatant (10,000g for 5 min) is applied to Sephadex G-50 superfine (1.2 × 26 cm; 18 ml/hr) equilibrated and run in 50 mM Tris-HC1, pH 7, 0.1 mM EDTA, and 0.1% (v/v) 2-mercaptoethanol to separate the labeled protein from unincorporated nucleotides. [32p]Casein fractions are pooled and stored at 4 ° (freeze-thawing would favor hydrolysis). Prior to being used as a substrate for PP2C activity, an aliquot of this stock solution is withdrawn and adjusted to 3 x 106 cpm/ml (equivalent to 3 nmol 32p/ml, Cerenkov radiation) by the addition of substrate buffer [50 mM Tris-HC1, pH 7, 0.1 mM EGTA, 1 mg/ml bovine serum albumin (BSA), 0.1% 2mercaptoethanol]. Then 30/zl 100 mg/ml BSA is added to 1 ml of this solution, to ensure that trichloroacetic acid (TCA) precipitation of the substrate remaining after phosphatase treatment is quantitative. PP2C assays are performed in 30-/xl reactions at 30 ° for 10 min (10/zl substrate to start the reaction, 10/zl enzyme diluted in substrate buffer, 5/~1 divalent cation solution, 5/M for other reagents). Final concentrations for a PP2C standard assay are 33 mM Tris-HC1, pH 7, 0.07% (v/v) 2-mercaptoethanol, 20 mM magnesium acetate, 1.3 mg/ml BSA, and 1 /xM [32p]casein with ~5 )< 104 cpm/assay tube (measured in the presence of 1 ml scintillant). Reactions are terminated by the addition of 200/.d 20% TCA and placed on ice for 5 min. After centrifugation at 10,000g (5 min), 200/zl of the supernatant is analyzed for 32p content. Radioactive Pi is converted and extracted as phosphomolybdic complex to discriminate phosphatase from unspecific protease activities. 18 Care must be taken to ensure that activity measurements are kept within the linear range of time and protein (maximally 25% release of Pi).
Separation of Retinal PP2C from Other Serine Threonine Protein Phosphatases To distinguish dephosphorylation of [3ap]casein carried by either PP2A or PP2C, assays need to be performed in duplicate, one in the absence and one in the presence of magnesium ions (e.g., 0.1 mM EDTA versus 20 mM Mg2+). Alternatively, 100 nM okadaic acid can be added to the Mg2+containing reactions. This tumor promotor completely blocks PP2A activity without affecting PP2C. One does not have to worry about eliminating PPI 18j. F. Antoniwand P. Cohen, Eur.
J. Biochem.
68, 45 (1976).
[381
PURIFICATION AND CHARACTERIZATION OF P P 2 C
573
activity when working with casein as a substrate for PP2C: [32p]casein is not dephosphorylated by PP1. Stepwise Purification by Column Chromatography Enzyme preparations are carded out at 4° with buffers containing 0.02% NaN3 and 0.1% (v/v) 2-raercaptoethanol. The isolation of PP2Ca and PP2C]3 as described here yields 2-6/~g PP2C proteins with a specific activity of 30-90 nraol Pi/min/mg. 13 Eighty bovine retinas are shaken for 90 sec in 80 ral sucrose medium [20 raM Tris-HC1, pH 7.5, 600 mM sucrose, 10 raM glucose, 0.2 raM EDTA, 0.1 raM phenylraethylsulfonyl fluoride (PMSF), 1 mM benzaraidine]. The soluble extract (48,000g, 30 min at 4°, followed by 100,000g, 1 hr) is applied to DEAE-Sephacel (2.5 X 10 cm; 80 ral/hr) equilibrated in buffer A (20 mM Tris-HCl, pH 7, 0.1 mM EDTA). The column is washed with buffer A containing 0.1 M NaC1 prior to elution of PP2C with buffer A containing 0.5 M NaC1. PP2C activity containing eluate is diluted 1 : 5 with buffer B (20 raM Tris-HC1, pH 7, 0.1 raM EDTA, 5% glycerol) and applied to heparin-Sepharose CL-6B equilibrated in buffer B (2.5 × 10 cra; 120 ral/hr). PP2C activity is recovered in the heparinSepharose flow-through and concentrated by ultrafiltration. PP2C is remarkably insensitive to short-terra exposure of 1 M NaCI (but not ammonium sulfate), thus enabling hydrophobic interaction chromatography. NaCI is added to yield a 1 M solution prior to application onto phenylSepharose CL-6B (1 × 2.6 cm) equilibrated in buffer C (20 ram Tris-HCl, pH 7, 1 raM EDTA) containing 1 M NaC1. PP2C activity is eluted with buffer C containing 20% glycerol (1 ml/rain) and concentrated on Mono Q HR5/5 run with buffer C containing 5% glycerol (0.5 ml/rain) using addition of 0.5 M NaCI for elution. Chromatography on Superdex 75 preparation grade 26/60 (1 ml/rain) is performed in buffer D (20 raM Tris-HC1, pH 7, 1 mM EDTA, 5% glycerol, 40 raM NaCI). MgCI2 (3 raM) is added to fractions containing PP2C activity. Isozyraes are separated on a 1-ral Blue-Sepharose column (Affi-Gel Blue in a HR 5/5 column or HiTrap) equilibrated in buffer D containing 3 raM MgC12 (0.75 ral/rain). A linear 60-ml gradient (2-0 ram Mg 2+) is required to separate PP2C~ (eluting at 1.5 raM Mg2+) from PP2Ca (0.5 ram Mg2+).19 Purification is summarized in Table I. Batch Procedure for Rapid Enrichment A simplified method has been developed to highly enrich PP2C proteins quickly, a6 One buffer is used throughout: 20 raM Tris-HC1, pH 7, l raM 19 S. Klumpp and D. Selke, Meth. M o t Biol. 93, 213 (1998).
574
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
TABLE I PURIFICATIONOF PP2C FROMBOVINE RETINAS
Fraction Supernatant DEAE-Sephace! Heparin-Sepharose Ultrafiltration Phenyl-Sepharose Mono Q Superdex 75 Blue-Sepharose Peak I (2C/3) Peak If (2Ca)
Volume (ml)
Total protein (mg)
Total activity (nmol/min)
170 625 482 44 31 4 15
654 284 135 124 34 11 0.39
73 74 40 20 18.5 12.0 1.5
13 10
0.006 0.002
0.17 0.18
Specific activity (nmol/min/mg)
Purification (-fold)
0.11 0.26 0.29 0.16 0.54 1.10 3.85 28 90
1 2.4 2.6 1.5 4.9 10 35
Recovery (%) 100 102 54 27 25 16 2.1
264 818
EDTA, 5% glycerol. The concentrations of NaCl for washing and elution are the same as those described for the column procedure. The beads (DEAE-Sephacel and heparin-Sepharose) are used with gentle rocking. Binding and elution times are 1 hr, respectively. Beads are pelleted by centrifugations using a swing-out rotor at 700g for 1 min at 4 °. The soluble retinal extract is applied to 50-ml DEAE-Sephacel. Heparin-Sepharose (10 ml) was sufficient for the next step. PP2C in the supernatant fraction is concentrated by Centriplus (Amicon, Danvers, MA) and further enriched using Mono Q H R 5/5 (0.7 M NaCl for elution). Gel filtration, as the final step, is performed as described earlier, yielding highly enriched PP2C free from other phosphatases. For assays dealing with the cation dependence and fatty acid stimulation of PP2C, EDTA in the ultimate column buffer was reduced to 0.1 raM. Purification of Recombinant PP2C Isozymes PP2Ca and PP2C/3 have been cloned from a bovine cDNA library. 2° They share high identity with PP2C isozymes from other mammalian species and tissues. For example, a comparison of PP2C from rat liver with the enzyme from bovine retina shows 5 and 26 substitutions in the amino acid sequence for a and/3, respectively. PP2C isozymes can be heterologously expressed. 13 His-tagged proteins are purified by affinity chromatography on Ni2+-NTA agarose. Enzyme activity of purified recombinant PP2C decreases on long-term storage (gone 20 D. Selke, S. Klumpp, B. Kaupp, and A. Baumann, Meth. Mol. Biol. 93, 243 (1997).
0.2 0.2
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
575
within 6 months at -80°). Enzyme activity in crude bacterial extracts, in contrast, is fairly stable (at least for 1 year). Therefore, a procedure has been developed to purify PP2C from small volumes: 300 txl lysate onto 50 /xl Ni2+-NTA agarose plugged into a Pasteur pipette, equilibrated in buffer E (20 mM Tris-HC1, pH 7.9, 5 mM imidazole, 0.5 M NaCI), and run by gravity. After washing in buffer E containing 60 mM imidazole, PP2C is eluted using buffer F (10 mM Tris-HCl, pH 7.9, 0.5 M imidazole, 0.25 M NaC1). Recovery of PP2C isozymes is 70%, respectively. A 20-ml culture of Escherichia coli normally yields 0.5 mg PP2C protein with a specific activity of 3-15 nmol Pi/min/mg. Expression of PP2Co~ routinely results in only about half of the amount of protein compared to PP2Cfl. Activity of both isozymes is strongly inhibited by imidazole (IC50 250 mM), therefore, immediate and at least fivefold dilution (substrate buffer) is recommended. General Comments on Purification and Analytics Separation of PP2C from PP1 is possible using chromatography on heparin-Sepharose: PP2C lacks affinity to heparin-Sepharose, thus, it can be quantitatively recovered in the flow-through fractions. Separation of PP2C from PP2A is achieved by gel filtration chromatography (Superdex 75 or Sephacryl S-200): the catalytic entities of PP2C ct and fl (~45 kDa) elute in clear distance to the high molecular weight PP2A holoenzymes. Separation of PP2C a and /3 isozymes can be accomplished by two distinct methods. Isozymes from rabbit liver are nicely separated on chromatography using ion-exchange resins. 4 This does not work for the retinal PP2C isozymes. Due to a few species- and tissue-specific amino acid substitutions, retinal PP2Ca and PP2Cfl--in contrast to PP2C isozymes from rabbit liver--end up with almost identical pI values. Separation of retinal PP2C isozymes is possible, however, on chromatography on Blue-Sepharosel9: both isozymes are retained by Cibachron Blue provided Mg 2+ ions are present. Decreasing the divalent cation concentration consequently elutes first fl, then a. In general, it is recommended that long-lasting dialysis steps be avoided. For short-term storage, e.g., overnight interruption during the purification process, freezing is best. Purified PP2C should be stored in aliquots at - 80°. Repetitive freeze-thawing of specifically recombinant PP2Ca should be avoided. Activity and protein "vanish." Adsorption to plastic vials might be the reason. This phenomenon is not observed with PP2C/3. Analyzing PP2C isozymes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bears two unexpected findings: (1) Unusual migration behavior: according to the amino acid composition, the molecular mass of PP2Ca is less compared to PP2C/3 (e.g., 382 versus 387 amino acids for
576
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
100" A
E =
mm
75'
E m
O
E
¢
50'
u s
>
mml
O
25'
"~'0"--0 jO/~'O/O O,
0--0,1
1
10
Mg 2+ (mMI FIG. 1. Mg 2÷ dependence of PP2C. Activity of recombinant PP2Ca was assayed under standard reaction conditions (©; 12.5 ng protein/tube) or in the presence of 0.5 mM oleic acid ( I ; 6.3 ng protein/tube). The shaded area marks the activation in the physiologic concentration range of free Mg z÷ (0.5-1.5 raM).
the enzymes from bovine retina). On denaturing electrophoresis, however, PP2Ca runs as a protein band above PP2Cfl. (2) For most proteins, silver staining is -10-fold more sensitive than Coomassie staining. Detection of PP2C isozymes by silver staining seems to be impaired. Characterization of Native and Recombinant PP2C Isozymes Mechanical disintegration of retinas (Dounce homogenizer) and subsequent centrifugation (100,000g for 60 miu) revealed that PP2C activity distributes equally well between the soluble and particulate fractions. Addition of detergents did not release further PP2C activity.
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
577
7
10' , A
"0 0
m
!
0 i
/
4'
E
i m
2'
~ 0
D/D
0
Fatty acid (mM) FIG. 2. Differential subcellular fatty acid composition affecting PP2C activity. Recombinant PP2Ca (6.3 ng/tube; specific activity 8 nmol Pi/min/mg) was assayed in the presence of 0.7 mM Mg2+ under otherwise standard reaction conditions. Fatty acids added were docosahexaenoic acid (0, 22:6) known to be concentrated in the photoreceptor outer segments, and palmitic acid (rT, 16:0) enriched in the neighboring pigment epithelium cells.
Except for the effect of fatty acids (vide infra), enzymatic properties of recombinant 2Co~ and 2C/3 are almost indistinguishable from one another and from their corresponding native counterparts. The optima for temperature and pH were 45°-55 ° (activation energy 70-90 kJ/mol) and pH 7.5-8 (Tris-HCl). A feature exhibited by all PP2C characterized to date is the requirement of unphysiologic high Mg 2+ concentrations for activity. A recent report describes that addition of unsaturated fatty acids results in high PP2C activity measurable at physiologic Mg 2+ concentrations. 16 Figure 1 shows the Mg 2+ dependence of PP2C in the absence versus presence of
578
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
oleic acid (18:1). Activity increases 10-fold and is highest at physiologic free Mg 2÷ levels (0.5-1.5 mM). Under those conditions, Ca 2÷ ions are inhibitory in the micromolar range. 16 The amount of fatty acids and the fatty acid composition are known to vary within cell compartments and from membrane to membrane. In vertebrate retinas, a striking concentration gradient exists between docosahexaenoic acid (22 : 6; major component in photoreceptor outer segments) and palmitic acid (16:0; found in the adjacent pigment epithelium cells) with important functional consequences. Supply of the photoreceptors with retinoids is based on the differential subcellular distribution of those longchain fatty acids: Docosahexaenoic acid--but not palmitic acid--induces release of 11-cis-retinal from a 140-kDa interphotoreceptor matrix retinoidbinding protein. 21 Within vertebrate retinas, PP2Ca and PP2Cfl isozymes are highly enriched in the photoreceptor outer segmentsJ 3 Figure 2 shows that docosahexaenoic acid--but not palmitic acid--increases the activity of recombinant PP2Ca up to 10-fold. The half-maximal activating concentration for docosahexaenoic acid was 350/zM. Stimulation of recombinant PP2Cfl and native PP2C was somewhat less. PP2C and docosahexaenoic acid are not only colocalized in rod outer segments, but are both associated with retinal degeneration processes. 13 Conclusions PP2C, a "black sheep" phosphatase (no activator, no inhibitor) had been considered to be 10-fold less compared to PP1 and PP2A. The rise in less-quantity amount of activity by fatty acids now brings PP2C onto stage as well. Fatty acids and Ca 2+ ions are established players in retinal signal transduction processes. We currently hypothesize regulation of PP2C at two levels: (1) Its basal activity is depending on the presence of unsaturated fatty acids. A long-term change in the fatty acid distribution and concentration as exemplified by apoptosis or retinitis pigmentosa will dramatically affect PP2C activity. (2) On top of that fatty acid sustained PP2C activity, shifts in the Ca 2+ ion concentration would account for short-term regulation of PP2C causing immediate changes in enzyme activity. Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (KI 601/8-1).
21 y. Chen, L. A. Houghton, J. T. Brenna, and N. Noy, J. BioL Chem. 271, 20507 (1996).
PHOTORECEPTOR PROTEIN PHOSPHATASES
[381
suitable basic species, the equilibrium is shifted from a closed structure, of the type shown in Fig. 5C, into an open conformation (Fig. 5B or 5D) in which the phosphate group is exposed for effective interaction with the phosphatase. The fact that the small phosphopeptides are efficiently hydrolyzed without requiring the presence of protamine is explained by invoking that with these substrates the ground state is populated by a large number of rapidly equilibrating structural forms from which the species containing unbridged structures are readily scavenged by the phosphatase. The hypothesis thus envisages that the stimulation of the activity of the two ROS phosphatases (catalytic subunit and holoenzyme) by basic amines may be a substrate-directed phenomenon as illustrated in the model of Fig. 5. For further comments on the model see Ref. 23. Acknowledgments This research was supported by the Wellcome Trust, Medical Research Council, and Ulverscroft Foundation.
[38] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f P r o t e i n Phosphatase
By S U S A N N E
Type 2C in Photoreceptors KLUMPP and DAGMAR SELKE
Introduction Increasing appreciation of the importance of protein phosphatases has generated considerable interest in methods for their separation, detection, and characterization. Phosphatases catalyzing the dephosphorylation of phosphoserine- and phosphothreonine-containing proteins are composed of two structurally distinct families (old nomenclature is given in parentheses)l'2: The PPP family, which includes PPP1 (PP1), PPP2 (PP2A), PPP3 (PP2B or calcineurin), etc.; and the PPM family of Mg2+- or Mn2+-dependent phosphatases including PPM1 (PP2C) enzymes. The presence of five distinct PP2C genes (c~,/3,y = FIN13, and ~-Wipl) has been reported in mammalian c e l l s . 3-7 PP2Ca and PP2C/3 are the major and best characterized isoforms 1 T. S. Ingebritsen and P. Cohen, Science 221, 331 (1983). 2 p. T. W. Cohen, Adv. Prot. Phosphatases 8, 371 (1994). 3 C. H. McGowan, D. G. Campbell, and P. Cohen, Biochim. Biophys. Acta 930, 279 (1987). 4 C. H. M c G o w a n and P. Cohen, Eur. J. Biochern. 166, 713 (1987). 5 S. M. Travis and M. J. Welsh, FEBS Lett. 412, 415 (1997).
METHODS IN ENZYMOLOGY,VOL. 315
Copyright © 2000 by AcademicPress All rights of reproduction in any form reserved. 0076-6879/00$30.00
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
571
with a molecular mass of 43-48 kDa (75% identity in primary structure). ~ Mouse PP2C/3 has been found to have five distinct isoforms, which are splicing variants originating from a single pre-mRNA. 9 The crystal structure of recombinant human 2Cot is known. 1° PP2C isoforms are ubiquitously expressed. The major differences of PP2C compared to the other Ser/Thr protein phosphatases include (1) strict requirement for Mg2+ or Mn 2+ ions for activityll; (2) inhibition by Ca 2+ ions12'13; (3) insensitivity toward okadaic acid, tautomycin, microcystin, etc., and insensitivity toward low molecular weight, acidic, and thermostable inhibitor proteins affecting PP1 and PP2A--thus, compounds to specifically enhance or inhibit PP2C activity are not available; and (4) monomeric structure: regulatory or targeting subunits referring localization and substrate specificity to the catalytic entity are unknown. With regard to regulation of PP2C activity, phosphorylation of the a isozyme by casein kinase II has been reported. 14'15 Both a and/3 isozymes were found to be sensitive to activation by unsaturated fatty acids. 16 Determination of Phosphatase Type 2C Activity PP2C activity is measured in the presence of magnesium ions using 32p-labeled casein as a substrate. Phosphorylation of bovine casein is performed slightly modifying the procedure of McGowan and CohenJ 7 Casein (10 mg) (Sigma, St. Louis, MO) and 2 mg cAMP-dependent protein kinase (Sigma) are incubated in the presence of 50/xM cAMP, I0 mM magnesium
6 M. A. Guthridge, P. Bellosta, N. Tavoloni, and C. Basilico, Mol. Cell, Biol. 17, 5485 (1997). 7 M. Fiscella, H. L. Zhang, S. Fan, K. Sakaguchi, S. Shen, W. E. Mercer, G. F. Van de Woude, P. M. O'Connor, and E. Appella, Proc. Natl. Acad. Sci. U.S.A. 94, 6048 (1997). D. J. Mann, D. G. Campbell, C. H. McGowan, and P. T. W. Cohen, Biochim. Biophys. Acta 1130, 100 (1992). S. Kato, T. Terasawa, T. Kobayashi, M. Ohnishi, Y. Sasahara, K. Kusuda, Y. Yanagawa, A. Hiraga, Y. Matsui, and S. Tamura, Arch. Biochem. Biophys. 318, 387 (1995). 10 A. K. Das, N. R. Helps, P. T. W. Cohen, and D. Barford, EMBO Z 15, 6798 (1996). 11M. D. Pato and R. S. Adelstein, J. Biol. Chem. 258, 7055 (1983). 12M. D. Pato and E. Kerc, Mol. Cell. Biochem. 101, 31 (1991). 13S. Klumpp, D. Selke, D. Fischer, A. Baumann, F. MUller, and S. Thanos, J. Neurosci. Res. 51, 328 (1998). t4 T. Kobayashi, S. Kanno, T. Terasawa, T. Murakami, M. Ohnishi, K. Ohtsuki, A. Hiraga, and S. Tamura, Biochem. Biophys. Res. Commun. 195, 484 (1993). t5 T. Kobayashi, K. Kusuda, M. Ohnishi, H. Wang, S. Ikeda, M. Hanada, Y. Yanagawa, and S. Tamura, FEBS Lett. 430, 222 (1998). 16 S. Klumpp, D. Selke, and J. Hermesmeier, FEBS Lett. 437, 229 (1998). 17 C. H. McGowan and P. Cohen, Methods Enzymol. 159, 416 (1988).
572
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
acetate, 200 ~ M adenosine triphosphate (ATP), and 37 MBq [y-3Zp]ATP (110 TBq/mmol). The phosphorylation reaction (1 ml) is incubated for 6-8 hr at 30° (incorporation rate 5-10%) and terminated by addition of 100/A containing 100 mM Na4P207, 100 mM EDTA, pH 7. The centrifugation supernatant (10,000g for 5 min) is applied to Sephadex G-50 superfine (1.2 × 26 cm; 18 ml/hr) equilibrated and run in 50 mM Tris-HC1, pH 7, 0.1 mM EDTA, and 0.1% (v/v) 2-mercaptoethanol to separate the labeled protein from unincorporated nucleotides. [32p]Casein fractions are pooled and stored at 4 ° (freeze-thawing would favor hydrolysis). Prior to being used as a substrate for PP2C activity, an aliquot of this stock solution is withdrawn and adjusted to 3 x 106 cpm/ml (equivalent to 3 nmol 32p/ml, Cerenkov radiation) by the addition of substrate buffer [50 mM Tris-HC1, pH 7, 0.1 mM EGTA, 1 mg/ml bovine serum albumin (BSA), 0.1% 2mercaptoethanol]. Then 30/zl 100 mg/ml BSA is added to 1 ml of this solution, to ensure that trichloroacetic acid (TCA) precipitation of the substrate remaining after phosphatase treatment is quantitative. PP2C assays are performed in 30-/xl reactions at 30 ° for 10 min (10/zl substrate to start the reaction, 10/zl enzyme diluted in substrate buffer, 5/~1 divalent cation solution, 5/M for other reagents). Final concentrations for a PP2C standard assay are 33 mM Tris-HC1, pH 7, 0.07% (v/v) 2-mercaptoethanol, 20 mM magnesium acetate, 1.3 mg/ml BSA, and 1 /xM [32p]casein with ~5 )< 104 cpm/assay tube (measured in the presence of 1 ml scintillant). Reactions are terminated by the addition of 200/.d 20% TCA and placed on ice for 5 min. After centrifugation at 10,000g (5 min), 200/zl of the supernatant is analyzed for 32p content. Radioactive Pi is converted and extracted as phosphomolybdic complex to discriminate phosphatase from unspecific protease activities. 18 Care must be taken to ensure that activity measurements are kept within the linear range of time and protein (maximally 25% release of Pi).
Separation of Retinal PP2C from Other Serine Threonine Protein Phosphatases To distinguish dephosphorylation of [3ap]casein carried by either PP2A or PP2C, assays need to be performed in duplicate, one in the absence and one in the presence of magnesium ions (e.g., 0.1 mM EDTA versus 20 mM Mg2+). Alternatively, 100 nM okadaic acid can be added to the Mg2+containing reactions. This tumor promotor completely blocks PP2A activity without affecting PP2C. One does not have to worry about eliminating PPI 18j. F. Antoniwand P. Cohen, Eur.
J. Biochem.
68, 45 (1976).
[381
PURIFICATION AND CHARACTERIZATION OF P P 2 C
573
activity when working with casein as a substrate for PP2C: [32p]casein is not dephosphorylated by PP1. Stepwise Purification by Column Chromatography Enzyme preparations are carded out at 4° with buffers containing 0.02% NaN3 and 0.1% (v/v) 2-raercaptoethanol. The isolation of PP2Ca and PP2C]3 as described here yields 2-6/~g PP2C proteins with a specific activity of 30-90 nraol Pi/min/mg. 13 Eighty bovine retinas are shaken for 90 sec in 80 ral sucrose medium [20 raM Tris-HC1, pH 7.5, 600 mM sucrose, 10 raM glucose, 0.2 raM EDTA, 0.1 raM phenylraethylsulfonyl fluoride (PMSF), 1 mM benzaraidine]. The soluble extract (48,000g, 30 min at 4°, followed by 100,000g, 1 hr) is applied to DEAE-Sephacel (2.5 X 10 cm; 80 ral/hr) equilibrated in buffer A (20 mM Tris-HCl, pH 7, 0.1 mM EDTA). The column is washed with buffer A containing 0.1 M NaC1 prior to elution of PP2C with buffer A containing 0.5 M NaC1. PP2C activity containing eluate is diluted 1 : 5 with buffer B (20 raM Tris-HC1, pH 7, 0.1 raM EDTA, 5% glycerol) and applied to heparin-Sepharose CL-6B equilibrated in buffer B (2.5 × 10 cra; 120 ral/hr). PP2C activity is recovered in the heparinSepharose flow-through and concentrated by ultrafiltration. PP2C is remarkably insensitive to short-terra exposure of 1 M NaCI (but not ammonium sulfate), thus enabling hydrophobic interaction chromatography. NaCI is added to yield a 1 M solution prior to application onto phenylSepharose CL-6B (1 × 2.6 cm) equilibrated in buffer C (20 ram Tris-HCl, pH 7, 1 raM EDTA) containing 1 M NaC1. PP2C activity is eluted with buffer C containing 20% glycerol (1 ml/rain) and concentrated on Mono Q HR5/5 run with buffer C containing 5% glycerol (0.5 ml/rain) using addition of 0.5 M NaCI for elution. Chromatography on Superdex 75 preparation grade 26/60 (1 ml/rain) is performed in buffer D (20 raM Tris-HC1, pH 7, 1 mM EDTA, 5% glycerol, 40 raM NaCI). MgCI2 (3 raM) is added to fractions containing PP2C activity. Isozyraes are separated on a 1-ral Blue-Sepharose column (Affi-Gel Blue in a HR 5/5 column or HiTrap) equilibrated in buffer D containing 3 raM MgC12 (0.75 ral/rain). A linear 60-ml gradient (2-0 ram Mg 2+) is required to separate PP2C~ (eluting at 1.5 raM Mg2+) from PP2Ca (0.5 ram Mg2+).19 Purification is summarized in Table I. Batch Procedure for Rapid Enrichment A simplified method has been developed to highly enrich PP2C proteins quickly, a6 One buffer is used throughout: 20 raM Tris-HC1, pH 7, l raM 19 S. Klumpp and D. Selke, Meth. M o t Biol. 93, 213 (1998).
574
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
TABLE I PURIFICATIONOF PP2C FROMBOVINE RETINAS
Fraction Supernatant DEAE-Sephace! Heparin-Sepharose Ultrafiltration Phenyl-Sepharose Mono Q Superdex 75 Blue-Sepharose Peak I (2C/3) Peak If (2Ca)
Volume (ml)
Total protein (mg)
Total activity (nmol/min)
170 625 482 44 31 4 15
654 284 135 124 34 11 0.39
73 74 40 20 18.5 12.0 1.5
13 10
0.006 0.002
0.17 0.18
Specific activity (nmol/min/mg)
Purification (-fold)
0.11 0.26 0.29 0.16 0.54 1.10 3.85 28 90
1 2.4 2.6 1.5 4.9 10 35
Recovery (%) 100 102 54 27 25 16 2.1
264 818
EDTA, 5% glycerol. The concentrations of NaCl for washing and elution are the same as those described for the column procedure. The beads (DEAE-Sephacel and heparin-Sepharose) are used with gentle rocking. Binding and elution times are 1 hr, respectively. Beads are pelleted by centrifugations using a swing-out rotor at 700g for 1 min at 4 °. The soluble retinal extract is applied to 50-ml DEAE-Sephacel. Heparin-Sepharose (10 ml) was sufficient for the next step. PP2C in the supernatant fraction is concentrated by Centriplus (Amicon, Danvers, MA) and further enriched using Mono Q H R 5/5 (0.7 M NaCl for elution). Gel filtration, as the final step, is performed as described earlier, yielding highly enriched PP2C free from other phosphatases. For assays dealing with the cation dependence and fatty acid stimulation of PP2C, EDTA in the ultimate column buffer was reduced to 0.1 raM. Purification of Recombinant PP2C Isozymes PP2Ca and PP2C/3 have been cloned from a bovine cDNA library. 2° They share high identity with PP2C isozymes from other mammalian species and tissues. For example, a comparison of PP2C from rat liver with the enzyme from bovine retina shows 5 and 26 substitutions in the amino acid sequence for a and/3, respectively. PP2C isozymes can be heterologously expressed. 13 His-tagged proteins are purified by affinity chromatography on Ni2+-NTA agarose. Enzyme activity of purified recombinant PP2C decreases on long-term storage (gone 20 D. Selke, S. Klumpp, B. Kaupp, and A. Baumann, Meth. Mol. Biol. 93, 243 (1997).
0.2 0.2
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
575
within 6 months at -80°). Enzyme activity in crude bacterial extracts, in contrast, is fairly stable (at least for 1 year). Therefore, a procedure has been developed to purify PP2C from small volumes: 300 txl lysate onto 50 /xl Ni2+-NTA agarose plugged into a Pasteur pipette, equilibrated in buffer E (20 mM Tris-HC1, pH 7.9, 5 mM imidazole, 0.5 M NaCI), and run by gravity. After washing in buffer E containing 60 mM imidazole, PP2C is eluted using buffer F (10 mM Tris-HCl, pH 7.9, 0.5 M imidazole, 0.25 M NaC1). Recovery of PP2C isozymes is 70%, respectively. A 20-ml culture of Escherichia coli normally yields 0.5 mg PP2C protein with a specific activity of 3-15 nmol Pi/min/mg. Expression of PP2Co~ routinely results in only about half of the amount of protein compared to PP2Cfl. Activity of both isozymes is strongly inhibited by imidazole (IC50 250 mM), therefore, immediate and at least fivefold dilution (substrate buffer) is recommended. General Comments on Purification and Analytics Separation of PP2C from PP1 is possible using chromatography on heparin-Sepharose: PP2C lacks affinity to heparin-Sepharose, thus, it can be quantitatively recovered in the flow-through fractions. Separation of PP2C from PP2A is achieved by gel filtration chromatography (Superdex 75 or Sephacryl S-200): the catalytic entities of PP2C ct and fl (~45 kDa) elute in clear distance to the high molecular weight PP2A holoenzymes. Separation of PP2C a and /3 isozymes can be accomplished by two distinct methods. Isozymes from rabbit liver are nicely separated on chromatography using ion-exchange resins. 4 This does not work for the retinal PP2C isozymes. Due to a few species- and tissue-specific amino acid substitutions, retinal PP2Ca and PP2Cfl--in contrast to PP2C isozymes from rabbit liver--end up with almost identical pI values. Separation of retinal PP2C isozymes is possible, however, on chromatography on Blue-Sepharosel9: both isozymes are retained by Cibachron Blue provided Mg 2+ ions are present. Decreasing the divalent cation concentration consequently elutes first fl, then a. In general, it is recommended that long-lasting dialysis steps be avoided. For short-term storage, e.g., overnight interruption during the purification process, freezing is best. Purified PP2C should be stored in aliquots at - 80°. Repetitive freeze-thawing of specifically recombinant PP2Ca should be avoided. Activity and protein "vanish." Adsorption to plastic vials might be the reason. This phenomenon is not observed with PP2C/3. Analyzing PP2C isozymes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bears two unexpected findings: (1) Unusual migration behavior: according to the amino acid composition, the molecular mass of PP2Ca is less compared to PP2C/3 (e.g., 382 versus 387 amino acids for
576
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
100" A
E =
mm
75'
E m
O
E
¢
50'
u s
>
mml
O
25'
"~'0"--0 jO/~'O/O O,
0--0,1
1
10
Mg 2+ (mMI FIG. 1. Mg 2÷ dependence of PP2C. Activity of recombinant PP2Ca was assayed under standard reaction conditions (©; 12.5 ng protein/tube) or in the presence of 0.5 mM oleic acid ( I ; 6.3 ng protein/tube). The shaded area marks the activation in the physiologic concentration range of free Mg z÷ (0.5-1.5 raM).
the enzymes from bovine retina). On denaturing electrophoresis, however, PP2Ca runs as a protein band above PP2Cfl. (2) For most proteins, silver staining is -10-fold more sensitive than Coomassie staining. Detection of PP2C isozymes by silver staining seems to be impaired. Characterization of Native and Recombinant PP2C Isozymes Mechanical disintegration of retinas (Dounce homogenizer) and subsequent centrifugation (100,000g for 60 miu) revealed that PP2C activity distributes equally well between the soluble and particulate fractions. Addition of detergents did not release further PP2C activity.
[38]
PURIFICATION AND CHARACTERIZATION OF P P 2 C
577
7
10' , A
"0 0
m
!
0 i
/
4'
E
i m
2'
~ 0
D/D
0
Fatty acid (mM) FIG. 2. Differential subcellular fatty acid composition affecting PP2C activity. Recombinant PP2Ca (6.3 ng/tube; specific activity 8 nmol Pi/min/mg) was assayed in the presence of 0.7 mM Mg2+ under otherwise standard reaction conditions. Fatty acids added were docosahexaenoic acid (0, 22:6) known to be concentrated in the photoreceptor outer segments, and palmitic acid (rT, 16:0) enriched in the neighboring pigment epithelium cells.
Except for the effect of fatty acids (vide infra), enzymatic properties of recombinant 2Co~ and 2C/3 are almost indistinguishable from one another and from their corresponding native counterparts. The optima for temperature and pH were 45°-55 ° (activation energy 70-90 kJ/mol) and pH 7.5-8 (Tris-HCl). A feature exhibited by all PP2C characterized to date is the requirement of unphysiologic high Mg 2+ concentrations for activity. A recent report describes that addition of unsaturated fatty acids results in high PP2C activity measurable at physiologic Mg 2+ concentrations. 16 Figure 1 shows the Mg 2+ dependence of PP2C in the absence versus presence of
578
PHOTORECEPTOR PROTEIN PHOSPHATASES
[38]
oleic acid (18:1). Activity increases 10-fold and is highest at physiologic free Mg 2÷ levels (0.5-1.5 mM). Under those conditions, Ca 2÷ ions are inhibitory in the micromolar range. 16 The amount of fatty acids and the fatty acid composition are known to vary within cell compartments and from membrane to membrane. In vertebrate retinas, a striking concentration gradient exists between docosahexaenoic acid (22 : 6; major component in photoreceptor outer segments) and palmitic acid (16:0; found in the adjacent pigment epithelium cells) with important functional consequences. Supply of the photoreceptors with retinoids is based on the differential subcellular distribution of those longchain fatty acids: Docosahexaenoic acid--but not palmitic acid--induces release of 11-cis-retinal from a 140-kDa interphotoreceptor matrix retinoidbinding protein. 21 Within vertebrate retinas, PP2Ca and PP2Cfl isozymes are highly enriched in the photoreceptor outer segmentsJ 3 Figure 2 shows that docosahexaenoic acid--but not palmitic acid--increases the activity of recombinant PP2Ca up to 10-fold. The half-maximal activating concentration for docosahexaenoic acid was 350/zM. Stimulation of recombinant PP2Cfl and native PP2C was somewhat less. PP2C and docosahexaenoic acid are not only colocalized in rod outer segments, but are both associated with retinal degeneration processes. 13 Conclusions PP2C, a "black sheep" phosphatase (no activator, no inhibitor) had been considered to be 10-fold less compared to PP1 and PP2A. The rise in less-quantity amount of activity by fatty acids now brings PP2C onto stage as well. Fatty acids and Ca 2+ ions are established players in retinal signal transduction processes. We currently hypothesize regulation of PP2C at two levels: (1) Its basal activity is depending on the presence of unsaturated fatty acids. A long-term change in the fatty acid distribution and concentration as exemplified by apoptosis or retinitis pigmentosa will dramatically affect PP2C activity. (2) On top of that fatty acid sustained PP2C activity, shifts in the Ca 2+ ion concentration would account for short-term regulation of PP2C causing immediate changes in enzyme activity. Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (KI 601/8-1).
21 y. Chen, L. A. Houghton, J. T. Brenna, and N. Noy, J. BioL Chem. 271, 20507 (1996).