©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mg-dependent, Ca-inhibitable Serine/Threonine Protein Phosphatase from Bovine Brain (*)

(Received for publication, June 5, 1995; and in revised form, August 8, 1995)

Yunxia Wang Francesca Santini (§) Kefeng Qin Charles Y. Huang (¶)

From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Mg-dependent serine/threonine protein phosphatases, also known as type 2C phosphatases (PP2C), belong to a gene family distinct from the other serine/threonine phosphatases and tyrosine phosphatases. Here we report the purification to apparent homogeneity of a novel Mg-dependent, Ca-inhibitable serine/threonine protein phosphatase from bovine brain. It is a type 2C enzyme in view of its Mg requirement, resistance to okadaic acid and calyculin A, inability to use phosphorylase a as substrate, and a segment of amino acid sequence typical of all PP2C type phosphatases known to date. However, it differs from the other PP2C enzymes, particularly the mammalian PP2Calpha and -beta isoforms, in that its molecular weight, 76,000, is considerably larger and that it is inhibited by Ca, NaF, and polycations, but not by orthovanadate. The Ca inhibition may not be related to its cellular regulation because of K values in the 20-90 µM range, but this property permits distinction of this enzyme from the other phosphatases. Although the precise physiological role of this phosphatase is not yet known, its ability to dephosphorylate a wide variety of phosphoproteins and its broad distribution, as shown by a survey of mouse tissues for its activity, suggest that it may serve an important cellular function.


INTRODUCTION

Protein phosphorylation-dephosphorylation is a universal mechanism by which numerous cellular events are regulated. Of the enzymes catalyzing the reversible reactions, it has become apparent that there may exist as many as 1,000 phosphatases which, like the kinases, are just as elaborately and rigorously controlled(1, 2, 3, 4, 5) . The serine/threonine-specific phosphatases have been classified into four main types according to their in vitro specificity for selected substrates and sensitivity to activators and inhibitors(6) . Sequence analyses revealed that they can be sorted into two major gene families. The first one includes type 1 (PP1), (^1)type 2A (PP2A), and type 2B (PP2B) phosphatases, which share 37-59% sequence identity (7) in their catalytic domains and are inhibited by okadaic acid(8) . The second family, the Mg-dependent phosphatases, also designated type 2C (PP2C), share little sequence similarity with the first family and are insensitive to okadaic acid.

The Mg-dependent phosphatases have been identified in animals(9, 10, 11, 12, 13, 14, 15) , plants(30) , and yeast(31) , and several have been purified to homogeneity(10, 11, 12, 32, 35) . cDNA sequences of PP2Calpha and -beta from mammalian sources showed >90% identity(16, 17, 18, 19, 34) , whereas those from yeast were 35% identical (13, 20, 33) and showed 21-24% identity with the mammalian ones. The mammalian PP2Calpha and -beta are monomeric cytosolic proteins with molecular weights in the 42,000-45,000 range, and yeast PP2Cs are 31,500-51,400 monomeric enzymes. PP2Cs of larger sizes, however, have been reported. A rabbit myosin light chain phosphatase has a molecular weight of 70,000(32) , and a bovine pyruvate dehydrogenase phosphatase is a dimer with a 50,000 catalytic subunit complexed to a 97,000 subunit(35) . The physiological roles of PP2Cs are unclear(1, 2) , although they have been implicated in the regulation of fatty acid and cholesterol biosynthesis (21) and heat shock response(13, 20) . Recently, a M(r) = 200,000 PP2C-like phosphatase from HeLa cells has been reported (15) which dephosphorylates the C-terminal region of RNA polymerase II. Conceivably, PP2Cs can exist in different molecular sizes and serve diverse biological functions.

We report here the purification to homogeneity and characterization of a novel Mg-dependent, Ca-inhibitable protein phosphatase (MCPP) from bovine brain. Although the Ca inhibition (K = 20-90 µM) may not be related to its cellular control, this unique property permits the differentiation of this PP2C-type phosphatase from other phosphatases during its isolation. While MCPP possesses many characteristics common to other PP2Cs, it is a monomer with a larger molecular weight of 76,000 and responds dissimilarly to several substrates and inhibitors.


EXPERIMENTAL PROCEDURES

Materials

Chemicals and biological materials were obtained from the following suppliers: DE52 ion exchanger and P81 phosphocellulose filter paper (Whatman); high resolution Sephacryl S-300 and Mono Q (Pharmacia Biotech Inc.); Affi-Gel Blue and Dowex AG 1-X8 (Bio-Rad); [-P]ATP (DuPont NEN), okadaic acid and calyculin A (Biomal Research Laboratory); p-nitrophenyl phosphate, histone H2B, histone H3, casein, MBP, porcine cAMP-dependent protein kinase catalytic subunit, heparin, protamine, polylysine, sodium orthovanadate, Kemptide (LRRASLG), and glycogen phosphorylase b (Sigma); syntide-2 (PLARTLSVAGLPGKK) (Bachem); tyrosine phosphatase, pp60 tyrosine kinase, and Raytide (Oncogene); gels for PAGE (Novex), PP1 and PP2A (UBI); and bovine brain cDNA library (Clontech). All other chemicals were of reagent grade. The following proteins were kind gifts from the following persons: phosphorylase b kinase, Dr. Gerald Carlson, University of Tennessee, Memphis; protein kinase C, Dr. Kuo-Ping Huang, NICHD, National Institutes of Health; mitogen-activated protein kinase and casein kinase 2, Dr. Lee Graves, University of Washington, Seattle; phosphatase inhibitor 1, Dr. Heng-Chun Li, Mount Sinai Medical School, New York; and recombinant phosphatase inhibitor-2, Dr. Anna de Paoli-Roach, Indiana University Medical School, Indianapolis.

Preparation of P-Labeled Protein Substrates

[P-Ser]Histone H2B was prepared by incubating 10 mg of the protein with 500 microunits of cAMP-dependent protein kinase catalytic subunit in 1 ml of solution containing 1.5 mM [-P]ATP (300 µCi), 5 mM MgCl(2), 50 mM Hepes, pH 7.0, at 30 °C for 2 h. Incorporation, usually 0.8-1 mol of P per mol of histone H2B, was measured by a thin layer chromatography method (23) . The reaction mixture was then passed through a Dowex AG 1-X8 column to remove free ATP. MBP, histone H1, and alpha-casein were phosphorylated similarly by cAMP-dependent protein kinase.

P-Syntide-2 and -Kemptide were prepared by the same procedure described above except that, after 2 h of incubation, the reaction mixtures were spotted on P-81 phosphocellulose filters, washed with 75 mM phosphoric acid, packed into a column, and then washed extensively with water and eluted with 5 N HCl. Incorporation of P was measured according to Roskoski(24) .

[P]Phosphorylase b kinase was prepared under conditions minimizing autophosphorylation by incubating 6 µM phosphorylase b kinase with 500 microunits of cAMP-dependent protein kinase catalytic subunit for 4 min at 30 °C in a 1-ml solution containing 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl(2), 0.2 mM [-P]ATP in 50 mM Hepes, pH 7.0. The phosphate incorporation (alpha and beta) was 2.0 mol/mol of protein (alphabeta).

[P[Phosphorylase a was prepared according to the method described by King et al.(25) .

P-Labeled histone H3 and -MBP were phosphorylated by protein kinase C according to the method of Masmoudi et al.(26) .

P-Labeled MBP was phosphorylated by mitogen-activated protein kinase by incubating 2 mg of MBP with 5 mM MgCl(2), 0.2 mM ATP (100 µCi), 30 µg of mitogen-activated protein kinase in 50 mM Tris buffer, pH 7.2, at 30 °C for 5 h. 0.3 mol of P/mol of MBP were incorporated.

P-Labeled beta-casein was phosphorylated by casein kinase 2 by incubating 339 µM beta-casein with 350 µM ATP (250 µCi), 10 mM MgCl(2), 0.1 M KCl, 10 µg of casein kinase 2, in 50 mM Hepes, pH 7.4, at 30 °C for 4 h. 0.5 mol of P/mol of protein was incorporated.

P-Labeled Raytide, MBP, and casein were phosphorylated by pp60 tyrosine kinase in the presence of 70 µM ATP (100 µCi), 11 mM MgCl(2), 70 µg/ml bovine serum albumin, 33 µM EDTA in 50 mM Hepes, pH 7.5, at 30 °C for 14 h. Incorporation of P was 0.1-0.2 mol/mol protein.

Phosphatase Assays

The activity of MCPP was routinely assayed in a 40-µl mixture containing 20 µMP-labeled histone H2B, 0.1 mM dithiothreitol, 1 mM EGTA, 50 mM KCl, 2.5 mg/ml bovine serum albumin, either with 2 mM MgCl(2) or without added MgCl(2) but with 2 mM CaCl(2) (EGTA keeps free Ca concentration at 1 mM), in 50 mM Hepes buffer, pH 7.8. The presence of CaCl(2) ensured that any MCPP activity in the latter case due to Mg carried over from enzyme and substrate preparations was completely inhibited. Thus, the difference between the -Ca and +Ca assays should reflect the activity of MCPP with some contributions from other forms of PP2Cs. The P(i) released was measured by an acid-molybdenum extraction method(28) . All other substrates were assayed in an identical manner. Assay using p-nitrophenyl phosphate was performed as described previously(22) . Specific activity of MCPP was expressed as the difference of moles of P(i) released per min per mg of protein in the absence and presence of Ca. Protein concentrations were determined by the method of Bradford (29) using bovine serum albumin as standard.

Dephosphorylation of alpha and beta Subunits of Phosphorylase b Kinase

The reaction mixture, in a final volume of 130 µl, contained 2 µM [P]phosphorylase b kinase, 50 mM KCl, 5 mM MgCl(2), 1 mM EGTA, 0.1 mM dithiothreitol, 50 mM Hepes buffer, pH 7.8, and 1 µg of purified MCPP at 30 °C. 15-µl aliquots were removed at different time intervals, processed, and run on SDS-PAGE 8-16% gradient gel. The gel was dried, and dephosphorylation was detected by a PhosphorImager (Molecular Dynamics). PP1 and PP2A were used as controls.

Purification of MCPP

In a typical preparation, 6 bovine brains (1130 g) were homogenized in a blender with 2 volumes per weight (2300 ml) of buffer A (50 mM Tris, pH 7.8, containing 0.5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 3 mM MgCl(2), 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM benzamidine-HCl). The homogenate was centrifuged at 1200 times g for 1 h, and the supernatant was filtered through 4 layers of cheesecloth. The extract was mixed with a 500-ml bed volume of DE52 pre-equilibrated in buffer A and gently stirred for 2 h. The DE52 was then collected on a 2-liter scintered glass funnel, resuspended in 500 ml of buffer A, and packed into a 5.5 times 43 cm column already packed with 500 ml of DE52. The column was washed with buffer A + 0.1 M NaCl and then eluted with a linear gradient of 0.1-0.6 M NaCl in the same buffer at a flow rate of 1.2 ml/min (Fig. 1A). The peak phosphatase activity fractions, assayed with histone H2B as described above, were pooled and dialyzed against buffer A. The dialyzed sample was loaded onto an Affi-Gel Blue column (5 times 15 cm) and washed with buffer A at a flow rate of 0.8 ml/min (Fig. 1B). The peak fractions from the wash were collected, concentrated in an Amicon stirred cell fitted with YM-30 membrane, and applied to a Sephacryl S-300 (high resolution) column (2.5 times 91 cm) equilibrated with buffer A + 0.1 M NaCl. The column was eluted with the same buffer at a flow rate of 0.33 ml/min (Fig. 1C). Peak activity fractions were pooled and loaded onto a Mono Q (HR 5/5) column equilibrated in buffer A in a Pharmacia FPLC system. The column was eluted with a linear gradient of 0.1-0.6 M NaCl (Fig. 1D). MCPP activity peak eluted at 0.44 M NaCl. When necessary, last traces of impurities were removed by applying the enzyme sample to a Superose 12 column in the FPLC system. All of the purification steps were performed at 4 °C.


Figure 1: Elution profiles of MCPP. The columns were eluted as described under Experimental Procedures. bulletbulletbulletbullet, absorbance at 280 nm; - - -, NaCl gradient; box, assay of MCPP by the ±Ca method; , MCPP and other PP2Cs detected by inhibiting PP1 and PP2A with 1 µM okadaic acid. A, DEAE-cellulose column. Each fraction = 12.5 ml. B, Affi-Gel Blue column. MCPP came out in the wash. Each fraction = 9 ml. C, high resolution Sephacryl S-300 column. Each fraction = 2.8 ml. D, Mono Q columns from FPLC. Each fraction = 0.5 ml.



Electrophoresis

Gel electrophoreses, nondenaturing or in SDS, were run according to the manufacturer's instructions on a minigel system (Novex). Samples used in SDS-PAGE were heated at 100 °C for 3 min in SDS sample buffer containing 5% beta-mercaptoethanol.

cDNA Sequence

The method of polymerase chain reaction was used for amplification of the coding gene of MCPP from bovine brain cDNA library (gt10 library). Polymerase chain reaction primers with BamHI sites were designed according to MCPP peptide sequences determined by Harvard Microchem. Other primers with SalI sites were on the vector gt10. The purified polymerase chain reaction products were digested with BamHI and SalI and then cloned into Bluescript II SK(+). The double-stranded DNA of recombinant clones containing polymerase chain reaction products were sequenced by a 373A DNA Sequencing System (ABI) to obtain cDNA sequence encoding MCPP. Details of the method will be described elsewhere. (^2)


RESULTS

Purification of MCPP

Table 1summarizes the results of a typical purification described under ``Experimental Procedures.'' The ±Ca assay used to identify MCPP turned out to be quite reliable. After the MCPP was purified and its properties were studied, we were able to compare the ±Ca assay with other assays using specific phosphatase inhibitors (cf. Table 4). As can be seen from Fig. 1A, at the DEAE-cellulose chromatography step, MCPP detected by the standard assay eluted with the main PP2C activity peak that was measured by an assay using 1 µM okadaic acid to inhibit both PP1 and PP2A. Calcineurin (PP2B) did not contribute much in this assay when Ca was present because exogenous calmodulin was not added and histone H2B is a poor substrate for this enzyme. After the Sephacryl S-300 step, MCPP activity can be measured readily by the ±Ca method. MCPP activity profile obtained by this method agreed well with that using 1 µM okadaic acid and 1 mM orthovanadate (see Table 3) to inhibit PP1, PP2A, and PP2Calpha and -beta. The peak fraction from the Mono Q step yielded the pure form of MCPP.







Purity, Molecular Weight, and Subunit Structure

As shown in Fig. 2, fraction 34 from the Mono Q step migrated predominantly as a single band of M(r) 76,000 in both 8-16% gradient SDS-PAGE and 4-20% gradient nondenaturing PAGE (overloaded). The gel patterns indicated that MCPP is a monomeric protein with >90% purity. When the 4-20% gel was sectioned and assayed, a sharp peak of MCPP activity corresponding to the M(r) = 76,000 band was observed (Fig. 3), confirming that the phosphatase activity is solely associated with this protein band.


Figure 2: Gel electrophoresis patterns of purified MCPP (fraction 34) from the Mono Q column step. Left panel, 8-16% SDS-PAGE: left lane, molecular weight markers; right lane, MCPP. Right panel, 4-20% nondenaturing PAGE: left lane, MCPP; right lane, molecular weight markers.




Figure 3: Identification of MCPP activity band. Fraction 34 from the Mono Q step was run on 4-20% nondenaturing PAGE in six lanes. One lane was stained with Coomassie Blue to mark the protein band. The other five were cut into strips, and the protein from each section was eluted and assayed for MCPP activity.



Activation and Inhibition by Various Cations

MCPP was found to require Mg for activity with a K(a) of 0.8 mM, which is considerably smaller than the 5 mM reported for rabbit PP2Cs(12) . The enzyme was totally inactive when Mg was removed by a chelator like EDTA. Mn could support MCPP activity as effectively as Mg, but the other cations tested, Ca, Co, Zn, Ni, Fe, and Al could not. All the cations tested were chloride salts, and all reagents had been passed through Chelex 100 columns. In the presence of 1 mM Mg, the percent inhibition observed with 1 mM cation in descending order was Zn, 98%; Fe and Al, 89%; Co, 86%; Ni and Ca, 85%. The inhibitory effect of these cations increased with decreasing Mg concentration. Although these cations all have similar inhibitory effects on MCPP, the inhibition by Ca distinguishes MCPP from most phosphatases. K(i) for Ca is on the order of 90 µM when histone H2B is the substrate. With MBP as substrate, however, K(i) for Ca is 20 µM. Calmodulin has no effect on this phosphatase.

pH Optimum of MCPP

The pH optimum of MCPP was determined by using histone H2B as substrate in the standard assay. MES buffer was used for pH values from 5.0-7.0 and Hepes buffer from pH 6.6-8.5. MCPP activity reached optimal values at pH values greater than 7.5, indicating that it is an alkaline protein phosphatase.

Substrate Specificity

The rates of MCPP-catalyzed dephosphorylation using various substrates and, whenever possible, at comparable concentrations (in terms of phosphoryl groups) are given in Table 2. None of the phosphotyrosyl Raytide, MBP, and casein served as substrate for MCPP. Nor was p-nitrophenyl phosphate, a widely used substrate for tyrosine phosphatases, dephosphorylated by MCPP. The data shown in Table 2showed that MCPP is a phosphoserine/threonine protein phosphatase with diverse specificity. However, MCPP appears to dephosphorylate mostly substrates phosphorylated by cAMP-dependent protein kinase and protein kinase C. Table 2also reveals that MCPP prefers basic proteins like MBP and histones as substrates, and MBP phosphorylated by cAMP-dependent protein kinase gave the highest specific activity of 2 µmol/min/mg. Phosphorylase b kinase, a substrate used by Ingebritsen and Cohen (6) to designate phosphatase types 1 and 2, was a poor substrate for MCPP. Phosphorylase a, a poor substrate for PP2Cs, was untouched by MCPP. In addition, there are several interesting protein kinase C-phosphorylated substrates of MCPP (not listed in Table 2because of inadequate quantitation): a leukemogenic protein called SET(38) , the microtubule-associated tau protein, and the 7.5-kDa neurogranin.



Effect of Known Phosphatase Inhibitors

The effects of different known inhibitors of protein phosphatases have been examined, and the results are summarized in Table 3. NaF, a common inhibitor of PP1, PP2A, and PP2B, but not of PP2Calpha and -beta, inhibits MCPP. Polylysine and protamine, which activate PP2A, are potent inhibitors of MCPP, although they do not inhibit PP1, PP2B, and PP2Calpha and -beta. Orthovanadate, a potent inhibitor of tyrosine phosphatase that inhibits PP1, PP2A, PP2C (2) at near millimolar levels, has no effect on MCPP at concentrations up to 1 mM. The phosphatase types 1 and 2A inhibitors okadaic acid and calyculin A (27) do not inhibit. Phosphatase (PP1) inhibitors 1 and 2 and heparin also have no effect.

Amino Acid Sequence Homology with Known PP2C-type Phosphatases

Partial cDNA sequence of MCPP obtained by us revealed a segment that is homologous to the same region found in every PP2C reported so far. Fig. 4compares a 24-residue segment of MCPP, which is 90 residues from the C terminus, with those from other sources. The results indicate that MCPP is a member of the PP2C gene family, but it is a new enzyme different from the mammalian PP2Calpha and -beta isoforms.


Figure 4: Comparison of amino acid sequences of a 24-residue segment deduced from the cDNA of MCPP with segments from 12 other PP2Cs. Residues identical with those of MCPP are underlined. Conservative substitutions (relative to MCPP) are in capital letters. The number given at the end of each sequence marks the position of the last residue. All alignments are obtained from the BLAST Server.



Distribution of MCPP in Different Tissues

Since MCPP was isolated from bovine brain, it is of interest to see whether this Ca-inhibitable enzyme or its isoforms may exist in other tissues and other animals. Therefore, we examined the distribution of MCPP activity in various mouse organs by using calyculin A to inhibit PP1 and PP2A and employing the standard ±Ca or ±polylysine assay with histone H2B as substrate to detect the presence of this type of phosphatase. As can be seen from Table 4, the Ca-inhibitable phosphatase activity can be demonstrated in the four selected mouse organs, brain, kidney, lung, and liver, and the results agree very well with those obtained from using polylysine to inhibit MCPP.


DISCUSSION

We have purified to homogeneity a new Mg-dependent protein phosphatase from bovine brain. It is named Ca-inhibitable protein phosphatase to differentiate it from other mammalian Mg-dependent phosphatases which have been called PP2Calpha, PP2Cbeta, or named after their substrates such as phosphofructokinase phosphatase(14) , myosin light chain phosphatase (32) , and pyruvate dehydrogenase phosphatase(35) . Although PP2C-type phosphatases have been identified in various sources, only a handful have been purified to homogeneity, and MCPP is the first 76-kDa enzyme obtained in pure form.

The catalytic and structural characteristics of MCPP indicate that it can be classified as a type 2C phosphatase. 1) It requires Mg (or Mn) for activity. 2) It catalyzes the dephosphorylation of phosphoseryl/threonyl residues of proteins and peptides phosphorylated by cAMP-dependent protein kinase and protein kinase C (Table 2). 3) It is insensitive to inhibitors like okadaic acid and calyculin A, heparin, and PP1 inhibitors 1 and 2. 4) It does not attack phosphorylase a. 5) A segment of its amino acid sequence (24 residues) is homologous to a region found in 12 other PP2C-type enzymes (Fig. 4). In this segment, MCPP is 54.58% identical with and 96% similar to 6 mammalian PP2Calpha and -beta isoforms; 46-54% identical with and 88-92% similar to 4 yeast, isolated or putative, PP2Cs. It should be of interest to note that a CDLLW motif similar to the CDGIW or CDGLW sequence of PP2Cs is found in practically every amino acid sequence of PP1, PP2A, and PP2B(7) .

MCPP, however, possesses properties that differ from PP2Calpha and -beta and other PP2Cs. 1) Its molecular weight, 76,000, is considerably larger than the M(r) = 42,000-51,000 mammalian and yeast enzymes, but comparable to the M(r) = 70,000 myosin light chain phosphatase (32) . 2) It is inhibited by polycations and F ion which do not inhibit PP2Calpha and -beta, although a Mg-dependent phosphatase from turkey gizzard smooth muscle also is inhibited by F(10) . 3) PP2Cs are inhibited by millimolar concentrations of orthovanadate, but MCPP is not. 4) PP2Cs preferentially dephosphorylate alpha subunit of phosphorylase b kinase, whereas MCPP has very low activity with this substrate and shows no preference for either alpha or beta subunit. The M(r) = 70,000 myosin light chain phosphatase also exhibited little activity with phosphorylase b kinase as substrate(32) . 5) MCPP is inhibited by Ca with K(i) in the 20-90 µM range, depending on the substrate used. The effect of Ca, however, is on the enzyme since the inhibitory effect was observed with every substrate tested so far. The shift in K(i) suggests that Ca is a noncompetitive inhibitor which affects both V(max) and K(m) for the substrates. Since the K(i) values are much higher than the intracellular Ca level of 0.1-1 µM, it appears that the Ca inhibition may not be physiologically important. However, regulation by Ca may not be excluded because, like the synergistic effect of glycogen on the Ca inhibition of glycogen-synthetase phosphatase(37) , the presence of another component may be involved in amplifying the Ca signal in vivo. In this regard, it is interesting to note that bovine pyruvate dehydrogenase phosphatase is activated by Ca with K(d) in the 24-62 µM range in the absence of EGTA buffer(36) .

The role of MCPP in the cell, like the other PP2Cs, is not clear at this time. The rather wide distribution of MCPP, as shown in the survey presented in Table 4, suggests that MCPP or its isoforms constitute a new subclass of PP2C and likely serve important cellular functions. It should be of interest to note that a SET protein encoded by a set gene (38) copurified with MCPP until the Mono Q chromatography step. In a case of acute undifferentiated leukemia, the set gene was fused to a can gene as a result of chromosomal translocation(38) . Phosphorylation of the SET protein by protein kinase C was blocked by the presence of trace amounts of MCPP, indicating that SET is an excellent substrate for the phosphatase. (^3)Since the set gene is expressed in all tissues of the mouse, particularly during embryogenesis, SET may play a key role in the cell and MCPP may regulate the function of SET. The fact that MCPP activity is highest with MBP (10 times better than any other substrates tested so far) may also imply a special function for this phosphatase in the brain.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 10107.

To whom correspondence should be addressed: Laboratory of Biochemistry, Bldg. 3, Rm. 218, NHLBI, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-6378; Fax: 301-496-0599.

(^1)
The abbreviations used are: PP1, PP2A, PP2B, PP2C, types 1, 2A, 2B, and 2C protein phosphatases; MCPP, Mg-dependent, Ca-inhibitable protein phosphatase; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; MES, 2-(N-morpholino)ethanesulfonic acid.

(^2)
K. F. Qin and C. Y. Huang, unpublished data.

(^3)
F. Santini, Y. Wang, and C. Y. Huang, unpublished data.


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