Protein Phosphatases 1,2A, and 2C Are Protein Histidine

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 25, Iasue of September 5, pp. 18513-18516,1993 Printed in U.S.A.

Protein Phosphatases 1,2A, and 2C Are Protein Histidine Phosphatases*

(Received for publication, April 7, 1993)

Younhee Kim$, Jianmin €hang$, Philip Coheng, and Harry R. MatthewsSIl From the $Department of Biological Chemistry, University of California at Davis, Davis, California 95616 and the $Department of Biochemistry, The University, Dundee, Scotland, DDl 4HN, United Kingdom

Eukaryotic cellular proteins contain phosphohistidine. To search for protein histidine phosphatases, protein histidine kinase from Saccharomyces cerevisiae was used to phosphorylate histone H4 on histidine at position 76 in the H4 amino acid sequence. Incubation of the phosphorylated histone H4 with either protein phosphatase 1,2A, or 2C resulted in extensive removal of phosphate from the phosphorylated histone. Thus, protein phosphatases 1,2A, and 2C are histidine phosphatases as well as serine/threonine phosphatases. Cal- cium/calmodulin-regulated protein phosphatase (protein phosphatase 2B) did not remove phosphate from phosphohistidine. The histidine phosphatase reaction was tested for a magnesium requirement and effects of inhibitor-1 and okadaic acid. In all cases, the protein phosphatases behaved as they do in their serine/threonine phosphatase activity. Extracts of the yeast, S. cerevisiae, contain protein histidine phosphatase activity. Quantitative measurement of phosphatase activity shows that the activity against phosphohistidine is a major activity of protein phosphatases 1,2A, and 2C.

Phosphohistidine, labeled in vivo, has been found in a number of proteins in eukaryotes, including nuclear proteins from rat liver (1, 2) and from the true slime mold, Physarum polycephalum (3). In rat liver membranes, a protein, p38, is phosphorylated on histidine by an endogenous kinase that is highly stimulated by addition of activated ras protein or glucagon and guanine nucleotides (4, 5). Phosphohistidine is unstable at low pH (6, 7) and is thus destroyed at some stage during most studies of protein phosphorylation. Phosphohis- tidine has a phosphoramidate bond (P-N), and hence phosphohistidine (and phospholysine and phosphoarginine) can be distinguished from phosphoserine and phosphothreonine because phosphohistidine is stable to strong alkali while phosphoserine and threonine are not. Where appropriate procedures have been used to preserve alkali-stable phosphorylation in proteins isolated from cells, relatively large amounts of alkali-stable phosphate have been measured, on the order of the amounts of phosphoserine or phosphothreonine (1, 3) and thus much larger than the amounts of phosphotyrosine. In 80 far as these experiments truly measure P-N bonds in proteins, they imply that phosphohistidine (and possibly phospholysine and phosphoarginine) is a quantitatively im-

This work was supported by American Cancer Society Grant BE 82H (to H. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence should be addressed Dept. of Biological Chemistry, University of California at Davis, Davis, CA 95616. Tel.: 916-752-3570; Fax: 916-752-3516.

portant component of cellular phosphorylated proteins under in vivo conditions.

Although phosphohistidine is acid-labile, it is stable at physiological pH (7). Thus, if histidine phosphorylation is to play a dynamic regulatory role analogous to that of serine, threonine, and tyrosine phosphorylation in eukaryotes or phosphohistidine in prokaryotes (8), then phosphohistidine should turn over, and there should be enzymes that add or remove the phosphate from the histidine residue. A protein histidine kinase has been isolated and purified from the yeast Saccharomyces cerevisiue (9), and protein histidine kinase activity has been measured in both mammalian (10, 11) and slime mold cells (12). I n vitro, the protein histidine kinase from yeast phosphorylates specifically one of the histidine residues in histone H4 and does not phosphorylate serine, threonine, or tyrosine (9). The histidine kinase activity is inhibited by genistein (13), a compound used to distinguish between protein tyrosine kinases and protein serine/threonine kinases, and thus it is possible that protein histidine kinase is responsible for some of the activities that have been attributed to phosphotyrosine on the basis of studies with genistein. When this study began, no method for removing the phosphate from phosphohistidine in eukaryotic proteins in vivo was known.

Three major families of protein phosphatase have so far been identified that dephosphorylate serine, threonine, and tyrosine residues. One of these includes the enzymes protein phosphatase-1 (PPl),’ protein phosphatase-2A (PPZA) (14), and the calcium/calmodulin-regulated enzyme termed protein phosphatase 2B (PPBB) (15-17) which are thought to act as protein serine/threonine phosphatases in vivo (18), although PP2A and PPPB also have the potential to dephosphorylate tyrosine residues under certain conditions. A further, magnesium-dependent enzyme, termed protein phosphatase 2C (PPBC), is also a protein serine/threonine phosphatase (18), but its amino acid sequence is unrelated to PPl/PP2A/PP2B (19). In contrast, the major protein tyrosine phosphatases in cells comprise an additional family of enzymes, whose struc- tures are unrelated to PPl/PPBA/PPBB or PP2C (20, 21). However, a subclass of the protein tyrosine phosphatase family, that includes cdc25 (the enzyme which dephosphorylates ~ 3 4 “ ~ ’ ) and an enzyme encoded in the genome of vaccinia virus, also have the potential to dephosphorylate phosphoserine and phosphothreonine residues (21).

Since there are no published data on protein histidine phosphatases, we have initially examined whether the known protein phosphatases that act on phosphoserine, phosphothreonine, and phosphotyrosine residues are also capable of acting on phosphohistidine. In this report we demonstrate

The abbreviation used is: PP, protein phosphatase.

18513

18514 Histidine Phosphatases

that PP1, PPZA, and PPZC are all active histidine phosphatases.

MATERIALS AND METHODS

Enzymes-Protein histidine kinase was purified from S. cereuisiae, as described, including the chromatography on MonoS (22). Histone H4 was isolated from total calf thymus histone (Sigma) by chromatography on a large Bio-Gel P-10 column (5 cm diameter X 1 m long) in 10 mM HCI. The catalytic subunits of PP1 and PP2A (Dr. D. Schelling) (23) and inhibitor-1 (Dr. M. Hubbard) (24) were purified from rabbit skeletal muscle at Dundee by the investigators indicated in parentheses. PPPC, also from rabbit skeletal muscle, was partially purified up to the Sephadex G-100 (25) step and provided by J. Corton and Dr. D. G. Hardie, University of Dundee. Calmodulin, PP2B, and the labeled substrate for PP2B were provided by Dr. C. Klee, NIH. Phosphorylase b and phosphorylase kinase were provided by K. Angelos and Dr. D. A. Walsh, University of California at Davis. One unit of protein phosphatase catalyzes the dephosphorylation of 1.0 pmol of phosphorylase a/min (PPl, PP2A) or of casein/min (PP2C). One milliunit is 1/1000 of 1 unit.

Labeled Substrate-Phosphorylase a was prepared from phosphorylase b, phosphorylase kinase, and [y-32P]ATP as described elsewhere (26).

Histone H4 was incubated with protein histidine kinase and [y- '*P]ATP in kinase buffer as previously described (27). The reaction was stopped by adding NaOH to a final concentration of 0.5 N and incubating at 60 'C for 30 min. For some experiments, a synthetic peptide substrate was used. The peptide was synthesized by Dr. I. Clark-Lewis at the University of British Columbia. It has the sequence of the amino acids 70 through 102 of histone H4 and is phosphorylated in a similar manner to intact H4. For some experiments, the kinase reaction volume and the stoichiometry of phosphorylation were increased by using a reaction volume of 0.5 ml containing 0.1 mg/ml histone H4, 0.1 ml of protein histidine kinase (2,200 units/ml (1 unit is incorporation of 1 pmol of phosphate in 15 rnin)), 0.1 mM [y-S2P]ATP (900 Ci/mol), 15 mM Tris, 20 mM MgC12, pH 7.5. After incubation for 1 h, 10 pl each of kinase and [y-32P]ATP were added, and incubation was continued for another hour. This was repeated twice more for a total incubation time of 4 h. The reaction was stopped by adding NaOH to a final concentration of 0.5 N and incubating at 60 "C for 45 min. The mixture was then dialyzed against 5% (v/v) glycerol in buffer A (50 mM Tris-C1, 0.1 mM EGTA, 0.1% 8-mercaptoethanol) adjusted to pH 8 overnight. It was further dialyzed twice against 5 % glycerol in buffer A adjusted to pH 7.5 and stored at -20 "C for up to 2 weeks. For the ultrafiltration assay, the [32P]histone H4 was further purified by ion-exchange chromatography on Bio-Rex 70 (28).

Phosphatase Assay-PP1 and PP2A were diluted into buffer A containing 1 mg/ml bovine serum albumin and adjusted to pH 7.5. An aliquot, 10 pl, of the diluted phosphatase was preincubated for 10 min at 30 "C with 10 pl of 0.03% (w/v) Brij-35 in buffer A adjusted to pH 7.5. PP2C was diluted in buffer A adjusted to pH 7.5, and an aliquot, 10 pl, of diluted PP2C was preincubated for 2 min at 30 "C with 10 pl of 60 mM magnesium acetate in buffer A adjusted to pH 7.5. For all protein phosphatases, the reaction was started by adding 10 p1 of [SZP]H4 (final H4 concentrations, given in the figures, were typically 1 p~ or less). The reaction mixture was incubated at 30 "C for 10 min and stopped by the addition of SDS loading buffer. The mixture was loaded onto a 17.5% polyacrylamide gel containing SDS (29) and electrophoresed, using 0.1 M sodium acetate in the anode buffer.

Inhibitor-1 was diluted in 0.03% (w/v) Brij-35 in buffer A (pH 7.5) for experiments with PPI or PP2A or in 60 mM magnesium acetate in buffer A (pH 7.5) for experiments with PP2C. Diluted protein phosphatase, 10 pl, was preincubated with 10 pl of diluted inhibitor- 1 for 2 min at 30 "C before starting the reaction as described above. Okadaic acid was a generous gift from Dr. Y. Tsukitani, Fujisaura Pharmaceutical Company, Tokyo, Japan. Okadaic acid was handled as described for inhibitor-1.

After electrophoresis, the gel was analyzed by autoradiography. In most cases the gel was never stained; in other cases, the gel was stained after autoradiography, using conventional procedures, or before autoradiography, using formalin fixation to avoid acidic conditions that remove phosphohistidine (22).

For quantitative data, an assay based on ultrafiltration was used. This has been described elsewhere (28); it uses a Microcon-3 (Amicon

Co.) centrifugal ultrafiltration device to separate released phosphate from protein.

RESULTS

The Gel Assay-A substrate containing phosphohistidine was obtained using a purified protein histidine kinase isolated from S. cereuisiue, histone H4 and [Y-~~PIATP. This kinase phosphorylates only histidine in the histone H4 substrate (9), specifically the histidine at position 75. To further rule out the presence of phosphoserine or phosphothreonine, the phosphorylated histone H4 was subjected to a partial alkaline hydrolysis in 0.5 N NaOH at 60 "C for 30 min, which is sufficient to remove the phosphate incorporated into histone H4 by the catalytic subunit of the cyclic AMP-dependent protein kinase (27). The hydrolysis causes limited proteolysis of histone H4 and accounts for the broad lower band seen in the autoradiographs. Fig. 1D shows how histone H4 runs when it has not been subjected to the alkaline hydrolysis before being used in the phosphatase reaction. The autoradiographs show only a portion of the gel (containing all the radioactivitity on the gel), and so the bands appear rather broad, due to the lack of context.

Phosphohistidine is very acid-labile, and thus phosphatase assays that require acid conditions for precipitation or meas- urements of free phosphate cannot be used to measure enzy- matic dephosphorylation of phosphohistidine. For these experiments, we have used an assay that measures the amount of remaining phosphorylated substrate, by subjecting the reaction mixture to gel electrophoresis after incubation. The remaining phosphorylated substrate is then detected by autoradiography. This assay can be made somewhat quantitative by quantitating the radioactivity in the band on the autora- diograph but a significant reduction in the substrate concentration must occur before any activity is measured, unlike assays in which the released phosphate is measured. Thus, the assay is less sensitive than conventional phosphatase assays. However, the sensitivity is quite sufficient for the experiments reported here and the autoradiographs provide a very positive indication that dephosphorylation has occurred. The standard control run for each experiment is an incubation without enzyme. Under these conditions, the phosphorylated substrate is completely stable and the controls are very reproducible.

Protein Histidine Phosphatase Activity-In the first phosphatase experiments, [32P]phosphorylase a was included in the reaction mixtures as a positive control. Thus, both ["PI phosphorylase a (phosphorylated on serine) and [32P]histone H4 (phosphorylated on histidine) were present in the same reaction mixture. After incubation, the reactions were stopped by addition of SDS sample buffer, and the mixtures were subjected to gel electrophoresis. The gel was autoradi- ographed, and an example of the results is shown in Fig. lA for PPZA. Similar results were obtained with PP1 (not shown). Both [32P]phosphorylase a and [32P]histone H4 were dephosphorylated by either PP1 or PPZA. Thus, PPI and PPZA are both protein serine/threonine phosphatases and protein histidine phosphatases. In subsequent experiments, the [32P]phosphorylase a was omitted, as shown for PPI and PPZC in Fig. hi. As an alternative substrate, a synthetic peptide corresponding to residues 70-102 of histone H4 was phosphorylated with protein histidine kinase (13), tested with PPI, and found to be quantitatively dephosphorylated (Fig. 1B). Since most of the phosphate is removed from ["PI histone H4 by any of PPl/PPZA/PPZC, it is clear that phosphate is being removed from phosphohistidine by the protein phosphatases, as this result could not arise from a minor undetected amount of phosphoserine or phosphothre-

Histidine Phosphatases 18515 PP1 At lower protein phosphatase concentrations, the dephos- + -

phorylation of phosphohistidine in histone H4 is dependent on the protein phosphatase concentration as shown in Fig. 2. For this experiment, we were exploring the lower limits of sensitivity for this assay, and thus the changes in the autoradiographs are more subtle than those in Fig. 1 where the basic phenomenon is displayed rather clearly. Fig. 2A shows an increase in activity (bands get fainter) as the PP1 concentration is increased from 0.5 milliunit/ml to 2.1 milliunits/ ml. Similarly, in Fig. 2B, the activity of PP2A is marginal at the lowest concentration of PPBA, 0.016 milliunit/ml, but reproducible activity is seen as the PP2A concentration increases from 0.048 to 1.3 milliunits/ml. With PPZC, the phosphatase activity is marginal, at best, at 0.003 milliunit/ ml but is reproducibly present at 0.012 milliunit/ml and increases as the PP2C concentration increases from 0.012 to

- t 0.8 milliunit/ml (Fig. 2C). Thus, for all three protein phos- + + phatases, the phosphatase activity is dependent on the con-

B

centration of the enzyme. Effects of M$+ or Inhibitor-I-The activity of PPZC as a

protein serine/threonine phosphatase is dependent on the presence of M T . PPZC also requires M F when it is acting as a protein histidine phosphatase. The titration with M P (Fig. 3) shows fairly flat histidine phosphatase activity with increasing [M?] in the range 5-30 mM. PP2c was inactive in the absence of M e and, in other experiments, 2 mM gave about half the activation observed with 5 mM Me.

PP1 A 0

PP1 PPPA PP2C + - + - + - phosphorylase

A

histone H4

calmodulin - + - + - + PPPB - - + + - -

D 1 2 3 4 5

BSA A H4

H4 PPPA - 0

FIG. 1. Protein phosphatases 1, 2A, and 2C remove phosphate from phosphohistidine. A, the phosphatase is indicated above each pair of lanes. For PPPA, [3ZP]phosphorylase a was included in addition to [32P]histone H4. B, [3'P]H4(70-102) was the substrate. C, PP2B (0.1 p ~ ) and calmodulin (0.1 p ~ ) were incubated as shown above each lane with either the peptide substrate (3.9 p ~ , lefthand lanes) or the [3ZPJhistone H4 substrate (1.8 p ~ , righthand lanes). D, histone H4 (10 p ~ ) was incubated with each of the phosphatases or the yeast cell extract. BSA indicates bovine serum albumin present in some of the buffers. Lone 1, no phosphatase; lane 2,0.67 milliunit/ ml PP1; lane 3, 0.10 milliunit/ml PP2A; lane 4, 0.15 milliunit/ml PP2C; lane 5, 1.2 mg/ml yeast cell extract protein. The stained gel is shown.

onine in the [32P]histone H4. Also, the enzyme preparations do not degrade the substrate (Fig. 1D).

Unlike PPl/PP2A/PPBC, PP2B was unable to dephosphorylate [32P]histone H4, although this preparation of PP2B was active against a synthetic substrate provided by Dr. C. Klee (Fig. IC). This experiment was carried out two more times, and no reproducible dephosphorylation of [32P]histone H4 was observed, even at higher concentrations of PP2B and calmodulin. Thus, PP2B does not use this particular labeled histone H4 as a substrate. Of course, this experiment does not indicate whether PP2B is ineffective because of a specificity for the phosphoamino acid or for the surrounding amino acid sequence.

B H4

PPPC A 0 C H4

FIG. 2. Dephosphorylation of phosphohistidine in histone H4 depends on the phosphatase concentration. The [32P]histone H4 concentration was 2.2 pht; phosphatase concentrations were ( le f t to right): 0.5, 1.1, 2.1, or 0 miIliunit/ml (PPl, panel A ) ; 0.016, 0.048, 0.14, 0.4, 1.3, or 0 milliunit/ml (PP2A, pone1 B ) ; 0.003, 0.012, 0.05, 0.2, 0.8, or 0 milliunit/ml (PP2C,panel C).


m M Mg" 0 5 10 15 20 25 30 is inhibited by inhibitor-1 (Fig. 4, B and C). This result confirms that PP1 is responsible for the dephosphorylation of phosphohistidine assuming that inhibitor-1 works in a

histone H4 similar manner against histidine dephosphorylation as it does against serinelthreonine dephosphorylation.

Okadaic Acid-Okadaic acid is a specific phosphatase inhibitor that is used to infer the involvement of PP1 or PPZA

FIG. 3. PP2C requires Me. [32P]Histone H4 (0.65 p ~ ) was in cellular processes. Okadaic acid, in the absence of phos- incubated with PPPC (0.67 milliunit/ml) and Me as indicated above phatase, has no effect when incubated with [32P]histone H4. each lane. When tested with PP1, no effect of okadaic acid was seen

with 1 or 10 nM okadaic acid, but 100 nM okadaic acid - - + + + + PP1 0 1 0 1 10 100 inhibitor-1

significantly inhibited the dephosphorylation of histidine (Fig. 5A). When tested with PPZA, even 1 nM okadaic acid showed a small but remoducible inhibition and almost com- plete inhibition was s'een at 10 nM okadaic acid (Fig. 5B). Thus, PPZA is much more sensitive to okadaic acid than is PP1. When tested with PPZC, okadaic acid was without effect at concentrations up to 10 p~ (Fig. 5C). These results are qualitatively similar to those obtained when these protein phosphatases are used with substrates containing phosphoserine or phosphothreonine.

Histidine Phosphatase Activity in Cell Extracts-A cell ex-

histone H4

A - - + + + + PP2A - - + + + + PP1 0 1 0 1 10 100 inhibitor-1 0 1 0 1 10 100 nM

B okadaic acid 1

histone H4 b r ~

histone H4

0 ,&

B - - + + + + PPPA

- - + + + + PP2C 0 30 0 30lO0300 inhibitor-1

C

histone H4

0 1 0 1 10 100 nM okadaic acid

histone H4

- - + + + + + pp2c c 0 10 0 10 100 lp00 nM

FIG. 4. Inhibitor-1 and protein phosphatases. Inhibitor-1 okadaic acid concentrations (nanomolar) are indicated above the lanes in each panel. Phosphatase and [32P]histone H4 concentrations were: PP1 8 milliunita/ml; 1.3 p~ H4; PP2A 2.6 milliunits/ml; 0.65 pM H4; PPPC 0.8 milliunit/ml; 2.6 pM H4.

histone H4

Inhibitor-1 is a protein inhibitor specific for PP1 when tested with serinelthreonine substrates. Fig. 4A shows that inhibitor-1 inhibits the activity of PPI against phosphohistidine. Partial inhibition is seen at an inhibitor concentration of 1 nM, and this is confirmed in three repeats of this experiment. Extensive inhibition is seen at 10 and 100 nM inhibitor- histone H4 concentration was o.65 pM; the okadaic acid concentra- FIG. 5. Okadaic acid and protein phosphatases. The [=PI

1. Inhibitor-1 was also tested against PP2A and PPZC, using phosphatase concentrations were 2.6 milliunits/ml ( P P I ) , 0.6 mil-

tions (nanomolar) are indicated above the lanes in each panel; the

the phosphohistidine substrate and neither PPZA nor PPZC Iiunit/mI (pP&t), 0.2 miIIiunit/ml (PP2C).

Histidine Phosphatases 18517

tract was prepared from S. cereuisiue and assayed with the [S'P]histone H4. At protein concentrations of 0.07 mg/ml and above (up to 1.15 mg/ml), the extract showed significant protein histidine phosphatase activity indicating the presence of this activity in cells as well as in vitro (Fig. 6). The activity is not due to PP2C since M$+ was not added. When MgZ+ was added, an increase in dephosphorylation was seen (Fig. 6), indicating that PP2C is also a significant histidine phosphatase under these conditions. To test whether the activity might be due to PPI and/or PPBA, okadaic acid was added to the extracts before the [3zP]histone H4 substrate. Partial inhibition of the phosphatase activity was seen (Fig. 6), indicating that PP1 and PPPA account for at least part of the protein histidine phosphatase activity in these extracts while also suggesting that other phosphatase(s) may be active.

Quantitatiue Reaction Rates-The gel assay used in the previous experiments is unsuitable for quantitative measure- ments of histidine phosphatase activity. We developed a new phosphatase assay that uses ultrafiltration to separate released phosphate from unchanged substrate at the end of the phosphatase reaction. A detailed description and validation of this method is presented elsewhere (28), but in essence it consists of incubation of the phosphatase with the 3'P-labeled substrate and any inhibitor or other effector for an appropriate length of time followed by dilution of the reaction with phosphate buffer and removal of the released phosphate by spinning the diluted reaction mixture through an ultrafilter that retains the protein but allows inorganic phosphate to pass through. The amount of [3'P]phosphate that passes through the filter is determined by liquid scintillation count- ing.

This ultrafiltration assay was used to determine the time course of dephosphorylation of [3'P]histone H4 with each of PP1, PPZA, and PP2C (Fig. 7). Even at the relatively high phosphatase concentrations used, the reaction rates are linear for at least 5 min, providing a method for determining initial rates of reaction,

The concentrations of phosphatase used in all the above experiments are quite reasonable in terms of their activity against phosphoserine in phosphorylase a, which is a good substrate for PP1 and PP2A (30). To obtain a more precise idea of the relative activity of these phosphatases against phosphohistidine and phosphoserine, a preparation of each phosphatase was assayed, on the same day, with both ["PI histone H4 and phosphorylase a. Under these conditions, phosphohistidine is at least as good a substrate as phosphoserine for both PP1 and PP2A (Table I). Thus, histidine phosphatase is a major activity of each of these phosphatases, confirming their ability to act as histidine phosphatases in vivo.

0.0- 0.0 0.5 1.0 1.5

extract protein, mglml

FIG. 6. Phosphatase activity in a yeast cell extract. The filled circles show the histidine phosphatase activity without added MgZ+ or okadaic acid. The open circle and square symbols show reactions carried out at 0.3 mg/ml extract but with added M2' (5 mM, open square) or added okadaic acid (1 nM, open circle; 5 pM, filled square).

PP2C / 0.6

0 5 10 15 20 Reaction Time (min)

FIG. 7. Time course of protein histidine phosphatase reaction. The enzyme and substrate concentrations were: PPI, 42 microunits/ml, [32P]histone H4,0.5 pM; PP2A, 3.1 microunita/ml, ["PI histone H4 0.37 p ~ ; PP2C 17 microunits/ml, [32P]histone H4 0.37 PM.

TABLE I Comparison of phosphatase activity with p2P]histone H4

(phosphohistidine) or phosphorylase a (phosphoserine) Phosphatase activity

Phosphatase [32P]histone H 4 , l gM

Phosphorylase a, 10 PM

pmollmin PP1 4.5 2.7 PP2A 6.9 7.6

DISCUSSION

Using a protein substrate containing [3'P]phosphohistidine as the sole phosphoamino acid, it has been shown that PP1, PPBA, and PP2C are protein histidine phosphatases. In contrast, Ca'+/calmodulin-regulated protein phosphatase (PPPB) does not dephosphorylate this substrate. In an earlier experiment with protein tyrosine phosphatase 1B provided by Dr. J. Dixon, dephosphorylation of [3'P]histone H4 was not observed.' The PPPB experiment has a good positive control for the activity of the phosphatase, ruling out fortuitous inactivation. The two negative results may be due to specificity either for the phosphoamino acid or for the amino acid sequence environment of the phosphoamino acid.

For PP1, PPBA, and PPZC, dephosphorylation of ["PI phosphohistidine in histone H4 was dependent on the amount of phosphatase added. With the gel assay, significant effects were seen with 0.5 milliunit/ml of PP1, 0.048 milliunit/ml of PPZA, or 0.012 milliunit/ml of PP2C. With the ultrafiltration assay, the time course of dephosphorylation was measured with 0.042 milliunit/ml (PPl), 3.1 milliunits/ml (PPZA), or 17 milliunits/ml (PPBC). These concentrations suggest that histidine phosphatase is a major activity of PP1, PP2A, and PPZC, at least as rapid as the serine phosphatase activity. A direct comparison of the reaction rate with [3'P]histone H4 and phosphorylase a for PP1 and PPPA confirmed the impor- tance of the histidine phosphatase activity (Table I). The quantitative comparison in Table I is valid only at the substrate concentrations used, but the [3'P]histone H4 concen-

J. Huang and H. R. Matthews, unpublished data.


tration was 1/10 of the phosphorylase a concentration and the phosphorylase a concentration was sufficient to give a reaction rate greater than V-/2. Thus, it is likely that at the same substrate concentration the preference for phosphohistidine would be even greater than shown in Table I. It is certainly quite clear that the histidine phosphatase activity is a major in vitro activity of these protein phosphatases.

The protein, inhibitor-1, specifically inhibited the activity of PP1 at concentrations down to 1 nM, and did not affect the activity of either PP2A or PP2C at concentrations up to 100 nM. Thus, the activity seen with PP1 against phosphohistidine is distinct from the activities seen with PP2A and PP2C. The activity of PP2C with the phosphohistidine substrate is completely dependent on the presence of M P . Neither the activity of PP1 nor the activity of PPPA required added M%+. Thus, the activity of PP2C with the phosphohistidine substrate is distinct from the activities shown by PP1 and by PP2A. Taken together, these data show that three distinct enzyme activities are capable of removing phosphate from phosphohistidine in a protein substrate.

The effects of inhibitor-1, okadaic acid, and Mg2+ mirror, at least qualitatively, their effects on serine/threonine dephosphorylation. Thus, okadaic acid at l nM inhibits PP2A while 100 nM is required for PP1; inhibitor-1 is completely specific for PP1. Thus, the histidine phosphatase activity of PPl/PP2A/PP2C is not easily distinguished from the serine/ threonine phosphatase activity.

The histidine phosphatase activity of PP1 and/or PP2A is expressed in cell extracts from the budding yeast, S. cerevisiae (Fig. 6). The inability of okadaic acid to inhibit all of the M%+-independent phosphatase activity may be due to the high protein concentration in the extract, which can interfere with inhibition by okadaic acid, or may be due to okadaic acid-resistant phosphatase. Further work is needed to distinguish these possibilities. S. cerevisiae also has a specific protein histidine kinase that has been purified and shown to phosphorylate exclusively histidine 75 in histone H4 in vitro (9). Proteins that contain phosphohistidine have been detected in a number of cells (3, 5), and identification of these proteins is now critical to further understanding of the role of phosphohistidine in eukaryotic cell proteins. There are two groups of reports in the literature on specific eukaryotic proteins that contain phosphohistidine: one group concerns nuclear proteins; the other concerns a plasma membrane protein. The first is the data from Smith and his colleagues showing phosphohistidine in histone H4 (1, 2). Nuclear proteins containing phosphohistidine were also found in Physa- rum but not identified (3). More recently, Das and his colleagues have shown histidine phosphorylation, stimulated by

the ras oncogene product, on a rat liver membrane protein,

The inactivation (or activation) of proteins following prein- cubation with PP1 and/or PPBA, and biological effects evoked in cells by inhibitors such as okadaic acid or by microinjection of PP1 or PP2A or transfection of their genes, is being used increasingly to suggest that physiological processes are regulated by serine/threonine phosphorylation. The present study shows that caution should be exercised when making such conclusions since some of these effects may be explained by changes in the histidine phosphorylation.

~ 3 8 (4,5).

Acknowledgments-We are grateful to Dm. J. Dixon and C. Klee for providing samples of protein phosphatases, Dr. D. A. Walsh and K. Angelos for samples of glycogen phosphorylase and phosphorylase kinase, Dr. I. Clark-Lewis for the peptide H4(70-102), and Drs. D. A. Walsh and M. Hanley for critical reading of the manuscript.

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Protein Phosphatases 1,2A, and 2C Are Protein Histidine