Specific dephosphorylation of phosphopeptides by the yeast alkaline phosphatase encoded by PHO8 gene

221

Biochimica et Biophysica Acta, 1177 (1993) 221-228 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13393

Specific dephosphorylation of phosphopeptides by the yeast alkaline phosphatase encoded by P H 0 8 gene Arianna Donella-Deana a, Sanja Ostoji6 b, Lorenzo A. Pinna and Slobodan Barbari6 b a

a

Dipartimento di Chimica Biologica and Centro per 1o Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Universitd di Padova, Padova (Italy) and b Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb (Croatia) (Received 19 January 1993)

Key words: Phosphohistone; Phosphopeptide; Dephosphorylation; (Saccharomyces cerevisiae)

Partially purified nonspecific phosphate-repressible alkaline phosphatase from Saccharomyces cerevisiae encoded by PH08 gene (rALPase), efficiently dephosphorylates phosphohistones and a variety of phosphopeptides. The pho8 mutant, constructed by disruption of the chromosomal counterpart of the PH08 gene, is lacking in phosphatase activity toward phosphopeptides, confirming that this activity is actually due to rALPase, rALPase activity tested on phosphopeptides is maximum in the pH range 6.5-7.5 and the K m values for these substrates are in the micromolar range, suggesting a possible physiological relevance of this enzyme as a protein phosphatase, rALPase dephosphorylates phosphotyrosyl more efficiently than phosphoseryl peptides, but is poorly active on phosphothreonyl peptides. Its specificity towards synthetic peptides and insensitivity to specific inhibitors and activators of authentic protein phosphatases indicate that rALPase differs from both Ser/Thr- and Tyr-specific protein phosphatases. This conclusion is consistent with the lack of homology with any class of known protein phosphatases.

Introduction

It has been previously reported that many nonspecific alkaline phosphatases, as well as acid phosphatases, from different sources could act on phosphoproteins [1,2]. It seems that a general property of these nonspecific acid and alkaline phosphatases is their higher efficiency toward proteins containing phosphotyrosyl residues as compared to those containing phosphoseryl or phosphothreonyl ones [1-3]. We have previously shown that purified nonspecific phosphate-repressible acid phosphatase from Saccharomyces cerevisiae [4] dephosphorylates very efficiently and specifically phosphohistones and several synthetic phosphopeptides in vitro [5-7], indicating a possible contribution of this enzyme to the overall protein phosphatase activity of yeast cells. Besides acid phosphatase isoenzymes, yeast S. cerevisiae contains two enzymes which hydrolyze p-

Correspondence to: A. Donella-Deana, Dipartimento di Chimica Biologica, Universit~ degli Studi di Padova, Via Trieste, 75, 35121 Padova, Italy. 1 This paper is dedicated to Professor P. Mildner on the occasion of his 75th birthday.

nitrophenylphosphate (pNPP) at alkaline pH. One is the so-called specific p-nitrophenylphosphatase (pNPPase) [8] and the other is the nonspecific repressible alkaline phosphatase (rALPase) [9]. The specific pNPPase is constitutively produced regardless of the inorganic phosphate concentration in the medium [9]. Recently, the structural gene P H 0 1 3 for the specific pNPPase was cloned and sequenced [10]. The physiological role of this enzyme has not yet been elucidated. The rALPase is a glycoprotein localized in the vacuole [11] and encoded by the P H 0 8 gene, which has been cloned [12] and sequenced [13]. The transcription of the P H 0 8 gene is regulated under the influence of inorganic phosphate present in the growth medium [8], through the action of several positive and negative regulatory proteins [14,15]. The physiological role of the enzyme is not clear at all. Interesting experimental findings have recently been presented, suggesting that rALPase could act as fructose-2,6-bisphosphate-6phosphatase [16]. This paper is the first report on the activity of yeast rALPase toward phosphoproteins and phosphopeptides. Clear and positive evidence is presented showing that rALPase can efficiently and specifically dephosphorylate different phosphopeptides and phosphoproteins in vitro. Kinetic properties and the speci-

222 ficity of the enzyme toward a variety of phosphopeptides have been examined and discussed in comparison with those of the authentic protein phosphatases. Materials and Methods

Yeast strains, plasmids and media The Saccharomyces cerevisiae strain YS18 (a, his311, his3-15, leu2-112, ura3-432, canR), kindly provided by Dr. W. Horz, was used as the wild-type strain. Escherichia coli JM103 was used as a host strain in the construction and propagation of plasmids. Plasmid pAL144, a generous gift from Dr. Y. Oshima to Dr. W. Horz, and plasmid pPZ [17] were kindly provided by W. Horz. Yeast cells were cultured at 30°C in YPD medium (2% Bacto peptone, 1% Bacto yeast extract, 2% glucose), from which inorganic phosphate has been removed (low-P i medium), ad described [18]. E. coli cells were cultivated in the complete Luria broth. For the selective media 2 5 / , g / m l tetracycline was added. Solid media contained 2% agar.

Materials Histone type II AS, angiotensin II (DRVYIHPF), okadaic acid, ealmodulin, phenylarsine oxide, a-naphtylphosphate, pNPP and Fast Red TR salt were all obtained from Sigma./3-casein was prepared as in Ref. 19. DEAE-Sepharose CL-6B and Utrogel AcA 22 were supplied by Pharmacia LKB Biotechnology, Restriction enzymes were purchased from Boehringer and zymolyase from Seikagaku Kogyo. All other chemicals were from Kemika (Zagreb). Peptides RRREEESEEEAA, DLDVPIPGRFDRRSVAAE and KKPLNRTLSVASLPGL were kindly supplied by Dr. H.E. Meyer (Ruhr-Universitat, Bochum), Dr. C.B. Klee (National Institutes of Health, Bethesda) and Dr. P. Cohen (University of Dundee), respectively. Other peptides were the generous gift from Drs. F. Marchiori and O. Marin (Universita di Padova). PP-1 and PP-2A were kindly supplied by Dr. J. Goris (University of Leuven) and PP-2B by Dr. C.B. Klee (National Institutes of Health, Bethesda).

RRREEESEEEAA, SEEEEE, TEEEEE were labelled by casein kinase 2 [21] and tyrosyl peptides by rat-spleen protein tyrosine kinase III [22]. Incubations (4 h) were stopped by adding 30% (v/v) acetic acid and [T-32p]ATP was removed by the ion exchange chromatography on a 2 x 0.5 cm Dowex AGIX8 column (Biorad) equilibrated with 30% (v/v) acetic acid. The eluate containing the phosphorylated substrates was lyophylised and dissolved in H20. The concentration of 32P-labelled substrates in the assays (calculated from specific radioactivity) was 4 /,M. No significant influence of the concentration of nonphosphorylated substrate on the dephosphorylation rate could be observed over the range 3-300/xM.

Enzyme assays The alkaline phosphatase assay with pNPP or anaphtylphosphate as substrate was performed at 30°C as described [23]. rALPase activity of colonies was detected by the staining method using a-naphtylphosphate as substrate [12]. The phosphatase activity of rALPase with phosphoproteins and phosphopeptides was assayed in 50/,1 of the incubation mixture containing 50 mM Tris-HC1 (pH 7), 5 mM MgC12, 5 mU of enzyme and 4 /,M 32p-labelled peptides, for 10 rain at 30°C. Reactions were stopped by the addition of 10% (w/v) trichloroacetic acid, and liberated [32p]phosphate was converted into its phosphomolybdic complex, extracted with isobutyl alcohol-toluene (1:1, v/v) and quantitated as in Ref. 24. The assay with each substrate was linear up to 10-20% phosphate release and dephosphorylation was kept within this limit to ensure that initial-rate conditions were met. One unit of enzyme activity is defined as the amount of enzyme which hydrolyzes 1/,mol of pNPP min-~ from 10 mM pNPP under standard assay conditions [23]. Average values from 5-10 determinations are shown and the standard error was always less than 14%. K m and Vm~x values were determined by double-reciprocal plots, constructed from the initial-rate measurements, fitted to the Michaelis-Menten equation. PP-1 and PP-2A were assayed as in Ref. 21, while PP-2B assay was as in Ref. 25.

Preparation of labelled phosphoproteins and phosphopeptides

Purification of rALPase

32p-labelled /3-casein was phosphorylated by casein kinase 2 [20]. After incubation for 4 h at 30°C, the reaction was stopped by the addition of 10% (w/v) TCA. Phosphoprotein was recovered by centrifugation, washed four times by resuspension in 10% (w/v) TCA, dissolved in 50 mM Tris-HC1 (pH 7) and dialysed overnight against water. Histone II AS, RRATVA and seryl peptides were phosphorylated by the incubation with the catalytic subunit of cyclic-AMP-dependent protein kinase from bovine cardiac muscle [21].

Yeast cells, grown to late log-phase in low-Pi medium in order to derepress rALPase, were harvested by centrifugation, washed in 0.1 M Tris-HCl/0.01 M MgCI 2 buffer (pH 9.0) and disrupted using glass beads in a Brown MSK homogenizer. Broken cells and other particulate material were removed by centrifugation and solid ammonium sulfate was slowly added to the supernatant up to 40% saturation. The solution was stirred gently overnight at 4°C, and after removal of precipitated proteins by centrifugation, the resulting

223 supernatant was brought up to 80% saturation with ammonium sulfate, stirred for 6 h and centrifuged. The sedimented material was dissolved in 0.02 M TrisHC1/0.01 M MgCI 2 buffer (pH 8.0) and dialyzed against the same solution over 72 h. The dialyzed enzyme solution was applied on DEAE-Sepharose 6B column (2.6 × 19 cm) equilibrated with the dialysis buffer. After washing off the column with one column volume of the starting buffer, bound proteins were first eluted with the starting buffer containing 0.075 M NaCI and then with 500 ml linear gradient of 0.09 M to 0.35 M NaCI. The fractions active on a-naphtylphosphate were pooled, concentrated by ultrafiltration and applied on an Ultrogel AcA 22 column (1.6 × 80 cm) equilibrated with 0.05 M Tris-HC1/0.001 M MgC12 buffer (pH 8.0). Proteins were eluted from the column with the same buffer and fractions containing rALPase were collected and concentrated.

Sall Bglll~

XhoI~

digested

with

digested with

IBgII andxhoI

IBg ITandSail

EcoRl

Xh o ~

Bglll I[

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'

tion

LEU~

Electrophoresis

Sacl EcoRI

The molecular weight determination of the partially purified rALPase was performed by gradient-gel electrophoresis as described [4], after detection of the enzyme by activity staining using a-naphtylphosphate as a specific substrate for rALPase [26].

PsiI~

OraI~

~nl

0~o~

DNA manipulation and analysis Transformation of yeast cells was as described by Ito et al. [27]. Yeast genomic DNA was isolated as described [28]. Plasmid DNA was isolated from E. coli according to the method described by Birnboim and Doly [29]. Southern blots were produced using Gene Screen Plus membranes and 1% agarose gels. DNA hybridization probe was prepared by the random hexamer priming method [30], using [a32p]dATP and dCTP. Other standard DNA methodology was performed according to Maniatis et al. [31].

Construction of the strain YSBK89 (pho8::LEU2) The construction of the plasmid pALBK89 used for the disruption of the PH08 gene was performed as shown in Fig. 1. Plasmid pAL144, containing 4 kb EcoRI-PstI yeast DNA fragment with PH08 gene subcloned in pBR322, was restricted with BgllI and XhoI to delete part of PH08 coding region, pALBK89 was then constructed by inserting the isolated 2.2-kb BgllI-SalI DNA fragment, containing LEU2 gene excised from pPZ, into digested pAL144. The resulting plasmid was digested with SacI and DraI and the obtained DNA fragments were transformed into YS18 strain to disrupt the chromosomal PH08 locus. Most of the obtained LEU + transformants, replicated onto minimal low-Pi medium, did not show rALPase activity by the staining method using a-naphtylphosphate as substrate. One of them (YSBK89) was selected and the

Fig. 1. Construction of plasmid pALBK89 used for disruption of the P H 0 8 gene. Construction of plasmid pALBK89 was performed as

described in Materials and Methods.

structure of its genomic DNA was analyzed by Southern hybridization. Results

Activity of the partially purified rALPase against phosphoproteins and phosphopeptides The nonspecific alkaline phosphatase was partially purified from derepressed yeast cells grown in low-P i medium by the procedure described in Materials and Methods. The nonspecific isoenzyme was separated from the so-called pNPPase by means of ion-exchange chromatography on a DEAE-Sepharose 6B. Two peaks of enzyme, active on pNPP, were resolved by this column, but while the second peak was also active on a-naphtylphosphate the first peak did not show any activity toward this substrate, consistent with the properties of the pNPPase (Fig. 2A). After the three purification steps the specific activity of rALPase was increased about 95-fold with a 14% yield. The molecular mass of the partially purified enzyme was determined by gradient-gel electrophoresis, after identification of the enzyme band by activity staining, as described in Materials and Methods, rALPase dis-

224 played an apparent M r of 130000, a value previously reported for the native enzyme [24] and in good agreement with the value calculated on the basis of the nucleotide sequence for the dimeric glycosylated enzyme [13]. Phosphoprotein phosphatase activity of alkaline phosphatase was first tested on 32p-labelled histones, phosphorylated with cAMP-dependent protein kinase, and on 32p-labelled casein. The results presented in Table I show that the enzyme dephosphorylates the phosphoseryl residues of histones but not the phosphothreonyl residue of casein. The activity of the enzyme was further examined with several synthetic phosphopeptides. As shown in Table I, alkaline phosphatase efficiently dephosphorylates the phosphoseryl peptide RRASVA, which represents the phosphorylation site of pyruvate kinase, but not the peptide RRA_TVA, in which Ser(P) was replaced by Thr(P). It may be worth mentioning that the maximum activity of rALPase (partially purified and therefore underestimated) to-

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100

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Fig. 2. DEAE-Sepharose 6B ion-exchange chromatography of proteins isolated from wild-type cells (A) and from pho8 mutant cells (B). Isolation of proteins and ion-exchange chromatography were performed as described in Materials and Methods. In collected fractions protein concentration ( ) and phosphatase activity on pNPP ( o o ) and on a-naphtylphosphate (e e) were measured. Concentration of NaCl in elution buffer was a shown

(. . . . . . ).

TABLE I Dephosphorylation rates o f phosphoproteins and phosphopeptides by rALPase

Enzyme activity is expressed as pmol of Pi min-1 released by one unit of rALPase. Phosphorylated residues are underlined. The concentration of substrates was 4 ~M. Substrates

Activity

[3Zp]Ser histone [32p]Thr/3-casein RR.A_SVA RRATVA SEEEEE !EEEEE DRVY_IHPF (angiotensin II)

61 <1 672 <1 625 166 2108

ward RRASVA resulted 57 nmol min -t mg -1 higher than that found for protein phosphatase 2A on the same phosphopeptide (24 nmolmin -1 mg -1) [21]. The enzyme preference for phosphoseryl over phosphothreonyl peptides was further demonstrated by the activity found on SEEEEE, which was much higher than that found on ! E E E E E . However, the rALPase showed the highest activity on the phosphotyrosyl peptide angiotensin II (Table I), indicating the preference of the enzyme for phosphotyrosyl rather than phosphoseryl substrates.

Genetic evidence that the dephosphorylation of phosphopeptides is catalyzed by alkaline phosphatase encoded by PH08 gene To have the incontrovertible proof that the activity of the purified enzyme preparation on phosphopeptides was actually due to the alkaline phosphatase encoded by PH08 gene, the yeast strain YSBK89 (pho8::LEU2) was constructed, in which the chromosomal PH08 gene was disrupted as described in Materials and Methods. The analysis of the structure of its genomic DNA by Southern hybridization, using the 1.5-kb 3zp-labelled EcoRI-BglII PH08 DNA fragment excised from pAL144 (see Fig. 1) as a probe, confirmed the replacement of the disrupted PH08 fragment for the chromosomal counterpart (not shown). The soluble fraction obtained from this mutant strain was subjected to two of the purification steps, consisting of (NH4)2SO 4 precipitation and DEAE-Sepharose 6B chromatography, used previously for the purification of the enzyme from the wild-type cells (see Materials and Methods). In a parallel experiment soluble proteins isolated from the wild-type strain YS18 were subjected to the same procedure. DEAE chromatography elution patterns of proteins from YS18 and YSBK89 are shown in Fig. 2A and B, respectively. The elution profile of the enzyme activity reported in Fig. 2B, shows only one peak of activity on pNPP and no activity on a-naphtylphosphate, confirming that con-

225 TABLE II

structed YSBK89 strain does not express rALPase. Protein fractions from mutant cells, corresponding to rALPase containing fractions from wild-type cells, i.e., eluted at the same ionic strength (fractions 92-112, Fig. 2B), were collected together and their activity was measured on the phosphopeptide RRASVA. While, as expected, the pool of fractions obtained from the wildtype strain actively dephosphorylated this phosphopeptide, the fractions isolated from the mutant did not show any activity even at a 50-times higher protein concentration. These results clearly demonstrate that the phosphatase activity measured toward phosphopeptides under the described conditions is due to the alkaline phosphatase encoded by the PH08 gene, i.e., n-M.2ase.

Rates of dephosphorylation of phosphoserylpeptides by rALPase Enzyme activity is expressed as pmol of Pi rain-1 released by one unit of rALPase. Phosphorylated residues are underlined. The concentration of peptides was 4/zM. Peptides a 1 2

A

RRRRAA_SVA

1086

5

RRRRAAAS__VA

1427 15 648

RRREEESEEEAA

7

RRLSISTES RR_SSLRA D L D V P I P G FDRRV_SVAAE R KKPLNRTLSVASLPGL NRTLSVA

8

9 IO 11

r

5

X"

916 976

Table III shows activities of rALPase towards phosphotyrosyl peptide substrates, most of which reproduce the sequences of phosphoacceptor sites of tyrosine protein kinases. All phosphopeptides tested were dephosphorylated at a rate higher than that found for phosphoseryl peptides (Table II). The broad specificity and similar rates of dephosphorylation shown by rALPase on phosphotyrosyl peptides, together with the highest activity displayed by the enzyme on the tripeptide [32p](Tyr)3 suggest that the most important determinant for this phosphatase might be the phosphotyrosine itself, and not primary or higher order structural features adjacent to phosphotyrosyl residue.

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204

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Peptides 1, 7, 8, 9 reproduce the phosphorylationsites of pyruvate kinase, phosphorylasekinase (a-subunit), ribosomal protein $6 and cAMP-dependent protein kinase regulatory subunit (Type II). Peptide 10 and its shortened derivative 11 are analogs of the N-terminal sequence of the glycogen synthase, where the serine not phosphorylated in vivo have been replaced by N and A, respectively.

a

In order to obtain information about the specificity of rALPase, its activity towards different phosphoseryl peptides was examined and the results obtained are summarized in Table II. The addition of N-terminal arginyl residues to the peptide RRASVA, as well as inclusion of alanine between arginine and serine, seems to improve the e n z y m e activity; the p e p t i d e R R R R A A A S V A was dephosphorylated more than 2fold faster than the peptide RRASVA. The enzyme also efficiently d e p h o s p h o r y l a t e d the p e p t i d e s RRI_~_ISTES a n d RRSSLRA, which reproduce the phosphorylation sites of phosphorylase kinase (a-subunit) and ribosomal protein $6, respectively. The peptide D L D L D V P I P G R F D R R V S V A E , representing the phosphorylation site of R-subunit of cAMP-dependent protein kinase (type II) was a relatively poor substrate. A similar dephosphorylation rate of KKPLNRTLSVA S L P G L (peptide n.10), which is related to glycogen synthase, and its shortened derivative (peptide n . l l ) was found, indicating that in this case the size of the peptide is not relevant.

951

1021

3 4 6

Specificity and kinetic properties of rALPase in reactions with phosphopeptides

Activity 636

RRA_SVA RRRASVA RRRRASVA

J 4

5

E

01 j

I

6

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7

8

9

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5

6

I

I

I

7

8

9

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I

I

I

I

I

6

7

8

9

10

pH

Fig. 3. Effect of pH on the activity of the purified rALPase on phosphopeptides DRVYHPF (A), RRASVA (B) and on pNPP (C). Enzyme activitywas measured in 50 mM Tris-acetate buffer (e) or in 100 mM Tris-HC1buffer (O), as described in Materials and Methods.

226 TABLE III

TABLE V

Rates of dephosphorylation of phosphotyrosyl peptides by rALPase

Influence of various effectors on the activity of rALPase

Enzyme activity is expressed as pmol of Pi m i n - l released by one unit of rALPase. Phosphorylated residues are underlined. The concentration of peptides was 4/zM.

Activities are expressed as percentages of those obtained in the absence of effectors. The concentration of substrates was 4 tzM. Effector

1 2 3 4

5 6 7 8 9 a

Peptides a

Activity

DRVY_IHPF AFLEDFFTSTEPQ_Y(IPGENL EPQ_YQPA EDNEYTA GVYAASG KDDEYNPA EKEYHAE EE_YAA [3ZP] (YYY)

2080 2100 3403

none MgCI 2 ZnCI 2 MnC12 EDTA orthovanadate pNPP

2829

2460 2501 2892 2868 6932

Peptides reproduce: 1, angiotensin 1I; 2, 3, 4, two major phosphoacceptor sites of pp60C-src; 5, 6, 7, tyrosine containing sequences of the protein kinases fes, fgr and EGF receptor, which are homologous to the main autophosphorylation site of pp60 c'src.

To better characterize the dephosphorylating activity of rALPase toward phosphopeptides, the pH optimum and kinetic parameters of the enzyme were examined. As shown in Fig. 3, the enzymatic activity toward pNPP exhibited a pH optimum between 8.5 and 9.0, while the maximum activity toward phosphopeptides RRASVA and DRVYIHPF was in the pH range 6.57.5. K m and Vm~, values, as well as the efficiency of the enzyme in reactions with several phosphotyrosyl and phosphoseryl peptides are shown in Table IV. K m values for both types of peptides were in the same range, from 4 to 20 /zM, whereas Vmax values, and therefore enzyme efficiencies toward phosphotyrosyl peptides, were 2.5-7-times higher than those found toward phosphoseryl ones. The influence of different effectors on the enzyme activity toward phosphopeptides RRASVA and DRVY_IHPF as well as pNPP was examined and the

Kinetic constants for rALPase with phosphopeptides Experimental conditions are detailed in Materials and Methods. For description of peptides see legends to Tables II and III.

DRVYIHPF AFLEDFFTSTEPQYQPGENL EPQY(;IPA RRA_SVA RRRRAA_SVA KKPLNRTLS_VAS LPGL NRTL_SVA

(7 mM) (0.5 mM) (6 mM) (1.2/xM) (100/zM) (800/zM)

DRVY_IHPF

RRA_SVA pNPP

100 109 111 98 21 9 <1

100 112 113 102 23 4 4

100 208 140 160 54 60 -

results obtained are shown in Table V. Mg z÷ ions and, albeit to a lesser extent Zn 2+ ions, stimulated the enzyme activity toward pNPP, but had no effect on that toward phosphopeptides. Orthovanadate and EDTA acted as inhibitors of both activities, being more effective on the peptides dephosphorylation. The addition of pNPP in the concentration range of the K m value of the enzyme for this substrate caused the complete inhibition of the peptide dephosphorylation. The effect of some known specific inhibitors and activators of the main classes of protein phosphatases was tested on the rALPase activity toward RRRRAASVA, which is substrate for the authentic protein phosphatases [21,32]. Okadaic acid, a powerful inhibitor of types 1 and 2A protein phosphatases [33], did not significantly decrease the dephosphorylation rate of RRRRAASVA by rALPase even at the concentration (0.1 tzM) that completely inhibited the dephosphorylation of this phosphopeptide by PP-1 and PP-2A protein phosphatases. Neither the tyrosine protein phosphatase inhibitor phenylarsine oxide [34,35], nor calmodulin and Ca z+ (or Ni2+), which are required for the maximum activity of type 2B protein phosphatase [36] significantly affected the rALPase activity (not shown). Discussion

TABLE IV

Peptides

Activity on:

Vmax (pmol min-I U - I )

Km (/xM)

Efficiency (Vm~x Km ~)

6640

8.5

781

4800

3.8

1262

14560

10.0

1456

2 680

12.8

209

6310 1768

20.1 5.6

314 315

2040

7.0

291

The regulation of the yeast rALPase synthesis by the presence of inorganic phosphate in the growth medium, as well as its activity towards a variety of phosphomonoesters, suggests that the formation of free phosphate is a physiological function of the enzyme. The dephosphorylating activity of the partially purified rALPase on phosphoproteins and phosphopeptides, reported herein, would indicate an additional role of this enzyme as a protein phosphatase. The clear-cut demonstration that the peptide dephosphorylation is indeed catalysed by rALPase, the P H 0 8 gene product, was provided by experiments showing that this activity is absent in a constructed p h o 8 mutant strain.

227 Some features of phosphopeptide dephosphorylation are different from those of pNPP dephosphorylation. The enzyme activity on phosphopeptide substrates is maximum between between pH 6.5 and 7.5, whereas that on pNPP shows the optimum in the pH range 8.5-9.0. This finding indicates that the protein phosphatase activity of rALPase could be physiologically relevant. The micromolar range of the apparent grn values of the enzyme for phosphopeptides is also consistent with a physiological relevance of the observed activity. In respect to pH-optimum and K m values, the activity of yeast rALPase resembles that shown on phosphoproteins by nonspecific alkaline phosphatase from higher organisms [1,3,37]. Moreover, the K m values displayed by rALPase are comparable to, or even lower than, those of some authentic protein phosphatases for the same, or similar, phosphoseryl peptides [21,32,38]. The results presented in this paper clearly show that rALPase is distinct from the authentic protein serine/threonine phosphatases, consistent with its lack of homology with this class of enzymes [39,40]. One of its distinguishing properties is the selectivity toward phosphoserine and phosphothreonine residues. It is well established that the four principal types of protein phosphatases (PP-1, PP-2A, PP-2C, and to a lesser extent PP-2B) prefer phosphothreonyl peptides over their phosphoseryl counterparts [21,32,41], while rALPase is rather poorly active toward phosphothreonyl peptides (Table I). The effect of specific inhibitors and activators of authentic protein phosphatases is another distinctive property. Okadaic acid, a very potent specific inhibitor of PP-1 and PP-2A from both mammalian and yeast cells [33,42], does not affect rALPase activity on phosphopeptides. In contrast with PP-2B, which is Ca2+/calmodulin-dependent [36] and with PP-2C, which exhibits an absolute requirement for Mg 2÷ [32,43], rALPase does not require any of these activators in the reaction with phosphopeptides (Table V, and data not shown). It is well known that certain membrane bound glycoproteins are protein tyrosine phosphatases [44]. The glycoprotein nature of yeast rALPase [23], its localization in the vacuolar membrane [11] and the preferred dephosphorylation of phosphotyrosyl substrates (Table IV), could be taken as an indication that yeast rALPase is a phosphotyrosyl protein phosphatase. However, some features of rALPase are different from those of phosphotyrosyl protein phosphatases, rALPase is strongly inhibited by EDTA, slightly activated by Zn 2+ (Table V) and not affected by phenylarsine oxide, while most of phosphotyrosyl protein phosphatases show optimum activity in the presence of EDTA [45], are generally inhibited by Zn 2+ [46] and in some cases are also inhibited by phenylarsine oxide [31,34]. Another distinguishing property is the lack of selectiv-

ity of rALPase for phosphotyrosyl peptide substrates (see Table III), in contrast to phosphotyrosyl protein phosphatases which appear to be more selective [47]. Most features of the phosphopeptide dephosphorylating activity of yeast rALPase resemble those of yeast-repressible nonspecific acid phosphatase encoded by PH05 gene [5-7]; however, some differences should be underlined. Acid phosphatase shows much higher efficiency for the phosphoseryl peptide, RRASVA, than for the phosphotyrosyl peptide DRVYIHPF [6], while rALPase displays an opposite preference (Table IV). In the reaction with phosphopeptides, both enzymes display a shift of pH optimum towards neutrality, but the pH optimum of rALPase is found in the pH range 6.5-7.5, while that of acid phosphatase is between pH 5 and 5.5 [7]. It may be interesting to note that the observed pH optima of the two enzymes are in agreement with the different pH values of the cellular compartments in which they are located; rALPase being a vacuolar [11], while acid phosphatase is an extracellular periplasmic protein [18]. This finding also suggests a possible physiological relevance of the apparent protein phosphatase activity of yeast nonspecific alkaline and acid phosphatases, which nevertheless remains to be assessed. However, it should be noted that rALPase may significantly contribute to the overall protein phosphatase activity assayed at the neutral pH in the intact yeast cells or crude extracts. The results presented in this paper also show how synthetic phosphopeptide substrates and specific inhibitors may be used to disclose the rALPase contribution to the overall protein dephosphorylating activity of the cell.

Acknowledgments This work was supported by AIRC, and Italian M.U.R.S.T. and C.N.R. (Target Project on Biotechnology and Bioinstrumentation to LAP) and by the Ministry of Sciences, Technology and Informatics of the Republic of Croatia (Grant 4-27-014 to SB). This study was also supported by a bilateral agreement on scientific cooperation between the Universities of Padova and Zagreb. SO is thankful to FEBS for a generous summer fellowship and Dr. W. Horz for the hospitality in his laboratory and advice during the construction of the YSBK89 strain.

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