1134( 14~921129-136 © 1992ElsevierSciencePublishemB,V, All rigb~ reserved016%4889/92/$(]5,00 Blochlmica et Biophysica Acla,

129

BBAMCR [3113

Identification of protein phosphatase activities in maize seedlings I z a b e l a J a g i e i t o ~, A r i a n n a D o n e l l a - D e a n a b, J a d w i g a S z c z e g i e l n i a k ", Lorenzo A. Pinna b and Gra~jna Muszyhska ~ and

# Dipartimento

a Institute of Biochemistry und Biophysk=~. Polish Academy of Sciences, Warsaw (Poland) di Chimlea BioloRtea and Centro per Io Studio della FLriologia Mitocondriale del C.N.It, Unlcer~ra di Padoco. Padocu Iltaly)

(Received IS October 199t) Key words: Phosphorylation;Pro:air, phosphalase; Maize seedling; phosphohism~e HI; PhOslphoea~imP'nosplmpq~ide Three phosphatases active on phosphoeasein (_PhosphoC_aseinPhosphatases) termed PCP-I, P(~P-il a d PCP-tI! were Lq31ated from maize seedlings by DEAE-celI,,Iose chromatography and were shown to display a different specificity toward a 1rudely of phosphnrylated substrates includinB ptqPP, phosphohistones, phosphowlase a and several plmsphopeptides containing either

phesphoserine or phosphoth~onine. PCP-] and PCP-ll hind to heparin-Scphar~C, retain a remarkable pNPP activity, are ancapable to dephosphoryiate phosphorylasc a, and display striking activity toward the acidic pho~hopeptide AS[32p~.EEEIE. They also by far prefer phosphosexyl peptide RRAS[3zP]VA over its phosphothreonyl derivative and are up.semitive to okadaic acid up to 1 p.M. These properties arc not consistent with the belonf~ng of PCP-1 and -II to any of the known classes of protein phosphatases and suggest that they are acidic pho~phatases. Conversely, PCP-II! is cS.~ntial[y free of pNPP activity; il readily dephosphorylates phosphohistone HI and phosphorylase a and it displays a striking preference toward the p h o ~ p h o t h ~ peptides (RRAT[~P]VA and RRREEET[~±P]EEEAA), while ihe phosphoseryl peptides (RRASI~2p]VA and AS[~P~EEEE) are very poor substrates of the enzyme. These properties together with the findings that PCP-HI does not bind to hepafiw Sepharose and is highly sensitive to okadaie acid (ICsn - 0.2 nM) allow to identify PcP-m wilh a plrot¢i~ pi~ephata.~ of the PP-ZA class. latmductlen Over the past few years accumulating lines of evidence strongly suggest that phosphorylation/dephosphorylation of proteins in plants is a widespread mechanism of intraeeliugar regulation of Browth and control of signal transduction [1-4]. There are indications that phospho~lation is involved in the regulation of nucleic acid synthesis either via modifications of non-enzymatic ehromatin proteins a n d / o r RNA polymerases [5-8] and in the regulation of plant protein synthesis [9,10l. It is also proved that at least seven specific en23'mes in plants are regulated by reversible phosphorylation [3]. The phosphorylation

Abbleviatioes: PKA, cyclicAMP-del~ndeat protein kinase; PP-I and pp-zA, 2B, 2C~ l~otein phusphutmestype 1 and type 2A, 2B, 2C; PCP. phosphocaseinphusphalase from maize scedlinB¢PMSF, phenylmethylsulphonylfluoride; pNPP. pnitrophnny] phosphate; DTT, dithiothgcitol;ICon,inhibitionconslanl(concentrationcausing 50% inhibition). Con~espomdence:(3. Mu~y6ska, Institute of Biochemistry~;0dBiophysics, Polish Academy of Sciences, 36 Rakowieck~ 5c. 02.532 Warsaw, Poland.

stalus and activity of some of them (chloroplastic pyrnvale Pi dikinase, phosphoenotpjmvate c a d a ~ l a s e , quinate dehydrogenase and s~,'rose-phc~hate synthese) are shown to be light dependent [4,11,12] confirming that light is the external signal in plants for protein phospimwlation events. Also, plant honmmal regulation seems to be affectod by protein p h o ~ h o rylation [13]. The phoep~rylation of plant proteins mediated in some cases by Ca2+/¢almodulin dependent protein kinases is the major mechanism by which second mesengers calcium and calmodulin regulate biochemical events inside the cell in response to external stimuli [14,15]. Presem[y, more data are available about plant prorein phosphatases ( ~ v e d in Refs. 2--4) although f-agmentary information is available about their physiological roles [2,3,12[- Protein phosphatases I~ve been identified in different tissues of both monm and dicotyledonous plants, hl cylosol as well as in the membrane fractions [16-24]. Plant protein phospharases exin'bit a remarkable similarity to definite types of animal protein pbesphatase in terms o f substxate specificity, requirement for divalent cations and sensitivity to specific inhibitors (protein inhibitor-1 and -2, okadaic ~¢id 8r~d microcysdn LR) [23,25,26]. Besides bioehemi-

130 cal similarities between PP-1 and PP.2A from mammals and plains, a high degree of primary structure identities between Brassica napus and rabbit protein phosphatases type-1 and -2A was elucidated by eDNA cloning [27]. The observation that the activity of specific casein kinases [28] and histone kinase(s) [29] is very pronounced in young maize seedlings prompted us to investigate the dephosphorylating system which is able to reverse the protein kinase activity. The aim of the present study was the identification and characterization of phosphatase activities present in maize seedlings extract and their comparison with typical classes of protein phosphatases, as have been classified in animal tissues [30].

Materials and Methods

Biological material. Maize (Zea mays) seeds, after soaking for 12 h, were grown for 72 h in the dark at 26°C. The apical parts of the seedlings were harvested. The material was placed directly into liquid N z and stored at - 7ff'C. Extraction and initial purification of the enzyme(s). 40 g of frozen maize seedlings were powdered with glass beads in the mortar and then homogenized with 2.5 volumes of buffer A. After 15 rain centrifugation at 12000 rpm, the supernatant was filtered through 'Miracloth" and precipitated to 60% of saturation by ammonium sulphate. The suspension was centrifuged for 45 rain at 12000 rpm. The collected precipitate was suspended and homogenized in buffer B, then centrifuged for 1 h at 45 000 rpm. 4°C. The supernatant was defined as 'high speed supematant'. "High speed supcrnatant' was treated with f'm~ volumes of 98% ethanol at 4°C and the precipitate was collected by centrifugation at 6500 rpm for 5 rain. The precipitate was then suspended in 60 mi of buffer B, homogenized and centrifuged at 16500 rpm for 15 rain. The supernatant was dialysed overnight against buffer C and applied onto a DEADcelIulose column (1.6 x 9.5 cat), previously equilibrated with the same buffer. The column was washed with buffer C and 3.2 ml fractions were collected. The breakthrough material, exhibiting the phosphatase activity was named PCP-I. Washing of the column was continued until the phmphatase activity and protein were completely eluted. Then, a linear gradient of 0-0.5 M NaC1 in buffer C of total volume 140 ml was used, The separately collected peaks (named PCP-II and PCP-III) were concentrated and desalted. All steps of enzyme extraction and further separation were carried out at 2-4°C. Cheraica~, Chemicals were purchased from the following sources: dietbylaminoethyl cellulose DE-52 and

ion exchange chromatography paper Cellulose Phosphate P-81 were from Whatman Binsystem Ltd. (Maidstone, U.K.); heparin-Sepharose CL-6B, PD-10 columns, Sephadex G-25M and Sephaeryl S-2GO were from Pharmacia (Uppsala, Sweden); EGTA, EDTA, Tristan-base, Mes, Pipes, PMSF, 2-mercaptoethanol, DTT, bovine serum albumin (BSA), histone H1 Type V S (lysine-rich subgroup fl), p-NPP and protein kinase catalytic subunit were from Sigma (St. Louis, Me); RRREEETEEEAA was obtained from Multiple Peptide System (San Diego, U.S.A.); dupeine, RRASVA, RRATVA and ASEEEEE were a generous gift from Dr. F. Marchiori (University of Padova, Italy); phesphorylase a and PP-2A (PCS-typc) were a gift from Dr. J. Gods (University of Louvcn, Belgium) [31]; okadaic acid derivative (prepared as in P,ef. 43) was a generous gift from Dr. Alastair Altken (National Institute for Medical Research, London, u.K.~, [7-~2P]ATP was prepared according to the procedure of Ref. 32 in the Institute of Biochemistry and Biophysics, Polish Academy of Sciences. Buffers. Buffer A contained 250 mM Tris-HCI (pH 7.8), 2 mM EDTA, [0 mM 2.mercaptoethanol,0.5 mM PMSF and 250 mM ammonium sulfate. Buffer B contained $00 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.25 mM PMSF and 0.1% (v/v) 2-mercaptoethanol. Buffer C contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0:25 mM PMSF, 0,1% 2-mereaptoethanol and 10% (v/v) glycerol. Buffer D as buffer C plus 0.5 M NaCi. Buffer E contained 100 mM Tris-HCl (pH 7.5), 0.01% Brij 35, 0.05 mM PMSF, 0,5 M NaCI and 10% (v/v) glycerol, Buffer F contained 100 mM Tris-HCl (pH 7.5), 0.5 mM DTT and BSA 1 mg/ml. Phosphorflation of peptides and proteins. Phosphorylase a, dupeine and synthetic peptides were routinely phusphorylated using phosphorylase kinase, ca. sein kinase type-2 or c-AMP-dependent protein kinasc under conditions previously described [33,34].

Preparation of 3ZP-labeledand nonradioactive phosphocaseine. Casein (2 rng/ml) was incubated with 3200 units of maize seedlings casein kinase-1 and 40 nine[ of [~,-32p]ATP (1400 cpm/pmol) for 2 h at 37°(3 in 250 pl of 40 mM Tris(pH 7.5) containing 8 mM MgCI 2. After phosphorylation the excess radioactive ATP was removed on a PE.-10 column. Nonradioactive phosphorylation of casein was performed under the same conditions, only unlabeled ATP was used as the phosphate donor.

Preparation of 32P-labded and nonradioactipephos. phohistone 111. Histone H1 (4A mg/ml) was incubated with 500 units of PK-A and 25 nmol of ['y-32P]ATP (3000 cpm/pmol) for 2 h at 3ff'C in 250/zl of 20 mM Mcs-NaOH (pH 6.5) containing 10 mM MgC] z. After phesphomylation the excess ATP was removed through filtration on a PD-10 column. Non-radioactive phosphorylation of histone H1 was performed under

131 the same conditions, only unlabeled ATP was used as the donor of phosphate. The background contamination of phosphorylated proteins by ATP was in the range of 3-6% of total content of the nucleotide. Phosphorylating units defimtiorc One trait will transfer I pmol of phosphate from [T-32P]ATP to hydrolyzed and partially dephosphorylated casein per rain at pH 6.5 at 3(YC and at 37°C for the PK catalytic subnait and maize seedlings casein kinase, respectively. Protein phosphatase assay procedures~ Dephospho~ rytation was performed with the following phosphorylated substrate: [32P]casein, [3~P]histone, [32P]phosphorylase a, [32P]clupeine or appropriate [-~2P]phosphopeptide. For routine assaying of phosphoprotein phosphatase activity, the [32p]casein (or [32plhistone HI) was mixed with nonradioactive phosphocasein (or nonradioactive phosphohistone HI) to give a specific radioactivity of 1~0 cpm/pmol of phosphorytated substrute. The concentration of 32-labelled substrates in the assays (calculated from the specific radioactivity) was 1 t~M. The phosphorylase a concentration was 10 pM. Each substrate was incubated at 37~C for 10 rain with maize phosphatase preparation in the reaction medium containing at the final volume (50 ~tl~. 20 mM Tris-HCl (pH 7.5), 0.t mM D'l*r, 0.2 mg/ml BSA, 0.02 mM EGTA, 0.05 mM PMSF and 0.02% ,8-mcrcapto-

ethanol. Procedure 1 was used routinely for determination of [32P]casein and [~2piphosphorylase a dephosphorylation. The reaction was terminated b,/the addition of 0.1 ml BSA (6 mg/m]) and 0.1 ml 20% TeA. The suspension was allowed to stand for 10 rain on ice and then was centrifuged at 13000 × g / 2 rain. The precipitate was removed and an aliquot of 200 ~1 of the supernatant was mixed with 2.5 ml of dioxane based scintillation fluid and counted for radioactivity. Procedure 2 was used for determination of [3ZP]histone dephosphorylation. The enzymatic reaction was stopped by adding 22 ~1 of glacial acetic acid to a final concentration of 30% and 50 ~1 aliqunts of the reaction mixture were spotted on P-81 paper discs. The discs were successively washed in 30% acetic acid (t~o times 20 ml), 15% acetic acid (once 20 ml) and acetone (once 20 ml), dried and counted for radioactMty in 5 ml toluene based scintillation fluid. Procedure 3. The phosphop~'ptide phosphatase activity was terminated by addition of "['CA to a final concentration of 5% and 32pt was converted into its pbosphomolybdi¢ complex, extracted with 1:1 (v/v) isobutyl alcohol-toluene and determined as in ReL 31. In the procedures 1 and 3, the amount of P~ liberated was taken for calculation of phosphatase activity, whereas in procedure 2 the degree of dephosphorylation was estimated by the subtraction of the sample after enzymatic treatment from the control sample (total [32p]proteit0.

The control incubation was performed with all the components except the enzymatic preparation under the described conditions. All phospbalase assays were carried out with enz ~ e concentrations such that the reaction was linear. One unit of phosphatase activity was defined as the amount of enzyme which releases 1 pmol Pi from protein substeate per rain at 37°C at pH 7.5. Specific activity was defined as units per mg of protein. Hydrolysis of p-nitroptwnyl phosphn~" Hydrolysis of p-nitrophenyl phosphate (pNPP) was performed in an incubation mixture of total volume 100/zl (containing 20 mM pNPP, 0.33 M Tris-HCI (pH 8.0) and the appropriate amount of enzyme) at pH 8.0 for l0 rain at 37~C. The reaction was stopped by adding [ ml of I M NgOH and product was measured at 410 am. Protein content. The protein content was determitied by the method of Bradford [35] using BSA as a standard. Gelfihralion. Gel filtration was performed either on FPLC (Pharmacia LKB) apparatus equipped with Superose 6HR 10/30 or on Sephacryl S-200 (1.6 X 82 cm) columns. The columns were quilibrated and developed with buffer E and calibrated with the folknving proreins: ribonuclease A (13.7 kDa)' ehymott3q~sinogen(25 kDa) ovalbumin (45 kDa)' bovine serum albumin (67 kDa). Fractions of I ml were collected. Results

Detection of phosphoprotein and phosphopeptide l~Tsohatase activitg in maize seedlings ~tracts Among the tested phosphoproteins [32P]phosphorylase a, [3~P]casein and a mixture of [32p]histon¢~ (historic H2a and H2b) are efficiently dephe6phorylated by the 'high speed supematant' of maize seedlings extract. Dephosphorylation of [~2P]clupein¢ is negligible. The same enz,~atic preparation very effectively dcph~l~aorylates phosphc~erine as well as phosphothreomne of acidic oligopeptides (AS[~2P] EEEEE and RRREEET[32p]EEEAA). Conversely, the dephcephorylation of bask: phosphoseryl peptides like RRLS[~2P]SLRA, RRLS[32p]ISTES, RRAS[32p]VA, RRRRAS[~2P]VA, RRRRAAAS[32p]VA r~roaeeds very slowly (Table !).

The influence of ethanol treatment on protein phos* phalase aet~L,i!ies Phosphatases present in maize seedlings high speed sopernatant are effectively (5-fold) purified by the ethanol treatment. The total phosphatase activity does not decrease after this step of purification and even protein phosphatase activity toward [x2p]histone HI as a substrate is relatively (40%) higher (Table II).

132 -i

ilpp-~llvll~

lOO z l l O ~ )

Fig.3x

1 I

I" r'"8

82.pi-I'OfeMIKI lopmxlO "a )

Protib (mg/ml)

0.011

o.0 [NaCI {M)

.................

°+'

A ........

°

/l/i

0,05

-

4.o

• ,!..6

+ ~'.o

,1.0 0'02 '

o SO

109 Frlcuon iluma#r Fig. !. Elution of [3~PJcascie (&), ['~zP~istone (o) and pNPP ( D ) phn~phatase aftivilies by chmmalograp~y oa DEAE-fc]IaI~¢.

' O.g

~.~',

~

IS

,

.

10

W

.

.

.

:!0

.

.

.

28

.

-

~0

a6

40

.

,

~

48

aO

0.0

El

Ffaotion number Flo,Sb

Resolution of protefi¢ phosphatase activities by DEAEcellulose ck~natography Chromatography of th© ethanol-prceipitalcd en~mc fraction on DEAE-cellulose gives rise to three main peaks of casein phosphalasc activity (Fig, 1) a heterogeneous peak (PCP-IA and PCP-IB) is eluted with breakthrough materia[, whereas two other peaks (PCP!I and PeP-Ill) are ¢luted by 0.05 M and 0.2 M NaCI, respectively, pNPPase activity is especially high in PCP-ll and quite detectable in PCP-I; however, it is neglig~le in PeP-Ill (see Table II).

0.t0 , l ~ l l l t 01~)/m]) o.s ~NaOl4M]

i

O~l

0 k. . . . . . . . . . . . . .

apparent m o l e c u l a r mass o f P C P - | l l was t o o low t o be

- 10 -8

0.04 -

~I -4

~

0.4):1 -

-'

.

.

0,14

a

. . . . . . -c D.

.

.

.

.

.

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I.

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P r o m ~ (n~tmO

,•

o,6 [N~u~(MI

'

...... ^ . . . .

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Fraction number

The apparent molecular masses determined by FPLC ~e[ filtration on Superose 6 HR were approx. 70 kDa, for both PCP-I and PCP-II (data not shown). The

~

". . . . . . . . . . . . . . .

0,O0

General properties of phcsphocasebz phosphatases

0.2- -

82PI-mleaaed (¢pm ,x 1Oral re ........

........

o u-o-*'"'"'"'"'+

- nt

"'""+'°'*"

s

0,04 •

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0JP6

2

0

45

EO

as

,~0

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ml pl ilUOll

ao

B5

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Fig. 2. Gel chromalogrnphy of maize seedling phosphoplole[~ phosphatase (PcP-nl) on a Sephacryl $-200 column. The protein molecular mass markets were: (.6.) BSA, 68 kDa; (B) ovalbuelln, 45 kDa; {C) chymolrypsyflogan, 25 kDa; (]3) ribonec]ease A, 13.7 kDa; Vo, blue Dextraa 2000. ~x, [~2P]casc[n phosphalase activity; o, [~=P]hislone HI p h ~ p h a t ~ e activily, • , protein (m$./ml).

S

tO

28

So

40

6o

410'

FruoUon number

Fig. 3. Elution profile of maize seedling phosphatas~s on hepariReSepharc~e columns. (a) PCP-IA; Co) PCP-ll; (c) PCP-I][+ The desalted material (appx~ox, I ms) from each peak after DEA~-¢ellulos¢ column warn separately applied onto a heparine-Sepharose column ( I . 2 x $ cm) previously cquUibrated with buffer C. The column was washed with 24 ml of the ~ buffer and 2 ml fraction~ ,,uqre collected. Then, n linear gradient 0-0,5 M HaCI (buffer D) ot mzal volume 80 ml was used. The f~ctions were tesled on the [~2p]casein (.'.), [3~P]hismne HI ( o ) and [z'zPlphosphorslase u ( x ) dephospho. wlaliag activity (the activity in the ease of the last substrata is 4-fold hisher).

133 :'l"rele~eO ( l ~ J A.

TABLE I

Detecelon of deollosphorylalin~ activities In maize seedling extract 'High speed supe.rnalanz' was the s o u r ~ of the enzyme ami every reaclion mixlur¢ contained 50 pmol of Pt it~corpmaled into tested

E ,1410~'~ e:l

"l

substrnte,

I¢~ S~bstrate

Acti,~ily

[ 3zP|Pho~phorylase a [ ~2P]Casein [32 P]Histones (H2a i H2b) ['~2P]Clupeine AS[32P]EEEEE RR].S[ 32p]sLRA RRLS[ 32p]ISTES R]~AS[ ~2P]VA RRRRAS[~2P]VA RRRRAAAS[~2 P]'VA RRS[3~ P]T[3;~P|VA RRREEE'~ ~ P]EEEAA

specific ( U / m 8 pmeein) $4i 12~ 56 4 479 21 8 2 45 57 101 34.5

relallve (is %) lflO ~_~ l0 I 89 4 2 < t 8 I) 19 64

eli

o e



,

02

10

accurately evaluated on Superose 6HR and was determined on a Sephacryl S-200 column to be approx. 28 kDa (Fig. 2). PCP-I and PCP-II bind to heparin-Sepharose and ate elutcd by 200 mM NaCI (Fig. 3a, b). In contrast, the major part of PCP-III activity does not bind to the gel. It should also be noted that the minor fraction of PCP-III which binds to heparin-Sepharos¢ is devoid of phosphorylase phosphatase activity, while the main peak eluted at the void volume retains the whole activity toward phosphorylase a (Fig. 3c). PCP-I and PCP-I! exhibit broad pH optimum profiles (4.0-5.6) for hydrolysis of pNPP, whereas their optimal pH for dephosphorylation of [~P]casein is shifted toward neutral values (pH 6.5-8.0) (Fig. 4a, b).

i



>, C.

lit*

OOll

Substrate ~'cO~c~

a

The analysis of substrate specificity (Table l i d shows that the acidic phosphopeptide AS[~ZP]EEEEE is the preferred substrate by PCP-ll, followed by RRREEET[32p]EEEAA, [32pkasein and RRAS[32P]VA. Dephosphorylatioft of [~P]histonea and RRAT[a2p]VA is ¢o~nparatively very low and phosphorylase a is not

r

t

a

Fig. 4. pH dependence o f the maize secdlinll phseldml~m~ activithut (separaled on DEAE.cellulme column) t o v a ~ : l = p k a ~ n ( z ) , [~P~iSlone HI ( 0 ) and pNPP ( Q ) e~ a subsqntc. (A) P O - I a (the protein ~nncentrntion was O.'r~l mg/rel); (B) PCP-ll (.moleia concentration 0.106 rag/rot) and (C) ][q::lP-11[ (pro~in omtcentnllion 0.542 ml/ml).

TABLE I!

~e ~ Substrate

of smc~#c and total protein pttosi~c~r ¢~'¢iui~es 'before" and ' Q~er' ethanol Ire~trr~ in n m ~ ~ Specific ~cftvill4U/m8 protein)

Tnta] acti~y (U)

'l~ore'

'before'

'after" ethanol txealment

[~¢P]l-listone H I [~P]Casein pNPP

249,4 76.4 6.6

t 918.0 440.0 34.,5

47 f',88 ]4616 1 254

~

'after" ethanol treatment

(%)

Rlattve a c d v ~

62297 15214 1191

139 104 95

134 TABLE lU

The substrate spe4cificity of maize zecdling phosphat~es sccar~ted on OEAE-celhalo.t¢ column The concentration calculated from "~2Pi incorporated, was 1 /zM for the synthetic phosphopeptides and 10 .aM fur phosphowlase a. The prol¢in concentration in PCP-IA, -IB, PCP-II and PCP-Ill tested fractions was O.]t)7 ( m s / m l ) ; 0.206 { m s / m l ) ; 0.502 ( m g / m l ) , resp~ctlvely.

0.468 { m s / r o D and

Phosphopro~einphospbataseactivity (U/ms protein) and p-NPP activity (E,4m/mg protein per rain) uP,

SoUstrnte

PC'P-IA

PCP-IB

O.0 208,5 51.0

O.0 471.0 66.2

0.0 18385.5 6094.6 404.6 137.0 2179.5

63.4 17.8

105.5

1593.7

289.1

20.3 1018.9

2ffL2 9610.4 34130.8 108.6

3289.,I. 2885.1 241.1 2.5

[3;~P]Phosphorylasc a [32p]Casein [S=P]Hislone HI RRAS[32 P]VA

RRAT[ a2P]VA RIRREEETt ~2P]EEEAA AS[32 PIEEEEE pNPP

809.4 2752.6

4105.0

11.3

t2.fl

PCP-[]

PCF'-Ill

dephosphorylated at all A similar efficiency order towards dephosphorylatioo of phosphoprotein and phosphopeptide substrates is exhibited by PCP-I, either -IA or -lB. The specificity of PCP-III is different and in some cases opposite: its activity toward AS[3ZP]EEEEE and pNPP is negligible in comparison to that of PCP-] and PCP-II, on the other hand, only PCP-III is able to dephosphorylate efficiently [32Plphosphorylase a, [32P]histon¢ H1, and the basic phosphothreouinc pepfide RRAT[a2p]VA. Its activity toward the phosphoseryl derivative RRAS [~2P]VA is approx, ll-fold lower. Such behaviour is reminiscent of animal PP-ZA [34]. /~¢liv;ty (~l

'~oo-~,- +'~-]t~.

=

=

~





B0

!. i"

o . . . .... . . . ....... . . . ........ . . . . . , ~ . . . ~ ~ , , ; o,1~1 OJ[~l 0.I 4. IC. [Okadaio aeidl InM]

Fig, 5, The comparison of the inhibilmy effect of okadaic a~d on

maize seedling protein phosphatase (PCP-III) with inhibition of protein phosphata;etype 2A from rabbit skeletal muscle. ,t, activlW of PCP-III; o, activiw of PP-2A; I , a¢ilvit3,'of PCP-IA, -IB and PCP-IL The inhibitow activitywas determinedafter 5 rain of preincubatioa in the presenceof okadaicacid, followedby the additionof RRAT[-"2PJVAas the substrale.

Moreover, as shown in Fig. 5, maize seedling PCP-III is readily inhibited by okadaie acid with an ICs0 value comparable to that of skeletal muscle PP-2A. The phosphocascin phosphateses (PCP-! and PCPII) from maize seedlings are conversely insensitive to up to three orders of magnitude higher concentrations of okadaic acid. Discussion

The whole [32P]casein phosphatase activity of crude ethanol treated extracts from maize seedlings is accounted for by at least three enzyme fractions (conventionally termed PCP-I, PCP-iI and PCP-I]I) which have been resolved and partia]ly purified by DEAEcellulose chromatography followed by heparin-Sepharose affinity chromatography and gel filtration. The identification of PCP-III as the catalytic subunit of a type-2A protein phosphatase is supported by the following findings: (a) It displays negligible activity toward pNPP, while it readily dephosphorylates phosphohistones and phos. phorylase a as expected for a protein phosphatase of either type-i or -2A [30]; (b) It does not bind to heparin-Sepharose, a distinctive property of PP-2A as opposed to PP-I [36]; (c) It exin'bits the typical phospbopeptide substrate specificity of PP-2A [33,34]; (d) It is inhibited by the same concentrations of okadaic acid as skeletal muscle PP-2A; (e) Its molecular mass is consistent with that of the catalytic subunit of animal PP-2A. On the other hand, neither PCP-I or PCP-II appear to belong to any class of typical S e r / T h r specific protein phosphatases, according to the criteria adopted for these enzymes from animal tissues. In particular, their molecular mass (70 kDa) after ethanol treatment is higher than expected for the catalytic subunits of PPl and PP2A; they do not display any requirement for either Ca 2÷ or trig 2÷ which are needed for the activity of PP2B and PP2C, respectively; their activity toward phosphohistones is neglisible and after heparin-Sepharose affinity chromatography they still retain n remarkable pNPPase activity whose pH optimum is shifted toward acidic values. The behaviour of PCP-I and PCP-II L~ opposite to PCP-III in that they both prefer the phosphosery] peptide RRAS[32PJVA over its phosphothreouyl homolog, a typical feature shared by all acid and alkaline phosphatasas tested so far (unpublished data in collaboration with C. Klee). Contrary to that, animal prtoein phosphatases exhibit a marked preference for phosphothreonyl- over phosphoseryl-substrates [33,34,37,38]. Another distinctive property of peaks I and II is their outstanding activity toward the very acidic phosphopeptide AS[32p]EEEEE0 which is typically unaffected by all four classes of

135 protein phosphatase [33,34,37] while being an excellent substrate for wheat germ acid phosphatase (unpublished data in collaboration with C. Klee). The conclusion that the protein/peptide phosphatase activity associated with PCP-I and -II is accounted for by acid phosphatases, rather than for typical protein phosphatases, is supported by its insensitivity to okadaic acid, a powerful and specific inhibitor of protein phosphatases type-1 and -2A (ICs0 for PP-2A is approx. 0.2 nM and for PP-1 approx. 20 nM [25]), but not of acid phosphatases which are not affected by up to 101~0-fold higher concentrations (5 pM) of this compound [23,25,26]. in the course of our experiments we could not find any clear-cut evidence for the presence of Wpe-I and type-2C protein phosphatases in the soluble fraction of maize seedling extracts. Evidently, it is not due to the loss of these enz3anes during the isolation procedure, especially not in the course of ethanol treatment. The protein phosphatas¢ type:l is cxpe~-'tedto bind to heparin-Sepharose [39]. The minor fraction of PCPIll which binds to hepatin-Sepharos¢ neither dephespho~lates phosphorylase a (Fig. 3c) nor is inh~ited by 0.l ~tM okadaic acid (not shown). The failure to detect any activity attributed to PP-1 does not mean that this enzyme is lacking in maize seedlings, It is quite possible, e,g., that PP-1 is mainly associated with the panicelate fraction, thus escaping detection in the soluble fraction employed for this stu~. This would be consistent with the observation that PP-I is responsible for approx. 75% of total protein phosphatase activity in the 16000 × g pellet from liver homogenates, and almost no PP-2A was found there [22]. Similar data were reported for FP-1 and -2A of Brassica napus seeds [23]. It should be also noted in this respect that the Brassica napus extracts exhibiting type-l, besides -2A phosphatase activily were obtained in the presence of 2% Triton X-100 [23], which was not included in the medium used in the present study for preparing maize seedling extracts. Recemly, two maize cDNAs, showing 70-80% homology to the rabbit PP-I catalytic domain were isolated (Smith, R.D. and Walker, J.C,, unpublished data). Also, the amino acid sequences deduced from Brassica napes PP-I and PP-2A-iike eDNA clones exhibit 72% and 79% overall identity to rabbit skeletal muscle PP! and PP2A catalytic sebunits, ~espectively [27]. Those findings might support the view, that the plant and mammalian phosphatases t,~3¢-1 and type-2A, as well as animal PP-2B, are members of the same serine/threonine protein phosphatases gone family

[401. As was mcntione~ above, we did not detect any activity of protein phosphatase type.. 2C. The addition of 20 mM Mg2÷ (in the presence of 5 p.M okadaic acid in the incubation mixture) does not increase protein pbosphatase activity in the 'high speed supernatant' which suggests the absence of PP-2C activity in maize

seedlings ~%ytosol(not sF.aw0. It has been previously reported that Mg2+-dependent okadaie add-insensitlve protein phosphatase (PP-2C) has not been identified in oilseed-rape seed extracts, contrary to the other plant tissues - pea leaf and wheat leaf extracts and carrot cells where the activity of PP-2C was detected |2], A partially purified phosphoprotein phosphatase from soybean hypocotyls []9] shows remarkable similarities with PeP-Ill from m;aze seedlings described here, as far as their chromatographic properties, pH dependence with [32p]histone-H] and apparent molecular mass after ethanol treatment (28 kDa) are concerned. Two other protein phosphatases which catalyse the deph~pho~lation of both phospl~hislone H1 and phosphocasein were extensively purified from wheat embryo [18]. Wheat germ enzymes are similar to mai2e seedlings PCP-III and soybean hypocotyls in protein phosphatases with ~spect to elution from DEAE-exchanger, optimum pH and lack of pNPPas¢ activity. However, ethanol precipitation was not included into the purification procedure of wheat-emb~o protein phosphatases. This probably accounts for its isolation as an heterogenous form with a molecular mass of approx. 197 kDa [18]. Most probably, maize seedling PCP-ilI as well as the other plant phosphohistone phnsphatases described so far have special physiological functions in cell g r o ~ h and proliferation. It is worth pointing out, thai they were isolated from fast growing plant tissues, namely seedlings, ~ l s and embryos in which histoue Idnase activities has been previously detected as well [5,29,41]. On the other hand, the role of the other two maize seedling fractkms exhibiting phospbucasein phesphetase activity, namely PEP-1 and PCP-II, remains unclear despite their ability to dephosphorslate certain phespho~l~ides. These ena3rmes clearly belong to the ela~ of acid phosphatases rather than to that of protein phesphatases. A novel observation was that these maize seedling acid phosphatases are able to bind to heparin-Sepharase which can be therefoTe utilized for their purification, as well as for their separatioa from PP-2A-I&e enzymes. Acid phosphatases are widely distributed in nature. In plants their activity increases with seed germination and seedling growth and is thmught to play a role in salubilization of macromdlecular organic phosphates. Plant cytoplasmatic acid phosphatases belong to multiple forms of enzymes with molecular masses in the range of 78-118 kDa, showing a strong affinity for p-nitrophenylphosphate [42].Their possible involvement in the dephospimrylation of proteins remains unanswered. Ackllowk,~n~ This work was supported by Italian M.U.R.S.T. and C.N.R. (Target prc~ct on Biotechnology and Bioin-

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Identification of protein phosphatase activities in maize seedlings.

Three phosphatases active on phosphocasein (PhosphoCasein Phosphatases) termed PCP-I, PCP-II and PCP-III were isolated from maize seedlings by DEAE-ce...
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