Rat liver cytosol phosphoprotein: Purification and enzymatic phosphorylation and dephosphorylation

27 °

BIOCHIMICAET BIOPHYSICAACTA

BBA 35803 RAT L I V E R CYTOSOL P H O S P H O P R O T E I N : P U R I F I C A T I O N AND ENZYMATIC P H O S P H O R Y L A T I O N AND D E P H O S P H O R Y L A T I O N

I,. A. PINNA, G. CLARI ANI) \:. MORET lslituto di Chimica Biologica delI'Universith di Padova and Centro per lo studio della /isiologia mitoco~zdriale del Consiglio Nazionale delle Ricerche, Padua (Italy)

(Received November 2oth, 197o)

SUMMARY A phosphoprotein fraction containing both phosphorylserine and phosphorylthreonine has been isolated from rat liver cytosol and purified up to Ioo-fold by DEAE-cellulose column chromatography followed by gel filtration through Sephadex G-2oo and G-ioo. The purified phosphoprotein, whose alkali labile phosphate content approximates 9 fig per mg protein, is resolved into at least five phosphorylated bands upon electrophoresis on cellulose-acetate strips, p H 7.2. The phosphorylation of cytosol phosphoprotein by [a2P]ATP is catalyzed by both cytosol and microsomal phosvitin kinase, free of any protamine kinase activity. A partial dephosphorylation of the a2P-labelled phosphoprotein can be accomplished through the reversal of the protein kinase reaction, using both ADP and GDP as phosphate acceptors. An ahnost complete release of previously incorporated a2p from the protein is catalyzed by a niitochondrial protein phosphatase. In contrast, no dephosphorylation could be observed with crude preparations of cytosol protein phosphatase.

INTRODUCTION Enzymes which catalyze the phosphate transfer from ATP to casein and phosvitin ("protein kinases") and the dephosphorylation of the same phosphoproteins ("protein phosphatases") have been described in a large number of different organisms and tissues 1-7. Likewise phosphoproteins have been observed in several biological sources (see ref. 8). However the relationship between phoshoproteins and the enzymes regulating their phosphate turnover are not yet completely known since the purification of endogenous phosphoproteins turned out to be a rather complicated enterprise. The previous finding that over 9o% of the liver protein kinase activity tested using casein and phosvitin as substrates, is present in the cytosol fraction 9, prompted us to undertake the isolation and the purification of phosphot~iochim. Biophys. Acta, 236 (I97 I) 270--278

RAT LIVER CYTOSOL PHOSPHOPROTEIN

271

proteins from rat liver cytosol. In a previous paper the preparation of a phosphoprotein fraction able to be actively phosphorylated by Ea2P]ATP in the presence of purified rat liver cytosol phosvitin kinase has been described 1°. In the present paper a method is described for a Io-fold purification of such a phosphoprotein fraction. Moreover the phosphorylation and dephosphorylation of such a phosphoprotein by cytosol, mitochondrial and microsomal enzymes have been investigated. MATERIALS AND METHODS

All Chemicals were from Sigma. Ion exchange resins were from Serva and gel filtration materials were Sephadex from Pharmacia. a2Pi was purchased from Radiochemical Centre, Amersham, England. Rat liver cytosol phosphoprotein fraction was prepared by a slight modification of a procedure already described1°: 4oo ml (equivalent to about 5 g prot¢:in) of the cytosol were dialyzed in o.o5 M Tris (pH 7.5) and then filtered through a DEAEcellulose column (5 cm × 13 cm) equilibrated with the same buffer. The column was washed with 6 vol. of the equilibrating buffer and with 3 vol. of the same buffer containing o.I M NaC1 in order to remove most of the non-phosphorylated proteins. The phosphoprotein fraction was then eluted from the column with o.o5 M Tris (pH 7.5) plus 0.2 M Nat1, and it was concentrated by Diaflo membrane ultrafiltration in order to obtain a protein concentration of about IO mg/ml. The phosphoprotein fraction was stored at --2o °. Purified phosvitin kinase free of activity toward protamine was prepared and assayed as already described 11. A unit of activity is defined as that amount of enzyme which catalyzes the transfer of I nmole 32p from I32P]ATP to I mg phosvitin in IO min at 37 °. Protamine kinase preparations free of phosvitin kinase activity were obtained as previously described 1°. The enzymatic phosphorylation of the phosphoprotein fraction by [s2P]ATP was assayed following the method previously described 1°. In order to test the "intrinsic" phosphorylation of the phosphoprotein fraction, the preincubation at 65 ° and the consequent addition of protein kinases were omitted. 32P-labelled phosphoprotein was prepared by incubating 5o IOO mg of the cytosol phosphoprotein fraction for 2 h at room temperature in the presence of i.o #mole [32P]ATP, prepared as described by GLYNN AND CHAPPEL12 with a specific radioactivity near to I . l O 7 counts/min per /~mole, IOO mM Tris buffer (pH 7.5), 8 mM MgC12 and 5 units of purified phosvitin kinase. At the end of incubation the sample was ice-cooled and dialyzed for at least 72 h against several changes of 2oo vol. of I.O mM Tris (pH 7.o) with continuous stirring. By such a procedure over 95 % of the radioactivity still present inside the sack at the end of the dialysis was accounted for by protein-bound ~P. The reversibility of the protein kinase reaction was tested by incubating aliquots of the labelled phosphoprotein containing IO ooo to 2o ooo counts/min in 1.5 ml of a medium containing 2oo mM Tris (pH 6.5), 8 mM MgC12, 3 units of purified phosvitin kinase and ioo mM ADP (or other nucleotide diphosphates) at 37 ° for 5 h. In control experiments ADP was omitted. Incubation was stopped by addition of o.2 ml HC10 4 and the sample was ice-cooled. After centrifugation the [s2P]ATP was preBiochim. Biophys. Acta, 236 (I971) 27o-278

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cipitated from the neutralized supernatant as barium salt, redissolved in 0.3 ml water by addition of Dowex 5 ° (H+-form) ion-exchange resin and finally isolated by paper electrophoresis on W h a t m a n 3 MM at pH 3.4 in 2o mM citrate buffer plus 3 mM zinc acetate (4 h, 2o V/cm). The radioactivity on the electrophoretic strip was determined with a Packard Radiochromatogram Scanner model 72Ol. As an alternative procedure, the HC1Q supernatant was shaken with 4oo mg Norit A, and tile adsorbed [a2p]ATP was evaluated as acid-labile radioactivity (i M HC1, io min, at I 0 0 °) according to the method described by RABINOWITZAND LIPMANN 1.

Enzymatic dephosphorylation of the cytosol phosphoprotein fraction was tested by incubating variable amounts of labelled phosphoprotein equivalent to 8ooo counts/min as protein-bound a2p in 2 ml of a medium containing: IOO mM Tris acetate (pH 6.5), 5 mM cysteine and crude preparations of proteinphosphatase either from mitochondria (I mg) or from cytosol (5 mg) obtained as already described ~a. In control experiments protein phosphatases were omitted. In some experiments tile labelled cytosol phosphoprotein was replaced either by phosvitin (I mg) or by Hammarsten casein (IO mg). After 6o min incubation at 37 ° the reaction was stopped by addition of 5o°/~ trichloroacetic acid (o.8 ml) and silico tungstic acid solution 14 (o. 9 ml) followed by centrifugation, a2Pi was determined in the supernatant by treatment with ammonium molybdate followed by isobutanoLbenzene extraction by the MARTIN AND DOTY15 procedure with a slight modification. Aliquots of the isobutanol benzene phase were counted in a thin-window Geiger counter. In the experiments with unlabelled phosphoproteins (casein and phosvitin), cold Pi extracted by the isobutanol-benzene phase as phosphomolybdic complex was determined colorimetrically after addition of SnC12. RESULTS

Phosphorylation of cytosol phosphoprotein The cytosol phosphoprotein fraction isolated as previously described by DEAEcellulose column chromatography 1° incorporates a2p when incubated in tile presence TABLE [ PHOSPHORYLATION

OF CYTOSOL

PHOSPHOPROTEIN

UNDER

DIFFERENT

CONDITIONS

E x p e r i m e n t a l conditions are described in the m e t h o d s section.

asp incorporated into the protein (counts/rain) Cytosol p h o s p h o p r o t e i n minus Mg 2+ plus E D T A plus o.I mM N-ethylmaleimide plus o.i mM cyclic AMP plus phosvitin (I ing) plus p r o t a m i n e (2 rag) Cytosol p h o s p h o p r o t e i n preheated 5 min at 65 ' plus cytosol phosvitin kinase plus inicrosomal phosvitin kinase plus p r o t a m i n e kinase

Biochim. Biophys. Acta, 236 (1971) 270-278

2080 o ~95 o i958 287oo 958o 23 ° 188o 173 o 34 °

RAT LIVER CYTOSOL PHOSPHOPROTEIN

273

of [32PIATP, even in the absence of added protein kinase (see Table I), thus suggesting that such a fraction is contaminated by protein kinase. This is confirmed by the finding that such a preparation is active also towards phosvitin and protamine (see Table I). That phosvitin kinase rather than protamine kinase is responsible for the phosphorylation of cytosol phosphoprotein is proved by the following observations: (i) N-Ethylmaleimide, which is known to inhibit the protamine kinase but not the phosvitin kinase activity ~6, was almost ineffective at inhibiting the labelling of cytosol phosphoprotein. (2) The phosphorylation of cytosol phosphoprotein was insensitive to 3',5'-AMP (Table I), which is known to stimulate the phosphorylation of histones by protamine kinase ~7. (3) The phosphorylation of cytosol phosphoprotein, prevented by 5 min incubation at 65 °, was restored by addition of purified cytosol phosvitin kinase (free of protamine kinase activity) while partially purified protamine kinase proved to be only slightly effective. Purified phosvitin kinase from rat liver microsomes could replace the cytosol enzyme (see Table I).

Purification and characterization of cytosol phosphoprotein In order to improve the purification of labelled phosphoprotein, several attempts have been made. In particular the procedure already used for the purification of nuclear phosphoprotein and consisting of filtration through Biorex 7 ° followed by adsorption on calcium phosphate geP, proved unsuccessful. Chromatography on phosphorylated cellulose (P-cellulose), as shown in Fig. I, separated labelled phosphoprotein from phosvitin kinase. However phosphoprotein

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Fig. i. S e p a r a t i o n of p h o s v i t i n kinase a c t i v i t y from labelled p h o s p h o p r o t e i n by p h o s p h o r y l a t e d cellulose c o l u m n c h r o m a t o g r a p h y . 5 ml of [32Plcytosol p h o s p h o p r o t e i n prepared as described in the m e t h o d s were c h r o m a t o g r a p h e d t h r o u g h a p h o s p h o r y l a t e d cellulose c o l u m n (2.o c m × i7.o cm) equilibrated w i t h o.o 5 M Tris (pH 7.o). A t the arrows the c o l u m n was eluted b y t h e equilibrating buffer c o n t a i n i n g t h e following increasing c o n c e n t r a t i o n s in NaCI: o . i , o.2, o.3, o.9 M. 4-ml fractions were collected.

Fig. 2. Gel filtration of [3~p]cytosol p h o s p h o p r o t e i n on S e p h a d e x G-2oo. 5 ml of [a2PJcytosol p h o s p h o p r o t e i n were applied to a S e p h a d e x G-2oo c o l u m n (2.o c m × 55.o cm) equilibrated with o.o 5 M Tris (pH 7.5) c o n t a i n i n g o. 7 M NaC1. F l o w rate: 6 ml/h. 3-mt fractions were collected.

Biochim. Biophys. Acta, 236 (1971) 270-278

274

L . A . PINNA #l ~/].

still remained contaminated by unlabelled proteins as evidenced by their unchanged specific radioactivity. More satisfactory results were obtained bv combined gel filtration on Sephadex G-2oo and G-Ioo at high ionic strength. By filtration on Sephadex G-moo two radioactive peaks were obtained (Fig. 2): the first eluted at the void volume together with most of the protein, the second was retarded by the gel and retained an average specific radioactivity about IO times higher than the first peak. Phosvitin kinase was eluted between the two radioactive phosphoprotein peaks without largely contaminating either of them (see Fig. 2). The second phosphoprotein peak from Sephadex G-2oo, accounting for about 7o~{, of the protein-bound radioactivity, was further purified by gel filtration on Sephadex G-ioo equilibrated with o. 7 M NaCI. Such a procedure improves the specific radioactivity of the preparation, as is clearly shown in I;ig. 3-

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Fig. 3. Purification of [32Plcytosol p h o s p h o p r o t e i n by gel filtration on Sephadex G-Ioo. Radioactive Peak I I from Sephadex G-2oo (see Fig. 2) was concentrated to 2 ml by ultrafiltration and filtered t h r o u g h a Sephadex G - I o o column (1.2 cm x 125.o cm) equilibrated with o.o 5 M Tris (pH 7.5) containing o. 7 M NaC1. Flow rate: lO ml/h. 3-ml fractions were collected. Fig. 4. Electrophoretic p a t t e r n of Sephadex G-ioo purified cytosol p h o s p h o p r o t e i n on cellulosepolyacetate strips (o.o2 M Tris buffer (pH 7.2) containing o.I mM m e r c a p t o e t h a n o l and o.5% Triton X - i o o ; 45 rain at 25o V). The proteins were stained with Nigrosin. The radioactivity was determined by the Packard C h r o m a t o s c a n n e r Modcq 72Ol.

It should be noted that direct filtration of the s2P-labelled protein on Sephadex G-Ioo, omitting the previous Sephadex G-2oo filtration, did not allow any satisfactory separation of the radioactive protein from the bulk of the proteins. The second phosphoprotein peak purified on Sephadex G-Ioo was still heterogeneous since it was resolved into at least five radioactive bands by electrophoresis on cellulose polyacetate strips (Fig. 4). Hiochim. Biophys. Acta, 236 (1971) 27o-278

275

RAT LIVER CYTOSOL PHOSPHOPROTEIN

Fig. 5- Electrophoretic detection of [32P]phosphoserine and [32P]phosphothreonine in acid hydrolvsates of purified labelled phosphoprotein. Electrophoresis was run in 20% formic acid (pH 1.4) for 60 rain at 45 V/cm on Whatman 3 MM paper. Carrier amino acids were detected by the ninhydrin reaction. Radioactivity was determined by counting the strip in the Packard Chromatoscanner Model 72oi.

To identify where incorporation of 32p occurred, the purified phosphoprotein was hydrolyzed in 2 M HC1 at I00 ° for I0 h. The hydrolysate, s u b m i t t e d to paper electrophoresis, showed three radioactive b a n d s identified respectively as a2Pi, [32p]serine a n d [32Plthreonine. The slowest radioactive b a n d , shown in Fig. 5, was converted into [a2Plserine a n d a2Pi u p o n prolonged hydrolysis. I t can be reasonably assumed t h a t E32p]serine accounted for over 9 0 % of the p r o t e i n - b o u n d radioactivity, considering t h a t during acid hydrolysis over 60% of the originally present p r o t e i n - b o u n d Ea2Plserine u n d e r w e n t dephosphorylation. I n d e e d the a m o u n t of 32pi recovered on paper electrophoresis fits with such a calculated figure. The total alkali-labile phosphate c o n t e n t of the purified phosphoprotein ranged TABLE II REVERSIBILITY

OF THE

PROTEIN

KINASE

REACTION

Experimental conditions described in the methods section. 3*p incorporated into nucleotides (counts/rain) E32P]Phosphoprotein (Peak II, G-2oo) plus ADP plus GDP plus UDP preheated 5 min 65 ° plus ADP preheated 5 rain 65 ° plus ADP plus phosvitin kinase ~3~PJPhosphoprotein (Peak I, G-2oo) plus ADP

I20

175o 132o 315 400 135o 1280

Biochim. Biophys. dcta, 236 (1971) 270-278

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276

from 7 to 9 #g/mg protein, a figure which is about IO times higher than that found in the phosphoprotein fraction obtained by the DEAE-cellulose procedure.

Reversibility of protein kinase reaction As shown in Table II, detectable amounts of [a2P]ATP were formed upon incubation of the 32P-labelled cytosol phosphoprotein with ADP in the presence of cytosol phosvitin kinase. GDP, unlike UDP, could replace ADP as a phosphate acceptor. Such a reversal of the protein kinase reaction took place with both the peaks into which the labelled phosphoprotein was resolved by Sephadex G-200 gel filtration (see Fig. 2), although it occurred more efficiently with the fraction with higher specific radioactivity (Peak II, see Table II). Apparently no more than 15 to 25% of the whole radioactive phosphate pool previously incorporated into the phosphoprotein through the forward protein kinase reaction could be transferred back to ADP through the reverse reaction. In fact prolonged incubation times and further addition of ADP and of fresh phosvitin kinase failed to increase the amount of [32P]ATP formed, after equilibrium had been reached (Fig. 6).

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Enzymatic dephosphorvlation of cytosol phosphoprotein by protein phosphatases Two different protein phosphatase activities are known to be present in rat liver: one is located in the mitochondria and the other in the cytosoP a. The actixdty of both these enzymes on cytosol phosphoprotein, compared with that on foreign phosphoproteins like casein and phosvitin, is shown in Table I I I . It can be seen that mitochondria were active on casein, phosvitin and cytosol 3~P-phosphoprotein. On the contrary tile cytosol preparation, which was able to dephosphorylate casein but not phosvitin, does not release any detectable amount of radioactivity from eytosol i82Pl phosphoproteins. DISCUSSION

The purification of a phosphoprotein fraction from rat liver cytosol described Biochim. Biophys. Acta, 236 (1971) 270-278

277

RAT LIVER CYTOSOL PHOSPHOPROTEIN TABLE III

ENZYMATIC OEPHOSPHORYLATION OF CYTOSOL PHOSPHOPROTEIN, CASEIN AND PHOSVITIN BY MITOCHONDRIAL AND CYTOSOL PREPARATIONS E x p e r i m e n t a l c o n d i t i o n s described in t h e m e t h o d s section.

% Alkali-labile P (or 32p) released Cy/osol protein phosphatase Ea2p~Phosphoprotein l a b e l l e d by "intrinsic" protein kinase [a2Pl P h o s p h o p r o t e i n l a b e l l e d by added phosvitin kinase after preheating Phosvitin Casein

Mi/ochondrial protein phosphatase

1. 5

70.0

0.8 o.o 45.3

92.0 75.0 65.0

in the present paper is relevant for understanding the composition and structure of such proteins as well as the metabolic significance of their phosphate turnover. Up to now the activities of enzymes, occurring in liver cells and catalyzing the protein phosphorylation and dephosphorylation, have been studied using as substrate foreign phosphoproteins like phosvitin and casein. The isolation and purification of the endogenous substrates of such enzymes, described in the present paper, make possible a more exhaustive investigation on the biological role of protein phosphorylation and dephosphorylation. The phosphorylation of the phosphoprotein is catalyzed by purified preparations of both cytosol and microsomal phosvitin kinases, completely free of any protamine kinase activity. The phosvitin kinase activity is constantly associated with the crude phosphoprotein preparations. However the purification procedures described in the present paper separate the phosvitin kinase activity from the phosphoprotein fraction. On the basis of our results two different mechanisms for the dephosphorylation of a2P-labelled phosphoprotein can be proposed : (I) the reversal of the protein kinase reaction and (2) the hydrolysis of protein-bound a2p catalyzed by a mitochondrial protein phosphatase. Apparently only a limited amount of the previously incorporated a2p can be transferred to ADP through the former way. The heterogeneity of the cytosol phosphoprotein fraction evidenced by gel eleetrophoresis, might account for this finding, assuming that not all the subfractions can undergo the reverse reaction. The protein phosphatase reaction promotes a practically complete release of the protein-bound a2p. It is very interesting that only the mitochondrial protein phosphatase is able to catalyze such a hydrolysis with all phosphoproteins examined, while the crude cytosol preparation is ineffective on cytosol phosphoprotein as well as on phosvitin. It is remarkable that the cytosol phosphoproteins incorporate phosphate by means of a soluble protein kinase located in the cytoplasm, while they undergo dephosphorylation through a reaction catalyzed by a particulate protein phosphatase bound to the mitochondrial membranes. The possible biological significance of such a phosphorylation-dephosphorylation mechanism is now under investigation. Biochim. Biophys. Acta, 236 (1971) 27o-278

L . A . PINNA et al.

278 ACKNOWLEDGEMENT

We wish to thank

Miss Carla Munari for valuable technical assistance.

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Socs. Meeting, Praha, z968. i o L. A. PINNA, (;. CLARI, V. MORET AN[) N. SILIPRANDI, F E n S Letters, 5 (I060) 77. I I B . ]~AGGIO, L. A. PINNA, V. MORET, N. SILIPRANDI, Biochim. Biophys. Aeta, 212 (197 o) 515 . 12 J. M. GLYNN AND J. I3. CHAPPEL, Biochem. J., 9o (1964) 147. 13 V. MORET, I,. A. PINNA AND G. MAGNI, II. J. Biochem., 19 (197o) 2o 4. 13 V. MORET, L. A. PINNA ANI) G. MAGNI, Ill J" Biochem., 19 (I97O) 2o4. 14 (). LINI)BERC~ AND L. F~RNSTER, in D. (;LICK, Methods of Biochemical Analysis, Vol. I I I, Int e rscience, New York, 1956, p. 8. 15 J. B. MARTIN AND O. M. DOTY, Anal. Chem., 2I (1949) 965. 16 T. A. LANGAN .aND l~. K. SMITtl, Federation Proc., 26 (19671 6o3. 17 T. A. [,ANGAN, Science, 162 (I968) 579-

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