Phosphorylation of rat liver cell sap protein on incubation with [32P]ATP

Bioehimica et Biopttysica Acta, 336 (1974) 140-150 ~'~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36612

PHOSPIIORYI. ATION OF RAT LIVER CELL SAP PROTEIN ON I N C U B A T I O N W I T H p2P]ATP

OLLE LJUNGSTROM and LORENTZ ENGSTROM ln.~1itate of Medical Chemistry, University of Uppsala, Uppsala (S~wden) (Received July 2nd, 1973)

SUMMARY I. The aim o f the present work was to obtain a n overall view o f the p2P]ATPdependent phosphate turnover o f the soluble rat liver p h o s p h o p r o t e i n s with phosphoryl groups bound to serine and th~eonine residues. R a t liver cell s a p was therefore incubated with [32P]ATP under different conditions. The protein t o o k up 32p to a considerable extent. The highest 3zp labelling o f the protein was o b t a i n e d on incubation with :5 m M [3~P]ATP for 1 h at 30 °C, and c o r r e s p o n d e d to an i n c o r p o r a t i o n of 55 nmoles o f phosphate into the cell sap protein from I g o f wet tissue. The total a m o u n t o f protein-bound phosphate o f this cell sap was 148 nmoles, as determined by chemical analysis. The ratio o f phosphorylserine ([~'P]Ser(P)) to p h o s p h o r y i threonine ([3zp]Thr(P)) isolated from acid hydrolysates o f [32P]ATP-incubated cell sap was 6-10. 2. F r o m the initial rates o f the 32p labelling it was concluded that the p h o s p h o r y l ation could not depend appreciably on the f o r m a t i o n o f inter~nediate p h o s p h o r y l enzymes. The rate was compatible with that o f protein kinas¢ reactions. 3. Cyclic 3 ' , 5 ' - A M P was shown to stimulate the 32p i n c o r p o r a t i o n . 4. The presence o f high phosphoprotein p h o s p h a t a s e activity was a p p a r e n t when all [3ZP]ATP had been consumed o r removed by gel c h r o m a t o g r a p h y . 5. It is assumed that rues! phosphoproteins o f rat liver cell sap are enzymes a n d other proteins which are regulatively phosDhorylated by protein kinases.

INTRODUCTION Phosphoproteins with phosphory[ groups b o u n d to serinc and thrconine residues are ,~idely dislributed in different eukaryotic cells a n d cell fractions [I-4]. Several ~ea rs ago a high rate of incorporation o f phosphate into intraceilular p h o s p h o proteins was demonstrated, indicating that these proteins have i m p o r t a n t metabolic functions [5]. Rat liver cell ,ap, like other ti ues [6], contains two diff,'-.'nt types o f protcia kinase activity, one 3f them stimulated by cyclic 3 ' , 5 ' - A M P a~,d the other not [7] AbbLeviatlons: Sc~(I ,, phosphorylserine: Thr(P), phosphorylthreon~,~e.

~41

In addition, there seem to be differences between the two types o f enzymes with regard to suhstrate specificity [6]. The endogenous protein substrates may be either enzymes which are regulatively phosphorytated by kinases and dephosphorylated by protein phosphatases [6, 8] or as yet unidentifi.'d proteins [9]. [UP]ATP-dependent phosphorylation o f serine and threonine residues of rat liver cell sap protein was described by Glomset in 1959 [10], Such a phosphorylation may, at least partially, be due to the presence of protein kinases and the eorresp, ading endogenous protein su,~strates. Since rat liver cell sap contains protein phospilatase activity [I I, 12], 3~p !a~lling of the protein on incubation with p2P]ATP may also reflect a turnover o f protein-hound phosphate without a net protein phosphorylation due to concomitant protein kinase and protein phosphatase reactions. A very rapid phosphorylation of rat liver cell sap protein occurs on incubation with 5- 10 -6 M [~P]ATP at 0 "C for 15 s [ 13]. However, this protein-hound phosphate is acid labile and not bound to serine or threonine residues. Most e f this rapid phosphorylation with acid-labile linkages seems to be due to the forrtat.on of two intermediate phosphoryl enzymes, A T P citrate lyase (EC 4.1.3.8) and nucleoside diphosphate kinase (EC 2.7.4.6) [14]. The content of protein-bound phosphate o f rat liver cell sap after removal o f all nucleic acid and phospholipids (e.g. accor,Hng to Langan and Lipman [I 5]) is not known. Nor is it possible from published data to estimate tl,e contribution of each known phosphoprotein to the total content and metabolism of the phosphoproteins o f rat liver cell sap. However, it seems reaoonable to state that the metabolic role o f the main part of these phosphoproteins is unknown. The aim e! the present investigation was to study the rate and extent of the phosphoryfation o f serine and threonine residues of rat liver cell sap protein on incubation with [UP]ATP ia an attempt to get some information on the function of intracellular phosphoprotei~'s. The following four problems were approached: (I) To what extent is ATP the immediate precursor to protein-bound phosphate ? A minimal value for the fraction o f ATP-derived, protein-bound phosphate would be obtained by comparing the maximal .~2p labelling_ from [~'P]ATP of cell sap protein with the total amount of protein-bound phosphate, as determined by chemical analysis. (2) How much o f the 32p labelling of the protein depends on the formation of intermediate phosphoryl enzymes? This may be estimated from the initial rate of the a2p labelling, since such phosphoryl enzymes usually appear in fractions of a second. On the other hand the rate o f phosphorylations catalyzed by protein kinases must be lower by several orders of magnitude. (3) How much of the phosphorylation is dependent on cycli.Y,5'-AMP ? (4) What conditions with regard, for example, to p : P ] A T P concentration, incubation time and pH are suilable for 3.,p labelling of liver cell sap proteins for further studies of their identity and function? EX PER I M ENTAL

~:/'

f T P ,,, rnlhesis

:'-I':P]AFP was prepared as described earlier [16]. Generally, i! ~as mixed with unlabelled A r P to give a specific radioactivity of 5000-10000 counts-rain-1. nmole- L

142

Analytical methods Radioactivity was measured on dried aliquots in aluminium cups as previously described [3]. Ninhydrin analyses were performed according to Moore and Stein [ 17]. P~ was determined according to Martin and Dory [18]. ~zp~was assayed by measuring the radioactivity of the organic phase.

Preparation of rat liver cell sap All steps were carried out at 0--4 °C, Livers from male Sprague-Dawley rats, with a body weight of 250-350 g and fed ad libitum with a standard laboratory food, were homogenized in 5 vol. of 0.25 M sucrose-10-+ M E D T A in a 50-ml PotterElvehjem homogenizer. The homogenate was centrifuged at l0 000 × g for 10 rain. The pellet was desearded, and the supernatant was centrifuged at 105 000 × g for 1 h in a Beckman L2-65B ultracentrifuge. The supernatant was chromatographed on a Sephadex 13-50 column in 0,02 M triethanolamine-acetic acid buffer (pH 7.4). The material eluted with the void volume was collected and used the same day. 1 ml of this cell sap corresponded to 0.17--0.20 g of original tissue.

Determination of protein-bound phosphate Protein-bound phosphate was assayed by the method o f Langan and Lipmann as described by Kleinsmith et al. [15] with slight modifications. Protein of 5 ml of the cell sap preparation (i.e. about 50 mg of protein) was precipitated with trichloroacetic acid to a final concentration of 5 % (w/v) and collected by centrifugation, The precipitate was dissolved in 4 ml of 0.1 M N a O H and reprecipitated with trichloroacetic acid. After centrifugation the precipitate was suspended in IO ml of 5 ~o trichloroacctic acid e~d heated to 90 °C for 15 rain. After washing the precipitate with 10 mi of 57~o trichloroacetic acid, it was extracted with l0 ml each of the following solvents: acetone, diethylether, chloroform-methanol (I / 1 ; v/v), chloroform~methanol-conc. HCI (200/100/1 : by vol+) and finally twice with diethylether. I mt of water was then added to the diethylether-moist precipitate, and the diethylether was evaporated on a water-bath by raising the temperature from. 30 °C to about 60 ~C. 0.1 ml of l0 M NaOH was added and the tube weighed. The sample was then heated to I00 ' C for 20 rain in order to split off protein+bound phosphate. After cooling, the volume was adjusted by addition of water to compensate for evaporation, After acidifying the sample with 0.3 ml of 4 M HCI, protein was precipitated with 0.3 ml of 70 0~, HCIO+ and the precipitated protein was collected by centrifugation. More protein was precipitated by addition to the supernatant of 0.5 ml of the silicotungstic reagent of Martin and Dory [I 8]. After eentrifugation, 1.8 ml of the final supcrnatant was sucked off'. 0.2 ml of 10 'Nl H2SO4 was then added, and P~ analysis was performed according to Martin and Dory [18]. The recovery of P+ was determined by adding a small amount of 32P+ to the samples before the precipitation witb HCIO 4. The recovery of the radioactivity measured in the organic phase was about 95 o+;~.The values given are corrected for losses.

D~.termination of protein-bound s2p after incubation ~" ceil sap with J'~P_fA7 P 0.9 ml of the ceil sap preparation was incubated with [3-'P]ATP under differeltt conditions. Mg -'~ wa~ added as magnesium acetate. All incubations were performed in dup icate. The final incubation volume was i.0 ml. The incubations were inter-

143 rupted by the addition of 3 ml of 10°,, t~:chtoroacetic acid After at least 5 n'.in at 0 C. the mixtures were centrifuged. A sample of the supernatants was used to determine the amount o f s2p, formed. To remove adsorbed [*zP]ATP and 3-p, from the precipitates, they were dissolved in 2 ml ofO.I M N a O H follo,~ed by precipitation *~'ith 2 ml o f 10°.i, trichloroacetic acid and eentrifugation. This procedure was repeated thr=c more times. Lipids were removed by the solvent system described for the detevnination of total protein phosphate, using 4 ml of each solvent. 1 ml of water was daen added and the diethylether evaporated. The protein was dissolved by adding N a O H to a final concentration o f 0.1 M and the radioactivity o f drie~a aliqt~ots was determined. When calculating the extent of the phosphoD'lation, a valuc for the specific radioactivity o f the protein-lzound phosphate which was the same as the v a l j e for that o f the [s2P]ATP added was used, In blank experiment.~ NaC.'-I was add-~d to a final concentration o f 0.1 M i rain before the addition o f [~-'P]ATP, which was immediately folloxvcd by addBion .f 3 ml o f t0~,, triehloroacetic acid. The protein precipitates were washed and extra~cted as described above. The a m o u n t o f adsorbed ~2P-labelled material was measured, and the values were subtracted from the corresponding values from the [3"P]ATP incubation experiments. All values given in Tables | and I l I - V refer to I g of liver {wet weightL

Isolation o f prote#~-bound : ~zp, Ser( P) and .:~P. Thr( P) [J'P]Ser{P) an,~ |3'P]ThrLP) were isolated from acid h~d'ol~,,at.~s of the final protein precipitate, essentially as described before [i, 3, 4]. 30/~motes of unlabelled ScrIP) and 15/~moles of unlabelled Thr(P) ~efe added before ~he acid hydrolysis in order to allow corrections for losses ,fflabelled phosphoamino acids during hydrolysis and c h r o m a t o g r a p h y . Recovery o f 3ZP-labelled phosphoprotcln in the ~32p ,4 TP incubation experiments In o r d e r to estimate the recovery o f 3-'P-labelled phosphoprotein durir.g the washing and extraction procedures, 0.9 ml of cell sap was incubated with I mM [~:P]ATP in the presence o f 1 0 r a m Mg ~'~ for 15 rain at 30 °C, One incubation mixture was inactivated By adding HCI to a final concentration o f 2 M, the other by adding 3 ml o f 10 ,°,~,tricL,Ioroacetic acid. In the former case unlabelled Ser(P) and Thr(P) references ~:cre added, and the total mixture was hydrolyzed in a sealed tube for 20 h at 100 ~C. In the latter case the mixture was centrifuged and the precipitate was washed and extracted as described for determination o f total protein-bound 3-,p. Unlabefled Ser(P~ and Thr(Pl references were a d d t d and the sample was hydrolyzed as described above, The a m o u n t of [~-'P]SerIP) isolated from the hydrolysate of the v, ashed and extracted sample by c h r o m a t o g r a p h y cn Dowex 50 and Dowex 1 was about 70-80",, of that obtained after incubation and hydrolysis in HCt. None of the values given for the ['~-'P]ATP incubation experiments are corrected for these losses. Demon¢trati, m o.f -xZP-labelling o f ,4 DP and est#~tation o f the change in the 3"-P-hJh,'tl;ng o f 3".p .4 TP during incubathm 1.8 ml of cell sap were inc':bated with 5 mM [~:P]ATP for 1, 5, 15 and 60 rain at 30 C in the p~esence o f 10 m M Mg z*. The supernatants ~ere extracted se~'eraJ times with diethylether and were then chromatographed on 10 ml Dowex t-X2

144 (formate) columns eluted by 150 ml linear gradients, consisting o f water to 4 M formic acid and of 4 M formic acid to 4 M formic acid containing 0.8 M a m m o n i u m formate. A D P was eluted after the start of the second gradient. The main part of each pooled [3=P]ATP fraction from the p r~'ceding chromatography was passed through a 10 ml Dowex 50-X8 (H +) c o l u m n to remo~e NH4 + and was then evaporated to dryness, The fractions were men dissolved in 2 ml of 50 m M Tris~ acetic acid buffer (pH 7.5) containing 5 m M magnesium acetate and 28 m M glucose and were incubated for 30 min at 30 °C with 0. ! mg o f crystalline yeast hexokinas¢ (EC 2.7.1.1) (Sigma type C-130). The hexokinase reaction was interrupted by the addition of 10 ml of I M formic acid. The reaction mixtures were chromatographed on 10 mi Dowex l-X2 (formate) columns, eluted with linear gradients o f 125 ml of 4 M for:nit acid to 125 ml of 4 M formic acid containing 0.8 M a m m o n i u m formate. The spo'ific radioactivity of the [ u P ] A D P fractions was determined. The specific radioactivity of the ~,-phosphoryl group of the original [J2P]ATP fractions was then calculated by subtracting the specific radioactivity values o f the p2P]ADP fractions from those of the [JZP]ATP fractions. RESULTS

Rate and extent of protein phosphoo'lation at different concentrations o f [3Zp'A TP Cell sap protein was phosphorylated to a considerable extent on incubation TABLE | PHOSPHORYLATION OF CELL SAP PROTEIN. DEPEND =-NCE ON P~P]ATP CONCENTRATION AND INCUBATION TIME Cell sap '.,as incubated with [sZP]ATPin the presence of t0 mM Mg~+ as described under Experimental. T:le [3zP]ATP concentration, incubation time and temperature were varied as indicated. Results fr,)m a typical experiment is given. [~±PIATPconcn (raM)

T e m p . Incubation time ~zp CC) Imin) incorporation

0.02

3o

0.1

30

~zp~formed (% of [~"P]ATP added)

(nmolcs,.g)

l.O

5.0

0.1

30

0

I 5 15 6O i 3 5 15 6O

1,3 1.4 1.0 0.5

I

3.9 9.7

S t5 6O I 5 I5 60 0.25

30 44 50 60

2.7

24

34 3.5

42

3.0

51

1.5

56

II

13 5.1 I1 17

39 0.2

45

8 21 34

43 I

II 17 35 I

145 w'.h ['~2P]ATP, as seen in Table I. The phosphorylation increased markedly with the [ ~ P ] A T P concentration. The initial rate of the reaction ~ as not extremely rapid; t ~a~, was most clearly shown by the fact that only 0.2-0.4 nmole of phosphate/g Fver ~as incorporated o n incubation with 0. ! m M [~P]ATP for 15 s at 0 °C. With 0.02 a n d 0.1 m M [3~P]ATP the maximal protein phosphorylaticn was obtained within 5 rain at 30 ~C. O n further incubatien up to 15 or 69 rain a substantial a m o u n t of protein-bound phosphate was released. This was probabl, due To phosphopTotein phosphatase reactions which would dominate over phosphor? lation reactions when most of the [s2P]ATP had be~n consumed, At higher [32p]ATP concentrations the maximal phosphorylation was obtained after longer incubation times. At a substrate concentration of 5 m M the maximal phosphorylation m; :ht not even have been obtained after 60 rain. The results in Table i l demonstrate that a de novo phosphorylation occurred o n incubation of ceil sap with unlabelted 5 m M A T P for 60 min at 30 °C. In addition it was shown that a considerable dephosphorylation of protein took place under the TABLE It TOTAL PROTEIN-BOUND PHOSPHATE 1N RAT LIVER CELL S/,P Protein-bound phosphate in cell sap was determined as described in the text. The cell sap was treated as indicated below. Each value is the mean of 5 determinations. The extreme values are given in parentheses. SampJe Untreated c¢11sap Cell sap incubated with 5 mM ATP in the presence of tO mM M$¢* for 60 rain at 30 °C Ceil sap incubated in the presence of 10 mM Mg2+ for 60 rain at 30 ~C

Protein-bound phosplrate (nmoles/g liver, wet weight) 118 (t0"7-125) 148 (142-161) 86 '83-88)

same conditions in the absence of ATP. It seems reasonable to assume that such a dephosphorylation also occurred in the presence of ATP. T , u s , during the experiments using [3'P]ATP, the 3~p labelling of protein would partly be the result of an exchange of protein-bound phosphate with [3'P]ATP due to concomitant kinase and phosphatase reactions.

Demonstrathm of A TPase and adenylate kinase activio" There was a rapid hydrolysis of [32P]ATP during the incubations, as estimated by the formation of ~:Pl (Table 1). However, on incubation with [s'P]ATP in a concentration of I m M or lower, the maximal recovery of S~P as szp~ was about 60",,. None of the remaining radioactivity was accounted for by [3Zp]ATP, as shown in experin nts where heat-inactivated incubation mixtures were chromatographed together ~ t h unlabelled A T P on 10 ml Dowex I-X2 columns, eluted with 4 M formic acid containing 0.25 M a m m o n i u m formate. Thus, other labelled products than szP t were a to formed during incubation.

146 It was obviously of interest to study if A D P became s'Polabelled during the reaction due to adenylate kinase activity. Cell sap was therefore incubat~--~lwith 5 r a m [azP]ATP for different times, as described under Experimental. The 3zp phosphorylation of the protein was determined in the usual way. A D P was isolated a n d was f o u n d to be considerably a2P-tabelled, confirming adenylate k i n a ~ activity. Since this would result in a decrease o f the a2p labelling o f the ),-phosphoryl g r o u p of ATP, the change of this labelling was further ~alyzedo

Decrease of 7-szP labelling of ,4 TP during incubation Correction of values for maximal protein phosphorylation The remaining [ u P ] A T P from the i n c u b a t i o n mixtures in the preceding experiment was isolated oy Dowex I-X2 chromatography. T h e specific radioactivity o f the y-phosphoryl groups was determined. The results are given in T a b l e I l l . D u r i n g incubation the specific radioactivity of the y-phosphoryl group of [s:P]ATP decreased. Since the protein phosphorylation was calculated with respect to the initial specifiL.

TABLE Ill DECREASE OF 7-a2P LABELLING OF ATP DURING INCUBATION. CORRECTION OF VALUES FOR PROTEIN PHOSPHORYLATION Cell sap was incubated with 5 mM [s~PIATPfor various tenghts of time as described in the text. The remaining [32plATPwas isolated by Dowex I-X2 chromatography and its specific radioactivity determined. It was then incubated with hexokinas¢ and glucose. The incubation mixtures were chromato graphed on Dow¢~ I-X2 and the specific radioactivity of the [azP]ADP formed was assayed. The specific radioactivities of t;3e ),-pi~osphorylgroups were then calculated by subtracting the [32P]ADP values from those of [aZPIATP.Protein phosphorylation was determined in the usual way, and values for protein phosphorylation were cotr~ted for the decrease of the specific radioactivity of the },phosphoryl groups of ATP, as described in the text. Vncubation • time (rain)

0 I 5 15 60

Specificradioactivity counts.min't-t~mole- ' [~2P]ADP

[azP]ATP

7-Phosphory] groups of [a2P]ATP

0 8000 28 000 103 000 123 000

253 000 253000 248 0 ~ 241 000 245 000

253 000 245000 220 0OD 138000 120 000

s2p incorporation ¢~nmoles/g)___. . . . . . . . . . . . . . . . . . Uncorrected Corrected value value

14 27 30 42

15 2g 32 55

radioactivity of [32P]ATP the apparent phosphorylation was lower than the true one. To better estimate the true protein phosphorylation with 5 mM [3~P]ATP the values for the specific radioactivity o f t h : 7-phosphoryt groups during each time interval was calculawed as the ,nean of those at tl~-- beginning and at the end of each time interval. By usir:g these revised values the ph¢,sphorylation during the time intervals could be roughl3 calculated and subsequeritly added (Table Ill). U n d e r these assumptions, the highest protein phospborylation oblained was 55 nmoles/g.

147

Variability of the methodfor measuring protehl phosphor ylation during, s:p. A TPincubaric,r in o r d e r to d e t e r m i n e the variability o f the p h o s p h a t e i n c o r p o r e t i o n values, 12 samples each f r o m the s a m e cell sap p r e p a r a t i o n s e r e i n c u b a t e d with 0.1 r,~:.~ and I m M [szP]ATP for 5 and 15 rain, respectively, at 30 "C and in the presence o f 10 r a m M ~ +. T h e samples were washed and e x t r a c t e d as described under Experimental. A t O. 1 m M [s2P]ATP the p h o s p h o r y l a t i o n a m o u n t e d to 3.9 -~ 0.2 and at I m M [ s ' P ] A T P to 14.2 ~ I.I n m o l e s o f 32p/g tissue (mean ± S.E.). W h e n experit,aental d a t a f r o m different cell sap p r e p a r a t i o n s were c o m p a r e d , the variability was m u c h greater but the n u m b e r of experimen,.s pcrforrned was not sufficient to p e r m i t statistical calculation. In fot, c experiments, however, the incorporation o f ~2p u n d e r the a b o v e c o n d i t i o n s a m o u n t e d to 1.2, 2.5, 2.6 and 3.5 n m o l e s / g tissue at 0.1 m M [3zP]ATP, and 14.6, 10.9, 8.7 and I I.I n m o l e s / g tissue, resl :cliveiy, at I m M [3zP]ATP.

Incubation of cell .sap with s,p, It was o f interest to find out whether s o m e o f the protein pho:;phorylation d e p e n d e d o n the m a j o r s2P-labelled d e g r a d a t i o n p r o d u c t o f [3zP]~TP, i.e. 3.~pi. F o r this p u r p o s e , cell sap was i n c u b a t e d at 30 :C with 0.1 m M s2p. for 5 rain (,r I m M s'P~ for 15 rain. The protein p h o s p h o r y l a t i o n o b t a i n e d amounte,~ to 0.3 and 0.8 n m o l e ; g liver, respectively. This s h o w s t h a t maximally 10 ~i, o f the p h o s p h o r y l a t i o n o c c u r r i n g d u r i n g i n c u b a t i o n with [s2P]ATP was d u e to 3:~Pt o r c o m p o u n d s formed f r o m it.

Reversibilio" of protein phosphorylation Actions of phosphoprotein phosphatases T a b l e IV shows t h e results o f a study o! the reversli~ility o f the protein phosp h o r y l a t i o n . Cell sap was incubated with [3~'P]ATP a n d the i n c u b a , i o n mixture was TABLE IV REVERSIBILITY OF Pi~,OTEIN PHOSPHORYLATION 18ral of cell sap were ~.~cubated with 1 raM [saP]ATP and l0 ram Mg z+ for .5 rain at 30 'C, After addition of EDTA to a ~,onccntration of 20 mM th~ i~cubation raixtur¢ was rapidly :ooled to 0 "C and immediately chror=.atographcd on Sephadex (, ~0 ~,~ ~t|2 Vl tdethanolamine-.acetic acid buffer (pH 7.4). Aliquots of 0.9 ral of the pooled fractiou~ ~xhi~tl appeared with the void w~lurae were incubated at 30 "C in the presence of 10 raM Mg :+ in a final volume of 1.0 ml for different leng.hs of time, with the additions listed below. Th,~ 3:p incorporation of a sample removed after initial incubation was 9,8 nraolcs/g. Incubation time train) 0 5

Addition

(nmol~.rg) None

15

5

ADP (1 raM)

t5

5 15 5 15

s=p mcorpot;tion

ATP (1 raM) [~2PIATP (I raM)

4.4 3,2 2.2 4.0 2.9 3,8 2.7 7.7 8,4

148

then chrcmatographed on Sephadex G-50 without previous inactivation of the material. It was found that considerable dephosphorylation occurred during chromatography and subsequent incubation at 30 °C with MSz+. On reincubation with [3zp]. ATP the protein became rephosphorylated. Reincubation with unlabelled ADP or ATP did not release the remaining labelled protein-bound phosphate, which demonstrates that the bound phosphate was not readily exchan6~,i in kinase "~aetions. When the native ~P-labelled protein was incubated with i0 mM Mgz+ for 15 rain at 30 °C and analyzed for ~zpi it was found that the decrease of protein-~ZP corresponded to a liberation of 3zp~. This indicates that protein-bound phosphate was split off by phosphoprotein phosphatases.

pH optimum of protein phosphorylation The optimum pH of the phosphorylation reaction was about 7; the phosphorylation decreased slightly when the pH was increased to 9 or decreased to 5-6 (Fig. 1)

5 o..

4

~c 2 !

~

o

4

5

6

7 pit

8

9

I0

Fig.rl. Phosphorylationof cell sap protein at different pH values. Cell sap (pH 7.4) was titrated with 1 ": ' MPacehc acid or 0.5 M Na~CO3and immediately incubated with I mM [32PlATPat 20 °C for 5 rain in the presence of 10raM Mgz+, as described under Experimental.

Effect of cyclic 3',5'-AMP on protein phosphorylation The effect of cyclic Y,5'-AMP on the protein phosphorylation is shown in Table V. At 0.1 and ! mM P2P]ATP the phosphate incorporation was considerably stimulated by 2.5.10 -s M cyclic 3',5'-AMP, especially on short incubations.

Isolation of {32PjSer(P) and {32PJThr(P) from {3'PjATP.incubated cell sap protein [32P]Ser(P) and [3~P]Thr(P) were isolated from acid hydrolysates of p'P]ATPincubated cell sap protein in addition to a large amount of ~P~ and small amounts of material e!uted from the Dowex 50-X8 columns in a position where phosphopeptides generally are eluted. 6-10 times more [3aP]Ser(p) than [3'P]Thr(P) was obtained after c¢~rrection for losses during hydrolysis. The results s h o ~ that most of the ~P was in fact protein-bound. This view is supported further from the finding that at east 8°,--90% of the 3zp was split off as ~2PI on incubation with 1 M NaOH for 15 rain at 100 °C [19].

149 TABLE V

EFFECT OF CYCLIC 3",5'-AMP ON PHOSPHORYLATION OF CELL SAP PROTEIN Cell sap was incubated with [32P]ATPat 30 ~C in the presence of I0 mM Mg2÷ and 2.5- I0- s I~| cyclic Y,5"-AMP, which was added immediately before the [3ZPIATP.Controls (in pall same as it, Table I) were incubated without cyclic 3",5'-AMP. [nP1ATP eoncn (raM) 0. I

Incubation time Cyclic ~P (rain) 3".5'-AMP incorporation (nraoles g) I

5 30 1.0

l

5

a-,p~ formed (% of [aZP]ATP)

÷

4.6

-~-

2.7 5.6 3.5 2.8 2.3

20 24 45 45 51 54

8.2 3.9

") 8

-~-

~ --

30 -

14 9.7 |2 12

24 21 50 46

DISCUSSION The present investigation clearly shows that ATP is a major precursor to r~rotein-bound phosphate of rat liver cell sap (Tables I and II). It seems reasonable to assume that the protein'°2P formed essentially derived from the 7-phosphoryl groups of [3"P]ATP. The quantitative data obtained are therefore given under the assumption that the specific radioactivity of the protein-bound phosphate was the same as that of the ?.-phosphate o f the [~'P]ATP used. However, due to the presence of adenylate kinase activity it is a p p a , e n t from Table Ii! that the true values were higher than those presented in Tables 1, IV and V. The variability of the assay of protein-i~ound a2p after incubation with [32p]. ATP, as estimated from duplicate determinations, was low. On the other hand, there were differences between various c¢11 sap preparations. The low initial velocity of the phosphorylation reaction and its non-reversibility on incubation of 32P-labelled proteiv with unlabelled substrate (Table IV) exclude the possibility that a large a m o u n t of intermediate phosphoryl enzymes was formed under the conditions used. The rate of the phosphorylation studied was essentially compatible with kinasecatalyzed reactions, i.e. similar to the rate of the regulative phosphorylation of enzymes (see ref. 6 for further references). Therefore, it is most probable that the phosphorylations observed in this study were regulative ph~,sphorylations of enzymes and other proteins catalyzed by protein kinases, Concomitant del,hosphorylation catalyzed by phosphoprotein phosphatases apparently occurred. In the present investigation, cyclic Y , 5 ' - A M P increased the protein phosphorylation considerably. ]'his means that cell sap contains considerable amounts of endo-

150 genous p r o t e i n s u b s t r a t e for cyclic Y , 5 ' - A M P - s t i m u l a t e d protein kinase activity. P h o s p h o r y l a s e kinase ( E C 2.7.1.38) a n d g l y c o g e n s y n t h e t a s e ( E C 2.4. I. t 1) are, as far as is k n o w n to date, the o n l y e n z y m e s in rat liver cell sap t h a t are such p r o t e i n k i n a s e substrates. It seems h i g h l y p r o b a b l e t h a t m o r e e n z y m e s o f this k i n d will be f o u n d . ACKNOWLEDGEMENTS ]'he present i n v e s t i g a t i o n was s u p p o r t e d by the Swedish M e d i c a l R e s e a r c h C o u n c i l (Project N o . . 5 0 - X ! 3). T h e skilful technical assistance o f Miss J i l | A n d e r s s o n and M r s G u n n e l B e r g s t r 6 m is g r a t e f u l l y a c k n o w l e d g e d . REFERENCES I A,gren, G, (1958) Acta Univ. Ups.. 2 Langan, T. A. (1967) Regulation of Nucleic Acid and Protein Biosynthesis. BBA Library, Vol. I0, pp. 233-242, Elsevier. Amsterdam 3 Forsberg, H., Zetterqvist, ~. and Engstr6m, L. 09691 Biochim. Biophys. Acta Igl, 171-175 4 Rask, L., Wllinder, O., Zetterqvist, O. and EngstrOm, L. (1970) Biochim. Biophys. Acta 221, 107-1t3 5 Ag3ren. G., de Verdict, C.-H. and Glomset, J. 0954) Acta Chem. Scand. 8, 1570-1578 6 Krebs, E. G. (1972) Current Topics in Cellular Regulation, Voi. 5, pp. 99-133, Academic Press, New York 7 Baggio, B., Pinna, L. A., Motet, V. and Siliprandi, N. ! 1970) Biochim. Biophys. Acta 212, 515-517 8 Sega L H. L. (1973) Science 180, 25-32 9 Pinna, L A., Clari, G. and Monet, V. (1971) Biochim. Biophys. Acta 236, 270--278 10 GIomset, J. A. (1959) Acta Soc. Med, Ups. 64, 236-243 i1 Magni, G., Caulini, G. and Moret, V. (1971) Biochim. Bioph)s. Acta 242, V23-128 ~,2 Meisler, M. H. and Langan, T. A. (1969) J. Biol. Chen~. 244, 4961-4968 13 Zetterqvist, ~. (1967) Biochim. Biophys. Acta 14t, 533-539 t4 M~rdh, S., Ljungstr6m, O., H/~gstedt, S. and Zetterqvist, ~. (1971) Biochim. Biophys. Acta 251, ~t 9-426 15 Langan, T. A. and Liprnann, F., referred to in Kleinsmith, L J., Allfrey. V. G. and Mirsky, A. E, (1966) Proc. Natl. Acad. Sci. U.S. 55, 1182-1189 16 Er~gstr0m, I. (1962) Ark. Kemi 19, 129-140 1"/ Moore, S. and Stein, W. H. (!954) J. P'31. Chem. 211,907-913 18 Marlir, J. B. and Duty, M. D, (1949) Anal. Chem. 21,965-967 19 Pabinogitz. M. and Lipmann, F. (1960) J. Biol. Chem. 235, IO43-1050