439
Biochimica et Biophysica Acta, 517 (1978) 439--446 © Elsevier/North-Holland Biomedical Press
BBA 99102 PHOSPHORYLATION OF CALF THYMUS RNA POLYMERASE II BY NUCLEAR CYCLIC 3',5'-AMP-INDEPENDENT PROTEIN KINASE
EVANGELIA G. KRANIAS and RICHARD A. JUNGMANN Department of Biochemistry, Northwestern University Medical School, Chicago, Ill. 60611 (U.S.A.)
(Received June 6th, 1977) (Revised manuscript receivedSeptember 28th, 1977)
Summary Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32p_ labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P/phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP.
Introduction DNA transcription can be regulated either by controlling the availability of the DNA template through nuclear repressor and activator proteins or nonhistone chromosomal proteins which bind to DNA, by changes of the levels of RNA polymerase (EC 2.7.7.6), or by modulation of the activity of RNA polymerases perhaps through phosphorylative modification. The latter possibility appears attractive, since protein kinase (EC 2.7.1.37) preparations achieve activation of nuclear RNA polymerases [1--5]. In view of this, it appears likely that functional modification of polymerase activity is achieved through direct phosphorylation of the core RNA polymerase. In this communication show that a nuclear cyclic AMP-independent protein kinase from calf thymus phosphorylates the homologous nucleoplasmic RNA
440 polymerase II. The main phosphate acceptor subunit and the nature of the phosphoester bonds generated are defined. Phosphorylation of the RNA polymerase II occurs concomitantly with stimulation of its enzymatic activity. Experimental
Materials. All biochemical reagents were purchased from Sigma Chemical Co. [~')2P]ATP, ammonium salt (2--10 Ci/mmol), [a-32P]UTP, ammonium salt (5--10 Ci/mmol), [5,6-3H2]UTP, tetrasodium salt (35--50 Ci/mmol) were from New England Nuclear. Phosphocellulose P-11 was from Whatman and was prepared as described [6]. DEAE-cellulose (DE-52), Bio-Gel HT hydroxyapatite, Dowex AG50W-5X (200--400 mesh), Sepharose 6B and calf thymus were obtained and prepared as described in the preceding paper [ 7]. Enzyme preparations. Nuclear cyclic AMP-independent protein kinase and RNA polymerase II were purified to homogeneity as described previously [7, 8]. Polyacrylamide gel electrophoresis under non-denaturing conditions revealed that RNA polymerase II obtained after phosphocellulose P-11 chromatography contained both the BI and BII forms of the enzyme [9]. No other contaminating proteins were detected. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed the characteristic banding pattern of RNA polymerase II subunits [9]. Under these denaturing conditions one contaminating protein band (I2) and in about 40% of the preparations two bands (I1, I2, see Fig. 4) were present in the RNA polymerase preparations [9]. The specific activity of RNA polymerase II varied between 50 and 200 units per mg RNA polymerase II. One unit is the amount of enzyme which catalyzes the incorporation of 1 nmol of UTP into acid-precipitable RNA in 15 min at 37°C. Assays of RNA polymerase and protein kinase activities. RNA polymerase assays were performed [8] in a total reaction volume of 0.15 ml. Under the experimental conditions incorporation of radioactive UMP into acid-insoluble material proceeded linearly up to 60 min incubation time. Protein kinase activity was assayed as described [7]. When RNA polymerase II was used as substrate, phosphorylations were carried out in 20 mM Tris/0.1 mM EDTA/5 mM MgC12/0.5 mM dithiothreitol/25% (v/v) glycerol, pH 7.4, (Buffer A), containing 40 mM {NH4)2SO4 for 15 rain at 35°C. Under the experimental conditions incorporation of 32p into RNA polymerase II proceeded linearly up to 15 min. Determination of [a2p]phosphoserine and [a2P]phosphothreonine. a~p. labeled phosphoserine and phosphothreonine were isolated after acid hydrolysis of a~P-labeled RNA polymerase II and chromatography on Dowex AG50W5X [81. Polyacrylamide gel electrophoresis. Gel electrophoresis and gel analysis were performed as previously described [8]. Determination of protein. Protein concentration was determined by the method of Lowry et al. [10] using crystalline bovine serum albumin as standard. Determination of radioactivity. Determination of radioactivity was carried out as described [8].
441 Results
Phosphorylation of calf thymus RNA polymerase H by nuclear cyclic AMPindependent protein kinase The nuclear cyclic AMP-independent protein kinase from calf thymus catalyzes the transfer of the terminal phosphate residue from [7-32P]ATP to RNA polymerase II (Fig. 1). There was no incorporation of 32p radioactivity into RNA polymerase II in the absence of protein kinase, and no autophosphorylation of the cyclic AMP-independent protein kinase preparations was observed. The degree of phosphorylation of RNA polymerase II in vitro varied with the different RNA polymerase preparations. Assuming a molecular weight of 700 000 for RNA polymerase II [9], an average value of 0.2 mol of [32P]phosphate incorporated per mol of RNA polymerase II at saturation levels was obtained. The phosphorylation reaction was routinely carried out in 40 mM (NH4)2SO4 because at this salt concentration the protein kinase exhibited optimum enzymatic activity [7].
DEAE-cellulose chromatography of a2P-labeled RNA polymerase H RNA polymerase II was incubated with cyclic AMP-independent protein kinase and [7-32P]ATP under conditions of optimum phosphorylation. After incubation the reaction mixture was dialyzed extensively and subsequently subjected to chromatography on DEAE-cellulose. Trichloroacetic acid-insoluble a2p radioactivity and RNA polymerase activity were assayed in the eluted fractions (Fig. 2). Fractions 33--45 from the DEAE-cellulose column containing 32P-label and RNA polymerase activity were pooled, dialyzed, and concentrated. The concentrated 32p-labeled RNA polymerase was used as enzyme source for polyacrylamide gel electrophoresis and acid hydrolysis.
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Protein Kinose (/~q) Fig. 1. E f f e c t o f increasing a m o u n t s o f calf t h y m u s nuclear cyclic A M P - i n d e p e n d e n t p r o t e i n kinase o n the i n c o r p o r a t i o n o f [ 3 2 p ] p h o s p h a t e i n t o R N A P o l y m e r a s e If. 7.5/~g o f R N A p o l y m e r a s e n w e r e i n c u b a t e d w i t h the i n d i c a t e d a m o u n t s o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinasc and 8 n m o l (4/~Ci) o f [ 7 -32 p] A T P in a t o t a l r e a c t i o n v o l u m e o f 0.4 m l as described u n d e r Experimental. T h e values s h o w n are the a r i t h m e t i c m e a n s o f three d e t e r m i n a t i o n s .
442
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Fig. 2. D E A E - c e l i u l o s e c h r o m a t o g r a p h i c e l u t i o n profile of 3 2 p r a d i o a c t i v i t y a n d R N A p o l y m e r a s e I I activity. 3 4 5 p g o f R N A p o l y m e r a s e II w e r e i n c u b a t e d w i t h 8 3 5 p g o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinase a n d 1 4 8 n m o l ( 2 . 1 5 m C i ) of [ 7 - 3 2 p ] A T P in a t o t a l r e a c t i o n v o l u m e o f 7.4 m l as d e s c r i b e d in E x p e r i m e n t a l . A t t h e e n d of t h e i n c u b a t i o n p e r i o d , 10 m l of ice-cold b u f f e r A w e r e a d d e d a n d t h e r e a c t i o n m i x t u r e was d i a l y z e d against 1 0 0 0 m l of b u f f e r A / 5 0 m M ( N H 4 ) 2 S O 4 w h i c h w a s c h a n g e d t h r e e t i m e s . T h e 32p-labeled protein fraction was c h r o m a t o g r a p h e d on DEAE-cellulose. The c o l u m n was washed with buffer A / 5 0 raM ( N H 4 ) 2 S O 4 a n d s u b s e q u e n t l y e l u t e d w i t h a 2 2 m l l i n e a r g r a d i e n t of 0 . 0 5 - - 0 . 5 M ( N H 4 ) 2 S O 4 in b u f f e r A (3 m l / h , 0 . 4 - m l f r a c t i o n s ) . A l i q u o t s of e a c h f r a c t i o n w e r e a s s a y e d f o r R N A p o l y m e r a s e a c t i v i t y a n d f o r 3 2 p r a d i o a c t i v i t y . F r a c t i o n s 3 3 - - 4 6 w e r e p o o l e d , d i a l y z e d against b u f f e r A / 5 0 m M ( N H 4 ) 2 S O 4 , c o n c e n t r a t e d , a n d u s e d as t h e s o u r c e o f 3 2 p - l a b e l e d R N A p o l y m e r a s e If.
Polyacrylamide gel electrophoresis of 32p-labeled RNA polymerase H 32p-labeled RNA polymerase II was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions on 5% polyacrylamide gels. Two forms of RNA polymerase II were visible in the gel (Fig. 3), as previously reported [9]. 32p radioactivity was associated with both enzyme bands. To determine the pattern of subunit phosphorylation 32p-labeled RNA polymerase II was subjected to SDS gel electrophoresis. The characteristic arrangement of six RNA polymerase II subunits [9] was obtained (Fig. 4). Using both 5% polyacrylamide gels (data not shown) and 5% acrylamide in the upper half and 10% acrylamide in the lower half (mixed gels) the 25 000 molecular weight subunit (B5) of RNA polymerase II was identified to be the phosphate acceptor protein (Fig. 4}.
Identification of the 32p-labeledphosphoamino acids 32P-labeled RNA polymerase II was subjected to acid and base hydrolysis; low stability of the phosphate-RNA polymerase linkages was observed after t r e a t m e n t of the 32p-labeled RNA polymerase II for 1 h at 37°C in 1 M NaOH, whereas high stability was observed after treatment for 1 h at 100°C in 0.1 M HC1. Ion-exchange chromatography of the hydrolyzed 32P-labeled RNA polymerase II shows that the protein contained more phosphoserine (70%) than phosphothreonine (30%).
443
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Fig. 3. Polyacrylamidc gel electrophoresis of phosphorylated R N A polymerase II under non-denaturing conditions. 15/zg of 32p-labeled R N A polymerase II (obtained from DEAE-cellulose, Fig. 2) were applied onto a 5 % polyacrylamide gel and subjected to electropboresis (4 h, 3 mA/gel, 4°C). The gel was stained, destained, scanned (550 nm), and sliced ( 1 - m m sections) for determination of 32p radioactivity.
Stimulation o f R N A polymerase H activity RNA polymerase II was preincubated with cyclic AMP-independent protein kinase and non-radioactive ATP. At the end of the preincubation period phosphorylation of RNA polymerase II was complete and the samples were processed for determination of RNA polymerase activity. Table I shows the correlation between RNA polymerase II activity and the amount of protein kinase present in the incubation samples. To determine the effect of specific inhibition of the protein kinase activity on its ability to stimulate RNA polymerase II, stimulation was checked in the presence of the ATP analogue adenylimidodiphosphate (AMP-PNP). AMP-PNP is an inhibitor of enzymes, e.g. protein kinase, that cleave the ~-~ linkage of nucleoside triphosphates and thus inhibits [32P]phosphate incorporation into substrate protein [11]. Table I shows that in the presence of AMP-PNP but absence of protein kinase RNA polymerase II activity was inhibited by 41% probably due to the reduced efficiency of AMP-PNP to act as substrate in the RNA polymerase assay. Furthermore, the stimulatory activity in the presence of protein kinase was reduced by 67% indicating that the protein kinase was not able to significantly stimulate the RNA polymerase II activity. Incubation of alkaline phosphatase from Escherichia coli with 32P-labeled RNA polymerase II resulted in a marked loss of 32p radioactivity and a c o m c o m i t a n t decrease of polymerase specific activity [8].
444
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Fig. 4. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of p h o s p h o r y l a t e d R N A p o l y m e r a s e II. 4 0 / ~ g of 3 2 p . l a b e l e d R N A p o l y m e r a s e lI ( o b t a i n e d f r o m D E A E - c e l l u l o s e , Fig. 2) w e r e a p p l i e d o n t o a m i x e d p o l y a c r y l a m i d e gel (5% a c r y l a m i d e in t h e u p p e r h a l f a n d 10% a c r y l a m i d e in the l o w e r h a l f ) c o n t a i n i n g 0 . 1 % SDS. E l e c t r o p h o r e s i s was c a r r i e d o u t ( 2 . 5 m A / g e l , 9 h, 25°C)~ A f t e r e l e c t r o p h o r e s i s t h e gel was stained, d e s t a i n e d , s c a n n e d ( 5 5 0 n m ) , a n d slided ( 2 - r a m s e c t i o n s ) for d e t e r m i n a t i o n o f 3 2 p r a d i o a c t i v i t y .
TABLE I E F F E C T OF A D E N Y L Y L I M I D O D I P H O S P H A T E ON T H E S T I M U L A T I O N OF C A L F T H Y M U S R N A P O L Y M E R A S E II BY N U C L E A R C Y C L I C A M P - I N D E P E N D E N T P R O T E I N K I N A S E A s s a y s w e r e p e r f o r m e d as d e s c r i b e d in E x p e r i m e n t a l . 3 p g o f R N A p o l y m e r a s e I I w e r e i n c u b a t e d w i t h t h e i n d i c a t e d a m o u n t s o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinase a n d 1.4 n m o l o f A T P or AMP-PNP in a t o t a l r e a c t i o n v o l u m e o f 70 # l o f p r o t e i n k i n a s e assay b u f f e r . I n c u b a t i o n s w e r e c a r r i e d o u t for 15 r a i n at 35°C a n d s u b s e q u e n t l y t h e s a m p l e s w e r e c o o l e d in ice f o r 20 rain. T o m e a s u r e t h e R N A p o l y m e r a s e a c t i v i t y o f t h e s a m p l e s , 6 n m o l (4 ~ C i ) o f [ ~ - 3 2 p ] U T P a n d all o t h e r r e a c t a n t s for t h e R N A p o l y m e r a s c assay ( 8 0 f~l) c o n t a i n i n g e i t h e r A T P o r AMP-PNP w e r e a d d e d . I n c u b a t i o n s w e r e c a r r i e d o u t f o r 6 0 rain at 37°C. E x p e r i m e n t a l d e t a i l s are g i v e n u n d e r E x p e r i m e n t a l . T h e v a l u e s s h o w n are t h e a r i t h m e t i c m e a n s o f t h r e e d e t e r m i nations. Cyclic AMPindependent p r o t e i n kinase (~g)
1.33 2.66 5.00 8.66
R N A p o l y m a r a s c II a c t i v i t y (pmol [a-32P] UMP/sample)
Stimulation *
ATP
AMP-PNP
ATP
AMP-PNP
215.68 295.28 370.26 464.45 499.81
127 144.32 153.67 157.48 168.91
-37 72 116 132
-16 21 24 33
* Relative to control (100%).
445 Discussion There has been evidence accumulating that phosphorylation of RNA polymerases may be one of the mechanisms which control transcription in both prokaryotes and eukaryotes [12--17]. In this study we have presented evidence that the nucleoplasmic RNA polymerase from calf thymus becomes phosphorylated in vitro by a nuclear protein kinase which is not regulated by cyclic AMP. The amount of [32P]phosphate incorporation under in vitro conditions into the various RNA polymerase preparations by an identical protein kinase preparation varied suggesting that the purified RNA polymerase II may already be phosphorylated in vivo to varying degrees. Both forms of the RNA polymerase were shown to incorporate phosphate. Hirsch and Martelo [5] reported that only one of the forms of the nucleolar RNA polymerase was phosphorylated in vivo in rat liver nuclei. The main phosphate acceptor subunit of the calf thymus RNA polymerase II was found to be the 25 000 dalton subunit and both phosphoserine and to a lesser extent phosphothreonine were formed. Rutter et al. [17] reported that the 25 000 molecular weight subunit of rat liver RNA polymerase II was the major site of the phosphorylative modification. We observed that phosphorylation of the 25 000 dalton subunit was accompanied by a stimulation of RNA polymerase activity. When the protein kinase activity was inhibited, no stimulation of RNA polymerase activity was observed (Table I). This suggests that phosphorylation of the B5 subunit may play a crucial role in determining the activity of the enzyme. Recently, Valenzuela et al. [18] using yeast RNA polymerase I showed that the molar ratio of the 24 000 dalton subunit, which may become phosphorylated [14,15] correlates with polymerase activity. Although we have not shown a relationship between the molar ratio of the B5 subunit and the RNA polymerase II activity in calf thymus, we tentatively conclude from our data that phosphorylation of this polypeptide subunit may have a possible role in the regulation of enzyme activity and therefore DNA transcription. We have previously shown that the B5 subunit was also the main phosphate acceptor subunit when nucleoplasmic RNA polymerase II from calf thymus was phosphorylated in vitro by a homologous nuclear protein kinase which is regulated by cyclic AMP [8]. This raises the question which of the nuclear protein kinases, the cyclic AMP-dependent or -independent or conceivably both, may achieve a biologically significant phosphorylation of RNA polymerase in vivo. Experiments are now in progress to determine if and which protein kinase phosphorylates RNA polymerase II in vivo and to identify the RNA polymerase II phosphate acceptor subunits. Acknowledgements We are grateful for the excellent technical assistance of Ms. Martha Bieber. This work was supported by National Science Foundation Grant PCM-7617213, and in part by the Research and Education Fund, Northwestern Memorial Hospital.
446
References 1 J u n g m a n n , R.A., Hiestand, P.C. and Schweppe, J.S. (1974) J. Biol. Chem. 249, 5444--5451 2 J u n g m a n n , R.A. and Kranias, E.G. (1976) in Advances in Biochemical Psychopharmacology (Costa, E., Giacobini, E. and Paoletti, R., eds.), Vol. 15, pp. 413--428, Raven Press, New Y ork 3 Martelo, O.J. and Hirsch, J. (1974) Biochem. Biophys. Res. Commun. 58, 1008--1015 4 Dahmus, M.E. (1976) Biochemistry 15, 1 8 2 1 - - 1 8 2 9 5 Hirsch, J. and Martelo, O.J. (1976) J. Biol. Chem. 251, 5408--5413 6 Gissinger, F. and Chambon, P. (1972) Eur. J. Biochem. 28, 277--282 7 Kranias, E.G. and J u n g m a n n , R.A. (1977) Biochim. Biophys. Acta 517, 447--456 8 Kranias, E.G., Schweppe, J.S. and Jungmann, R.A. (1977) J. Biol. Chem. 252, 6 7 5 0 - - 6 7 5 8 9 Kedinger, C. and Chambon, P. (1972) Eur. J. Biochem. 28, 283--290 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 11 Yo unt, R.G., Babcock, D., Ballantyne, W. and Ojala, D. (1971) Biochemistry 10, 2484--2489 12 Martelo, O.J., Woo, S.L.C. and Davie, E.W. (1974) J. Mol. Biol. 87, 685--696 13 ZiUig, W., Fujiki, H., Blum, W., Janekovie, D., Schweiger, M., Rahmsdorf, H.J., Ponta, H. and HirschKauffman, M. (1975) Proe. Natl. Acad. Sci. U.S. 72, 2 5 0 6 - - 2 5 1 0 14 Bell, G.I., Valenzuela, P. and Rutter, W.J. (1976) Nature 2 6 1 , 4 2 9 - - 4 3 1 15 Bell, G.I., Valenzuela, P. and Rutter, W.J. (1977) J. Biol. Chem. 252, 3082--3091 16 Buhler, J.M., Iborra, F., Sentenac, A. and Fromageot, P. (1976) FEBS Lett. 71, 37---41 17 Rutter, W.J., Morris, P.W., Goldberg, M. and Morris, R.W. (1973) in The Biochemistry of Gene Expression in Higher Organisms (Pollack, J.K. and Lee, J.W., eds.), pp. 89--104, Reidel Publishing Co., Boston 18 Valenzuela, P., Bell, G.I. and Rutter, W.J. (1976) Biochem. Biophys. Res. Commun. 71, 26--31
Biochimica et Biophysica Acta, 517 (1978) 439--446 © Elsevier/North-Holland Biomedical Press
BBA 99102 PHOSPHORYLATION OF CALF THYMUS RNA POLYMERASE II BY NUCLEAR CYCLIC 3',5'-AMP-INDEPENDENT PROTEIN KINASE
EVANGELIA G. KRANIAS and RICHARD A. JUNGMANN Department of Biochemistry, Northwestern University Medical School, Chicago, Ill. 60611 (U.S.A.)
(Received June 6th, 1977) (Revised manuscript receivedSeptember 28th, 1977)
Summary Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32p_ labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P/phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP.
Introduction DNA transcription can be regulated either by controlling the availability of the DNA template through nuclear repressor and activator proteins or nonhistone chromosomal proteins which bind to DNA, by changes of the levels of RNA polymerase (EC 2.7.7.6), or by modulation of the activity of RNA polymerases perhaps through phosphorylative modification. The latter possibility appears attractive, since protein kinase (EC 2.7.1.37) preparations achieve activation of nuclear RNA polymerases [1--5]. In view of this, it appears likely that functional modification of polymerase activity is achieved through direct phosphorylation of the core RNA polymerase. In this communication show that a nuclear cyclic AMP-independent protein kinase from calf thymus phosphorylates the homologous nucleoplasmic RNA
440 polymerase II. The main phosphate acceptor subunit and the nature of the phosphoester bonds generated are defined. Phosphorylation of the RNA polymerase II occurs concomitantly with stimulation of its enzymatic activity. Experimental
Materials. All biochemical reagents were purchased from Sigma Chemical Co. [~')2P]ATP, ammonium salt (2--10 Ci/mmol), [a-32P]UTP, ammonium salt (5--10 Ci/mmol), [5,6-3H2]UTP, tetrasodium salt (35--50 Ci/mmol) were from New England Nuclear. Phosphocellulose P-11 was from Whatman and was prepared as described [6]. DEAE-cellulose (DE-52), Bio-Gel HT hydroxyapatite, Dowex AG50W-5X (200--400 mesh), Sepharose 6B and calf thymus were obtained and prepared as described in the preceding paper [ 7]. Enzyme preparations. Nuclear cyclic AMP-independent protein kinase and RNA polymerase II were purified to homogeneity as described previously [7, 8]. Polyacrylamide gel electrophoresis under non-denaturing conditions revealed that RNA polymerase II obtained after phosphocellulose P-11 chromatography contained both the BI and BII forms of the enzyme [9]. No other contaminating proteins were detected. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed the characteristic banding pattern of RNA polymerase II subunits [9]. Under these denaturing conditions one contaminating protein band (I2) and in about 40% of the preparations two bands (I1, I2, see Fig. 4) were present in the RNA polymerase preparations [9]. The specific activity of RNA polymerase II varied between 50 and 200 units per mg RNA polymerase II. One unit is the amount of enzyme which catalyzes the incorporation of 1 nmol of UTP into acid-precipitable RNA in 15 min at 37°C. Assays of RNA polymerase and protein kinase activities. RNA polymerase assays were performed [8] in a total reaction volume of 0.15 ml. Under the experimental conditions incorporation of radioactive UMP into acid-insoluble material proceeded linearly up to 60 min incubation time. Protein kinase activity was assayed as described [7]. When RNA polymerase II was used as substrate, phosphorylations were carried out in 20 mM Tris/0.1 mM EDTA/5 mM MgC12/0.5 mM dithiothreitol/25% (v/v) glycerol, pH 7.4, (Buffer A), containing 40 mM {NH4)2SO4 for 15 rain at 35°C. Under the experimental conditions incorporation of 32p into RNA polymerase II proceeded linearly up to 15 min. Determination of [a2p]phosphoserine and [a2P]phosphothreonine. a~p. labeled phosphoserine and phosphothreonine were isolated after acid hydrolysis of a~P-labeled RNA polymerase II and chromatography on Dowex AG50W5X [81. Polyacrylamide gel electrophoresis. Gel electrophoresis and gel analysis were performed as previously described [8]. Determination of protein. Protein concentration was determined by the method of Lowry et al. [10] using crystalline bovine serum albumin as standard. Determination of radioactivity. Determination of radioactivity was carried out as described [8].
441 Results
Phosphorylation of calf thymus RNA polymerase H by nuclear cyclic AMPindependent protein kinase The nuclear cyclic AMP-independent protein kinase from calf thymus catalyzes the transfer of the terminal phosphate residue from [7-32P]ATP to RNA polymerase II (Fig. 1). There was no incorporation of 32p radioactivity into RNA polymerase II in the absence of protein kinase, and no autophosphorylation of the cyclic AMP-independent protein kinase preparations was observed. The degree of phosphorylation of RNA polymerase II in vitro varied with the different RNA polymerase preparations. Assuming a molecular weight of 700 000 for RNA polymerase II [9], an average value of 0.2 mol of [32P]phosphate incorporated per mol of RNA polymerase II at saturation levels was obtained. The phosphorylation reaction was routinely carried out in 40 mM (NH4)2SO4 because at this salt concentration the protein kinase exhibited optimum enzymatic activity [7].
DEAE-cellulose chromatography of a2P-labeled RNA polymerase H RNA polymerase II was incubated with cyclic AMP-independent protein kinase and [7-32P]ATP under conditions of optimum phosphorylation. After incubation the reaction mixture was dialyzed extensively and subsequently subjected to chromatography on DEAE-cellulose. Trichloroacetic acid-insoluble a2p radioactivity and RNA polymerase activity were assayed in the eluted fractions (Fig. 2). Fractions 33--45 from the DEAE-cellulose column containing 32P-label and RNA polymerase activity were pooled, dialyzed, and concentrated. The concentrated 32p-labeled RNA polymerase was used as enzyme source for polyacrylamide gel electrophoresis and acid hydrolysis.
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Protein Kinose (/~q) Fig. 1. E f f e c t o f increasing a m o u n t s o f calf t h y m u s nuclear cyclic A M P - i n d e p e n d e n t p r o t e i n kinase o n the i n c o r p o r a t i o n o f [ 3 2 p ] p h o s p h a t e i n t o R N A P o l y m e r a s e If. 7.5/~g o f R N A p o l y m e r a s e n w e r e i n c u b a t e d w i t h the i n d i c a t e d a m o u n t s o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinasc and 8 n m o l (4/~Ci) o f [ 7 -32 p] A T P in a t o t a l r e a c t i o n v o l u m e o f 0.4 m l as described u n d e r Experimental. T h e values s h o w n are the a r i t h m e t i c m e a n s o f three d e t e r m i n a t i o n s .
442
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Fig. 2. D E A E - c e l i u l o s e c h r o m a t o g r a p h i c e l u t i o n profile of 3 2 p r a d i o a c t i v i t y a n d R N A p o l y m e r a s e I I activity. 3 4 5 p g o f R N A p o l y m e r a s e II w e r e i n c u b a t e d w i t h 8 3 5 p g o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinase a n d 1 4 8 n m o l ( 2 . 1 5 m C i ) of [ 7 - 3 2 p ] A T P in a t o t a l r e a c t i o n v o l u m e o f 7.4 m l as d e s c r i b e d in E x p e r i m e n t a l . A t t h e e n d of t h e i n c u b a t i o n p e r i o d , 10 m l of ice-cold b u f f e r A w e r e a d d e d a n d t h e r e a c t i o n m i x t u r e was d i a l y z e d against 1 0 0 0 m l of b u f f e r A / 5 0 m M ( N H 4 ) 2 S O 4 w h i c h w a s c h a n g e d t h r e e t i m e s . T h e 32p-labeled protein fraction was c h r o m a t o g r a p h e d on DEAE-cellulose. The c o l u m n was washed with buffer A / 5 0 raM ( N H 4 ) 2 S O 4 a n d s u b s e q u e n t l y e l u t e d w i t h a 2 2 m l l i n e a r g r a d i e n t of 0 . 0 5 - - 0 . 5 M ( N H 4 ) 2 S O 4 in b u f f e r A (3 m l / h , 0 . 4 - m l f r a c t i o n s ) . A l i q u o t s of e a c h f r a c t i o n w e r e a s s a y e d f o r R N A p o l y m e r a s e a c t i v i t y a n d f o r 3 2 p r a d i o a c t i v i t y . F r a c t i o n s 3 3 - - 4 6 w e r e p o o l e d , d i a l y z e d against b u f f e r A / 5 0 m M ( N H 4 ) 2 S O 4 , c o n c e n t r a t e d , a n d u s e d as t h e s o u r c e o f 3 2 p - l a b e l e d R N A p o l y m e r a s e If.
Polyacrylamide gel electrophoresis of 32p-labeled RNA polymerase H 32p-labeled RNA polymerase II was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions on 5% polyacrylamide gels. Two forms of RNA polymerase II were visible in the gel (Fig. 3), as previously reported [9]. 32p radioactivity was associated with both enzyme bands. To determine the pattern of subunit phosphorylation 32p-labeled RNA polymerase II was subjected to SDS gel electrophoresis. The characteristic arrangement of six RNA polymerase II subunits [9] was obtained (Fig. 4). Using both 5% polyacrylamide gels (data not shown) and 5% acrylamide in the upper half and 10% acrylamide in the lower half (mixed gels) the 25 000 molecular weight subunit (B5) of RNA polymerase II was identified to be the phosphate acceptor protein (Fig. 4}.
Identification of the 32p-labeledphosphoamino acids 32P-labeled RNA polymerase II was subjected to acid and base hydrolysis; low stability of the phosphate-RNA polymerase linkages was observed after t r e a t m e n t of the 32p-labeled RNA polymerase II for 1 h at 37°C in 1 M NaOH, whereas high stability was observed after treatment for 1 h at 100°C in 0.1 M HC1. Ion-exchange chromatography of the hydrolyzed 32P-labeled RNA polymerase II shows that the protein contained more phosphoserine (70%) than phosphothreonine (30%).
443
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Fig. 3. Polyacrylamidc gel electrophoresis of phosphorylated R N A polymerase II under non-denaturing conditions. 15/zg of 32p-labeled R N A polymerase II (obtained from DEAE-cellulose, Fig. 2) were applied onto a 5 % polyacrylamide gel and subjected to electropboresis (4 h, 3 mA/gel, 4°C). The gel was stained, destained, scanned (550 nm), and sliced ( 1 - m m sections) for determination of 32p radioactivity.
Stimulation o f R N A polymerase H activity RNA polymerase II was preincubated with cyclic AMP-independent protein kinase and non-radioactive ATP. At the end of the preincubation period phosphorylation of RNA polymerase II was complete and the samples were processed for determination of RNA polymerase activity. Table I shows the correlation between RNA polymerase II activity and the amount of protein kinase present in the incubation samples. To determine the effect of specific inhibition of the protein kinase activity on its ability to stimulate RNA polymerase II, stimulation was checked in the presence of the ATP analogue adenylimidodiphosphate (AMP-PNP). AMP-PNP is an inhibitor of enzymes, e.g. protein kinase, that cleave the ~-~ linkage of nucleoside triphosphates and thus inhibits [32P]phosphate incorporation into substrate protein [11]. Table I shows that in the presence of AMP-PNP but absence of protein kinase RNA polymerase II activity was inhibited by 41% probably due to the reduced efficiency of AMP-PNP to act as substrate in the RNA polymerase assay. Furthermore, the stimulatory activity in the presence of protein kinase was reduced by 67% indicating that the protein kinase was not able to significantly stimulate the RNA polymerase II activity. Incubation of alkaline phosphatase from Escherichia coli with 32P-labeled RNA polymerase II resulted in a marked loss of 32p radioactivity and a c o m c o m i t a n t decrease of polymerase specific activity [8].
444
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Fig. 4. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of p h o s p h o r y l a t e d R N A p o l y m e r a s e II. 4 0 / ~ g of 3 2 p . l a b e l e d R N A p o l y m e r a s e lI ( o b t a i n e d f r o m D E A E - c e l l u l o s e , Fig. 2) w e r e a p p l i e d o n t o a m i x e d p o l y a c r y l a m i d e gel (5% a c r y l a m i d e in t h e u p p e r h a l f a n d 10% a c r y l a m i d e in the l o w e r h a l f ) c o n t a i n i n g 0 . 1 % SDS. E l e c t r o p h o r e s i s was c a r r i e d o u t ( 2 . 5 m A / g e l , 9 h, 25°C)~ A f t e r e l e c t r o p h o r e s i s t h e gel was stained, d e s t a i n e d , s c a n n e d ( 5 5 0 n m ) , a n d slided ( 2 - r a m s e c t i o n s ) for d e t e r m i n a t i o n o f 3 2 p r a d i o a c t i v i t y .
TABLE I E F F E C T OF A D E N Y L Y L I M I D O D I P H O S P H A T E ON T H E S T I M U L A T I O N OF C A L F T H Y M U S R N A P O L Y M E R A S E II BY N U C L E A R C Y C L I C A M P - I N D E P E N D E N T P R O T E I N K I N A S E A s s a y s w e r e p e r f o r m e d as d e s c r i b e d in E x p e r i m e n t a l . 3 p g o f R N A p o l y m e r a s e I I w e r e i n c u b a t e d w i t h t h e i n d i c a t e d a m o u n t s o f cyclic A M P - i n d e p e n d e n t p r o t e i n kinase a n d 1.4 n m o l o f A T P or AMP-PNP in a t o t a l r e a c t i o n v o l u m e o f 70 # l o f p r o t e i n k i n a s e assay b u f f e r . I n c u b a t i o n s w e r e c a r r i e d o u t for 15 r a i n at 35°C a n d s u b s e q u e n t l y t h e s a m p l e s w e r e c o o l e d in ice f o r 20 rain. T o m e a s u r e t h e R N A p o l y m e r a s e a c t i v i t y o f t h e s a m p l e s , 6 n m o l (4 ~ C i ) o f [ ~ - 3 2 p ] U T P a n d all o t h e r r e a c t a n t s for t h e R N A p o l y m e r a s c assay ( 8 0 f~l) c o n t a i n i n g e i t h e r A T P o r AMP-PNP w e r e a d d e d . I n c u b a t i o n s w e r e c a r r i e d o u t f o r 6 0 rain at 37°C. E x p e r i m e n t a l d e t a i l s are g i v e n u n d e r E x p e r i m e n t a l . T h e v a l u e s s h o w n are t h e a r i t h m e t i c m e a n s o f t h r e e d e t e r m i nations. Cyclic AMPindependent p r o t e i n kinase (~g)
1.33 2.66 5.00 8.66
R N A p o l y m a r a s c II a c t i v i t y (pmol [a-32P] UMP/sample)
Stimulation *
ATP
AMP-PNP
ATP
AMP-PNP
215.68 295.28 370.26 464.45 499.81
127 144.32 153.67 157.48 168.91
-37 72 116 132
-16 21 24 33
* Relative to control (100%).
445 Discussion There has been evidence accumulating that phosphorylation of RNA polymerases may be one of the mechanisms which control transcription in both prokaryotes and eukaryotes [12--17]. In this study we have presented evidence that the nucleoplasmic RNA polymerase from calf thymus becomes phosphorylated in vitro by a nuclear protein kinase which is not regulated by cyclic AMP. The amount of [32P]phosphate incorporation under in vitro conditions into the various RNA polymerase preparations by an identical protein kinase preparation varied suggesting that the purified RNA polymerase II may already be phosphorylated in vivo to varying degrees. Both forms of the RNA polymerase were shown to incorporate phosphate. Hirsch and Martelo [5] reported that only one of the forms of the nucleolar RNA polymerase was phosphorylated in vivo in rat liver nuclei. The main phosphate acceptor subunit of the calf thymus RNA polymerase II was found to be the 25 000 dalton subunit and both phosphoserine and to a lesser extent phosphothreonine were formed. Rutter et al. [17] reported that the 25 000 molecular weight subunit of rat liver RNA polymerase II was the major site of the phosphorylative modification. We observed that phosphorylation of the 25 000 dalton subunit was accompanied by a stimulation of RNA polymerase activity. When the protein kinase activity was inhibited, no stimulation of RNA polymerase activity was observed (Table I). This suggests that phosphorylation of the B5 subunit may play a crucial role in determining the activity of the enzyme. Recently, Valenzuela et al. [18] using yeast RNA polymerase I showed that the molar ratio of the 24 000 dalton subunit, which may become phosphorylated [14,15] correlates with polymerase activity. Although we have not shown a relationship between the molar ratio of the B5 subunit and the RNA polymerase II activity in calf thymus, we tentatively conclude from our data that phosphorylation of this polypeptide subunit may have a possible role in the regulation of enzyme activity and therefore DNA transcription. We have previously shown that the B5 subunit was also the main phosphate acceptor subunit when nucleoplasmic RNA polymerase II from calf thymus was phosphorylated in vitro by a homologous nuclear protein kinase which is regulated by cyclic AMP [8]. This raises the question which of the nuclear protein kinases, the cyclic AMP-dependent or -independent or conceivably both, may achieve a biologically significant phosphorylation of RNA polymerase in vivo. Experiments are now in progress to determine if and which protein kinase phosphorylates RNA polymerase II in vivo and to identify the RNA polymerase II phosphate acceptor subunits. Acknowledgements We are grateful for the excellent technical assistance of Ms. Martha Bieber. This work was supported by National Science Foundation Grant PCM-7617213, and in part by the Research and Education Fund, Northwestern Memorial Hospital.
446
References 1 J u n g m a n n , R.A., Hiestand, P.C. and Schweppe, J.S. (1974) J. Biol. Chem. 249, 5444--5451 2 J u n g m a n n , R.A. and Kranias, E.G. (1976) in Advances in Biochemical Psychopharmacology (Costa, E., Giacobini, E. and Paoletti, R., eds.), Vol. 15, pp. 413--428, Raven Press, New Y ork 3 Martelo, O.J. and Hirsch, J. (1974) Biochem. Biophys. Res. Commun. 58, 1008--1015 4 Dahmus, M.E. (1976) Biochemistry 15, 1 8 2 1 - - 1 8 2 9 5 Hirsch, J. and Martelo, O.J. (1976) J. Biol. Chem. 251, 5408--5413 6 Gissinger, F. and Chambon, P. (1972) Eur. J. Biochem. 28, 277--282 7 Kranias, E.G. and J u n g m a n n , R.A. (1977) Biochim. Biophys. Acta 517, 447--456 8 Kranias, E.G., Schweppe, J.S. and Jungmann, R.A. (1977) J. Biol. Chem. 252, 6 7 5 0 - - 6 7 5 8 9 Kedinger, C. and Chambon, P. (1972) Eur. J. Biochem. 28, 283--290 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 11 Yo unt, R.G., Babcock, D., Ballantyne, W. and Ojala, D. (1971) Biochemistry 10, 2484--2489 12 Martelo, O.J., Woo, S.L.C. and Davie, E.W. (1974) J. Mol. Biol. 87, 685--696 13 ZiUig, W., Fujiki, H., Blum, W., Janekovie, D., Schweiger, M., Rahmsdorf, H.J., Ponta, H. and HirschKauffman, M. (1975) Proe. Natl. Acad. Sci. U.S. 72, 2 5 0 6 - - 2 5 1 0 14 Bell, G.I., Valenzuela, P. and Rutter, W.J. (1976) Nature 2 6 1 , 4 2 9 - - 4 3 1 15 Bell, G.I., Valenzuela, P. and Rutter, W.J. (1977) J. Biol. Chem. 252, 3082--3091 16 Buhler, J.M., Iborra, F., Sentenac, A. and Fromageot, P. (1976) FEBS Lett. 71, 37---41 17 Rutter, W.J., Morris, P.W., Goldberg, M. and Morris, R.W. (1973) in The Biochemistry of Gene Expression in Higher Organisms (Pollack, J.K. and Lee, J.W., eds.), pp. 89--104, Reidel Publishing Co., Boston 18 Valenzuela, P., Bell, G.I. and Rutter, W.J. (1976) Biochem. Biophys. Res. Commun. 71, 26--31
