Phosphorylation of Rat C6 Glioma Cell DNA-dependent RNA Polymerase I1 in Vivo IDENTIFICATION OF PHOSPHORYLATED SUBUNITS AND MODULATION OF PHOSPHORYLATION BY ISOPROTERENOL AND N6,02’-DIBUTYRYL CYCLIC AMP*

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase I1 subunits. RNA polymerase I1 in control and isoproterenol-stim- ulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear ex- tracts with hen anti-calf RNA polymerase I1 antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase I1 was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase I1 subunits of 214,000,180,000,140,000,35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase I1 subunit were incorporated.

Evidence has accumulated suggesting that covalent phosphorylative modification of enzymes and other regulatory proteins represents a fundamental ubiquitous regulatory mechanism in eukaryotes (1,2). Among cellular organelles, the nucleus contains the highest concentration of phosphoproteins (3,4) whose phosphate groups are of relatively short half-life and are rapidly turning over. Phosphorylation of selected nuclear proteins has been implicated in the regulation of gene activity primarily on the basis of a temporal correlation between activation of nuclear protein kinase(s) and a concomitant elevation of the synthesis of several RNA species (for review see Ref. 5 ) .
The possibility of a functional regulation of RNA polym-*This research was supported by National Institutes of Health Grant GM 23895 and by the Research and Education Fund, Northwestern Memorial Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Based on the established regulatory function of phosphoproteins in many biological systems, phosphorylative modification of RNA polymerase I1 can reasonably be expected to be involved in regulating certain functions of the polymerase. However, attempts to demonstrate a correlation between phosphorylationJdephosphorylation and modulation of polymerase function(s) have met with variable success (6-8, l l , 12, 17-19). Thus, the question whether phosphorylation of RNA polymerase I1 subunit(s) represents a post-translational modification of constitutive nature or, alternativeIy, serves a regulatory role and modulates RNA polymerase functions remains unsolved.
Rat C6 glioma cells respond to catecholamine and cyclic AMP stimulation by rapidly modulating lactate dehydrogenase A subunit mRNA transcription by RNA polymerase I1 (20). Therefore, this cell system provides a convenient model to test the effects of RNA polymerase I1 phosphorylation/ dephosphorylation on RNA polymerase function specifically during the period of lactate dehydrogenase mRNA synthesis. As an initial step towards this goal, we have investigated whether RNA polymerase I1 subunits become selectively phosphorylated after catecholamine treatment of glioma cells. munotitration. Ouchterlony double-diffusion analysis using rabbit anti-hen IgG as secondary antibody showed that the interaction of serum from immunized hens with highly purified calf thymus RNA polymerase I1 as well as with nuclear extracts from nonstimulated and stimulated C6 glioma cells produced a single continuous precipitin line. In contrast, no precipitin band was observed with preimmune serum (results not shown).

Characterization
Since a preliminary assay showed no detectable inhibitory effect of the hen antiserum on RNA polymerase I1 activity, rabbit anti-hen IgG was used as secondary antibody to precipitate the soluble primary immunocomplex. After removal of the immunoprecipitate by centrifugation, the supernatant fraction was assayed for residual polymerase activity. As shown in Fig. 1, calf thymus as well as glioma cell RNA polymerase I1 activity progressively declined after addition of increasing amounts of hen antiserum, indicating that the inhibition of polymerase activity was due to a specific antibody-antigen interaction.
As an additional evaluation of the reactivity and specificity of the hen antiserum, immunoprecipitation of "'1-labeled calf thymus RNA polymerase I1 was performed with hen antiserum and preimmune serum. The immunocomplexes were precipitated with rabbit anti-hen IgG and analyzed by SDSpolyacrylamide gel electrophoresis and autoradiography. The immunoprecipitate obtained after incubation of hen antiserum shows a distinct labeling pattern (Fig. 2B, scan I ) , and six "'I-labeled polymerase subunits can be identified. In contrast, the immunocomplex formed with preimmune serum and l"I-labeled RNA polymerase shows no such distinct labeling pattern (Fig. 2B, scan I I ) . The autoradiogram shows a nonpolymerase peptide of an apparent M, = 85,000 which coprecipitated with hen antiserum, indicating that this peptide is tightly associated with calf thymus RNA polymerase I1 and coprecipitates with the antigen.
The titer of the hen antiserum was determined by incubation of '"1-labeled calf thymus RNA polymerase I1 with varying amounts of hen antiserum. Hen preimmune serum was added as supplement to the hen antiserum to keep the total amount of hen immunoglobulins constant (47.5 pg/ assay) throughout the assays. As shown in Fig. 3, the amount of antigen precipitated depended on the antiserum concentration. Half-maximal binding of '2'I-labeled RNA polymerase I1 was at an antiserum dilution of 1:200, which was equivalent to 0.475 pg of hen immunoglobulins and which corresponded to a ratio of '2'I-labeled antigen to hen antibody of 1:44 (w/ w). At 70% of the maximal precipitation, the amounts of labeled antigen and hen antibody were 10.8 ng and 2.1 pg, respectively.

Effect of Isoproterenol on RNA Polymerase II Subunit Phos-
phorylution-To investigate the phosphorylation of RNA polymerase I1 subunits, it appeared necessary to immunoprecipitate RNA polymerase I1 from the nuclear protein extract rapidly and with a minimum of experimental manipulation in order to avoid artifactual dephosphorylation and proteolysis which might occur despite the presence of inhibitors of protease and phosphatase.
Glioma cells were prelabeled with Na2HV2PO, in phosphate-free medium. Stimulation of cells with isoproterenol in the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine was carried out for 1 h. RNA polymerase I1 was then immunoprecipitated from the nuclear protein extract with chicken anti-calf RNA polymerase I1 antiserum and rabbit anti-hen IgG. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 4). Despite the relatively high background density, the phosphorylation of a peptide corre- Due to the coprecipitation of large amounts of nonspecific radioactivity, it became necessary to partially purify a2Plabeled RNA polymerase I1 prior to immunoprecipitation. Removal of nonspecific '*P was attempted by ribonuclease treatment of nuclear extracts followed by ammonium sulfate precipitation and heparin-Sepharose chromatography.' RNA polymerase I1 was then isolated with hen antibody-conjugated Sepharose beads. Using this modified experimental approach, the effect of isoproterenol on RNA polymerase I1 phosphorylation was investigated. In addition to the 214-kDa subunit, increased isoproterenol-mediated ["P]phosphate incorporation into five subunits of smaller size occurred, e.g. the 180-, 140-, 35-, 28-, and 16.5-kDa subunits (Fig. 5). Under the After heparin-Sepharose chromatography, RNA polymerase I1 preparations from control and isoproterenol-stimulated cells exhibited similar specific activities as measured in a conventional polymerase assay (8). Recoveries of RNA polymerase I1 at this stage were comparable between stimulated and unstimulated cells.
Glioma cell RNA polymerase I1 subunit phosphorylation after isoproterenol stimulation. Glioma cells were labeled for 5 h with Na2H"'P04 (5 mCi/roller bottle). Stimulation of cells was carried with 10 p~ isoproterenol for 30 min before the end of the S-h labeling period. After the addition of unlabeled glioma cells as carrier, nuclei were isolated. Nuclear extracts were incubated a t 37 "C for 10 min with 10 pg/ml ribonuclease A and 1.0 pg/ml ribonuclease T1. RNA polymerase I1 was then precipitated with ammonium sulfate and subjected to heparin-Sepharose column chromatography as described under "Experimental Procedures." Eluted RNA polymerase was pooled and adsorbed with hen antibody-conjugated Sepharose beads. Adsorbed "P-labeled RNA polymerase I1 was dissolved in SDS-sample buffer and applied onto a polyacrylamide gel consisting of 7.5% acrylamide in the upper half (5.5 cm) and 12.5% acrylamide in the lower half (5.5 cm) of the gel. Electrophoresis was carried out a t 110 V for 6 h a t room temperature. Radioactivity was identified by autoradiography of the gel. A, immunoprecipitated RNA polymerase from mock-stimulated glioma cells; B , RNA polymerase I1 from glioma cells stimulated for 30 min. experimental conditions, no "P-labeling of subunits from unstimulated control cells was observed, but phosphorylation of the subunits from isoproterenol-stimulated cells can clearly be identified after a 30-min stimulation time.
In order to minimize artifactual experimental variations between unstimulated and stimulated cells, we additionally employed a double-isotope labeling method to identify the phosphorylated subunits. Na,H"PO, and Na,"3P04 were used to prelabel the intracellular ATP pools. Mock-stimulated control cells were prelabeled with Na2HR3P04, whereas cells to be stimulated were prelabeled with Na,HR2P04. After isoproterenol stimulation, ?'P-labeled stimulated cells were combined with the "P-labeled unstimulated cells. "P-and 32Plabeled RNA polymerase I1 was partially purified from the RNase-treated nuclear extracts and immunoprecipitated. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis, and 32P/3:'P radioactivity was determined in sliced gel sections. The results are illustrated in Fig. 6. The amount of "'P radioactivity/gel slice did not vary appreciably throughout the gel. However, D2P radioactivity increased markedly after stimulation in gel slices, corresponding to the location of RNA polymerase I1 subunits 214, 180, 140,35, 28, and 16.5 kDa. The increase of ["P]phosphate incorporation into the subunits after isoproterenol stimulation can also be assessed by comparing in each gel slice the ratio of counts/ min '"P uersus disintegrations/min "P incorporated (see Fig.   7). This method of analysis confirms the selective incorporation of phosphate into the six polymerase subunits after isoproterenol stimulation. In about 30% of the experiments, were isolated. Nuclear extracts were incubated a t 37 'C for 10 min with 10 pg/ml ribonuclease A and 1.0 pg/ml ribonuclease T1. RNA polymerase I1 was subsequently partially purified by ammonium sulfate precipitation and heparin-Sepharose column chromatography.
32P/"P-labeled RNA polymerase II was immunoadsorbed with hen antiserum-conjugated Sepharose beads. The polymerase was eluted from the Sepharose beads by boiling for 5 min with SDS-sample buffer, after which it was applied onto a polyacrylamide gel consisting of 7.5% acrylamide in the upper half and 12.5% acrylamide in the lower half of the gel. Electrophoresis was carried out a t 100 V for 6 h a t room temperature. After electrophoresis, the gel was stained, destained, and sliced into 2-mm sections which were counted to determine the level of 32P and "P incorporated. Panels A-E, cells stimulated with isoproterenol for 0,s. 15,30, and 60 min, respectively. To test whether dibutyryl cAMP mimics the action of isoproterenol, glioma cells were stimulated with the cAMP analogue. Phosphate incorporation into RNA polymerase I1 subunits was assessed by the double 32P and ' "P isotope labeling method. The results are shown in methionine incorporation into total protein but did not affect the isoproterenol-mediated phosphorylation of the six subunits (data not shown). Based on these findings, de nouo protein synthesis is not required for isoproterenol to achieve its effect, indicating that phosphorylation of RNA polymerase I1 subunits is a post-translational modification.
Analysis of Phosphoamino Acids-To identify the phosphate acceptor amino acid, [32P]RNA polymerase I1 was isolated by immunoabsorption to hen antibody-conjugated Sepharose beads. Electrophoretically separated subunits were subjected to partial acid hydrolysis, and [32P]phosphoamino acids were separated by high voltage paper electrophoresis. As shown in Fig. 9

DISCUSSION
Structural and functional modification of nonhistone chromosomal proteins through phosphorylation/dephosphorylation has been suggested as one of the epigenetic mechanisms involved in the regulation of gene expression in eukaryotes. This hypothesis is based on established temporal correlations between phosphorylation/dephosphorylation and gene activation (5,32). The possibility of a regulation of RNA polymerase I1 function via phosphorylation/dephosphorylation has been considered by a number of researchers. However, studies attempting to correlate phosphorylative modification and modulation of polymerase function have not yielded uniform results. While some investigators reported that in vitro phosphorylation of RNA polymerase I1 leads to an increase in  Fig. 6. Cells labeled with "P, were treated with 10 /IM isoproterenol or 1 mM dihutyryl CAMP. "Plaheled control cells were mock stimulated. Incuhation was continued for 45 min hefore cell harvest. Control cells labeled with 3RP were combined with an identical number of "'P-labeled stimulated cells. Isolation of nuclei, partial purification, immunoadsorption, and SDSpolyacrylamide gel electrophoresis were carried out as described in legend of Fig. 6. The gel was sliced into 2-mm gel sections and the amount of "lP and "P in each gel slice were determined. The values represent the average of four separate experiments.  9. Analysis of [s2P]phosphoamino acids by paper highvoltage electrophoresis. Cells were labeled with Na2H3*P0, and stimulated for 0.5, and 30 min with 10 p~ isoproterenol as described in the legend of Fig. 7. RNA polymerase was subjected to SDS-gel electrophoresis. Partial acid hydrolysates of gel slices containing the 214-kDa subunit were fractionated by paper electrophoresis at pH 2.4 (1% formic acid, 4% acetic acid; 500 V; 16 h) as described under "Experimental Procedures." [32P]Phosphoamino acids were visualized by autoradiography and identified by comigration of authentic standards. h n e A, control cells; lune B, cells stimulated for 5 min; lune C, cells stimulated for 30 min. enzymatic activity (5,8,18,19) or RNA chain initiation ( l l ) , others failed to find such correlations (6,10,14).
So far, no studies on RNA polymerase I1 phosphorylation have been carried out a t times of physiological transition such as development or hormonal stimulation leading to altered gene expression and synthesis of specific gene products. Studies in such experimental systems would allow examination of a possible correlation between RNA polymerase I1 phosphorylation and function. The experimental system used by us is ideally suited for such studies. However, in order to determine if glioma cell RNA polymerase I1 undergoes phosphorylative modification in uiuo after isoproterenol stimulation, it was necessary to develop a procedure for the purification of RNA polymerase I1 from small quantities of cells. The procedure is based on the fact that antiserum against calf thymus RNA polymerase I1 cross-reacts efficiently with RNA polymerase I1 of other mammalian species (25,33). We have applied an immunoprecipitation procedure which avoided a lengthy purification and allowed the isolation and identification of RNA polymerase I1 from a relatively small number of cells in a reproducible fashion. Application of this procedure resulted in the isolation of immunoprecipitated glioma cell RNA polymerase I1 which had polypeptide subunits of 214, 180, 140, 35, 28, and 16.5 kDa. In several experiments, a 240-kDa subunit was also identified. This subunit structure is similar to that of calf thymus (220, 180, 140, 34, 25, and 16.5 kDa) and rat liver (220, 214, 180, 140, 34, 25, and 16.5 kDa) RNA polymerase I1 described by Kedinger et al. (34). Our attempts to selectively immunoprecipitate ['"PI RNA polymerase I1 directly from nuclear extracts were not successful because the coprecipitation of nonspecific "'P-labeled material caused a high background in the autoradiographs and prevented identification of several "'P-labeled subunits. The contaminants may be minor but are probably very highly labeled. Of various purification procedures evaluated, it appeared to be important to treat "'P-labeled nuclear extracts with ribonuclease. This led to an efficient removal of contaminants (probably mostly ["'PIRNA).
T o investigate the in uiuo sites of RNA polymerase I1 phosphorylation, the enzyme was isolated from confluent unstimulated and stimulated glioma cells that had been labeled with isotope for several hours. RNA polymerase I1 from unstimulated cells contained only low levels of "'P, and prolonged exposures (longer than the ones used here) were required to detect this level of radioactivity by autoradiography. However, isoproterenol treatment of glioma cells led to a marked incorporation of ["'P]phosphate into six RNA polymerase I1 subunits in a time-dependent fashion. Chemical analysis of ["'Plpolymerase subunits indicated that the incorporation of phosphorus occurred primarily on serine in all six subunits. This is in agreement with our previous studies which identified serine as the principal phosphate acceptor of calf thymus RNA polymerase I1 in vitro (8). The temporal pattern of phosphate addition was similar for all six subunits, but whereas phosphorylation of the 214-and 35-kDa subunits remained slightly elevated 2 h after stimulation, the level of phosphorylation of the other four subunits had declinied to control levels by that time.
In cell culture, the stoichiometry of the phosphate incorporation into the individual RNA polymerase subunits is difficult to determine with accuracy, but it is possible to obtain approximate estimated values. We have previously shown that glioma cell cultures equilibrated with Na2H"P04 will incorporate %'Pi into ["'PIATP linearly up to 2 h of labeling time to reach a specific activity of about 400 cpm of "P/pmol of ATP. After 2 h, the specific activity of ["'PIATP remained constant for an additional 4-h period and was not altered by isoproterenol or dibutyryl cAMP treatment of the cells (24). Using this experimentally determined value, it can be estimated that there were about two phosphate groups incorporated per 140-kDa subunit.
The relative disparity in the labeling of the individual subunits (Fig. 5) suggests that the 214-, 180-, 28-, and 16.5-kDa subunits incorporate one phosphate group/subunit and the 35-kDa subunit only 0.5 phosphate group/subunit. Several lines of evidence indicate a post-translational mechanism of RNA polymerase I1 phosphorylation. First, inhibition of protein synthesis by cycloheximide had no discernible effect on the isoproterenol-mediated phosphorylations. Second, nuclear CAMP-dependent protein kinase as well as nuclear CAMP-independent kinase NII phosphorylate in vitro all the subunits which are labeled in U~U O .~ Finally, our results strongly suggest that the phosphorylative modifications of the RNA polymerase I1 subunits are mediated via CAMP-dependent protein kinase. This reasoning is based on the finding that dibutyryl cAMP mimicked and (RS)-propranolol, a padrenergic antagonist, blocked the action of isoproterenol.
The major question of whether isoproterenol-mediated phosphorylation of RNA polymerase I1 has any functional significance in the C6 glioma cell remains unsettled. The rat C6 glioma cell responds to p-adrenergic agonists with an activation of its adenyl cyclase leading to a rapid transient rise of cAMP levels (35) and activation of nuclear CAMPdependent protein kinase (36,37). This is followed by an increased rate of transcription of lactate dehydrogenase A subunit mRNA (20) and increased activity of a number of enzymes such as cAMP phosphodiesterase (38), ornithine decarboxylase (39), 2':3'-cAMP-3' phosphohydrolase (40,41), and phosphorylase a (42). Catecholamines or dibutyryl cAMP also potentiate the glucocorticoid induction of glycerol phosphate dehydrogenase (43) and increase the levels of S-100 protein in nonconfluent cell cultures (44) and of @-nerve growth factor (45,46).
It is of interest that the phosphorylation of glioma cell RNA polymerase I1 subunits occurs concomitantly with the phosphorylation of several other chromosomal (24,47,48) and cytoplasmic (49) proteins. Thus, there is a strong physiological correlate between the pattern ofphosphorylation of several glioma cell proteins and altered gene expression after catecholamine stimulation. It is tempting, therefore, to assume that a phosphorylative and functional modification of RNA polymerase I1 may play a role in the altered pattern of gene expression in glioma cells. Having demonstrated a catecholamine-and CAMP-mediated structural modification of RNA polymerase 11, it is now necessary to identify the physiological role of the phosphorylation. The introduction of negative charges onto the RNA polymerase I1 molecule could modulate inter-as well as intramolecular interactions. Therefore, it is necessary to measure the effect of RNA polymerase phosphorylation on several parameters such as its binding affinity for S.-K. Lee, J. S. Schweppe, and R. A. Jungmann, unpublished observations. specific regulatory gene sequences, correct initiation and termination of transcription, and enzyme half-life.