Nucleolus-Associated Phosphoprotein Kinases of Normal and Regenerating Liver and Novikoff Hepatoma Ascites Cells1

Home , Phosphoprotein

[CANCER RESEARCH 38, 3421-3426, October 1978] 0008-5472/78/0038-0000$02.00 Nucleolus-associated Phosphoprotein Kinases of Normal and Regenerating Liver and Novikoff Hepatoma Ascites Cells1

Mark O. J. Olson,2 Sally Hatchett,3 Ruth Allan, T. C. Hawkins,4 and Harris Busch

Nuclear Protein Laboratory, Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT induce hepatic nucleolar hypertrophy. In all of these sys tems, an increase in RNA polymerase I activity (1, 2, 23, 36) Phosphoprotein kinase activities of cell nuclei are is concurrent with the increases in nucleolar size. changed by various alterations in cellular physiology. For The complex process of ribosome biogenesis requires evaluation of the effects on the nucleolus-associated systems for regulation of rates of transcription and for the enzymes, the activities and Chromatographie profiles of coordination of the many steps in preribosome assembly nucleolar cyclic nucleotide-independent phosphoprotein and processing. One such system may involve protein kinases were compared in normal and regenerating rat phosphorylation, which has been suggested to be a regu liver and Novikoff hepatoma ascites cells. Incorporation latory factor in gene transcription (19). The nucleolus con of radioactivity from [y-'?P]ATP into casein, nucleolar tains numerous phosphorylated proteins (24-26), some of protein, or histone was measured by filter disc assays. which may regulate or coordinate nucleolar processes (3, With casein as the substrate, the pH activity curve was 16). The nucleolus also appears to contain the kinases for biphasic, with peaks near pH 7 and 9. Activity was maxi phosphorylation of most of these phosphoproteins (12-14, mal in the presence of 0.1 to 0.15 M NaCI at either pH. 16, 17, 35) and phosphatases for their dephosphorylation Divalent cations were required for activity. MgCL (10 to 20 (25). mm)was the most effective; CaCI,, MnCL, and CoCI. were The fractionation of nuclear protein kinases has been poor substitutes, and ZnCI was inhibitory. generally limited to studies on whole nuclei or chromatin. Nucleoli of two proliferating tissues, Novikoff hepatoma Nuclear chromatin contains at least 6 (28) and as many as and 18-hr regenerating liver, had kinase activities (casein 12 or more (18) distinct kinase activities. One protein kinase substrate) 3- to 4-fold greater per unit DMA than normal was reported to be tumor specific (32). The level of nuclear liver. Phosphoprotein kinase activity from Novikoff hepa protein kinase activity varies with the phase of the cell cycle toma nucleoli was fractionated into five major peaks on (9) and is enhanced during chemical carcinogenesis (6). columns of DEAE-Sephadex. All peaks phosphorylated Since nucleolus-associated protein kinase of normal and casein, but nucleolar protein was an effective acceptor tumor cells has not been extensively studied, this study was for only three peaks (K6, K7, and K8), which eluted at initiated to determine for 3 tissues with varying rates of (NH,) SO, concentrations greater than 0.2 M. None of the nucleolar activity: (a) the total amount of nucleolar phos peaks was capable of phosphorylating histone. The Chro phoprotein kinase activity; (b) the number of different matographie profiles of regenerating liver nucleoli were species of enzymes present; and (c) the distribution of similar to those of Novikoff hepatoma. However, in the activity in the Chromatographie fractions of nucleolar ki pattern from normal liver one early-eluting peak (K3) was nases. In addition, substrate specificity and pH, ionic reduced or absent. Proteins C23 and C2, previously strength, and metal ion optima were determined for Novi identified as phosphorylated nucleolar proteins, were the koff hepatoma nucleolar protein kinases. major bands phosphorylated by Peaks K6, K7, and KB. These studies indicate a correlation of increased phos MATERIALS AND METHODS phoprotein kinase activity with the elevated nucleolar synthetic activity of proliferating tissues. Animals and Tissues. Albino rats used in these studies were obtained from the Holtzman Co. (Madison, Wis.). For INTRODUCTION partial hepatectomy 3 major lobes of the livers were surgi cally removed under ether anesthesia according to the The synthetic activity of the nucleolus varies according to procedure of Higgins and Anderson (15). The remaining the needs of the cell in which it is contained. For example, livers were excised 18 hr later. Immediately after the rats cells of rapidly growing tumors have enlarged nucleoli, were killed by decapitation, the normal or regenerating probably reflecting increased preribosome production (4). livers were perfused with 0.13 M NaCI:0.005 M KCI:0.008 M Various stimuli, such as partial hepatectomy (2, 30, 31), MgCI, containing 0.1 mM PMSF.5 For Novikoff hepatoma thioacetamide injection (20), or methionine deprivation (2), preparations the ascites cells were transplanted 6 days prior to the experiments. The tumor cells were drained from the abdominal cavity, filtered through cheesecloth, and 1Supported by the USPHS Cancer Program Project Grant CA-10893, P3. washed several times with the 0.13 M NaCI:0.005 M 2To whom requests for reprints should be addressed. KCI:0.008 M MgCI,-PMSF solution. 3 Present address: IBM. Office Products Division, 3825 DaComa, Houston Isolation of Nucleoli. Nucleoli from normal and regener- Texas 77092. 4 Present address: University of Houston, College of Pharmacy, Houston, Texas 77004. â€¢¿Theabbreviationsused are: PMSF, phenylmethylsulfonylfluoride: DTT, Received April 4, 1978: accepted July 18, 1978. dithiothreitol.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. M. O. J. Olson et al. ating liver and Novikoff hepatoma nuclei were isolated by washed as described by Valenzuela ef al. (34). The precipi the sucrose calcium procedure by means of sonic oscilla tated protein was dissolved in sample buffer (34), boiled for tion (4, 26). Purified nucleoli were suspended in a solution 3 min, and applied to the 7.5% polyacrylamide gel-sodium containing 50% glycerol:0.05 M Tris;0.1 HIM PMSF;0.1 mw dodecyl sulfate system of Laemmli (21). After staining with DTT, pH 7.5, and stored at -70Â° prior to use. Coomassie blue (34), the gels were sliced into 1-mm slices, Extraction of Kinase Activity. Pelleted nucleoli were sus dried, and counted in the scintillation counter. pended at 0-4Â° in deionized water at a concentration of 2 mg protein per ml, estimated by absorbance at 260 and 280 RESULTS nm in 6 M guanidine hydrochloride (22). To this was added an equal volume of 0.7 M NaCI;0.02 M Tris;0.2 HIM PMSF, Extraction of Phosphoprotein Kinase. Phosphoprotein pH 7.5. The mixture was homogenized briefly, stirred in the kinase activity measured with casein as substrate was cold for 30 min, and centrifuged at 6000 x g for 20 min. The readily extractable from Novikoff hepatoma nucleoli with supernatant solution, which represented more than 90% of dilute 0.9% NaCI solution. Earlier studies indicated that the extractable activity (casein substrate), was used for all nearly 75% of the activity could be extracted with combined subsequent studies. dilute NaCI solution. Earlier studies indicated that nearly Phosphoprotein Kinase Assay. Protein kinase was as 75% of the activity could be extracted with combined sayed by a filter disc method similar to that of Corbin and NaCI as solvent. Table 1 illustrates that 1 extraction with Reimann (8). The reaction mixtures (150 /*!) contained 10 mw 0.35 M NaCI at pH 7.5 solubilized an amount of activity that MgCI,, 0.25 mM ATP, 24 mM 2,2-bis(hydroxymethyl)-2,2',2"- was greater (143%) than the total activity of suspended nitriloethanol (pH 7.0), 6 mM DTT, 70 mM NaCI, protein unextracted nucleoli, possibly due to the removal of inhibi substrate, enzyme as either a 0.35 M extract of nucleoli or tors or competing substrates. A second and third extraction column fractions, and trace quantities (0.01 to 0.1 /uCi) of with the same buffer removed only 11 and 4%, respectively, [?-32P]ATP (100 to 3000 Ci/mmol) obtained from either ICN of the control activity. Chemical Radioisotope Division (Irvine, Calif.) or New Eng Subsequent extractions with higher concentrations of land Nuclear (Boston, Mass.). The protein substrate con NaCI (0.6 M and 1.0 M) after preextraction with 0.35 M NaCI sisted of 1 of the following: casein, 2 mg/ml (Sigma Chem released only small amounts of activity (Table 1). Therefore, ical Co., St. Louis, Mo.), acid-soluble nucleolar proteins, 1 1 extraction with 0.35 M NaCI was used as the standard mg/ml (26), or whole calf thymus histone, 1 mg/ml (Sigma). procedure to solubilize the bulk of the phosphoprotein After incubation for various times at 37', aliquots of the kinase activity. mixture were spotted on Whatman no. 3 MM cellulose filter Optimal Activity Conditions. Various parameters were discs. The discs were washed twice in 15% trichloroacetic surveyed to optimize the assay conditions. The kinase acid: 2 mw ATP:1 mw NaH,PO,: and 34 mM Na,P,O7, exhibited peaks of activity near pH 9, as well as near pH 7 followed by 1 wash with 95% ethanol and a final wash in (Chart 1). However, the pH 9 activity was highly unstable, ether. The filter discs were counted in a scintillation counter and activity was routinely measured only at pH 7. Protein with the use of Formula 947 (New England Nuclear) as kinase activity for casein was maximal at NaCI concentra scintillation fluid. Comparisons of activities in nucleoli of tions between 0.1 and 0.2 M for both pH optima (Chart 2). various tissues were based on nucleolar DNA content deter The enzyme activity was maximal with MgCI., at 10 to 20 mined according to the method of Burton (5). mM, whereas MnCI,, CoC!,, and CaCL at 4 to 20 mM DEAE-Sephadex Chromatography. The 0.35 M NaCI ex maintained approximately 10% of the maximal activity and tract of nucleoli (3 to 12 ml) was dialyzed at 0-4Â°for 18 hr ZnCI, was completely inhibitory at all concentrations tested against a solution containing 25% glycerol;0.1 mM (Chart 3). When enzyme activity was measured as in Chart EDTA;0.1 mM DTT;0.1 mM PMSF;0.05 M Tris, pH 7.5 (Buffer 3 at 10 mM MgCI., in the presence of either 1 /J.M cyclic 1). The dialyzed extract was applied to columns (0.9 x 45 cm) of DEAE-Sephadex A-25-120 (Sigma) previously equili Table 1 brated with Buffer 1. The columns were eluted at a flow rate Extraction of phosphoprotein kinase from Novikoff hepatoma of 30 ml/hr, first with 50 ml of Buffer 1 and then by 20 ml of nucleoli Buffer 2 [Buffer 1 containing 0.1 M (NH,),SO,]. A linear Nucleoli at a concentration of 1 mg protein per ml were extracted initially with 0.35 M NaCI:0.01 M Tris (pH 7.5) containing 0.1 mw gradient consisting of 80 ml of Buffer 2 and 80 ml of Buffer PMSF as described in the text. Subsequent extractions with the 3 [Buffer 1 containing 0.28 M (NH4),,SO,] was then applied to same or higher salt concentrations were done similarly, with the complete the elution. Fractions of 2.5 ml were collected. same volume of extradant. All assays (30 min with casein sub Aliquots (30 fj.i) of every other tube were analyzed for strate) were based on 30-/J aliquots of supernatants or pellets protein kinase activity by 30-min assays as described above. suspended in identical volumes of buffer. Polyacrylamide Gel Electrophoresis. For determination Total cpm incorpo rated x 10 3 of the specificity of protein phosphorylation by peaks of Extradant kinase activity, the assay mixture with nucleolar protein was nucleoli)0.35None (whole scaled up 5-fold, and the unlabeled ATP concentration was extraction)0.35M NaCI (first extraction)0.35M NaCI (second reduced to 0.05 mM. The nucleolar protein substrate in this extraction)PelletM NaCI (third case consisted of the crude enzyme extract, which was extractions0.6after 3 concentrated 5-fold and boiled for 3 min. After incubation NaCI1M NaCI after 0.35 M the protein was precipitated with 3 ml of 10% trichloroacetic NaCIPellet.0 M NaCI after 0.6 M acid with 20 Â¿igpolyadenylic acid added as carrier and after 1 M NaCI14.220.21.60.50.73.00.70.310014211452152

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. Nucleolar Phosphoprotein Kinases adenosine 3':5'-monophosphate or cyclic guarÃosme 3':5'- monophosphate, no significant alteration in activity was seen. Thus the phosphoprotein kinase activity was routinely measured with casein in the presence of 10 mw MgCL and 0.15 M NaCI at pH 7.0. Nucleolar Phosphoprotein Kinase Activities of Normal and Regenerating Liver and Novikoff Hepatoma. For com parisons of protein kinase activities, the extracted enzymes of normal and regenerating liver and Novikoff hepatoma nucleoli were assayed with the use of casein as a substrate (Table 2). Since the protein content of nucleoli varies from tissue to tissue, the activities were based on the DNA content of the nucleoli before extraction. Regenerating liver and Novikoff hepatoma had similar activities (300 to 350 pmol/min/pg DNA). Both tissues had 3 to 4 times the 4567 8 1 10 1 pH activity of normal liver. DEAE-Sephadex Chromatography of Nucleolar Phos Chart 1. pH-activity profile of nucleolar phosphoprotein kinase of Novikoff hepatoma. Assays with casein substrate were run for 30 min as described in phoprotein Kinase. For assessment of the heterogeneity of text. The reaction buffer was 2.2-bis(hydroxymethyl)-2,2',2"-nitriloethanol the kinase activity, a system of fractionation on DEAE- (pH 4 to 7) or 0 024 M Tris (pH 7.5 to 11). All data points are relative to the pH Sephadex was developed. The system used combined step- 9.0 value (850 cpm incorporated) and are the means of duplicate analyses wise and gradient elution with ammonium sulfate (Chart 4). The activity was resolved reproducibly into 5 major compo nents (Peaks 1, 3, 6, 7, and 8) and approximately 3 minor components (Peaks 2, 4, and 5) with casein as a substrate. pH 1.0 Peak 5 was variable in size and appeared to be highly unstable. When 0.4 N H.SO.-extracted nucleolar protein (40 to 60% of the total nucleolar protein) was used as substrate,

Table 2 Phosphoprotein kinase activities of nucleoli of various tissues Rate of incorporation" Tissue source (pmol/min/pg DNA) NormalliverRegenerating Â±26ft353 liver (18 hr) Â±21 Novikoff hepatoma80 303 Â±51100441 379 " The initial rate was calculated from 10-min time points of the assay (described in text) with casein substrate and crude enzyme 0.2 0 ) 0Â« 05 06 NaCI IM] extract. The values were based on the original content of DNA in the nucleoli before extraction. Chart 2. Effect of NaCI concentration on nucleolar phosphoprotein kinase * Mean Â±S.D. derived from at least 3 separate preparations of of Novikoff hepatoma. Samples containing identical amounts of enzymes nucleoli from each tissue. and substrate (casein) were assayed as described in the text with varying concentrations of NaCI with 0.024 M 2.2-bis(hydroxymethyl)-2,2'-2"-nitriloethanol at pH 7.0 or with 0.024 MTris at pH 9.0. All data points are relative to the 0.1 Mpoint at pH 9.0 (600 cpm incorporated)

40 50 W FRACTION NUMBER ! a 6 8 10 12 14 16 18 20 [ M** ]. mM Chart 4. Chromatography of nucleolar phosphoprotein kinase of Novikoff hepatoma on DEAE-Sephadex. The crude nucleolar extract (6 ml) was Chart 3. Effect of divalent cations on nucleolar phosphoprotein kinase of applied as described in the text to a column (0.9 x 45 cm) of DEAE-Sephadex Novikoff hepatoma. Samples containing identical amounts of enzyme and A-25-120 equilibrated with 0.05 M Tris (pH 7.5) buffer containing 25% substrate (casein) were assayed at pH 7.0 as described in the text with glycerol, 0.1 mM EDTA, 0.1 rtiM DTT, and 0.1 mw PMSF. The column was varying concentrations of chloride salts to divalent metals. All data points are eluted with the gradient of (NHJ..SO,, ( ). Protein kinase activity was relative to the activity with 16 mM MgCI. (2850 cpm incorporated). [M ]. measured on 30-^1 aliquots from alternate tubes with casein, histone. or concentration of divalent cation. nucleolar protein as substrate.

OCTOBER 1978 3423

Peaks 6, 7, and 8 had the greatest activity (Chart 4). No 32P RECENERATINC 11 VtR NUCIEOLI incorporation into the nucleolar protein substrate was ob served for Peak 3, which was the major peak of activity toward casein. Histone was a poor substrate for any enzyme peak of the Chromatographie profile (Chart 4). 0.3 â€ž¿ z

When the crude protein kinase from normal liver was 0.2 ^

subjected to chromatography on DEAE-Sephadex under Wl 0.1S conditions identical with those used for Novikoff hepatoma nucleoli, a similar separation resulted (Chart 5). Peaks of 10 20 30 10 SO 60 70 SO Â«0 100 activity toward casein eluted from the column at the same FRACTION NUMBER ionic strengths required to elute Peaks 1,6,7, and 8 of the Chart 6. Chromatography of nucleolar phosphoprotein kinase of regen erating rat liver on DEAE-Sephadex. The crude nucleolar extract (3 ml) was Novikoff column. Peak 6 was somewhat reduced in activity run on a column of DEAE-Sephadex under conditions identical with those of relative to Peaks 7 and 8. The major difference between the Chart 4. Elution with the (NHJ..SO, gradient is indicated in the chart ( ). Novikoff hepatoma and liver profiles was the absence of Protein kinase activity was assayed on 30-Â¿iIaliquots from alternate tubes, with casein (â€¢{asthe substrate. Due to differences in ATP-specific activities. activity in the position of Peak 3. As in the tumor pattern, ordinate scales are not directly comparable in Charts 4, 5, and 6. the activity in normal liver against nucleolar protein was found predominantly in Peaks 6, 7, and 8; all other peaks exhibited little or no activity to this substrate. The crude extract of protein kinase activity from regener ating liver nucleoli also showed the characteristic pattern of separation on DEAE-Sephadex (Chart 6), with major peaks eluting at Positions 1, 3, 6, 7, and 8. In this case the relative activity of Peak 3 was intermediate between the activities of Novikoff hepatoma and normal liver nucleoli. Thus, in addition to the differences in total phosphoprotein kinase activity, Peak 3 varied from tissue to tissue. Specificity of Phosphorylation. Since Peaks 6, 7, and 8 were the only kinase peaks capable of labeling nucleolar proteins, it was of interest to determine which nucleolar proteins served as [32P] phosphate acceptors. When aliquots from the peak tubes of Peaks 6, 7, and 8 were incubated with the nucleolar protein substrate in the pres ence of [y-32P]ATP, 2 major protein bands were labeled as shown by gel-sodium dodecyl sulfide electrophoresis (Chart 7). These bands comigrated with the stained bands of proteins C2 and C23, which were also labeled in vitro in 0 10 20 30 40 50 60 70 80 90 whole nucleoli (16). Only traces of radioactivity were found GEL SLICE NUMBER in other bands, including protein B23, which was previously Chart 7. Electrophoretic analyses of proteins labeled by DEAE-Sephade separated peaks of phosphoprotein kinase. Aliquots (150 /

protein kinase activity. The 2 dividing tissues contained larger amounts of activity, possibly reflecting higher rates of nucleolar synthetic activity in Novikoff hepatoma and regenerating liver. The 3- to 4-fold increase in protein kinase activity 18 hr after partial hepatectomy was similar to the increase in RNA polymerase I activity (approximately 3- fold) observed in 17-hr regenerating liver (4) and was 4-fold greater in Novikoff hepatoma (7) than in normal liver nu

30 Â« Â» 60 cleoli. Although the increase in the 2 activities may be FRACTION NUMBER unrelated, it is possible that the protein kinase acts to Chart 5. Chromatography of nucleolar phosphoprotein kinase of normal rat liver on DEAE-Sephadex. The crude nucleolar extract (3 ml) was run on a modulate polymerase activity or that the increase in kinase column of DEAE-Sephadex under conditions identical with those of Chart 4. activity is a response to the enhanced activity of polymer Elution with (NH<):SO4is indicated in the chart (O). Protein kinase activity ase. If the latter case is correct, phosphorylation might be was assayed on 30-Â¿tlaliquotsfrom alternate tubes using casein or nucleolar protein as substrate. Because of differences in ATP-specific activities, concerned with preribosomal particle assembly, as pro ordinate scales are not directly comparable in Charts 4, 5, and 6. posed earlier (27).

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Chromatography of the extracted kinase on DEAE-Seph- kinases in the nucleolus must await answers from further adex columns showed that 1 peak of activity (Peak 3) was studies. increased in the 2 active tissues, hepatoma and regenerat ing liver. However, Peak 3 accounts for only approximately ACKNOWLEDGMENTS 10% of the total column activity of these 2 tissues. There The authors wish to thank R. K. Busch for supplying the animals and fore, the increase in Peak 3 activity by itself cannot account Karl Guetzow for assistance in performing partial hepatectomies. for the 3- to 4-fold increase in total activity (Table 2), and

activity must be enhanced in all species of the protein REFERENCES kinase. Since the activity of Peak 3 was enhanced in both the tumor and the normal proliferating tissue, the phenom 1. Andersen, M. W., Ballai, N. R., and Busch, H. Nucleoli of Thioacetamide- Treated Liver as a Model for Studying Regulation of Preribosomal RNA enon appears to be related to growth rate or rate of Synthesis. Biochem. Biophys. Res. Commun., 87: 129-135. 1977. ribosome synthesis rather than to tumor specificity. 2. Bailey, R. P., Vrooman, M. J., Sawai, K., Tsukada, K.. Short, J., and Peaks 6, 7, and 8 and, to a small extent, Peak 5 were the Lieberman, I. Amino Acids and Control of Nucleolar Size, the Activity of RNA Polymerase I, and DNA Synthesis in Liver. Proc. Nati. Acad. Sei. U. only peaks from the DEAE-Sephadex profile capable of S., 73. 3201-3205, 1976. phosphorylating nucleolar protein substrates. Since pro 3. Ballai, N. R., Kang, Y. J., Olson, M. O. J., and Busch, H. Changes in Nucleolar Proteins and Their Phosphorylation Patterns during Liver teins C23 and C2 were the only major proteins labeled with Regeneration. J. Biol. Chem., 250. 5921-5925. 1975. Peaks 6, 7, and 8, it is probable that these are the natural 4. Busch, H., and Smetana, K. The Nucleolus, pp. 449-453. New York: substrates for the enzymes. Although Peak 3 was variable Academic Press, Inc., 1970. 5. Burton, K. Determination of DNA Concentration with Diphenylamine. in casein kinase activity from tissue to tissue, the natural Methods Enzymol., 12 Suppl. 13: 163-166, 1968. protein substrate is not known. It is possible that the correct 6. ChiÃ¹, J.-F., Craddock, S., and Hnilica. L. S. Nonhistone Chromatin Protein Phosphorylation during Azo-Dye Carcinogenesis. Federation substrate is not present in the mixture of extracted nucleo European Biochem. Soc. Letters, 33: 247-350, 1973. lar proteins used for the assay. Alternatively, the enzyme in 7. Choi, Y. C., Mauritzen, C. M., Taylor, C. W., and Busch. H. Rate Peak 3 may be in a form that is inactive against the natural Constants for Nucleolar Processing of High Molecular Weight RNA. Physiol. Chem. Phys.,3. 116-128, 1973. substrate. 8. Corbin, J. D., and Reimann, E. M. Assay of Cyclic AMP-Dependent The phosphorylation of protein C23 is of special interest Protein Kinases. Methods Enzymol., 38: 287-290. 1974. because this protein was recently found to contain phos- 9. Costa, M., FÃ¼ller,D.J. M., Russell, D. H., and Gerner, E. W. Cytoplasmic and Nuclear Protein Kinases during the Cell Cycle. Biochim. Biophys. phorylated regions rich in acidic amino acids (23). The Acta. 479: 416-426. 1977. function of additional negative charges due to the phospho 10. Desjardins, P. R., Lue, P. F., Liew, C. C., and Cornali, A. H. Purification and Properties of Rat Liver Nuclear Protein Kinases. Can. J. Biochem.. rylation of an already acidic cluster is unclear. However, the 50: 1249-1259, 1972. highly negatively charged regions are potential sites of 11. Farron-Furstenthal. F. Protein Kinases in Hepatoma, and Adult and Fetal interaction with the corresponding basic regions of other Liver of the Rat. I. Subcellular Distribution. Biochem. Biophys. Res. proteins, suggesting a role in the regulation of nucleolar Commun., 67: 307-314, 1975. 12. Fugassa, E., Gallo, G., Pertica, M.. Voci, A., and Orunesu, M. Increased structure. Activity of Rat Liver Nucleolar Protein Kinase following Triiodothyronine The presence of multiple nuclear phosphoprotein kinases Administration. Biochim. Biophys. Acta, 485: 350-356, 1977. 13. Gamo, S., and Lindeil, T. J. Presence of Two Protein Kinases in Highly has been observed by several workers. Approximately 5 Purified Rat Liver Nucleoli. Life Sci., 15: 2179-2187, 1975. species were observed in the nuclei of rat liver and hepa 14. Grummt, I. Studies on the Phosphorylation of Nucleolar Proteins. toma (11) and in Chinese hamster ovary cells (9). Thomson Federation European Biochem. Soc. Letters. 39. 125-128, 1974. 15 Higgins. G. M., and Anderson, R. M. Experimental Pathology of the ef al. (32) also resolved nuclear phosphoprotein kinase Liver. Arch. Pathol., 12: 186-202, 1931. activity toward endogenous protein substrates into approxi 16. Kang, Y. J., Olson, M. O. J., and Busch, H. Phosphorylation of Acid- soluble Proteins in Isolated Nucleoli of Novikoff Hepatoma Ascites Cells. mately 5 fractions. Although these studies were performed J. Biol. Chem., 249: 5580-5583, 1974. on whole nuclei, they agree with our data on the complexity 17. Kang. Y. J., Olson, M. 0. J.. Jones, C., and Busch, H. Nucleolar of kinase activity. Earlier studies by Kish and Kleinsmith Phosphoproteins of Normal Rat Liver and Novikoff Hepatoma Ascites Cells. Cancer Res.,35: 1470-1475, 1975. (18) suggested the presence of at least 12 chromatin ki 18. Kish, V. M., and Kleinsmith. L. J. Nuclear Protein Kinases. J. Biol. nases. However, their assay system involved autophosphor- Chem..249: 750-760. 1974. ylation of endogenous protein, which is likely to detect 19. Kleinsmith, L. J., Stein, J., and Stein, G. Dephosphorylation of Nonhis tone Proteins Specifically Alters the Pattern of Gene Transcription in enzyme-substrate complexes, as well as distinct enzymes. Reconstituted Chromatin. Proc. Nati. Acad. Sei. U. S., 73. 1174-1178, Other workers have divided nuclear protein kinases into 1976. 20. Koulish. S., and Kleinfeld. R. G. The Role of the Nucleolus. I. Tritiated essentially 2 major ionic species (10, 28, 33). Peaks 1 and 3 Cytidine Activity in Liver Parenchymal Cells of Thioacetamide-Treated of the nucleolar kinase elute at low, but distinctly different Rats. J. Cell Biol., 23. 39-51, 1964. ionic strengths. Peaks 6, 7, and 8 have similar substrate 21. Laemmli, U. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227. 680-685. 1970. specificities and elute together when a steeper gradient is 22. Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring used. Thus 2 major ionic classes of enzyme that can be Proteins. Methods Enzymol., 3. 447-454, 1957. further subfractionated also appear to exist in nucleoli. 23. Mamrack, M. D., Olson, M. O. J., and Busch, H. Negatively Charged Phosphopeptides of Nucleolar Nonhistone Proteins from Novikoff Hep The mechanism by which phosphoprotein kinase activity atoma Ascites Cells. Biochem. Biophys. Res. Commun.. 76 150-157, increases in nucleoli remains to be determined. Since these 1977. enzymes appear not to be regulated by cyclic nucleotides, 24. Olson, M. 0. J.. Ezrailson, E. G., Guetzow, K., and Busch. H. Localiza tion and Phosphorylation of Nuclear, Nucleolar and Extranucleolar Non de novo synthesis is the simplest explanation. However, histone Proteins of Novikoff Hepatoma Ascites Cells. J. Mol. Biol., 97: activation of preexisting inactive molecules by posttransla- 611-619, 1975. 25. Olson, M. O. J., and Guetzow. K. Phosphoprotein Phosphatase Activity tional modification of proenzymes cannot be ruled out. of Novikoff Hepatoma Nucleoli. Biochem. Biophys. Res. Commun., 70: These questions and the precise role of the phosphoprotein 717-722, 1976.

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26. Olson, M. O. J., Orrick, L. R.. Jones, C., and Busch. H Phosphorylation tion and Growth, pp. 45-59. Princeton: Princeton University Press. 1956. of Acid-soluble Nucleolar Proteins of Novikoff Hepatoma Ascites Cells in 32. Thomson. J A.. ChiÃ¹. J.-F , Sakuma, K., and Hnilica, L. S. Nuclear Vivo. J. Biol. Chem.,249. 2823-2827. 1974. Protein Phosphokinases in Normal and Neoplastia Tissues. Cancer Res.. 27. Olson, M. O. J.. Prestayko, A. W.. Jones, C. E., and Busch. H. Phospho 37 3266-3272. 1977. rylation of Proteins of Ribosomes and Nucleolar Preribosomal Particles 33 Thornburg, W.. and Lindell. T. J Purification of Rat Liver Nuclear from Novikoff Hepatoma Ascites Cells J. Mol. Biol., 90: 161-168, 1974. Protein Kinase Nil. J. Biol. Chem.. 252. 6660-6665. 1977. 28. Rikans, L. E., and Ruddon, R. W. Partial Purification and Properties of a 34. Valenzuela. P.. Weinberg. F., Bell. G., and Rutter, W. J. Yeast DNA Chromatin-Associated Phosphoprotein Kinase from Rat Liver Nuclei. Dependent RNA Polymerase I A Rapid Procedure for the Large Scale Biochim. Biophys. Acta, 422. 73-86. 1976. Purification of Homogeneous Enzyme. J. Biol. Chem.. 25). 1464-1470. 29 Rothblum. L I., Mamrack, P. M., Kunkle, H. M., Olson. M. 0. J.. and 1976. Busch. H. Fractionation of Nucleoli Enzymatic and Two-Dimensional 35. Wilson, M J , and Ahmed, K. Localization of Protein Phosphokmase Polyacrylamide Gel Electrophoretic Analysis. Biochemistry. 76. 4716- Activities in the Nucleolus Distinct from Extra-Nucleolar Regions in Rat 4721,1977. Ventral Prostate Nuclei Exptl. Cell Res.. 93. 261-266, 1975. 30. Stowell, R. E. Alterations in Nucleic Acids during Hepatoma Formation 36 Yu, F.-L. Increased Levels of Rat Hepatic Nuclear Free and Engaged in Rats-Fed p-Dimethylaminoazobenzene. Cancer, 2. 121-131, 1949. RNA Polymerase Activities during Liver Regeneration Biochem. Bio 31. Swift. H.. Rebhun, L.. Rasch. E , and Woodard, J. The Cytology of phys. Res. Commun., 64: 1107-1115, 1975. Nuclear RNA. In: D. Rudnick (ed.). Cellular Mechanisms in Differentia

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. Nucleolus-associated Phosphoprotein Kinases of Normal and Regenerating Liver and Novikoff Hepatoma Ascites Cells

Mark O. J. Olson, Sally Hatchett, Ruth Allan, et al.

Cancer Res 1978;38:3421-3426.

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