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Non-histone protein phosphorylation in normal and neoplastic rat liver chromatin
Printed in Sweden Copyright 8 1975 by Academic Press, Inc. All rights of reproduction in my form rmerv’d
NON-HISTONE
Experimental
Cell Research 91 (1975) 200-206
PROTEIN
PHOSPHORYLATION
NEOPLASTIC
RAT
LIVER
IN NORMAL
AND
CHROMATIN
JEN-FU CHIU, W. P. BRADE, JUDY THOMSON, YU-HUI TSAI and L. S. HNILICA Department of Biochemistry, M. D. Anderson Hospital
The University of Texas System Cancer Center, and Tumor Institute, Houston, Tex. 77025, USA
SUMMARY Hepatectomy or exposure of rats to a hepatocarcinogenic azo dye, N,N-dimethyl-p-(m-tolylazo)aniline or 3’ MDAB resulted in a sharp increase of the activity of cytoplasmic and chromatinbound phosphoprotein kinases in liver. While in the regenerating rat liver both enzyme groups increased nearly equally, in the 3’-MDAB-treated animals, there was much greater increase of the cytoplasmic kinases as compared with the chromatin-bound enzymes. The activation of phosphoprotein kinases was accompanied by an increase of the ability of liver chromatin to template for the in vitro RNA synthesis. This transcriptional activation was attributed to a partial destabilization of chromatin, resulting probably from the increased phosphorylation of chromatin proteins. The phosphorylation patterns of non-histone proteins differed in the liver chromatins of sham-operated, regenerating and 3’-MDAB-treated rats.
It was suggested that the phosphorylation of nuclear non-hi&one proteins may be a part of the mechanism regulating gene expression in higher organisms [l-4]. Nuclear phosphoproteins from various sources were found to be highly heterogeneous and tissuespecific by electrophoretic criteria [2, 51, to bind preferentially to homologous DNA [2, 3, 6, 71, and to stimulate the transcription by RNA polymerase of isolated DNA or chromatin [2, 6, 8, 93. The extent of nuclear non-histone protein phosphorylation could be correlated to the transcriptional activity of various tissues [lo-131. We show here that neoplastic transformation is accompanied by intensive phosphorylation of several non-histone proteins in chromatin. METHODS Male Fisher rats (15&200 g) were maintained on a diet containing 0.06 % of N,N-dimethyl-p-(m-tolylazo)Exptl
Cell Res 91 (1975)
aniline or 3’-MDAB [14]. Male albino SpragueDawley rats (150-200 g) fed normal laboratory diet were hepatectomized according to the method of Higgins & Anderson [15]. No livers were removed in sham-operated animals. All operations were performed between 8 and 9 a.m. Unless stated otherwise, all preparative work was performed at 24°C. To isolate nuclei, livers were homogenized in 10 vol of 0.32 M sucrose+ 5 mM MgCl, and centrifuged at 900 g for 10 min. The supernatant fluid was collected and centrifuged at 20000 g for 60 min. The activity of cytoplasmic phosphokinases was determined in the supernatants from this centrifugation. The sediment from the first centrifugation was homogenized in 10 vol of 2.2 M sucrose + 5 mM MgCl, and centrifuged at 70 000 g for 60 min. Chromatin was isolated from the pellet by following the method of Wilhelm et al. 1161. The phosphoprotein kinase activity was measured by the modified method of Kamiyama & Dastugue [4]. The reaction mixture contained 20 pmoles Tris-HCI buffer, pH 7.5, 4 nmoles [y-““PI ATP (spec. act. 0.23 Ci/nmole), 5 pmoles MgCl,, 25 pmoles NaCl and a given amount of cytoplasmic or nuclear enzyme extract or isolated chromatin. The final volume was adjusted with distilled water to 0.25 ml. The mixture was incubated at 37°C for 10 min and the reaction was terminated by addition of ice-cold 10 % trichloroacetic acid. The precipitate was washed 3 times with cold 5 % trichloroacetic acid. dissolved in NCS solubilizer (Amersham-Searle, inc.) and, after mixing
Non-histone protein phosphorylation
201
Fig. 1. Abscissa: (a) chromatin (L&gDNA); (b) MgCl, (mM x 10-r); (c) NaCl (mM); ordinate: enzyme activity (cpm x 10m4). Incorporation of label from [y-S2P]ATP into chromatin proteins in dependence on
with Bray’s scintillation fluid [17], its radioactivity was determined in a scintillation spectrometer. To study the in vivo phosphorylation of nuclear proteins, groups of 5-6 animals were injected intraperitoneally with 2 mCi/kg of S2P-orthophosphate (carrier-free, Schwarz-Mann, Inc.) and sacrificed 60 min thereafter. Livers were removed immediately after the sacrifice and chromatin was prepared from isolated nuclei. Individual chromatin preparations were extracted twice with 20-25 vol of ice-cold 0.25 M HCl to remove histones. The insoluble material was then extracted with 10 vol of 0.2 M HCl in chloroform-methanol (1 : 1, v/v) and 10 vol of 0.2 M HCl in chloroform-methanol (2 : 1, v/v). The extracted residue was finally dissolved in a solution containing 1 % (w/v) sodium dodecyl sulfate and 1 % (v/v) 2-mercaptoethanol in 50 mM Tris-HCI buffer, pH 8.0. The solubilized chromatin samples were centrifuged at 105 000 g (18°C) for 24 h as was described by Elgin & Bonner [ 181. The supernatant fluid containing essentially all the chromatin proteins was dialysed against deionized water and lyophilized. Aliquots (1 mg) of dry protein were dissolved in 1 ml of 8 M urea in 0.01 M sodium phosphate buffer pH 8.0 containing 1 g/l sodium dodecyl sulfate and 1 ml/l 2-mercaptoethanol and electrophoresed in 10 % polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue and scanned, after destaining, at 600 nm. Details of the electrophoretic, staining and scanning procedures were described by Wilhelm et al. [16]. The scanned gels were cut in 1 mm thick slices which were depolymerized in 30 % H,O, and counted in Bray’s scintillation fluid [17] using a scintillation spectrometer.
RESULTS It was shown [8, 191that isolated rat liver chromatin can incorporate the y-terminal phosphate of ATP into its proteins. The chromatin-bound phosphokinases from the normal rat livers are partially characterized in fig. la-c. The incorporation of y-terminal 32P of ATP into phosphoproteins is proportional to the chromatin concentration over a DNA range of 25-125 pg (fig. la).
For maximum activity, Mg2+ and Na+ were required in the incubation medium (20 and 75 mM, respectively, fig. 1b, c). Cyclic AMP in concentrations from 1.0 x lo-* M to 1.0 x 1O-2 M did not stimulate the activity of protein phosphokinase in liver chromatin under experimental conditions described in the Methods. It did stimulate, however, the cytoplasmic enzymes (20-30 %, in normal liver and over 100% in 6 h regenerating rat liver). Since Johnson & Allfrey [20] reported that CAMP stimulates the in vivo phosphorylation of some nuclear non-histone proteins it is possible that some regulatory co-factors or phosphokinase sub-units were lost from the nuclei during their isolation. However, because the cytoplasmic phosphoprotein kinases were stimulated by CAMP it is possible that the phosphoprotein fractions Table 1. Chromatin bound and cytoplasmic phosphoprotein kinase activities in control and hepatectomized rat livers, expressed as 32P incorporated (pmolelmg protein)
Sample
Chromatin bound enzyme activity
Sham 5.1 Hours after regeneration 1 6 7.0 12 7.5 18 1.3
%
Cytoplasmic enzyme activity %
100.0
22.7
100.0
122.8 131.3 128.1
23.4 29.6 30.7 32.4
103.1 130.4 135.2 142.9
Exptl
Cell Res 91 (1975)
202
Jen-Fu Chiu et al.
Table 2. Chromatin bound and cytoplasmic phosphoprotein kinase activities in control and 3’-MDAB rat livers, expre.wed as 32P incorporated (pmoleslmg protein)
cells to be several times higher than in normal liver. Concomitant to the activation of proteinphosphorylating enzymes was the change in templating capacity of chromatin in the Chromatin livers of animals in both experimental groups. bound Cytoplasmic The increase, recorded at 6 and 12 h after enzyme enzyme Sample activity activity % % hepatectomy and its return to nearly normal values at 18 h is presented in fig. 2. As in the hepatectomized animals, the templatControl 6.2 100.0 21.5 100.0 Days on ing capacity of liver chromatin from rats 3’-MDAB fed the 3’-MDAB containing diet increased diet 2 106.5 31.9 148.4 markedly (fig. 3). The increasepeaked around 15 66.: 104.8 47.5 220.9 47 days and then dropped to about 130% of 28 11:5 185.5 67.2 312.6 36 10.3 166.6 91.5 425.6 the controls at the end of this experiment 41 8.4 135.8 55.1 256.4 (96 days). 96 5.7 91.4 33.6 156.1 It was shown by Brown & Coffey [22-241 that the in vitro addition of polyanions to isolated nuclei or chromatin increased their which could be stimulated by CAMP in vivo capacity to template for the in vitro DNA [20] were phosphorylated by the cytoplasmic replication. According to our observations enzymes. [25] the addition of polyanions to isolated The activities of chromatin-associated chromatin produced its decondensation which phosphoprotein kinases from the livers of was reflected in a weaker association of hepatectomized, sham-operated, and 3’- histones with DNA and its increased capacity MDAB-fed rats are shown in tables 1 and 2. to template for the in vitro DNA or RNA There was only a small increase of kinase synthesis. Therefore, we used the DNA activity in the livers of hepatectomized polymerase assay as a probe to determine animals as compared with the considerable whether the increased RNA templating increase in the 3’-MDAB group (table 2). capacity of chromatin in the 3’-MDAB An even greater increase was observed in the animals can be the result of limited deconcytoplasmic fractions. The activities of cyto- densation of chromatin produced by the plasmic phosphoprotein kinases increased phosphorylation of its nonhistone proteins. 3040% in the hepatectomized animals Chromatin samples which were used to determine the in vitro RNA templating (table 1) and more than 4-fold in the 3’-MDAB treated rats (table 2). Becauseaqueous media activity (fig. 3) were also assayed for their were employed in isolating nuclei, the high ability to support the in vitro replication of phosphoprotein kinase activities observed in DNA. As can be seen in fig. 4, the capacity the cytoplasm might have originated in the of chromatin samples to template for DNA nuclei and leaked out into the cytoplasm synthesis in vitro closely resembles the during the isolation process. The data shown increase detected for the in vitro synthesis of in table 2 are- in good agreement with the RNA. This suggests that the template report of Granner [21], who found the changes of chromatin observed in our protein kinase activity in HTC hepatoma experiments may be the manifestation of Exptl
Cell Res 91 (1975)
Non-h&one
10
20
30
Figs 2-4. Abscissa: (pg DNA) chromatin; ordinate: cpm x 10-3. Fig. 2. Ternplating capacity for the in vitro RNA synthesis of regenerating rat liver chromatin. The reaction mixture (final vol, 0.25 ml) consisted of 0.2 pmoles ATP, 0.2 pmoles GTP, 0.2 pmoles CTP, 0.05 pmoles [3H]UTP, 10 pmoles Tris-HCl, pH 8.0, 30 pmoles KCI, 0.025 pmoles dithiothreitol, 0.025 ymoles EDTA and 0.625 pmoles MnCl,. The chromatin DNA concentration in each assay was as indicated together with 15 units of E. coli polymerase (spec. act. 60&700 units/mg protein). The assays were incubated at 37°C for 10 min and the reaction was terminated by adding 1 ml of cold 10 % trichloroacetic acid (TCA). The precipitated RNA was collected on filter paper (Whatman 3 MM) discs, washed with TCA and sodium pyrophosphate solutions; dried with absolute ethanol and counted in a toluene based scintillation counting liquid. e-0, sham operated; V-7, 6h regenerating; [I-[], 12 h regenerating; and, m-m, 18 h regenerating livers. Fig. 3. Ternplating capacity for the in vitro RNA synthesis of control and 3’-MDAB rat liver chromatin. The assay conditions were described in the caption to fig. 2. o--0, controls; v--V, 2 days; o--o, 15 days; A-A, 28 days; O-O, 36 days; A-A, 47 days; +-+, 96 days on 3’-MDAB diet. Fig. 4. Ternplating activity of control and 3’-MDAB rat liver chromatin for the in vitro DNA synthesis. The reaction mixture (final vol, 0.4 ml) consisted of 20 pmoles Tris-HCI buffer, pH 7.4; 2 pmoles
protein phosphorylation
203
partial decondensation of chromatin resulting from the increased phosphorylation of some of its non-histone proteins. The in vitro studies were complemented by measurements of the in vivo incorporation of [32P]orthophosphate into the non-histone proteins of liver chromatin. As revealed by electrophoretic analysis, there was only a moderate increase in the phosphorylation of non-histone proteins in liver chromatin of the hepatectomized rats. As is illustrated in fig. 5, the changes were mostly confined to the high molecular weight region of the gels. Exposure of rats to the hepatocarcinogen 3’-MDAB for 28 days increased the phosphorylation of chromatin nonhistone proteins significantly (fig. 6). In addition to the increased phosphorylation of the high molecular weight proteins there was a considerable activity associatedwith 2-3 major bands in the lower molecular weight area of the gels. The increase in phosphorylation pattern of 3’-MDAB liver chromatin was quantitatively different from that observed in the hepatectomized animals (figs 5, 6).
DISCUSSION Administration of carcinogens to the experimental animals results in the proliferation of cells in the target tissues and ensuing cellular transformation to cancerous phenotype. Chemicals which stimulate cells to dithiothreitol; 20 nmoles each of dATP, dGTP and dCTP with 1 pCi [SH]dTTP (52 Ci/mmole), the indicated amount of chromatin and 5 units of E. coli DNA polymerase (spec. act. 200 units mg). After incubation at 37°C for 30 min, the reaction was terminated by adding 2 ml of cold 10% TCA. The precipitate was collected by centrifugation and washed 3 times with 10 ml of 5 % TCA + 1 % sodium pyrophosphate solution. Finally, the precipitates were dissolved each in 0.1 ml NCS and 10 ml of Bray’s scintillation liquid [17]. The radioactivity was determined using a scintillation spectrometer. V-V, controls; l -•, 24 days; U-B, 47 days; O-U, 96 days on 3’-MDAB diet. Exptl
Cell Res 91 (1975)
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Jen-Fu Chiu et al.
6
proliferate, frequently enhance the action of carcinogens. According to Farber [26], chemical carcinogenesiscan be regarded as a selection of cells first for resistance against the cytotoxicity of carcinogen, then for resistance and growth and finally, for malignancy and invasiveness.During this schedule, there is a qualitative change in the cellular phenotype after which the cells cannot return to their orginal state if the carcinogenic stimulus was discontinued. From our studies illustrated in table 2 and figs 3 and 4, it appears that the qualitative change to cancerous phenotype is accompanied by substantial modifications of the chromatin templates. The carcinogenic stimulus first Exptl
Cell Res 91 (1975)
gel length (mm); ( %). (““P) distribution in 1 mm slices of polyacrylamide gel electrophoretogram of liver chromatin proteins. Sham operated (o-o); 6 h regenerating (+-+); and 12 h regenerating (A'-...A) rat livers. Individual samples were electrophoresed in separate gels and the results were superimposed. Fig. 6. Radioactivity (““P) distribution in 1 mm slices of polyacrylamide gel electrophoretograms of liver chromatin proteins from control rats (o-o) and rats on 3’-MDAB diet for 28 days (O......O). Individual samples were electrophoresed in separate gels and the results were superimposed. Figs 5-6. Abscissa: ordinate: radioactivity Fig. 5. Radioactivity
activates enzymatic phosphorylation of nuclear non-histone proteins with ensuing activation of the transcriptional activity of chromatin. As judged from our results with DNA polymerase (fig. 4), the increased phosphoprotein phosphorylation seemsto change the condensed state of chromatin, opening new sites for the polymerizing enzymes. A considerable part of this effort seems to be spent on the qualitative reprogramming of cellular phenotype because in rapidly proliferating, hepatectomized rat livers, the phosphorylation of non-histone proteins is increased to a much smaller extent (table 1, fig. 5). However, even this limited increase produced a substantial activation of chro-
Non-h&one matin to template for the in vitro RNA transcription (fig. 2). Indeed, as can be seen by comparing figs 2 and 3, the schedule of template activation of chromatin in hepatectomized livers is very similar to that observed in the 3’-MDAB animals. It appears as if much more effort, reflected in the phosphorylation of nuclear non-histone proteins, were spent on the qualitative transformation of cellular phenotype as compared with the activation of mitotic activity. The interpretation of our results indicating that the phosphorylation of nuclear nonhistone proteins appears to be associated with the reprogramming of restrictions affecting the transcription of chromatin is in good agreement with the reports of Ahmed & Ishida [27]. These authors found that the activity of chromatin-bound phosphoprotein kinases in rat submandibular salivary glands increased substantially after a simple injection of isoproterenol. This coincided temporally with the enhanced phosphorylation of nuclear non-histone proteins. Since it is believed that hormones can selectively activate the cellular genom, the results of Ahmed & Ishida [27] can be interpreted to indicate that nuclear non-histone protein phosphorylation is a required step in the activation of selected genes. Although the compensatory hypertrophy of rat liver is primarily orientated to the rapid replacement of lost tissue, it is also accompanied, especially in the early regenerative period, by an activation of specific transcriptional sites in chromatin [28, 291. It is possible that a part of the increase in phosphoprotein kinase activity and the ability of chromatin to template for in vitro RNA synthesis reflects the activation of selected genes, inactive in the resting hepatocyte. The isolation and characterization of chromatin phosphoproteins which are phosphorylated coincident with the activation of cellular
protein phosphorylation
205
genome may provide information leading to the understanding of the mechanisms restricting or activated individual genes in eukaryotic organisms. The authors are indebted to Mrs Catherine Craddock for her skilful technical assistance. The work was suooorted bv grants from The Robert A. Welch Foundation (Gil 3& and the USPHS (CA-07746). J-F. C. is a Rosalie B. Hite Postdoctoral Fellow. W. P. B. is a recipient of a Deutsche Forschungsgemeinschaft Fellowship.
REFERENCES 1. Langan, T A, Regulation of nucleic acid and protein biosynthesis (ed V V Koningsberger & L Bosch) pp. 223-242. Elsevier, Amsterdam (1967). 2. Teng, C S, Teng, C T & Allfrey, V G, J biol them 246 (1971) 3595. 3. Kleinsmith, L J, Heidema, J & Carroll, A, Nature 226 (1970) 1025. 4. Kamiyama, M & Dastugue, B, Biochem biophys res commun 44 (1971) 29. 5. Platz, R D, Kish, V M & Kleinsmith, L J, FEBS lett 12 (1970) 38. 6. Shea, M & Kleinsmith, L J, Biochem biophys res commun 50 (1973) 473. 7. Wakabayashi, K, Wang, S, Hord, G & Hnilica, L S, FEBS lett 32 (1973) 42. 8. Kamiyama, M, Dastugue, B & Kruh, J, Biochem biophys res commun 44 (1971) 1345. 9. Kamiyama, M & Wang, T Y, Biochim biophys acta 228 (1971) 563. 10. Kleinsmith, L J, Allfrey, V G & Mirsky, A E, Science 154 (1966) 780. 11. Gershey, E C & Kleinsmith, L J, Biochim biophys acta 194 (1960) 519. 12. Turkington, R W & Riddle, M, J biol them 224 (1966) 6040. 13. Ahmed, K & Ishida, H, Mol pharmacol 7 (1971) 323. 14. Chiu, J F, Craddock, C, Getz, S & Hnilica, L S, FEBS lett 33 (1973) 247. 15. Higgins, G M & Anderson, R M, Arch path01 12 (1931) 186. 16. Wilhelm, J A, Ansevin, A T, Johnson, A W & Hnilica, L S, Biochim biophys acta 272 (1972) 220. 17. Bray, G A, Anal biochem 1 (1960) 279. 18. Elgin, S C R & Bonner, J, Biochemistry 9 (1970) 4440. 19. Takeda, M, Yamamura, H & Ohga, Y, Biochem bioohvs res commun 42 (1971) 103. 20. John&n, E M & Allfrey, V-G, Arch biochem biophys 152 (1972) 786. 21. Granner, D K, Biochem biophys res commun 46 (1972) 1516. 22. Brown, D G & Coffey, D S, Science 171 (1971) 176. E.xptl
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23. - Biochem biophys res commun 42 (1971) 326. 24. - J biol them 247 (1972) 7674. 25. Getz, S, Ansevin, A T & Hnilica, L S. In preparation. 26. Farber, E, Cancer res 33 (1973) 2537. 27. Ahmed, K & Ishida, H, Fed proc 32 (1973) 476.
Exptl
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28. Church, R B & McCarthy, (1967) 459. 29. - Ibid 23 (1967) 477.
B J, J mol biol 23
Received June 24, 1974 Revised version received August 19, 1974
NON-HISTONE
Experimental
Cell Research 91 (1975) 200-206
PROTEIN
PHOSPHORYLATION
NEOPLASTIC
RAT
LIVER
IN NORMAL
AND
CHROMATIN
JEN-FU CHIU, W. P. BRADE, JUDY THOMSON, YU-HUI TSAI and L. S. HNILICA Department of Biochemistry, M. D. Anderson Hospital
The University of Texas System Cancer Center, and Tumor Institute, Houston, Tex. 77025, USA
SUMMARY Hepatectomy or exposure of rats to a hepatocarcinogenic azo dye, N,N-dimethyl-p-(m-tolylazo)aniline or 3’ MDAB resulted in a sharp increase of the activity of cytoplasmic and chromatinbound phosphoprotein kinases in liver. While in the regenerating rat liver both enzyme groups increased nearly equally, in the 3’-MDAB-treated animals, there was much greater increase of the cytoplasmic kinases as compared with the chromatin-bound enzymes. The activation of phosphoprotein kinases was accompanied by an increase of the ability of liver chromatin to template for the in vitro RNA synthesis. This transcriptional activation was attributed to a partial destabilization of chromatin, resulting probably from the increased phosphorylation of chromatin proteins. The phosphorylation patterns of non-histone proteins differed in the liver chromatins of sham-operated, regenerating and 3’-MDAB-treated rats.
It was suggested that the phosphorylation of nuclear non-hi&one proteins may be a part of the mechanism regulating gene expression in higher organisms [l-4]. Nuclear phosphoproteins from various sources were found to be highly heterogeneous and tissuespecific by electrophoretic criteria [2, 51, to bind preferentially to homologous DNA [2, 3, 6, 71, and to stimulate the transcription by RNA polymerase of isolated DNA or chromatin [2, 6, 8, 93. The extent of nuclear non-histone protein phosphorylation could be correlated to the transcriptional activity of various tissues [lo-131. We show here that neoplastic transformation is accompanied by intensive phosphorylation of several non-histone proteins in chromatin. METHODS Male Fisher rats (15&200 g) were maintained on a diet containing 0.06 % of N,N-dimethyl-p-(m-tolylazo)Exptl
Cell Res 91 (1975)
aniline or 3’-MDAB [14]. Male albino SpragueDawley rats (150-200 g) fed normal laboratory diet were hepatectomized according to the method of Higgins & Anderson [15]. No livers were removed in sham-operated animals. All operations were performed between 8 and 9 a.m. Unless stated otherwise, all preparative work was performed at 24°C. To isolate nuclei, livers were homogenized in 10 vol of 0.32 M sucrose+ 5 mM MgCl, and centrifuged at 900 g for 10 min. The supernatant fluid was collected and centrifuged at 20000 g for 60 min. The activity of cytoplasmic phosphokinases was determined in the supernatants from this centrifugation. The sediment from the first centrifugation was homogenized in 10 vol of 2.2 M sucrose + 5 mM MgCl, and centrifuged at 70 000 g for 60 min. Chromatin was isolated from the pellet by following the method of Wilhelm et al. 1161. The phosphoprotein kinase activity was measured by the modified method of Kamiyama & Dastugue [4]. The reaction mixture contained 20 pmoles Tris-HCI buffer, pH 7.5, 4 nmoles [y-““PI ATP (spec. act. 0.23 Ci/nmole), 5 pmoles MgCl,, 25 pmoles NaCl and a given amount of cytoplasmic or nuclear enzyme extract or isolated chromatin. The final volume was adjusted with distilled water to 0.25 ml. The mixture was incubated at 37°C for 10 min and the reaction was terminated by addition of ice-cold 10 % trichloroacetic acid. The precipitate was washed 3 times with cold 5 % trichloroacetic acid. dissolved in NCS solubilizer (Amersham-Searle, inc.) and, after mixing
Non-histone protein phosphorylation
201
Fig. 1. Abscissa: (a) chromatin (L&gDNA); (b) MgCl, (mM x 10-r); (c) NaCl (mM); ordinate: enzyme activity (cpm x 10m4). Incorporation of label from [y-S2P]ATP into chromatin proteins in dependence on
with Bray’s scintillation fluid [17], its radioactivity was determined in a scintillation spectrometer. To study the in vivo phosphorylation of nuclear proteins, groups of 5-6 animals were injected intraperitoneally with 2 mCi/kg of S2P-orthophosphate (carrier-free, Schwarz-Mann, Inc.) and sacrificed 60 min thereafter. Livers were removed immediately after the sacrifice and chromatin was prepared from isolated nuclei. Individual chromatin preparations were extracted twice with 20-25 vol of ice-cold 0.25 M HCl to remove histones. The insoluble material was then extracted with 10 vol of 0.2 M HCl in chloroform-methanol (1 : 1, v/v) and 10 vol of 0.2 M HCl in chloroform-methanol (2 : 1, v/v). The extracted residue was finally dissolved in a solution containing 1 % (w/v) sodium dodecyl sulfate and 1 % (v/v) 2-mercaptoethanol in 50 mM Tris-HCI buffer, pH 8.0. The solubilized chromatin samples were centrifuged at 105 000 g (18°C) for 24 h as was described by Elgin & Bonner [ 181. The supernatant fluid containing essentially all the chromatin proteins was dialysed against deionized water and lyophilized. Aliquots (1 mg) of dry protein were dissolved in 1 ml of 8 M urea in 0.01 M sodium phosphate buffer pH 8.0 containing 1 g/l sodium dodecyl sulfate and 1 ml/l 2-mercaptoethanol and electrophoresed in 10 % polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue and scanned, after destaining, at 600 nm. Details of the electrophoretic, staining and scanning procedures were described by Wilhelm et al. [16]. The scanned gels were cut in 1 mm thick slices which were depolymerized in 30 % H,O, and counted in Bray’s scintillation fluid [17] using a scintillation spectrometer.
RESULTS It was shown [8, 191that isolated rat liver chromatin can incorporate the y-terminal phosphate of ATP into its proteins. The chromatin-bound phosphokinases from the normal rat livers are partially characterized in fig. la-c. The incorporation of y-terminal 32P of ATP into phosphoproteins is proportional to the chromatin concentration over a DNA range of 25-125 pg (fig. la).
For maximum activity, Mg2+ and Na+ were required in the incubation medium (20 and 75 mM, respectively, fig. 1b, c). Cyclic AMP in concentrations from 1.0 x lo-* M to 1.0 x 1O-2 M did not stimulate the activity of protein phosphokinase in liver chromatin under experimental conditions described in the Methods. It did stimulate, however, the cytoplasmic enzymes (20-30 %, in normal liver and over 100% in 6 h regenerating rat liver). Since Johnson & Allfrey [20] reported that CAMP stimulates the in vivo phosphorylation of some nuclear non-histone proteins it is possible that some regulatory co-factors or phosphokinase sub-units were lost from the nuclei during their isolation. However, because the cytoplasmic phosphoprotein kinases were stimulated by CAMP it is possible that the phosphoprotein fractions Table 1. Chromatin bound and cytoplasmic phosphoprotein kinase activities in control and hepatectomized rat livers, expressed as 32P incorporated (pmolelmg protein)
Sample
Chromatin bound enzyme activity
Sham 5.1 Hours after regeneration 1 6 7.0 12 7.5 18 1.3
%
Cytoplasmic enzyme activity %
100.0
22.7
100.0
122.8 131.3 128.1
23.4 29.6 30.7 32.4
103.1 130.4 135.2 142.9
Exptl
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Jen-Fu Chiu et al.
Table 2. Chromatin bound and cytoplasmic phosphoprotein kinase activities in control and 3’-MDAB rat livers, expre.wed as 32P incorporated (pmoleslmg protein)
cells to be several times higher than in normal liver. Concomitant to the activation of proteinphosphorylating enzymes was the change in templating capacity of chromatin in the Chromatin livers of animals in both experimental groups. bound Cytoplasmic The increase, recorded at 6 and 12 h after enzyme enzyme Sample activity activity % % hepatectomy and its return to nearly normal values at 18 h is presented in fig. 2. As in the hepatectomized animals, the templatControl 6.2 100.0 21.5 100.0 Days on ing capacity of liver chromatin from rats 3’-MDAB fed the 3’-MDAB containing diet increased diet 2 106.5 31.9 148.4 markedly (fig. 3). The increasepeaked around 15 66.: 104.8 47.5 220.9 47 days and then dropped to about 130% of 28 11:5 185.5 67.2 312.6 36 10.3 166.6 91.5 425.6 the controls at the end of this experiment 41 8.4 135.8 55.1 256.4 (96 days). 96 5.7 91.4 33.6 156.1 It was shown by Brown & Coffey [22-241 that the in vitro addition of polyanions to isolated nuclei or chromatin increased their which could be stimulated by CAMP in vivo capacity to template for the in vitro DNA [20] were phosphorylated by the cytoplasmic replication. According to our observations enzymes. [25] the addition of polyanions to isolated The activities of chromatin-associated chromatin produced its decondensation which phosphoprotein kinases from the livers of was reflected in a weaker association of hepatectomized, sham-operated, and 3’- histones with DNA and its increased capacity MDAB-fed rats are shown in tables 1 and 2. to template for the in vitro DNA or RNA There was only a small increase of kinase synthesis. Therefore, we used the DNA activity in the livers of hepatectomized polymerase assay as a probe to determine animals as compared with the considerable whether the increased RNA templating increase in the 3’-MDAB group (table 2). capacity of chromatin in the 3’-MDAB An even greater increase was observed in the animals can be the result of limited deconcytoplasmic fractions. The activities of cyto- densation of chromatin produced by the plasmic phosphoprotein kinases increased phosphorylation of its nonhistone proteins. 3040% in the hepatectomized animals Chromatin samples which were used to determine the in vitro RNA templating (table 1) and more than 4-fold in the 3’-MDAB treated rats (table 2). Becauseaqueous media activity (fig. 3) were also assayed for their were employed in isolating nuclei, the high ability to support the in vitro replication of phosphoprotein kinase activities observed in DNA. As can be seen in fig. 4, the capacity the cytoplasm might have originated in the of chromatin samples to template for DNA nuclei and leaked out into the cytoplasm synthesis in vitro closely resembles the during the isolation process. The data shown increase detected for the in vitro synthesis of in table 2 are- in good agreement with the RNA. This suggests that the template report of Granner [21], who found the changes of chromatin observed in our protein kinase activity in HTC hepatoma experiments may be the manifestation of Exptl
Cell Res 91 (1975)
Non-h&one
10
20
30
Figs 2-4. Abscissa: (pg DNA) chromatin; ordinate: cpm x 10-3. Fig. 2. Ternplating capacity for the in vitro RNA synthesis of regenerating rat liver chromatin. The reaction mixture (final vol, 0.25 ml) consisted of 0.2 pmoles ATP, 0.2 pmoles GTP, 0.2 pmoles CTP, 0.05 pmoles [3H]UTP, 10 pmoles Tris-HCl, pH 8.0, 30 pmoles KCI, 0.025 pmoles dithiothreitol, 0.025 ymoles EDTA and 0.625 pmoles MnCl,. The chromatin DNA concentration in each assay was as indicated together with 15 units of E. coli polymerase (spec. act. 60&700 units/mg protein). The assays were incubated at 37°C for 10 min and the reaction was terminated by adding 1 ml of cold 10 % trichloroacetic acid (TCA). The precipitated RNA was collected on filter paper (Whatman 3 MM) discs, washed with TCA and sodium pyrophosphate solutions; dried with absolute ethanol and counted in a toluene based scintillation counting liquid. e-0, sham operated; V-7, 6h regenerating; [I-[], 12 h regenerating; and, m-m, 18 h regenerating livers. Fig. 3. Ternplating capacity for the in vitro RNA synthesis of control and 3’-MDAB rat liver chromatin. The assay conditions were described in the caption to fig. 2. o--0, controls; v--V, 2 days; o--o, 15 days; A-A, 28 days; O-O, 36 days; A-A, 47 days; +-+, 96 days on 3’-MDAB diet. Fig. 4. Ternplating activity of control and 3’-MDAB rat liver chromatin for the in vitro DNA synthesis. The reaction mixture (final vol, 0.4 ml) consisted of 20 pmoles Tris-HCI buffer, pH 7.4; 2 pmoles
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partial decondensation of chromatin resulting from the increased phosphorylation of some of its non-histone proteins. The in vitro studies were complemented by measurements of the in vivo incorporation of [32P]orthophosphate into the non-histone proteins of liver chromatin. As revealed by electrophoretic analysis, there was only a moderate increase in the phosphorylation of non-histone proteins in liver chromatin of the hepatectomized rats. As is illustrated in fig. 5, the changes were mostly confined to the high molecular weight region of the gels. Exposure of rats to the hepatocarcinogen 3’-MDAB for 28 days increased the phosphorylation of chromatin nonhistone proteins significantly (fig. 6). In addition to the increased phosphorylation of the high molecular weight proteins there was a considerable activity associatedwith 2-3 major bands in the lower molecular weight area of the gels. The increase in phosphorylation pattern of 3’-MDAB liver chromatin was quantitatively different from that observed in the hepatectomized animals (figs 5, 6).
DISCUSSION Administration of carcinogens to the experimental animals results in the proliferation of cells in the target tissues and ensuing cellular transformation to cancerous phenotype. Chemicals which stimulate cells to dithiothreitol; 20 nmoles each of dATP, dGTP and dCTP with 1 pCi [SH]dTTP (52 Ci/mmole), the indicated amount of chromatin and 5 units of E. coli DNA polymerase (spec. act. 200 units mg). After incubation at 37°C for 30 min, the reaction was terminated by adding 2 ml of cold 10% TCA. The precipitate was collected by centrifugation and washed 3 times with 10 ml of 5 % TCA + 1 % sodium pyrophosphate solution. Finally, the precipitates were dissolved each in 0.1 ml NCS and 10 ml of Bray’s scintillation liquid [17]. The radioactivity was determined using a scintillation spectrometer. V-V, controls; l -•, 24 days; U-B, 47 days; O-U, 96 days on 3’-MDAB diet. Exptl
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proliferate, frequently enhance the action of carcinogens. According to Farber [26], chemical carcinogenesiscan be regarded as a selection of cells first for resistance against the cytotoxicity of carcinogen, then for resistance and growth and finally, for malignancy and invasiveness.During this schedule, there is a qualitative change in the cellular phenotype after which the cells cannot return to their orginal state if the carcinogenic stimulus was discontinued. From our studies illustrated in table 2 and figs 3 and 4, it appears that the qualitative change to cancerous phenotype is accompanied by substantial modifications of the chromatin templates. The carcinogenic stimulus first Exptl
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gel length (mm); ( %). (““P) distribution in 1 mm slices of polyacrylamide gel electrophoretogram of liver chromatin proteins. Sham operated (o-o); 6 h regenerating (+-+); and 12 h regenerating (A'-...A) rat livers. Individual samples were electrophoresed in separate gels and the results were superimposed. Fig. 6. Radioactivity (““P) distribution in 1 mm slices of polyacrylamide gel electrophoretograms of liver chromatin proteins from control rats (o-o) and rats on 3’-MDAB diet for 28 days (O......O). Individual samples were electrophoresed in separate gels and the results were superimposed. Figs 5-6. Abscissa: ordinate: radioactivity Fig. 5. Radioactivity
activates enzymatic phosphorylation of nuclear non-histone proteins with ensuing activation of the transcriptional activity of chromatin. As judged from our results with DNA polymerase (fig. 4), the increased phosphoprotein phosphorylation seemsto change the condensed state of chromatin, opening new sites for the polymerizing enzymes. A considerable part of this effort seems to be spent on the qualitative reprogramming of cellular phenotype because in rapidly proliferating, hepatectomized rat livers, the phosphorylation of non-histone proteins is increased to a much smaller extent (table 1, fig. 5). However, even this limited increase produced a substantial activation of chro-
Non-h&one matin to template for the in vitro RNA transcription (fig. 2). Indeed, as can be seen by comparing figs 2 and 3, the schedule of template activation of chromatin in hepatectomized livers is very similar to that observed in the 3’-MDAB animals. It appears as if much more effort, reflected in the phosphorylation of nuclear non-histone proteins, were spent on the qualitative transformation of cellular phenotype as compared with the activation of mitotic activity. The interpretation of our results indicating that the phosphorylation of nuclear nonhistone proteins appears to be associated with the reprogramming of restrictions affecting the transcription of chromatin is in good agreement with the reports of Ahmed & Ishida [27]. These authors found that the activity of chromatin-bound phosphoprotein kinases in rat submandibular salivary glands increased substantially after a simple injection of isoproterenol. This coincided temporally with the enhanced phosphorylation of nuclear non-histone proteins. Since it is believed that hormones can selectively activate the cellular genom, the results of Ahmed & Ishida [27] can be interpreted to indicate that nuclear non-histone protein phosphorylation is a required step in the activation of selected genes. Although the compensatory hypertrophy of rat liver is primarily orientated to the rapid replacement of lost tissue, it is also accompanied, especially in the early regenerative period, by an activation of specific transcriptional sites in chromatin [28, 291. It is possible that a part of the increase in phosphoprotein kinase activity and the ability of chromatin to template for in vitro RNA synthesis reflects the activation of selected genes, inactive in the resting hepatocyte. The isolation and characterization of chromatin phosphoproteins which are phosphorylated coincident with the activation of cellular
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genome may provide information leading to the understanding of the mechanisms restricting or activated individual genes in eukaryotic organisms. The authors are indebted to Mrs Catherine Craddock for her skilful technical assistance. The work was suooorted bv grants from The Robert A. Welch Foundation (Gil 3& and the USPHS (CA-07746). J-F. C. is a Rosalie B. Hite Postdoctoral Fellow. W. P. B. is a recipient of a Deutsche Forschungsgemeinschaft Fellowship.
REFERENCES 1. Langan, T A, Regulation of nucleic acid and protein biosynthesis (ed V V Koningsberger & L Bosch) pp. 223-242. Elsevier, Amsterdam (1967). 2. Teng, C S, Teng, C T & Allfrey, V G, J biol them 246 (1971) 3595. 3. Kleinsmith, L J, Heidema, J & Carroll, A, Nature 226 (1970) 1025. 4. Kamiyama, M & Dastugue, B, Biochem biophys res commun 44 (1971) 29. 5. Platz, R D, Kish, V M & Kleinsmith, L J, FEBS lett 12 (1970) 38. 6. Shea, M & Kleinsmith, L J, Biochem biophys res commun 50 (1973) 473. 7. Wakabayashi, K, Wang, S, Hord, G & Hnilica, L S, FEBS lett 32 (1973) 42. 8. Kamiyama, M, Dastugue, B & Kruh, J, Biochem biophys res commun 44 (1971) 1345. 9. Kamiyama, M & Wang, T Y, Biochim biophys acta 228 (1971) 563. 10. Kleinsmith, L J, Allfrey, V G & Mirsky, A E, Science 154 (1966) 780. 11. Gershey, E C & Kleinsmith, L J, Biochim biophys acta 194 (1960) 519. 12. Turkington, R W & Riddle, M, J biol them 224 (1966) 6040. 13. Ahmed, K & Ishida, H, Mol pharmacol 7 (1971) 323. 14. Chiu, J F, Craddock, C, Getz, S & Hnilica, L S, FEBS lett 33 (1973) 247. 15. Higgins, G M & Anderson, R M, Arch path01 12 (1931) 186. 16. Wilhelm, J A, Ansevin, A T, Johnson, A W & Hnilica, L S, Biochim biophys acta 272 (1972) 220. 17. Bray, G A, Anal biochem 1 (1960) 279. 18. Elgin, S C R & Bonner, J, Biochemistry 9 (1970) 4440. 19. Takeda, M, Yamamura, H & Ohga, Y, Biochem bioohvs res commun 42 (1971) 103. 20. John&n, E M & Allfrey, V-G, Arch biochem biophys 152 (1972) 786. 21. Granner, D K, Biochem biophys res commun 46 (1972) 1516. 22. Brown, D G & Coffey, D S, Science 171 (1971) 176. E.xptl
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23. - Biochem biophys res commun 42 (1971) 326. 24. - J biol them 247 (1972) 7674. 25. Getz, S, Ansevin, A T & Hnilica, L S. In preparation. 26. Farber, E, Cancer res 33 (1973) 2537. 27. Ahmed, K & Ishida, H, Fed proc 32 (1973) 476.
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28. Church, R B & McCarthy, (1967) 459. 29. - Ibid 23 (1967) 477.
B J, J mol biol 23
Received June 24, 1974 Revised version received August 19, 1974