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Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation
172
Biochimica et Biophysica Acta 952 (1988) 172 180
Elsevier BBA 33048
Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation Jorma Palvimo and Pekka H. M~ienp~iii Department of Biochemistry, University of Kuopio, Kuopio (Finland)
(Received2 October 1987)
Key words: High mobilitygroup protein; Protein phosphorylation; Histone H1; DNA; (Calf thymus) We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobilitygroup proteins H M G 14 and H M G 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone HI. Without in vitro phosphorylation, H M G 14 and H M G 1 7 eluted from double-stranded DNA-columns at 200 mM NaCl. H M G 14 was released from single-stranded DNA-column at 300 mM NaCl and from HI-column at 130 mM NaCI, whereas the corresponding values for H M G 17 were 230 mM and 20 mM, respectively. Phosphorylation of H M G 14 and H M G 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCI, respectively) for single-stranded DNA, whereas H M G 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCi) ahead of the unpbosphorylated protein. H M G 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCI) with the unphospborylated protein. Interestingly, phosphorylation of H M G 14 by NII-kinase increased considerably its affinity for histone H I and the phospborylated protein eluted at 200 mM NaCl. Pbosphorylation of H M G 14 by A-kinase did not alter its interaction towards histone HI. These results indicate that modification of H M G 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H I , and that H M G 17 has much weaker affinity for single-stranded DNA and histone H I than H M G 14.
Introduction
The high-mobility-group (HMG) proteins are a family of well-characterized non-histone proteins extractable from nuclei and chromatin with 0.35 M NaC1 [1]. The low-molecular-weight H M G proteins H M G 14 and H M G 17 (100 and 89 aminoacid residues, respectively, for proteins from calf
Abbreviations: HMG, high-mobility-group; A-kinase, cAMPdependent protein kinase; NIl-kinase, nuclear protein kinase II. Correspondence: J. Palvimo, Department of Biochemistry, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland.
thymus) are characterized by asymmetrical charge distribution. Two-thirds of H M G 17 and half of H M G 14 are highly basic in their amino-terminal ends. In contrast, the carboxy-terminal regions have an overall negative charge [2]. The central region of H M G 17 contains six helix-destabilizing proline residues which may contribute to a lack of ordered tertiary structure observed in solution [3,4]. It seems that more ordered conformations of H M G 14 and H M G 17 are generated by binding of these proteins to chromatin [5]. The domain structures of these H M G proteins suggest that they participate in multiple interactions with chromatin components in their native state. H M G 14 and H M G 17 have been implicated in
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (Biomedical Division)
173 modifying the chromatin structure of transcriptionally active genes [6]. Several kinds of postsynthetic modifications of these HMG proteins, e.g., ADP-ribosylation and phosphorylation, have been described [7-9]. Post-translational modifications of chromosomal proteins are thought to play an important role in regulation of chromatin structure. HMG 14 from calf thymus can be phosphorylated by both cyclic nucleotide-dependent and -independent protein kinases. The major high-affinity site phosphorylated by cyclic nucleotide-dependent protein kinases is Ser-6 at the basic amino-terminal region of the molecule [10,11]. Thyrotropin stimulates phosphorylation of HMG 14 in vivo at the site catalyzed by cAMPdependent protein kinase in vitro [12]. In contrast, casein kinase II, which is similar to nuclear protein kinase II, phosphorylates HMG 14 predominantly at Ser-89 in the acidic carboxy-terminal region [8,13]. Walton and Gill [14] have shown that purified casein kinase II catalyzes phosphorylation of hHMG 14a2 at a site which is phosphorylated in vivo in HeLa cells. It seems probable that phosphorylation of these proteins affects their interactions with DNA and other chromatin components, e.g., histones. We have shown in a preliminary study that phosphorylation of H M G 14 by cGMP-dependent protein kinase decreases its affinity for single-stranded DNA [15]. Affinity chromatography was used in this study to examine the effects of phosphorylation of HMG 14 by A-kinase and NII-kinase on its binding properties towards DNA and histone H1. We also compared binding properties of unphosphorylated and phosphorylated HMG 17 with those of H M G 14. Materials and Methods
HMG 14 and HMG 17 were purified from calf thymus by chromatography on CM-Sephadex [16]. Histone H1 was purified from calf thymus using the method of Johns [17]. The endogenous, alkalilabile phosphate content of purified chromatin proteins was determined using the method of Chen et al. [18]. The purified H M G 14, HMG 17 and H1 contained 0.08, 0.03 and 0.20 mol of alkali-labile phosphate per mol of protein, respectively. The catalytic subunit of cAMP-dependent pro-
tein kinase was purified to apparent homogeneity from bovine heart according to Reiman et al. [19]. Nuclear protein kinase was purified from isolated rat liver chromatin. The 0.4 M NaCl-extract of chromatin was dialyzed, fractionated on DEAESephadex, dialyzed and chromatographed on a phosvitin-casein-Sepharose 4B affinity column [20]. Reaction buffers for phosphorylation reactions were as described in Ref. 8. DNA-agarose containing 0.66 mg calf thymus single-stranded DNA per ml agarose was purchased from Bethesda Research Laboratories. Double-stranded DNA-cellulose was from Sigma (1 mg calf thymus DNA per ml cellulose) or from Pharmacia (1.14 mg DNA per ml cellulose). Single-stranded DNA regions were removed from double-stranded DNA-columns by passing S1 nuclease through the columns according to Isackson et al. [21]. Sigmacell-cellulose was used for blank columns. Electrophoretically pure histone H1 was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to instructions provided by the manufacturer. Usually, 7 g (wet weight) of prewashed activated Sepharose was coupled with 100 mg of histone H1 which was dissolved in a sufficient volume of 0.4 M sodium carbonate buffer (pH 9.0) to provide a thick slurry upon the addition of the wet Sepharose. Coupling was accomplished by stirring the slurry overnight at 4°C. Substituted Sepharose was then washed with 100 ml of the coupling buffer and 1000 ml of 2 M NaCI/0.2 M Tris-HC1 (pH 8.0) in several aliquots during 1 h. After packing, the columns were washed overnight with 1 M NaC1/10 mM TrisHC1 (pH 7.5) before equilibrating with the starting buffer (0.02 M NaC1/10 mM Tris-HC1 (pH 7.5)). The resulting Sepharose contained 5 mg of covalently coupled histone H1 per ml Sepharose. The reactive groups in blank Sepharose 4B were blocked with Tris-HC1 buffer (0.2 M, pH 8.0). Acetic acid/urea or SDS-polyacrylamide gel electrophoresis and autoradiography were performed as described in Ref. 8. Results
Binding of phosphorylated HMG 14 to singlestranded DNA The effect of phosphorylation of HMG 14 by
174
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Fraction no. Fig. 1. Chromatography of HMG 14 phosphorylated by NII-kinase (A) or A-kinase (B) on single-stranded DNA-agarose columns. H M G 14 (0.1 mg) was phosphorylated by NlI-kinase (0.4 ~g) in the presence of 30 /~M [32p]ATP for 90 min at 3 0 ° C (A), or by A-kinase (1.4 t.tg) for 30 min (B). Free radioactivity was separated from phosphoprotein by gel filtration on Sephadex G-25 at 4 ° C (0.9×25 cm) with 0.2 M NaC1/1 mM Tris-HC1 (pH 7.5) (starting buffer). 0.4 mg of carrier H M G 14 was mixed with each phosphorylated sample, and they were applied to two parallel single-stranded DNA-agarose columns (containing 3.3 mg of single-stranded DNA), which were pre-equilibrated with the starting buffer. The gradient (100 ml for two columns) was made using a single gradient maker and introduced into two parallel columns by way of a three-way connector and a dual-channel peristaltic pump. The columns were first washed with 1 bed vol. of starting buffer and eluted at 4 ° C with 100 ml of a linear gradient of 0.2 to 0.6 M NaC1/1 mM Tris-HCl (pH 7.5). Effluents from the columns were collected simultaneously with a single fraction collector. Aliquots of indicated gradient fractions (1 ml) were electrophoresed in acetic acid/urea gels. Stained gels and autoradiograms of the gels are shown on the right. Protein was detected by measuring the absorbance at 220 nm (11), and radioactivity as Cerenkov radiation (D) from 1 ml fractions. The salt concentration in gradient fractions ( . . . . . . ) was determined by measuring the conductivity with a Philips PW 9501/01 conductivity meter.
175
NII-kinase or A-kinase on its affinity for singlestranded DNA was studied using affinity chromatography on single-stranded DNA-agarose. Without in vitro phosphorylation, HMG 14 eluted from the column at 300 mM NaC1 (Figs. 1 and 2),
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176
from the DNA-column at a slightly lower salt concentration (290 mM NaC1) (Fig. 1A). Because these in vitro phosphorylated molecules represent only a small portion of total HMG 14, their contribution to the A220 profile is negligible. When HMG 14 was phosphorylated by A-kinase to a stoichiometry of about 1 mol phosphate per mol
protein, the phosphorylated protein was visible as a peak in the absorbance profile cochromatographing with the radioactivity peak at 270 mM NaC1 (Fig. 1B). The peak corresponding to unphosphorylated HMG 14 eluted at 300 mM NaC1 as in Fig. 1A. When HMG 14 was phosphorylated by A-kinase using a lower concentration of en-
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Fig. 3. Chromatography of H M G 14 phosphorylated by NII-kinase (A) or A-kinase (B) on parallel double-stranded DNA-cellulose columns (4 ml). HMG 14 was phosphorylated by NIl-kinase or A-kinase as described in Fig. 1 and Fig. 2, respectively. 0.05 M NaC1/1 mM Tris-HCl (pH 7.5), was used as a starting buffer in this experiment.
177
zyme and a shorter reaction time, the phosphorylated protein was not visible as a peak in the A z20 profile (Fig. 2B), but the peak of radioactivity eluted at the same NaC1 concentration (270 mM) as shown in Fig. lB. The concentration was clearly
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2O Fraction no. Fig. 4. Chromatography of H M G 14 phosphorylated by NlI-kinase (A) or A-kinase (B) on parallel histone H1-Sepharose columns (4 ml). H M G 14 was phosphorylated as in Fig. 3. The columns were eluted with 100 ml of a linear gradient of 0.02 M to 1.0 M NaC1/10 mM Tris-HC1 (pH 7.5). Aliquots of indicated gradient fractions were electrophoresed in acetic acid/urea gels. Stained gels and autoradiograms of the gels are shown on the right.
178
molecules applied to the columns. The affinity of unphosphorylated HMG 17 for single-stranded DNA was compared to that of HMG 14 (Fig. 2). Interestingly, HMG 17 was released from the DNA-column at a considerably lower ionic strength (230 mM NaC1) than HMG 14. HMG 17 can be phosphorylated in vitro by A-kinase using longer reaction times and higher enzyme concentrations than those required for HMG 14. The stoichiometry of the reaction is low, being less than 10% of that achieved with HMG 14 under identical conditions. The phosphorylated HMG 17 eluted at the beginning of the salt gradient (Fig. 2A). Possible degradation of the HMG proteins during phosphorylation reaction and chromatography was examined by electrophoresis. The proteins were found to comigrate with the untreated proteins during chromatography, indicating that degradation did not take place.
shown in Fig. 4. Unphosphorylated HMG 14 eluted from the column at 130 mM NaC1/10 mM Tris-HC1 (pH 7.5). This agrees with the results of Espel et al. [23]. Using chemical cross-linking, they detected interaction between histone H1 and HMG 14, which was abolished above 120 mM NaC1/25 mM triethanolamine. Phosphorylation of HMG 14 by A-kinase was without effect on its binding to histone H1, but phosphorylation by NII-kinase increased its affinity for H1 considerably. The phosphorylated protein eluted at 200 mM NaC1 (Fig. 4A). HMG 14 was not retained by the blank Sepharose column (data not shown). HMG 17 did not bind to histone H1-Sepharose under conditions used in this study (20 mM NaC1) (data not shown). This indicates a profound difference between binding of HMG 14 and HMG 17 to histone H1. Discussion
Binding of phosphorylated HMG 14 to doublestranded DNA Isackson and Reeck [22] have reported previously that HMG 14 and HMG 17 from chicken erythrocytes fail to bind to double-stranded DNA-cellulose at 200 mM NaC1/1 mM Tris-HCl (pH 7.5), and that they bind preferentially to single-stranded DNA. Fig. 3 shows that unphosphorylated HMG 14 was released from S1 nuclease-treated double-stranded DNA-cellulose at 200 mM NaCI/1 mM Tris-HC1 (pH 7.5), when a linear salt gradient of 50-600 mM NaC1 was used. Interestingly, unphosphorylated HMG 17 also eluted at an identical salt concentration (data not shown). No significant differences between the binding of HMG 14 phosphorylated by NII-kinase or A-kinase to double-stranded DNA-cellulose were observed (Fig. 3). The phosphorylated proteins eluted only slightly ahead of the unphosphorylated species. Neither HMG 14 nor HMG 17 were retained by the cellulose matrix itself (data not shown). The affinity of phosphorylated HMG 14 for histone HI The elution profiles from histone H1-Sepharose of non-radioactive carrier HMG 14 and HMG 14 phosphorylated by NII-kinase or A-kinase are
The results of DNA-column chromatography shown here agree with previous findings [15,22], indicating that both HMG 14 and HMG 17 possess preferential affinity for single-stranded DNA. Both proteins are eluted from double-stranded DNAcellulose at 200 mM NaC1/1 mM Tris-HC1 (pH 7.5). Interestingly, HMG 17 is released from single-stranded DNA-column at a markedly lower salt concentration than HMG 14 (230 mM and 300 mM NaC1, respectively). It has been reported that HMG 14 and HMG 17 elute from phosphocellulose in reversed order compared to the results from our single-stranded DNA-column experiments [24]. The present results suggest that single-stranded DNA-column does not merely act as a simple ionic exchanger for low-Mr HMG proteins. It was somewhat surprising that HMG 14 and HMG 17, a pair of closely related HMG proteins both thought to be involved in similar functions in maintaining the conformation of active chromatin [6], exhibit a widely differential binding to histone H1. HMG 17 does not bind to histone H1-Sepharose in 20 mM NaC1/10 mM Tris-HCl (pH 7.5), whereas HMG 14 elutes as a sharp peak at 130 mM NaC1. These results confirm the results of Espel et al. [23] obtained using chemical cross-lin-
179
king. The same authors have also shown that, in addition to histone H1, HMG 14 interacts also with histones H2A and H2B, and that DNA seems to be involved in the attachment of HMG 14 to nucleosomes in reconstituted chromatin [25]. It is interesting that HMG 14 binds to histone H1, since both HMG 14 and histone H1 are basic proteins [26]. HMG 14 has, however, an asymmetrical charge distribution, since its carboxy-terminal half is predominantly negatively charged. It is thus possible that HMG 14 interacts through this region with histone H1. The carboxy-terminal region of HMG 17 is less negatively charged, but it is probably not the only determinant of the molecule responsible for its interaction with histone H1. N M R studies on the binding of calf thymus H M G 14 to DNA have shown that the DNAbinding region of HMG 14 resides between residues 17 and 60 [27]. The present results indicate that phosphorylation of Ser-6, which is located at the basic amino-terminal region of the molecule, by cAMP-dependent protein kinase clearly weakens the interaction between the DNA-binding amino-acid residues of HMG 14 and the negatively charged phosphate groups of DNA. Phosphorylation of Ser-89 at the acidic carboxylterminal region of the molecule by cyclic nucleotide-independent nuclear protein kinase II [8,13] has only a weak effect on its binding to singlestranded DNA. In contrast to binding to single-stranded DNA, no clear differences between binding to doublestranded DNA of HMG 14 phosphorylated by A or NII-kinases were observed. The acidic carboxy-terminal region of HMG 14 can presumably interact with other chromosomal proteins. The results of experiments with histone HI-columns confirm this, since phosphorylation of Ser-6 in HMG 14 was without effect on its binding to histone H1, whereas phosphorylation of Ser-89 had a strong effect. Thus, addition of a negative charge by phosphorylation with the NIIkinase enhanced the interaction between the carboxy-terminal region of HMG 14 and histone H1. The relatively high NaC1 concentration (200 mM) required to dissociate the phosphorylated H M G 14 from histone H1 suggests that this modification could be physiologically important. Phos-
phorylation of HMG 14 (or HMG 14-like proteins) by casein kinase II, which is similar to NII-kinase, has been observed in vivo [8,14]. The phosphorylation of HMG 14c~2 increases during mitosis when gene transcription is suppressed and the chromosomes are condensed [14]. It is possible that the modification of HMG 14 by NII-kinase affects the activation/inactivation-state of chromatin by enhancing the binding of HMG 14 to histone H1. Taken together, these results suggest that phosphorylation of HMG 14 at different sites by various protein kinases activated through distinct hormonal or cell cycle-dependent signals occurs and may be functionally important.
Acknowledgements This work was supported in part by grants from the Sigrid Jus61ius Foundation, Finland, and the Research Council for Natural Sciences, the Academy of Finland, We thank Ms. Hanna Eskelinen for skilful technical assistance.
References 1 Einck, L. and Bustin, M. (1985) Exp. Cell Res. 156, 295-310. 2 Goodwin, G.H., Nicolas, R.H., Wright, C.A. and Zavou, S. (1985) in Chromosomal Proteins and Gene Expression (Reeck, G.R., Goodwin, G.H. and Puigdom~nech, P., eds.), pp. 221-238, Plenum Press, New York. 3 Abercrombie, B.D., Kneale, G.G., Crane-Robinson, C., Bradbury, E.M., Goodwin, G.H., Walker, J.M. and Johns, E.W. (1978) Eur. J. Biochem. 84, 173-177. 4 Gary, P.D., King, D.S., Crane-Robinson, C., Bradbury, E.M., Rabbani, A., Goodwin, G.H. and Johns, E.W. (1980) Eur. J. Biochem. 112, 577-588. 5 Bradbury, E.M. (1983) Ciba Found. Symp.~93, 246-266. 6 Weisbrod, S. (1982) Nature (Lond.) 297, 289-295. 7 Laland, S.G., Lung, T. and Holtlund, J. (1985) in Chromosomal Proteins and Gene Expression (Reeck, G.R., Goodwin, G.H. and Puigdom~nech, P., eds.), pp. 239-247, Plenum Press, New York. 8 Palvimo, J., Pohjanpelto, P., Linnala-Kankkunen, A. and Maenpaa, P.H. (1986) Biochem. Biophys. Res. Commun. 134, 617-623. 9 Palvimo, J., Pohjanpelto, P., Linnala-Kankkunen, A. and Maenpaa, P.H. (1987) Biochim. Biophys. Acta 909, 21-29. 10 Walton, G.M., Spiess, J. and Gill, G.N. (1982) J. Biol. Chem. 257, 4661-4668. 11 Palvimo, J., Linnala-Kankkunen, A. and M~ienp~i~i, P.H. (1983) Biochem. Biophys. Res. Commun. 110, 378-382. 12 Walton, G.M., Gill, G.N., Cooper, E. and Spaulding, S.W. (1983) J. Biol. Chem. 259, 601-607.
180 13 Walton, G.M., Spiess, J. and Gill, G.N. (1985) J. Biol. Chem. 260, 4745-4750. 14 Walton, G.M. and Gill, G.N. (1983) J. Biol. Chem. 258, 4440-4446. 15 Palvimo, J., Linnala-Kankkunen, A. and M~ienp~i~i, P.H. (1985) Biochem. Biophys. Res. Commun. 133, 343-346. 16 Goodwin, G.H., Rabbani, A., Nicolas, R.H. and Johns, E.W. (1977) FEBS Lett. 80, 413-416. 17 Johns, E.W. (1976) in Subnuclear Components (Birnie, G.D., ed.), pp. 187-208, Butterworth, London. 18 Chen, P.S., Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. 19 Reimann, E.M. and Beham, R.A. (1983) Methods Enzymol. 99, 51-55. 20 Inoue, A., Tei, Y., Qi, S., Higashi, Y., Yukioka, M. and Morisawa, S. (1984) Biochem. Biophys. Res. Commun. 123, 398-403.
21 Isackson, P.J., Fishback, J.L., Bidney, D.L. and Reeck, G.R. (1979) J. Biol. Chem. 254, 5569-5572. 22 Isackson, P.J. and Reeck, G.R. (1981) Nucl. Acids Res. 9, 3779-3791. 23 Espel, E., Bernues, J., Querol, E., Martinez, P., Barris, A. and Lloberas, J. (1983) Biochem. Biophys. Res. Commun. 117, 817-822. 24 Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337-342. 25 Espel, E., Bernues, J., Perez-Pons, J.A. and Querol, E. (1985) Biochem. Biophys. Res. Commun. 132, 1031 1037. 26 Nicolas, R.H. and Goodwin, G.M. (1982) in The HMG Chromosomal Proteins (Johns, E.W., ed.), pp. 41-68, Academic Press, London. 27 Walker, J.M. (1982) in The HMG Chromosomal Proteins (Johns, E.W., ed.), pp. 69-87, Academic Press, London.
Biochimica et Biophysica Acta 952 (1988) 172 180
Elsevier BBA 33048
Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation Jorma Palvimo and Pekka H. M~ienp~iii Department of Biochemistry, University of Kuopio, Kuopio (Finland)
(Received2 October 1987)
Key words: High mobilitygroup protein; Protein phosphorylation; Histone H1; DNA; (Calf thymus) We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobilitygroup proteins H M G 14 and H M G 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone HI. Without in vitro phosphorylation, H M G 14 and H M G 1 7 eluted from double-stranded DNA-columns at 200 mM NaCl. H M G 14 was released from single-stranded DNA-column at 300 mM NaCl and from HI-column at 130 mM NaCI, whereas the corresponding values for H M G 17 were 230 mM and 20 mM, respectively. Phosphorylation of H M G 14 and H M G 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCI, respectively) for single-stranded DNA, whereas H M G 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCi) ahead of the unpbosphorylated protein. H M G 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCI) with the unphospborylated protein. Interestingly, phosphorylation of H M G 14 by NII-kinase increased considerably its affinity for histone H I and the phospborylated protein eluted at 200 mM NaCl. Pbosphorylation of H M G 14 by A-kinase did not alter its interaction towards histone HI. These results indicate that modification of H M G 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H I , and that H M G 17 has much weaker affinity for single-stranded DNA and histone H I than H M G 14.
Introduction
The high-mobility-group (HMG) proteins are a family of well-characterized non-histone proteins extractable from nuclei and chromatin with 0.35 M NaC1 [1]. The low-molecular-weight H M G proteins H M G 14 and H M G 17 (100 and 89 aminoacid residues, respectively, for proteins from calf
Abbreviations: HMG, high-mobility-group; A-kinase, cAMPdependent protein kinase; NIl-kinase, nuclear protein kinase II. Correspondence: J. Palvimo, Department of Biochemistry, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland.
thymus) are characterized by asymmetrical charge distribution. Two-thirds of H M G 17 and half of H M G 14 are highly basic in their amino-terminal ends. In contrast, the carboxy-terminal regions have an overall negative charge [2]. The central region of H M G 17 contains six helix-destabilizing proline residues which may contribute to a lack of ordered tertiary structure observed in solution [3,4]. It seems that more ordered conformations of H M G 14 and H M G 17 are generated by binding of these proteins to chromatin [5]. The domain structures of these H M G proteins suggest that they participate in multiple interactions with chromatin components in their native state. H M G 14 and H M G 17 have been implicated in
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (Biomedical Division)
173 modifying the chromatin structure of transcriptionally active genes [6]. Several kinds of postsynthetic modifications of these HMG proteins, e.g., ADP-ribosylation and phosphorylation, have been described [7-9]. Post-translational modifications of chromosomal proteins are thought to play an important role in regulation of chromatin structure. HMG 14 from calf thymus can be phosphorylated by both cyclic nucleotide-dependent and -independent protein kinases. The major high-affinity site phosphorylated by cyclic nucleotide-dependent protein kinases is Ser-6 at the basic amino-terminal region of the molecule [10,11]. Thyrotropin stimulates phosphorylation of HMG 14 in vivo at the site catalyzed by cAMPdependent protein kinase in vitro [12]. In contrast, casein kinase II, which is similar to nuclear protein kinase II, phosphorylates HMG 14 predominantly at Ser-89 in the acidic carboxy-terminal region [8,13]. Walton and Gill [14] have shown that purified casein kinase II catalyzes phosphorylation of hHMG 14a2 at a site which is phosphorylated in vivo in HeLa cells. It seems probable that phosphorylation of these proteins affects their interactions with DNA and other chromatin components, e.g., histones. We have shown in a preliminary study that phosphorylation of H M G 14 by cGMP-dependent protein kinase decreases its affinity for single-stranded DNA [15]. Affinity chromatography was used in this study to examine the effects of phosphorylation of HMG 14 by A-kinase and NII-kinase on its binding properties towards DNA and histone H1. We also compared binding properties of unphosphorylated and phosphorylated HMG 17 with those of H M G 14. Materials and Methods
HMG 14 and HMG 17 were purified from calf thymus by chromatography on CM-Sephadex [16]. Histone H1 was purified from calf thymus using the method of Johns [17]. The endogenous, alkalilabile phosphate content of purified chromatin proteins was determined using the method of Chen et al. [18]. The purified H M G 14, HMG 17 and H1 contained 0.08, 0.03 and 0.20 mol of alkali-labile phosphate per mol of protein, respectively. The catalytic subunit of cAMP-dependent pro-
tein kinase was purified to apparent homogeneity from bovine heart according to Reiman et al. [19]. Nuclear protein kinase was purified from isolated rat liver chromatin. The 0.4 M NaCl-extract of chromatin was dialyzed, fractionated on DEAESephadex, dialyzed and chromatographed on a phosvitin-casein-Sepharose 4B affinity column [20]. Reaction buffers for phosphorylation reactions were as described in Ref. 8. DNA-agarose containing 0.66 mg calf thymus single-stranded DNA per ml agarose was purchased from Bethesda Research Laboratories. Double-stranded DNA-cellulose was from Sigma (1 mg calf thymus DNA per ml cellulose) or from Pharmacia (1.14 mg DNA per ml cellulose). Single-stranded DNA regions were removed from double-stranded DNA-columns by passing S1 nuclease through the columns according to Isackson et al. [21]. Sigmacell-cellulose was used for blank columns. Electrophoretically pure histone H1 was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to instructions provided by the manufacturer. Usually, 7 g (wet weight) of prewashed activated Sepharose was coupled with 100 mg of histone H1 which was dissolved in a sufficient volume of 0.4 M sodium carbonate buffer (pH 9.0) to provide a thick slurry upon the addition of the wet Sepharose. Coupling was accomplished by stirring the slurry overnight at 4°C. Substituted Sepharose was then washed with 100 ml of the coupling buffer and 1000 ml of 2 M NaCI/0.2 M Tris-HC1 (pH 8.0) in several aliquots during 1 h. After packing, the columns were washed overnight with 1 M NaC1/10 mM TrisHC1 (pH 7.5) before equilibrating with the starting buffer (0.02 M NaC1/10 mM Tris-HC1 (pH 7.5)). The resulting Sepharose contained 5 mg of covalently coupled histone H1 per ml Sepharose. The reactive groups in blank Sepharose 4B were blocked with Tris-HC1 buffer (0.2 M, pH 8.0). Acetic acid/urea or SDS-polyacrylamide gel electrophoresis and autoradiography were performed as described in Ref. 8. Results
Binding of phosphorylated HMG 14 to singlestranded DNA The effect of phosphorylation of HMG 14 by
174
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Fraction no. Fig. 1. Chromatography of HMG 14 phosphorylated by NII-kinase (A) or A-kinase (B) on single-stranded DNA-agarose columns. H M G 14 (0.1 mg) was phosphorylated by NlI-kinase (0.4 ~g) in the presence of 30 /~M [32p]ATP for 90 min at 3 0 ° C (A), or by A-kinase (1.4 t.tg) for 30 min (B). Free radioactivity was separated from phosphoprotein by gel filtration on Sephadex G-25 at 4 ° C (0.9×25 cm) with 0.2 M NaC1/1 mM Tris-HC1 (pH 7.5) (starting buffer). 0.4 mg of carrier H M G 14 was mixed with each phosphorylated sample, and they were applied to two parallel single-stranded DNA-agarose columns (containing 3.3 mg of single-stranded DNA), which were pre-equilibrated with the starting buffer. The gradient (100 ml for two columns) was made using a single gradient maker and introduced into two parallel columns by way of a three-way connector and a dual-channel peristaltic pump. The columns were first washed with 1 bed vol. of starting buffer and eluted at 4 ° C with 100 ml of a linear gradient of 0.2 to 0.6 M NaC1/1 mM Tris-HCl (pH 7.5). Effluents from the columns were collected simultaneously with a single fraction collector. Aliquots of indicated gradient fractions (1 ml) were electrophoresed in acetic acid/urea gels. Stained gels and autoradiograms of the gels are shown on the right. Protein was detected by measuring the absorbance at 220 nm (11), and radioactivity as Cerenkov radiation (D) from 1 ml fractions. The salt concentration in gradient fractions ( . . . . . . ) was determined by measuring the conductivity with a Philips PW 9501/01 conductivity meter.
175
NII-kinase or A-kinase on its affinity for singlestranded DNA was studied using affinity chromatography on single-stranded DNA-agarose. Without in vitro phosphorylation, HMG 14 eluted from the column at 300 mM NaC1 (Figs. 1 and 2),
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176
from the DNA-column at a slightly lower salt concentration (290 mM NaC1) (Fig. 1A). Because these in vitro phosphorylated molecules represent only a small portion of total HMG 14, their contribution to the A220 profile is negligible. When HMG 14 was phosphorylated by A-kinase to a stoichiometry of about 1 mol phosphate per mol
protein, the phosphorylated protein was visible as a peak in the absorbance profile cochromatographing with the radioactivity peak at 270 mM NaC1 (Fig. 1B). The peak corresponding to unphosphorylated HMG 14 eluted at 300 mM NaC1 as in Fig. 1A. When HMG 14 was phosphorylated by A-kinase using a lower concentration of en-
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Fig. 3. Chromatography of H M G 14 phosphorylated by NII-kinase (A) or A-kinase (B) on parallel double-stranded DNA-cellulose columns (4 ml). HMG 14 was phosphorylated by NIl-kinase or A-kinase as described in Fig. 1 and Fig. 2, respectively. 0.05 M NaC1/1 mM Tris-HCl (pH 7.5), was used as a starting buffer in this experiment.
177
zyme and a shorter reaction time, the phosphorylated protein was not visible as a peak in the A z20 profile (Fig. 2B), but the peak of radioactivity eluted at the same NaC1 concentration (270 mM) as shown in Fig. lB. The concentration was clearly
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178
molecules applied to the columns. The affinity of unphosphorylated HMG 17 for single-stranded DNA was compared to that of HMG 14 (Fig. 2). Interestingly, HMG 17 was released from the DNA-column at a considerably lower ionic strength (230 mM NaC1) than HMG 14. HMG 17 can be phosphorylated in vitro by A-kinase using longer reaction times and higher enzyme concentrations than those required for HMG 14. The stoichiometry of the reaction is low, being less than 10% of that achieved with HMG 14 under identical conditions. The phosphorylated HMG 17 eluted at the beginning of the salt gradient (Fig. 2A). Possible degradation of the HMG proteins during phosphorylation reaction and chromatography was examined by electrophoresis. The proteins were found to comigrate with the untreated proteins during chromatography, indicating that degradation did not take place.
shown in Fig. 4. Unphosphorylated HMG 14 eluted from the column at 130 mM NaC1/10 mM Tris-HC1 (pH 7.5). This agrees with the results of Espel et al. [23]. Using chemical cross-linking, they detected interaction between histone H1 and HMG 14, which was abolished above 120 mM NaC1/25 mM triethanolamine. Phosphorylation of HMG 14 by A-kinase was without effect on its binding to histone H1, but phosphorylation by NII-kinase increased its affinity for H1 considerably. The phosphorylated protein eluted at 200 mM NaC1 (Fig. 4A). HMG 14 was not retained by the blank Sepharose column (data not shown). HMG 17 did not bind to histone H1-Sepharose under conditions used in this study (20 mM NaC1) (data not shown). This indicates a profound difference between binding of HMG 14 and HMG 17 to histone H1. Discussion
Binding of phosphorylated HMG 14 to doublestranded DNA Isackson and Reeck [22] have reported previously that HMG 14 and HMG 17 from chicken erythrocytes fail to bind to double-stranded DNA-cellulose at 200 mM NaC1/1 mM Tris-HCl (pH 7.5), and that they bind preferentially to single-stranded DNA. Fig. 3 shows that unphosphorylated HMG 14 was released from S1 nuclease-treated double-stranded DNA-cellulose at 200 mM NaCI/1 mM Tris-HC1 (pH 7.5), when a linear salt gradient of 50-600 mM NaC1 was used. Interestingly, unphosphorylated HMG 17 also eluted at an identical salt concentration (data not shown). No significant differences between the binding of HMG 14 phosphorylated by NII-kinase or A-kinase to double-stranded DNA-cellulose were observed (Fig. 3). The phosphorylated proteins eluted only slightly ahead of the unphosphorylated species. Neither HMG 14 nor HMG 17 were retained by the cellulose matrix itself (data not shown). The affinity of phosphorylated HMG 14 for histone HI The elution profiles from histone H1-Sepharose of non-radioactive carrier HMG 14 and HMG 14 phosphorylated by NII-kinase or A-kinase are
The results of DNA-column chromatography shown here agree with previous findings [15,22], indicating that both HMG 14 and HMG 17 possess preferential affinity for single-stranded DNA. Both proteins are eluted from double-stranded DNAcellulose at 200 mM NaC1/1 mM Tris-HC1 (pH 7.5). Interestingly, HMG 17 is released from single-stranded DNA-column at a markedly lower salt concentration than HMG 14 (230 mM and 300 mM NaC1, respectively). It has been reported that HMG 14 and HMG 17 elute from phosphocellulose in reversed order compared to the results from our single-stranded DNA-column experiments [24]. The present results suggest that single-stranded DNA-column does not merely act as a simple ionic exchanger for low-Mr HMG proteins. It was somewhat surprising that HMG 14 and HMG 17, a pair of closely related HMG proteins both thought to be involved in similar functions in maintaining the conformation of active chromatin [6], exhibit a widely differential binding to histone H1. HMG 17 does not bind to histone H1-Sepharose in 20 mM NaC1/10 mM Tris-HCl (pH 7.5), whereas HMG 14 elutes as a sharp peak at 130 mM NaC1. These results confirm the results of Espel et al. [23] obtained using chemical cross-lin-
179
king. The same authors have also shown that, in addition to histone H1, HMG 14 interacts also with histones H2A and H2B, and that DNA seems to be involved in the attachment of HMG 14 to nucleosomes in reconstituted chromatin [25]. It is interesting that HMG 14 binds to histone H1, since both HMG 14 and histone H1 are basic proteins [26]. HMG 14 has, however, an asymmetrical charge distribution, since its carboxy-terminal half is predominantly negatively charged. It is thus possible that HMG 14 interacts through this region with histone H1. The carboxy-terminal region of HMG 17 is less negatively charged, but it is probably not the only determinant of the molecule responsible for its interaction with histone H1. N M R studies on the binding of calf thymus H M G 14 to DNA have shown that the DNAbinding region of HMG 14 resides between residues 17 and 60 [27]. The present results indicate that phosphorylation of Ser-6, which is located at the basic amino-terminal region of the molecule, by cAMP-dependent protein kinase clearly weakens the interaction between the DNA-binding amino-acid residues of HMG 14 and the negatively charged phosphate groups of DNA. Phosphorylation of Ser-89 at the acidic carboxylterminal region of the molecule by cyclic nucleotide-independent nuclear protein kinase II [8,13] has only a weak effect on its binding to singlestranded DNA. In contrast to binding to single-stranded DNA, no clear differences between binding to doublestranded DNA of HMG 14 phosphorylated by A or NII-kinases were observed. The acidic carboxy-terminal region of HMG 14 can presumably interact with other chromosomal proteins. The results of experiments with histone HI-columns confirm this, since phosphorylation of Ser-6 in HMG 14 was without effect on its binding to histone H1, whereas phosphorylation of Ser-89 had a strong effect. Thus, addition of a negative charge by phosphorylation with the NIIkinase enhanced the interaction between the carboxy-terminal region of HMG 14 and histone H1. The relatively high NaC1 concentration (200 mM) required to dissociate the phosphorylated H M G 14 from histone H1 suggests that this modification could be physiologically important. Phos-
phorylation of HMG 14 (or HMG 14-like proteins) by casein kinase II, which is similar to NII-kinase, has been observed in vivo [8,14]. The phosphorylation of HMG 14c~2 increases during mitosis when gene transcription is suppressed and the chromosomes are condensed [14]. It is possible that the modification of HMG 14 by NII-kinase affects the activation/inactivation-state of chromatin by enhancing the binding of HMG 14 to histone H1. Taken together, these results suggest that phosphorylation of HMG 14 at different sites by various protein kinases activated through distinct hormonal or cell cycle-dependent signals occurs and may be functionally important.
Acknowledgements This work was supported in part by grants from the Sigrid Jus61ius Foundation, Finland, and the Research Council for Natural Sciences, the Academy of Finland, We thank Ms. Hanna Eskelinen for skilful technical assistance.
References 1 Einck, L. and Bustin, M. (1985) Exp. Cell Res. 156, 295-310. 2 Goodwin, G.H., Nicolas, R.H., Wright, C.A. and Zavou, S. (1985) in Chromosomal Proteins and Gene Expression (Reeck, G.R., Goodwin, G.H. and Puigdom~nech, P., eds.), pp. 221-238, Plenum Press, New York. 3 Abercrombie, B.D., Kneale, G.G., Crane-Robinson, C., Bradbury, E.M., Goodwin, G.H., Walker, J.M. and Johns, E.W. (1978) Eur. J. Biochem. 84, 173-177. 4 Gary, P.D., King, D.S., Crane-Robinson, C., Bradbury, E.M., Rabbani, A., Goodwin, G.H. and Johns, E.W. (1980) Eur. J. Biochem. 112, 577-588. 5 Bradbury, E.M. (1983) Ciba Found. Symp.~93, 246-266. 6 Weisbrod, S. (1982) Nature (Lond.) 297, 289-295. 7 Laland, S.G., Lung, T. and Holtlund, J. (1985) in Chromosomal Proteins and Gene Expression (Reeck, G.R., Goodwin, G.H. and Puigdom~nech, P., eds.), pp. 239-247, Plenum Press, New York. 8 Palvimo, J., Pohjanpelto, P., Linnala-Kankkunen, A. and Maenpaa, P.H. (1986) Biochem. Biophys. Res. Commun. 134, 617-623. 9 Palvimo, J., Pohjanpelto, P., Linnala-Kankkunen, A. and Maenpaa, P.H. (1987) Biochim. Biophys. Acta 909, 21-29. 10 Walton, G.M., Spiess, J. and Gill, G.N. (1982) J. Biol. Chem. 257, 4661-4668. 11 Palvimo, J., Linnala-Kankkunen, A. and M~ienp~i~i, P.H. (1983) Biochem. Biophys. Res. Commun. 110, 378-382. 12 Walton, G.M., Gill, G.N., Cooper, E. and Spaulding, S.W. (1983) J. Biol. Chem. 259, 601-607.
180 13 Walton, G.M., Spiess, J. and Gill, G.N. (1985) J. Biol. Chem. 260, 4745-4750. 14 Walton, G.M. and Gill, G.N. (1983) J. Biol. Chem. 258, 4440-4446. 15 Palvimo, J., Linnala-Kankkunen, A. and M~ienp~i~i, P.H. (1985) Biochem. Biophys. Res. Commun. 133, 343-346. 16 Goodwin, G.H., Rabbani, A., Nicolas, R.H. and Johns, E.W. (1977) FEBS Lett. 80, 413-416. 17 Johns, E.W. (1976) in Subnuclear Components (Birnie, G.D., ed.), pp. 187-208, Butterworth, London. 18 Chen, P.S., Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. 19 Reimann, E.M. and Beham, R.A. (1983) Methods Enzymol. 99, 51-55. 20 Inoue, A., Tei, Y., Qi, S., Higashi, Y., Yukioka, M. and Morisawa, S. (1984) Biochem. Biophys. Res. Commun. 123, 398-403.
21 Isackson, P.J., Fishback, J.L., Bidney, D.L. and Reeck, G.R. (1979) J. Biol. Chem. 254, 5569-5572. 22 Isackson, P.J. and Reeck, G.R. (1981) Nucl. Acids Res. 9, 3779-3791. 23 Espel, E., Bernues, J., Querol, E., Martinez, P., Barris, A. and Lloberas, J. (1983) Biochem. Biophys. Res. Commun. 117, 817-822. 24 Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337-342. 25 Espel, E., Bernues, J., Perez-Pons, J.A. and Querol, E. (1985) Biochem. Biophys. Res. Commun. 132, 1031 1037. 26 Nicolas, R.H. and Goodwin, G.M. (1982) in The HMG Chromosomal Proteins (Johns, E.W., ed.), pp. 41-68, Academic Press, London. 27 Walker, J.M. (1982) in The HMG Chromosomal Proteins (Johns, E.W., ed.), pp. 69-87, Academic Press, London.