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Importance of substrate conformation in the phosphorylation of chromatin-associated proteins by exogenous protein kinase C
Cellular Signalling Vol. 4 No. 2, pp. 145-151, 1992.
0898-6568/92 $5.00+ .00 © 1992 Pergamon Press pie
Printed in Great Britain,
IMPORTANCE OF SUBSTRATE CONFORMATION IN THE PHOSPHORYLATION OF CHROMATIN-ASSOCIATED PROTEINS BY EXOGENOUS PROTEIN KINASE C ALESSANDROTESTORI, LEIGH A. BURGOYNEand ANDREW W. MURRAY* School of Biological Sciences, The Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia
(Received 20 October 1991; and accepted 29 October 1991)
Abstract--Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytesfrom primary cultures exhibited PKC-dependent phosphorylation of chromatin-associatedproteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsivenesswas particularly obvious in the decondensedchromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC. Key words: Protein kinase C, phosphorylation, chromatin.
(PKC), a lipid-regulated family of enzymes known to be involved in numerous and diverse cellular processes [4]. PKC is present in the nuclei of cells [5-8] and has been shown to phosphorylate a number of nuclear proteins in vitro, including transcription factors [9] and chromatin-associated enzymes such as topoisomerases I and II [10-13]. These facts suggest that PKC might represent a key regulator of nuclear processes including the modulation of chromatin conformation and gene transcription. This paper examines the major factors which modulate the phosphorylation of nuclear proteins by exogenously added protein kinase C in nuclei isolated from a variety of cell types. In these studies chromatin, prepared in the well characterized conditions of 'Buffer-A' in which DNA integrity and chromatin structure is known to be well maintained [14-20], was expanded and contracted by manipulating the low levels of polyamines normally used in this buffer system. The results show that chromatin-associated proteins are only phosphorylated by exogenous PKC when the
INTRODUCTION PROTEIN phosphorylation is a major mechanism for modulating cellular processes, particularly those involving the transduction of signals through the interior of the cell [l]. The phosphorylation of nuclear proteins has long been suspected to be one of the processes driving changes in nuclear conformation during the cell cycle [1,2]. However, the great complexity of the phosphorylation profiles of nuclei and their extreme sensitivity to almost any change in the nuclear environment has created a need for standardized preparation procedures and a need to determine the major variables which effect nuclear phosphorylation. One variable appears to be the degree of chromatin condensation [3]. Another obvious, major variable is the complement of nuclear protein kinases and a class that has received considerable attention is protein kinases C
*Author to whom correspondenceshould be addressed. 145
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c h r o m a t i n is in an e x p a n d e d state a n d that nuclei f r o m different cell types differ greatly in their responsiveness to exogenous P K C . MATERIALS AND METHODS PKC purification Rat brain PKC was purified by adsorption to and elution from inside-out red cell vesicles [21] and was further purified by DE-52 cellulose chromatography [22]. Peak fractions of Ca2+/phosphatidylserinedependent activity were pooled and stored at 4°C. The purity of these preparations has been described in a previous paper [23]. The major PKC isozymes PKC-ct, PKC-fl and PKC-7 have all been found in these preparations [24], although their relative proportions have not been established. Ce//s HL-60 cells were grown in RPMI 1640 media, Swiss 3T3 and HeLa cells were grown in Dulbecco's modification of Eagles' medium (DMEM) media and primary lymphocytes were grown in McCoy's 311 medium. Cells were harvested in late log phase and suspended in PBS (phosphate-buffered saline: 0.87% NaCI, 10 mM Na2HPO4, pH 7.5). Nuclear preparations All buffers used in the preparation of nuclei were based on the basic salts/mercaptoethanol of buffer-A [14, 15] and were essentially modifications of this buffer. Their final pH was always 7.5. For convenience the base solution (15mM (0.1%) 2-mercaptoethanol, 15 mM NaOH, 60 mM KCI) is herein referred to as 'buffer A'. All washes were 1 ml for 5 min at 2°C. PBS-washed cells (19mg wet weight of pellet) were washed once in A-HEPES-polyamines buffer (buffer A containing 15mM HEPES, 0.15mM spermine and 0.5mM spermidine) with 0.1 mM EDTA, 0.05 mM EGTA, 4 m M MgCI 2, 0.1% bovine serum albumin and 40/~g/ml phenyi methyl sulphonyl fluoride (PMSF). The cells were then stripped of their membranes by washing once with 0.05% Triton X-100 in the above solution but with 0.5% bovine serum albumin and in the absence of PMSF. The samples were then washed into the appropriate ionic environment (see below).
A-HEPES-polyamines with the addition of 0.1% bovine serum albumin and were then resuspended in 120#1 of A-HEPES-polyamines, 0.1 mM EDTA. For environment L (low polyamines), nuclei were washed once in A-phosphate (buffer A containing 75 mM NaH2PO4, 0.1% bovine serum albumin) and once in A - H E P E S (buffer A containing 15 mM HEPES, 0.1 mM EDTA) and were finally resuspended in 120#l of A - H E P E S buffer. This wash-extraction of the multivalent cations causes the nuclei to decondense so that they become intractable gels. DNAase-I (Boehringer Mannheim) and micrococcal nuclease (Pharmacia) were used as structural disturbing agents in some experiments. When DNAase-I was used, 5 mM MgCl 2 and 2 #g of the nuclease were added and the tubes were incubated at 37°C for 30 min. When micrococcal nuclease was used, 3 mM CaCl 2 and 10 units of the nuclease were added and the tubes were incubated at 37°C for 30 min. Phosphorylation assays In a typical reaction an aliquot of nuclei or decondensed nuclear fraction (approximately 40 #g protein content as estimated by a modification of the method of Lowry [25]) was incubated in 60 #1 of the buffer appropriate to its ionic environment, plus 5 mM MgC12 and 40 nM of 7-[32p]-ATP (4000Ci/mmol), 40#g/ml phosphatidylserine and 2.5 mM CaC12. PKC (75 ng protein in 5 #1 of 20 mM Tris, pH 7.5, 2 mM EDTA, 5 mM EGTA) or buffer was added and incubations carried out at 30°C for 3 min. Calcium independent activity was assayed in incubations containing 5 mM EGTA and no calcium or phosphatidylserine. Reactions were stopped by the addition of 20 #1 of a solution containing 10% glycerol, 2% SDS, 62.5 mM Tris-HC1 pH 6.8, 5% 2-mercaptoethanol, 0.00125% (w/v) Bromophenoi Blue, followed by incubation for 3 min in a boiling water bath. The lysate was incubated at room temperature for 30 min with 2/~g DNAase-I and 1 #g RNAase. A. Despite the presence of SDS, the residual activity of the nucleases was enough to greatly reduce the vicosity of samples. Aliquots containing ca. 13 pg of protein were then analysed on polyacrylamide gels. Electrophoresis was performed following the method of Laemmli [26], using 12% SDS-acrylamide gels.
Experimental conditions for structural comparisons Two ionic environments were used, generating two levels of nuclear condensation. These differed with respect to polyamine concentration; environment H (high polyamines) consisted of nuclei washed twice in
RESULTS C h r o m a t i n p r e p a r a t i o n s , differing in their level o f c o n d e n s a t i o n , were p h o s p h o r y l a t e d
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FIG. 1. Phosphorylation of chromatin-associated proteins in chromatin prepared from primary lymphocytes by exogenously added PKC. Lanes 1-4: Expanded chromatin (L conditions). Lanes 5-8: Condensed chromatin (H conditions). Lanes 1 and 5: phosphorylation by endogenous enzymes in the presence of 2.5 mM Ca ~+, 40/~g/ml phosphatidylserine. No exogenous PKC was added. Lanes 2 and 6: phosphorylation under the same conditions in the presence of exogenously added PKC (25 ng protein). Lanes 3 and 7: phosphorylation by the endogenous enzymes in the presence of 5 mM EGTA and no calcium or phosphatidyiserine. Lanes 4 and 8: pbosphorylation in the presence of exogenously added PKC (25 ng protein), 5 mM EGTA and no calcium or phosphatidylserine. All phosphorylation reactions were for 3 min at 30°C and used y-[32p]-ATP as a source of phosphate. Each lane contained approximately 13/~g protein from chromatin preparations. Lane 9 : 5 ng of PKC radioactivity labelled by autophosphorylation. FIG. 2. Phosphorylation of chromatin prepared from 3T3 cells by exogenously added PKC. Chromatin (11/~g protein) was induced to expand by removing the polyamine stabilization (refer to text). Lane h endogenous phosphorylation in the presence of 2.5 mM Ca 2+, 40/~g/ml phosphatidylserine (control). Lane 2: the same reaction but with the addition of purified PKC (25 ng protein). Lane 3: control with 5 mM EGTA and no calcium or phosphatidylserine. Lane 4: addition of PKC (25 ng protein) with 5 mM EGTA and no calcium or phosphatidylserine.
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FIG. 3. Modulation of chromatin phosphorylation patterns by nucleases. Expanded chromatin (see Materials and Methods) was used in these experiments. Lanes l-4: endogenous chromatin phosphorylation (14 pg protein) in the presence of 3 mM Ca*+, 40 pg/ml phosphatidylserine, the same reaction but with the addition of 25 ng PKC, endogenous phosphorylation in the presence of 5 mM EGTA and no calcium or phosphatidylserine, addition of 25 ng PKC in the presence of 5 mM EGTA and no calcium or phosphatidylserine. Lanes 5-8: the above sequence of incubations carried out using nuclei pre-treated with micrococcal nuclease. Lanes 9-12: the above sequence of incubations carried out using nuclei pre-treated with DNAase I. Lane 13: histone Hl phosphorylated with PKC.
148
Phosphorylation of chromatin-associated proteins in vitro by exogenously added protein kinase C utilizing ~-[32p]-ATP as a source of phosphate. Proteins in nuclei prepared from 3T3 fibroblasts or primary lymphocytes were phosphorylated by PKC and the extent of phosphorylation and the patterns observed were dependent on the condensational state of the nuclei (Figs 1 and 2). Stable, condensed nuclei (environment H) from primary lymphocytes exhibited only a low endogenous level of protein phosphorylation and little additional phosphorylation was induced by exogenous PKC. Expansion of the primary lymphocyte nuclei was forced by reducing the polyamine concentration [3] and was clearly observable as an increase in the viscosity of the samples (environment L). This had little effect on the endogenous phosphorylation of these nuclei but greatly enhanced phosphorylation of many nuclear proteins when exogenous PKC was added (Fig. 1, lane 2). Condensed 3T3 nuclei were not phosphorylated by exogenous PKC (not shown). However, following expansion of the 3T3 nuclei, protein phosphorylation by the endogenous kinases increased sharply and a few protein species were also phosphorylated in response to PKC, in a Ca2+/phospholipid dependent fashion (Fig. 2). Proteins in nuclei prepared from HL-60 and HeLa cells were not phosphorylated by the addition of PKC under any of the preparation conditions used (data not shown). However, histones added to these preparations were phosphorylated by the exogenous PKC in a calcium/phospholipiddependent manner (data not shown). Thus the failure of PKC to phosphorylate chromatinassociated proteins in these particular cell types was not due to inactivation of the kinase. The predominantly nuclear localization of the proteins phosphorylated in primary lymphocyte nuclei by exogenously added PKC was deduced from the effects of DNAse-I and micrococcal nuclease treatment. Nuclease digestion of nuclei prior to their phosphorylation by the endogenous enzymes, produced completely different protein phosphorylation patterns (Fig. 3). These results indicate that the phosphorylations which were observed are
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most likely to be linked to DNAase-sensitive structures. DISCUSSION It is clear from these data that expansion of chromatin, induced by lowering the polyamine concentration, dramatically increased the phosphorylation of chromatin proteins by exogenous PKC. A trivial explanation is that polyamines have a strong inhibitory effect on PKC activity [27]. However, this is not likely to be an explanation of the present results, as PKC activity in vitro, using H1 histone as a substrate, was unaffected by the concentration of polyamines used in these studies (data not shown). In addition, it was observed that nuclease treatment of the nuclei prior to their phosphorylation, dramatically altered the response of chromatin proteins to PKC, without any change in the polyamine concentration. We therefore propose that the eondensational state of the chromatin is central in determining the phosphorylation of chromatinassociated proteins by exogenous PKC. This is the first in vitro study of exogenous PKC-mediated phosphorylation of chromatinassociated proteins conducted under conditions in which the integrity and the structure of chromatin have been maintained. Previous studies have focused mainly on in vitro phosphorylation by PKC of individually purified nuclear proteins [9-13] or on phorbol esterinduced phosphorylation of proteins that copurify with the nuclear fraction [28, 29] and of other, better characterized species such as histones [30, 31], high mobility group proteins [32, 33] and nucleosomes [33]. It is interesting that intact HL-60 cells do not exhibit phorbol ester-induced phosphorylation of nuclear proteins [28]. By contrast, phorbol ester treatment of 3T3 cells induces phosphorylation of several nuclear proteins [28]. Thus these in vivo results [28] are consistent with the in vitro data presented in this paper. The fact that different cell lines exhibit different responses to exogenous PKC is intriguing. It is unlikely to be due to changes in
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the substrate proteins as, at least some of these seem to be the highly conserved and essential proteins of the nucleus. One plausible explanation is that HL-60 and HeLa cells possess a higher level of endogenous PKC activity which maintains PKC substrates in a highly phosphorylated state, as proposed by other investigators [28]. Detailed examination of the effects of the nucleases allows two useful logical eliminations to be made. The first relates to the question of cytoplasmic contaminations and the second relates to the possibility of the nucleases actually acting via the alteration of free polyamine levels. Cytoplasmic contamination is always a problem with relatively 'native' chromatin or nuclear preparations [34]. However, the two nucleases employed extensively modulated the phosphorylation patterns, indicating that the phosphorylation targets were truly DNA-associated at the time of phosphorylation. Moreover, as the modulations caused by the two nucleases were quite unlike each other in detail, it is most unlikely that the nuclease effects were actually due to slight increases in the free polyamine levels following D N A destruction.
REFERENCES 1. Hunter T. (1987) Cell 50, 823-829. 2. Dunphy W. G. and Newport J. W. (1988) Cell 55, 925-928. 3. Testori A., Skinner J. D., Murray A. W. and Burgoyne L. A. (1991) Biochem. biophys. Res. Commun. 180, 329-333. 4. Nishizuka Y. (1986) Science 233, 305-312. 5. Hagiwara M., Uchida C., Usuda N., Nagata T. and Hidaka H. (1990) Biochem. biophys. Res. Commun. 168, 161-168. 6. Capitani S., Girard P. R., Mazzei G. J., Kuo J.F., Berezney R. and Manzoli F.A. (1987) Biochem. biophys. Res. Commun. 142, 367-375. 7. Masmoudi A., Labourdette G., Mersel M., Huang F. L., Huang K.-P., Vincendon G. and Malviya A.N. (1989) J. biol. Chem. 264, 1172-1179. 8. Rogue P., Labourdette G., Masmoudi A., Yoshida Y., Huang F. L., Huang K.-P., Zwiller J., Vincendon G. and Malviya A.N. (1990) J. biol. Chem. 265, 4161-4165.
9. Yamamoto K. K., Gonzalez G. A., Biggs III W. H. and Montminy M. R. (1988) Nature 334, 494-499. 10. Samuels D. S., Shimizu Y. and Shimizu N. (1989) Fedn Eur. biochem. Socs Lett. 259, 57-60. 11. Pommier Y., Kerrigan D., Hartman K. D. and Glazer R.I. (1990) J. biol. Chem. 265, 9418-9422. 12. Sahyoun N., Wolf M., Besterman J., Hsieh T.-S., Sander M., Levine III H., Chang K.-J. and Cuatrecasas P. (1986) Proc. natn. Acad. Sci. U.S.A. 83, 1603-1607. 13. Rottmann M., Schroder H. C., Gramzow M., Renneisen K., Kurelec B., Dorn A., Friese U. and Muller W. E. G. (1987) Eur. molec. Biol. Org. J. 6, 3939-3944. 14. Burgoyne L. A., Waqar M. A. and Atkinson M. R. (1970) Biochem. biophys. Res. Commun. 39, 254-259. 15. Hewish D. R. and Burgoyne L. A. (1973) Biochem. biophys. Res. Commun. 52, 504-510. 16. Sedat J. W. and Manuelidis U (1978) Cold Spring Harbor Syrup. Quant. Biol. 42, 331-350. 17. Filipski J., Leblanc J., Youdale T., Sikorska M. and Walker P. R. (1990) Eur. molec. Biol. Org. J. 9, 1319-1327. 18. Boulikas T. (1990) J. biol. Chem. 265, 4638-4647. 19. Belmont A. S., Braunfeld M. B., Sedat J. W. and Agard D. A. (1989) Chromosoma 98, 129-143. 20. Paddy M. R., Belmont A. S., Saumweber H., Agard D. A. and Sedat J. W. (1990) Cell 62, 89-106. 21. Wolf M., Cuatrecasas P. and Sayhoun N. (1985) J. biol. Chem. 260, 15,718-15,722. 22. Tapley P. M. and Murray A. W. (1984) Biochem. biophys. Res. Commun. 118, 835-841. 23. Testori A., Hii C. S. T., Fournier A., Burgoyne L.A. and Murray A.W. (1988) Biochem. biophys. Res. Commun. 156, 222-227. 24. Clark K. J., Isaac E. L. and Murray A.W. (1991) Biochem. Int. 23, 243-247. 25. Harrington C. R. (1990) Anal. Biochem. 186, 285-287. 26. Laemmli U. (1970) Nature 227, 680-685. 27. Mezzetti G., Monti M. G. and Moruzzi M. S. (1988) Life Sci. 42, 2293-2298. 28. Thomas T. P., Talwar H. S. and Anderson W. B. (1988) Cancer Res. 48, 1910-1919. 29. Macfarlane D. E. (1986) J. biol. Chem. 261, 6947-6953. 30. Butler A. P., Byus C. V. and Slaga T. J. (1986) J. biol. Chem. 261, 9421-9425. 31. Patskan G. J. and Baxter C. S, (1985) J. biol. Chem. 260, 12,899-12,903. 32. Kimura K., Katoh N., Sakurada K. and Kubo S. (1985) Biochem. J. 227, 271-276.
Phosphorylation of chromatin-associatedproteins 33. Ramachandran C., Yau P., Bradbury E.M., Shyamala G., Yasuda H. and Walsh D.A. (1984) J. biolo Chem. 259, 13,495-13,503.
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34. Van Holde K. E. (1988) in Chromatin (Rich A., Ed.). Springer Vedag, New York, 181-218.
0898-6568/92 $5.00+ .00 © 1992 Pergamon Press pie
Printed in Great Britain,
IMPORTANCE OF SUBSTRATE CONFORMATION IN THE PHOSPHORYLATION OF CHROMATIN-ASSOCIATED PROTEINS BY EXOGENOUS PROTEIN KINASE C ALESSANDROTESTORI, LEIGH A. BURGOYNEand ANDREW W. MURRAY* School of Biological Sciences, The Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia
(Received 20 October 1991; and accepted 29 October 1991)
Abstract--Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytesfrom primary cultures exhibited PKC-dependent phosphorylation of chromatin-associatedproteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsivenesswas particularly obvious in the decondensedchromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC. Key words: Protein kinase C, phosphorylation, chromatin.
(PKC), a lipid-regulated family of enzymes known to be involved in numerous and diverse cellular processes [4]. PKC is present in the nuclei of cells [5-8] and has been shown to phosphorylate a number of nuclear proteins in vitro, including transcription factors [9] and chromatin-associated enzymes such as topoisomerases I and II [10-13]. These facts suggest that PKC might represent a key regulator of nuclear processes including the modulation of chromatin conformation and gene transcription. This paper examines the major factors which modulate the phosphorylation of nuclear proteins by exogenously added protein kinase C in nuclei isolated from a variety of cell types. In these studies chromatin, prepared in the well characterized conditions of 'Buffer-A' in which DNA integrity and chromatin structure is known to be well maintained [14-20], was expanded and contracted by manipulating the low levels of polyamines normally used in this buffer system. The results show that chromatin-associated proteins are only phosphorylated by exogenous PKC when the
INTRODUCTION PROTEIN phosphorylation is a major mechanism for modulating cellular processes, particularly those involving the transduction of signals through the interior of the cell [l]. The phosphorylation of nuclear proteins has long been suspected to be one of the processes driving changes in nuclear conformation during the cell cycle [1,2]. However, the great complexity of the phosphorylation profiles of nuclei and their extreme sensitivity to almost any change in the nuclear environment has created a need for standardized preparation procedures and a need to determine the major variables which effect nuclear phosphorylation. One variable appears to be the degree of chromatin condensation [3]. Another obvious, major variable is the complement of nuclear protein kinases and a class that has received considerable attention is protein kinases C
*Author to whom correspondenceshould be addressed. 145
146
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c h r o m a t i n is in an e x p a n d e d state a n d that nuclei f r o m different cell types differ greatly in their responsiveness to exogenous P K C . MATERIALS AND METHODS PKC purification Rat brain PKC was purified by adsorption to and elution from inside-out red cell vesicles [21] and was further purified by DE-52 cellulose chromatography [22]. Peak fractions of Ca2+/phosphatidylserinedependent activity were pooled and stored at 4°C. The purity of these preparations has been described in a previous paper [23]. The major PKC isozymes PKC-ct, PKC-fl and PKC-7 have all been found in these preparations [24], although their relative proportions have not been established. Ce//s HL-60 cells were grown in RPMI 1640 media, Swiss 3T3 and HeLa cells were grown in Dulbecco's modification of Eagles' medium (DMEM) media and primary lymphocytes were grown in McCoy's 311 medium. Cells were harvested in late log phase and suspended in PBS (phosphate-buffered saline: 0.87% NaCI, 10 mM Na2HPO4, pH 7.5). Nuclear preparations All buffers used in the preparation of nuclei were based on the basic salts/mercaptoethanol of buffer-A [14, 15] and were essentially modifications of this buffer. Their final pH was always 7.5. For convenience the base solution (15mM (0.1%) 2-mercaptoethanol, 15 mM NaOH, 60 mM KCI) is herein referred to as 'buffer A'. All washes were 1 ml for 5 min at 2°C. PBS-washed cells (19mg wet weight of pellet) were washed once in A-HEPES-polyamines buffer (buffer A containing 15mM HEPES, 0.15mM spermine and 0.5mM spermidine) with 0.1 mM EDTA, 0.05 mM EGTA, 4 m M MgCI 2, 0.1% bovine serum albumin and 40/~g/ml phenyi methyl sulphonyl fluoride (PMSF). The cells were then stripped of their membranes by washing once with 0.05% Triton X-100 in the above solution but with 0.5% bovine serum albumin and in the absence of PMSF. The samples were then washed into the appropriate ionic environment (see below).
A-HEPES-polyamines with the addition of 0.1% bovine serum albumin and were then resuspended in 120#1 of A-HEPES-polyamines, 0.1 mM EDTA. For environment L (low polyamines), nuclei were washed once in A-phosphate (buffer A containing 75 mM NaH2PO4, 0.1% bovine serum albumin) and once in A - H E P E S (buffer A containing 15 mM HEPES, 0.1 mM EDTA) and were finally resuspended in 120#l of A - H E P E S buffer. This wash-extraction of the multivalent cations causes the nuclei to decondense so that they become intractable gels. DNAase-I (Boehringer Mannheim) and micrococcal nuclease (Pharmacia) were used as structural disturbing agents in some experiments. When DNAase-I was used, 5 mM MgCl 2 and 2 #g of the nuclease were added and the tubes were incubated at 37°C for 30 min. When micrococcal nuclease was used, 3 mM CaCl 2 and 10 units of the nuclease were added and the tubes were incubated at 37°C for 30 min. Phosphorylation assays In a typical reaction an aliquot of nuclei or decondensed nuclear fraction (approximately 40 #g protein content as estimated by a modification of the method of Lowry [25]) was incubated in 60 #1 of the buffer appropriate to its ionic environment, plus 5 mM MgC12 and 40 nM of 7-[32p]-ATP (4000Ci/mmol), 40#g/ml phosphatidylserine and 2.5 mM CaC12. PKC (75 ng protein in 5 #1 of 20 mM Tris, pH 7.5, 2 mM EDTA, 5 mM EGTA) or buffer was added and incubations carried out at 30°C for 3 min. Calcium independent activity was assayed in incubations containing 5 mM EGTA and no calcium or phosphatidylserine. Reactions were stopped by the addition of 20 #1 of a solution containing 10% glycerol, 2% SDS, 62.5 mM Tris-HC1 pH 6.8, 5% 2-mercaptoethanol, 0.00125% (w/v) Bromophenoi Blue, followed by incubation for 3 min in a boiling water bath. The lysate was incubated at room temperature for 30 min with 2/~g DNAase-I and 1 #g RNAase. A. Despite the presence of SDS, the residual activity of the nucleases was enough to greatly reduce the vicosity of samples. Aliquots containing ca. 13 pg of protein were then analysed on polyacrylamide gels. Electrophoresis was performed following the method of Laemmli [26], using 12% SDS-acrylamide gels.
Experimental conditions for structural comparisons Two ionic environments were used, generating two levels of nuclear condensation. These differed with respect to polyamine concentration; environment H (high polyamines) consisted of nuclei washed twice in
RESULTS C h r o m a t i n p r e p a r a t i o n s , differing in their level o f c o n d e n s a t i o n , were p h o s p h o r y l a t e d
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FIG. 1. Phosphorylation of chromatin-associated proteins in chromatin prepared from primary lymphocytes by exogenously added PKC. Lanes 1-4: Expanded chromatin (L conditions). Lanes 5-8: Condensed chromatin (H conditions). Lanes 1 and 5: phosphorylation by endogenous enzymes in the presence of 2.5 mM Ca ~+, 40/~g/ml phosphatidylserine. No exogenous PKC was added. Lanes 2 and 6: phosphorylation under the same conditions in the presence of exogenously added PKC (25 ng protein). Lanes 3 and 7: phosphorylation by the endogenous enzymes in the presence of 5 mM EGTA and no calcium or phosphatidyiserine. Lanes 4 and 8: pbosphorylation in the presence of exogenously added PKC (25 ng protein), 5 mM EGTA and no calcium or phosphatidylserine. All phosphorylation reactions were for 3 min at 30°C and used y-[32p]-ATP as a source of phosphate. Each lane contained approximately 13/~g protein from chromatin preparations. Lane 9 : 5 ng of PKC radioactivity labelled by autophosphorylation. FIG. 2. Phosphorylation of chromatin prepared from 3T3 cells by exogenously added PKC. Chromatin (11/~g protein) was induced to expand by removing the polyamine stabilization (refer to text). Lane h endogenous phosphorylation in the presence of 2.5 mM Ca 2+, 40/~g/ml phosphatidylserine (control). Lane 2: the same reaction but with the addition of purified PKC (25 ng protein). Lane 3: control with 5 mM EGTA and no calcium or phosphatidylserine. Lane 4: addition of PKC (25 ng protein) with 5 mM EGTA and no calcium or phosphatidylserine.
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FIG. 3. Modulation of chromatin phosphorylation patterns by nucleases. Expanded chromatin (see Materials and Methods) was used in these experiments. Lanes l-4: endogenous chromatin phosphorylation (14 pg protein) in the presence of 3 mM Ca*+, 40 pg/ml phosphatidylserine, the same reaction but with the addition of 25 ng PKC, endogenous phosphorylation in the presence of 5 mM EGTA and no calcium or phosphatidylserine, addition of 25 ng PKC in the presence of 5 mM EGTA and no calcium or phosphatidylserine. Lanes 5-8: the above sequence of incubations carried out using nuclei pre-treated with micrococcal nuclease. Lanes 9-12: the above sequence of incubations carried out using nuclei pre-treated with DNAase I. Lane 13: histone Hl phosphorylated with PKC.
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Phosphorylation of chromatin-associated proteins in vitro by exogenously added protein kinase C utilizing ~-[32p]-ATP as a source of phosphate. Proteins in nuclei prepared from 3T3 fibroblasts or primary lymphocytes were phosphorylated by PKC and the extent of phosphorylation and the patterns observed were dependent on the condensational state of the nuclei (Figs 1 and 2). Stable, condensed nuclei (environment H) from primary lymphocytes exhibited only a low endogenous level of protein phosphorylation and little additional phosphorylation was induced by exogenous PKC. Expansion of the primary lymphocyte nuclei was forced by reducing the polyamine concentration [3] and was clearly observable as an increase in the viscosity of the samples (environment L). This had little effect on the endogenous phosphorylation of these nuclei but greatly enhanced phosphorylation of many nuclear proteins when exogenous PKC was added (Fig. 1, lane 2). Condensed 3T3 nuclei were not phosphorylated by exogenous PKC (not shown). However, following expansion of the 3T3 nuclei, protein phosphorylation by the endogenous kinases increased sharply and a few protein species were also phosphorylated in response to PKC, in a Ca2+/phospholipid dependent fashion (Fig. 2). Proteins in nuclei prepared from HL-60 and HeLa cells were not phosphorylated by the addition of PKC under any of the preparation conditions used (data not shown). However, histones added to these preparations were phosphorylated by the exogenous PKC in a calcium/phospholipiddependent manner (data not shown). Thus the failure of PKC to phosphorylate chromatinassociated proteins in these particular cell types was not due to inactivation of the kinase. The predominantly nuclear localization of the proteins phosphorylated in primary lymphocyte nuclei by exogenously added PKC was deduced from the effects of DNAse-I and micrococcal nuclease treatment. Nuclease digestion of nuclei prior to their phosphorylation by the endogenous enzymes, produced completely different protein phosphorylation patterns (Fig. 3). These results indicate that the phosphorylations which were observed are
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most likely to be linked to DNAase-sensitive structures. DISCUSSION It is clear from these data that expansion of chromatin, induced by lowering the polyamine concentration, dramatically increased the phosphorylation of chromatin proteins by exogenous PKC. A trivial explanation is that polyamines have a strong inhibitory effect on PKC activity [27]. However, this is not likely to be an explanation of the present results, as PKC activity in vitro, using H1 histone as a substrate, was unaffected by the concentration of polyamines used in these studies (data not shown). In addition, it was observed that nuclease treatment of the nuclei prior to their phosphorylation, dramatically altered the response of chromatin proteins to PKC, without any change in the polyamine concentration. We therefore propose that the eondensational state of the chromatin is central in determining the phosphorylation of chromatinassociated proteins by exogenous PKC. This is the first in vitro study of exogenous PKC-mediated phosphorylation of chromatinassociated proteins conducted under conditions in which the integrity and the structure of chromatin have been maintained. Previous studies have focused mainly on in vitro phosphorylation by PKC of individually purified nuclear proteins [9-13] or on phorbol esterinduced phosphorylation of proteins that copurify with the nuclear fraction [28, 29] and of other, better characterized species such as histones [30, 31], high mobility group proteins [32, 33] and nucleosomes [33]. It is interesting that intact HL-60 cells do not exhibit phorbol ester-induced phosphorylation of nuclear proteins [28]. By contrast, phorbol ester treatment of 3T3 cells induces phosphorylation of several nuclear proteins [28]. Thus these in vivo results [28] are consistent with the in vitro data presented in this paper. The fact that different cell lines exhibit different responses to exogenous PKC is intriguing. It is unlikely to be due to changes in
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the substrate proteins as, at least some of these seem to be the highly conserved and essential proteins of the nucleus. One plausible explanation is that HL-60 and HeLa cells possess a higher level of endogenous PKC activity which maintains PKC substrates in a highly phosphorylated state, as proposed by other investigators [28]. Detailed examination of the effects of the nucleases allows two useful logical eliminations to be made. The first relates to the question of cytoplasmic contaminations and the second relates to the possibility of the nucleases actually acting via the alteration of free polyamine levels. Cytoplasmic contamination is always a problem with relatively 'native' chromatin or nuclear preparations [34]. However, the two nucleases employed extensively modulated the phosphorylation patterns, indicating that the phosphorylation targets were truly DNA-associated at the time of phosphorylation. Moreover, as the modulations caused by the two nucleases were quite unlike each other in detail, it is most unlikely that the nuclease effects were actually due to slight increases in the free polyamine levels following D N A destruction.
REFERENCES 1. Hunter T. (1987) Cell 50, 823-829. 2. Dunphy W. G. and Newport J. W. (1988) Cell 55, 925-928. 3. Testori A., Skinner J. D., Murray A. W. and Burgoyne L. A. (1991) Biochem. biophys. Res. Commun. 180, 329-333. 4. Nishizuka Y. (1986) Science 233, 305-312. 5. Hagiwara M., Uchida C., Usuda N., Nagata T. and Hidaka H. (1990) Biochem. biophys. Res. Commun. 168, 161-168. 6. Capitani S., Girard P. R., Mazzei G. J., Kuo J.F., Berezney R. and Manzoli F.A. (1987) Biochem. biophys. Res. Commun. 142, 367-375. 7. Masmoudi A., Labourdette G., Mersel M., Huang F. L., Huang K.-P., Vincendon G. and Malviya A.N. (1989) J. biol. Chem. 264, 1172-1179. 8. Rogue P., Labourdette G., Masmoudi A., Yoshida Y., Huang F. L., Huang K.-P., Zwiller J., Vincendon G. and Malviya A.N. (1990) J. biol. Chem. 265, 4161-4165.
9. Yamamoto K. K., Gonzalez G. A., Biggs III W. H. and Montminy M. R. (1988) Nature 334, 494-499. 10. Samuels D. S., Shimizu Y. and Shimizu N. (1989) Fedn Eur. biochem. Socs Lett. 259, 57-60. 11. Pommier Y., Kerrigan D., Hartman K. D. and Glazer R.I. (1990) J. biol. Chem. 265, 9418-9422. 12. Sahyoun N., Wolf M., Besterman J., Hsieh T.-S., Sander M., Levine III H., Chang K.-J. and Cuatrecasas P. (1986) Proc. natn. Acad. Sci. U.S.A. 83, 1603-1607. 13. Rottmann M., Schroder H. C., Gramzow M., Renneisen K., Kurelec B., Dorn A., Friese U. and Muller W. E. G. (1987) Eur. molec. Biol. Org. J. 6, 3939-3944. 14. Burgoyne L. A., Waqar M. A. and Atkinson M. R. (1970) Biochem. biophys. Res. Commun. 39, 254-259. 15. Hewish D. R. and Burgoyne L. A. (1973) Biochem. biophys. Res. Commun. 52, 504-510. 16. Sedat J. W. and Manuelidis U (1978) Cold Spring Harbor Syrup. Quant. Biol. 42, 331-350. 17. Filipski J., Leblanc J., Youdale T., Sikorska M. and Walker P. R. (1990) Eur. molec. Biol. Org. J. 9, 1319-1327. 18. Boulikas T. (1990) J. biol. Chem. 265, 4638-4647. 19. Belmont A. S., Braunfeld M. B., Sedat J. W. and Agard D. A. (1989) Chromosoma 98, 129-143. 20. Paddy M. R., Belmont A. S., Saumweber H., Agard D. A. and Sedat J. W. (1990) Cell 62, 89-106. 21. Wolf M., Cuatrecasas P. and Sayhoun N. (1985) J. biol. Chem. 260, 15,718-15,722. 22. Tapley P. M. and Murray A. W. (1984) Biochem. biophys. Res. Commun. 118, 835-841. 23. Testori A., Hii C. S. T., Fournier A., Burgoyne L.A. and Murray A.W. (1988) Biochem. biophys. Res. Commun. 156, 222-227. 24. Clark K. J., Isaac E. L. and Murray A.W. (1991) Biochem. Int. 23, 243-247. 25. Harrington C. R. (1990) Anal. Biochem. 186, 285-287. 26. Laemmli U. (1970) Nature 227, 680-685. 27. Mezzetti G., Monti M. G. and Moruzzi M. S. (1988) Life Sci. 42, 2293-2298. 28. Thomas T. P., Talwar H. S. and Anderson W. B. (1988) Cancer Res. 48, 1910-1919. 29. Macfarlane D. E. (1986) J. biol. Chem. 261, 6947-6953. 30. Butler A. P., Byus C. V. and Slaga T. J. (1986) J. biol. Chem. 261, 9421-9425. 31. Patskan G. J. and Baxter C. S, (1985) J. biol. Chem. 260, 12,899-12,903. 32. Kimura K., Katoh N., Sakurada K. and Kubo S. (1985) Biochem. J. 227, 271-276.
Phosphorylation of chromatin-associatedproteins 33. Ramachandran C., Yau P., Bradbury E.M., Shyamala G., Yasuda H. and Walsh D.A. (1984) J. biolo Chem. 259, 13,495-13,503.
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34. Van Holde K. E. (1988) in Chromatin (Rich A., Ed.). Springer Vedag, New York, 181-218.