Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 862-871
December 31, 1990
DNA BINDING ACTIVITY OF CASEIN KINASE II Odile FILHOL, Claude COCHET & Edmond M. CHAMBAZ Unit6 INSERM 244, DBMS/BRCE, Centre d'Etudes Nucl6aires, 85X, F-38041 Grenoble Cedex, France Received October 31, 1990
Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations ; duplex ~ phage DNA exhibited at least two types of binding sites and the high affinity system (Kd _=_ 6 x 1013M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its o~ subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors. ~ 1990 Academic Press, Inc.
Casein kinase II (CKII) serine-threonine protein kinase various eucaryotic sources and two different subunits with an
is an ubiquitous enzyme belonging to the family (1). CKII has been purified from shown to be an oligomeric protein made of o~2132 stoichiometry (2). The ~ subunit has
been shown to bear the catalytic site whereas the B subunit is the target of a self-phosphorylation reaction and appeared to be required for optimal enzymatic activity (3, 4). CKII has been isolated either from soluble tissue fractions or from nuclear extracts in which it was termed Nil kinase. Distinctive properties of CKII include use of GTP as well as ATP as phosphate donor, activation by polyamines and inhibition by micromolar concentrations of heparin (5, 6). The biological functions and mechanisms of control of CKII in living cell remain to be fully understood. A number of potential cytoplasmic substrates have been characterized including enzymes of glycogen and 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
862
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lipid metabolism, as well as cytoskeleton proteins and factors involved in the protein synthesis machinery (1, 6, 7). On the other hand, a number of nuclear components including HMG proteins (8) RNA polymerase II (9), topoisomerase II (10), nucleolin (11), oncoproteins such as Myc, Myb, ErbA, E7 and SV40 proteins (12-16) and recently SRF and P53 proteins have been described as CKII substrates mostly following in vivo studies (17, 18). The possible implication of CKII in the regulation of nuclear activities is further suggested by recent reports showing that the enzyme may be activated in response to several mitogens such as serum (19), insulin (20, 21), insulin like growth factor I (21) and epidermal growth factor (20, 22, 23). CKH may thus be examined as a possible component in the transduction of the mitogenic signals from the plasma membrane to the cell nucleus (24). In this communication, we present evidence that purified casein kinase II selectively associates with double stranded DNA in vitro. This association involves high affinity binding sites and results in an apparent enzyme activation which may be explained by prevention of the kinase denaturation in vitro. In addition, CKII-DNA interaction was shown to specifically involve the o~ subunit of the kinase.
MATERIALS AND METHODS
Chemicals y32p-ATP and cz32p-dCTP (3,000 Ci/mmol) was from Amersham (UK). Nucleotides, polynucleotides, proteins, polyvinylpyrolidone, EDTA, were from Sigma Chemicals (St Louis, MO), while sucrose was purchased from Merck (Germany). Ficoll was from Pharmacia, and nitrocellulose filter disks were furnished by Millipore Co. Nick translation kit, calf thymus DNA, Lambda DNA and tRNA were from Boehringer (Germany).
Sucrose
density
gradient
analysis
Samples of casein kinase II in a final volume of 0.12 ml were analyzed by sedimentation in the absence or in the presence of calf thymus DNA, as indicated, in linear 5 - 25 % sucrose density gradients (4,4 ml) in 10 mM Tris HC1 pH 7.4 buffer containing 2.5 mM MgC12, 0.1 mM CaC12 and 0.15 M NaC1. Centrifugation was carried out at 200,000 g for 3.5 hours at 4°C in a 20.60 Kontron centrifuge. Fractions of 0.3 ml were collected and assayed for casein kinase II activity, as previously described (5). DNA was detected in the fractions by recording the optical density at 260 nm.
DNA binding assays DNA binding activity of casein kinase II was measured by the nitrocellulose filter disk assay (25, 26) using 32p labeled calf thymus DNA 863
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or Coliphage ~ DNA. Both DNA were previously labeled by nick translation (27) yielding a specific activity of (0.5 - 1) 108 cpmAtg DNA. Prior to the assay, the nitrocellulose filters (HA 45 from Millipore) were soaked in DNA binding buffer (10 mM Tris-HC1, pH 7.4), containing 0.1 mg/ml bovine serum albumin and 10 mM MgC12 for 30 min at 22°C. Binding reactions were run in 50 Ixl of the binding buffer in the presence of 0.5-2 ng of 32p labeled DNA and purified casein kinase II which was diluted from a stock solution in binding buffer at the very last moment. Reaction was initiated by the addition of the enzyme and carried out at 22°C for the indicated periods of time. It was stopped upon addition of 500 ~tl of chilled binding buffer immediatly followed by filtration of the reaction mixture through a nitrocellulose membrane. The membrane was washed twice with 3 ml of binding buffer, dried and counted for its radioactivity content in 5 ml of aquasol II scintillation fluid.
Western blotting and DNA binding activity of the casein kinase II s u b u n i t s The two subunits of casein kinase II (2 ~tg) were separated by SDS PAGE and then transfered onto nitrocellulose filters by the bidirectional diffusion method described by Laemmli (28). This transfer yielded two mirror images each containing equal amounts of proteins on each filter. One filter was treated by Coomassie blue staining and the other one was preincubated for 30 min at room temperature in 50 ml renaturation buffer (10 mM Tris HCI pH 7, containing 1 mM EDTA, 0.02 % BSA, 50 mM NaC1, 0.02 % polyvinylpyrrolidone, 0,02 % Ficoll). This buffer was removed and 20 ml of binding buffer containing 150 ng of 32p labeled DNA (3x106 cpm) was added. The filter was incubated under continuous shaking for one hour at 22°C and then washed three times for 30 min in 70 ml of binding buffer containing O.1 M NaC1. The dried filter was exposed to autoradiography using hyperfilm MP films from Amersham.
RESULTS
Interaction of casein kinase II with DNA in vitro We first performed sucrose density gradient analysis of casein kinase II previously incubated with calf thymus DNA. The experiment depicted in Figure 1 shows that in an isotonic ionic strength medium (0.15 M NaC1), the enzyme sedimented as a 13 S macromolecule, a value which is higher than expected with regard to its known molecular weight (140 kDa). Furthermore, the recovery of the enzyme activity following centrifugation was strikingly low. By contrast, when casein kinase II was run in the presence of DNA, it was observed that the enzyme co-sedimented with DNA and that the enzyme activity was recovered to nearly 100 %. (Figure 1). This observation strongly suggested an interaction between casein kinase II and DNA resulting in a complex in which the enzyme activity was 864
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
.c ~
/
A
12. v0 >-
B
E tO LO
/
> t--
o< 150, nl CO
5 1.5 0.8 >5 5 25
Ixg/ml Ixg/ml Ixg/ml tx g / m 1 ng/ml ng/ml
Samples of CK II (5ng) were incubated for 30 min with 10ng/ml of 32p labeled ~, DNA under standard conditions in the presence of different polynucleotides at a concentration that gives 50% of DNA-binding inhibition (IDs0). calculated to r e p r e s e n t a binding capacity of about 1 mol CKII per mol of DNA. Modulation
Several
of
agents
casein
have
activity. M a g n e s i u m
kinase
been
II
DNA
described
as
interaction.
modulators
of
casein
kinase
II
and p o l y a m i n e s are potent activators of CK II in vitro
(6, 3). As illustrated in F i g u r e 2 (panel B) submillimolar concentrations of spermine when
increased
assayed
the
interaction
between
casein
by the n i t r o c e l l u l o s e filter binding A
kinase
assay.
II
and
Similarly,
DNA MgC12
B
~!!~iii~i!i¸i~!~
i~ iii ll ii .....
Fi~,ure 3 . D N A binding activity of the casein kinase II subunits The two CK II subunits were separated by SDS-PAGE, and transfered onto two nitrocellulose filters by the bidirectional diffusion method. One filter was stained with Coomassie blue and the other incubated with 32p labeled DNA as described in Material and Methods. A : Coomassie blue stained filter. B : Autoradiography of the homologous filter incubated with 32p DNA.
867
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
stimulated the DNA binding activity of the enzyme at concentrations previously reported to optimally support casein kinase II activity as well as its nuclear uptake in vitro (29, 30). Heparin which is a well characterized selective inhibitor of casein kinase II (31, 6) inhibits binding of the enzyme to DNA (Figure 2, panel C). Identification of the casein kinase II subunit involved in DNA binding Since casein kinase II is an oligomeric protein made of two different ~ and B subunits, with an ot2B2 stoichiometry (2), it was of interest to examine whether one of the subunit may be selectively involved in the CK II-DNA interaction. The two subunits can be separated by SDS-PAGE (2, 5). Following electrophoresis, the polypeptides were transfered onto nitrocellulose filters. After a renaturation step, the filters were incubated with 32P-labeled DNA. Subsequent autoradiography revealed that only the subunit was able to bind DNA under these conditions (Figure 3). Although these experiments demonstrate that at least, part of the DNA binding affinity of casein kinase II resides in a domain of the o~ subunit, they do not exclude the possibility that the B subunit might participate in this process when the enzyme is under its native, oligomeric form.
DISCUSSION The ubiquitous casein kinase II has been found distributed both in the cytoplasmic and the nuclear compartments in a number of eucaryotic cells examined so far (1, 6). The observation that the kinase concentrates in the nuclear (30) or in the nucleolar (32, 33) compartments when the cells are stimulated to proliferate, together with a number of reported potential nuclear CKII protein targets (8-18), strongly suggests that the enzyme may be examined as a transducer in the mechanism of action of growth factors and the control of nuclear activities (24). The present report shows that purified CKII interacts with genomic DNA in vitro. This interaction resulted in an association which was clearly demonstrated by the co-sedimentation of a DNA-CKII complex upon centrifugation analysis. Purified CKII diluted in an isotonic medium (i.e. 0.15 M NaC1 b u f f e r ) h a s been shown to form aggregates in which the kinase is inactivated (34). It has been observed that higher salt concentration (i.e. 0.5 M NaC1) was required to prevent this aggregation and to preserve the kinase activity (35). In the presence of DNA, the kinase aggregation appeared to be prevented by its binding to the polynucleotide and CKII activity could be fully recovered when DNA concentration was optimal. 868
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Binding of CKII to DNA could be characterized in some detail ; kinetic analysis disclosed that duplex DNAs show a high affinity for CKII, exhibiting at least two types of binding sites. Interestingly, in the case of ~. phage DNA, the high affinity system was calculated to represent maximum binding capacity of about 1 mol CKII per mol DNA. This would suggest that the bacteriophage DNA may be an interesting simplified model to search for specific recognition sequences for CKII. This limited number of high affinity binding sites also strongly support the idea of a specific DNA-CKII interaction. This is in agreement with displacement experiments which showed that optimum CKII binding required double stranded DNA and that various polynucleotides including single stranded DNA were ineffective. These observations ruled out a CKII interaction involving only a non specific affinity for polyanions. By contrast, the polyanionic structure of heparin was able to inhibit the CKII-DNA interaction. However, heparin is a selective inhibitor of CKII activity and heparin-like structures have been shown to associate with purified CKII (36). The present data give an experimental support to a recent study in which homologies were observed between the catalytic domain of the CKII subunit and the putative D N A binding domain of nuclear factor I, on the basis of data bank aminoacid sequences (37). The present observations are of special interest in view of the current ideas concerning the possible role of CKII in the control of nuclear activities, especially in response to cell growth factors (24). The requirement of a double stranded DNA for CKII association and the saturable CKII binding capacity exhibited by the model genomic DNAs examined, suggest that CKII-DNA interaction may have a biological significance in vivo. This would be in line with previous observations showing that CKII is actively taken up by nuclear preparations in vitro to become strongly associated with chromatin structures (30). Such nuclear CKII association may involve both binding to DNA and to nuclear proteins, the later being possibly themselves DNA-binding proteins. It was found in the present study that polyamines induced an increase in the CKII-DNA association ; this effect should, at least partly, explain the previous observation that these polycations markedly stimulated the CKII uptake and its association with intact nuclei (30). The present observations are in line with the hypothesis t h a t casein kinase II may be examined as a possible component in the transduction of mitogenic signals from the cell membrane to the nucleus. It remains to be examined whether specific DNA recognition sequences are involved and whether this interaction may direct a specific CKII targeting in chromatin ultimately playing a role in the control of specific gene expression related to cell proliferation processes. 869
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgments : This work was supported by the INSERM (U 244), the Commissariat ~t l'Energie Atomique (DBMS), the Fondation pour la Recherche M6dicale, the GEFLUC and the Ligue Nationale Fran~aise contre le Cancer. We are grateful to S. Lidy for typing the manuscript.
1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22.
Edelman, A. M., Blumenthal, D. K. and Krebs, E. G. (1987) Ann. Rev. Biochem. 56, 567-613. Cochet, C. and Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406. Cochet, C. and Chambaz, E. M. (1983) Mol. Cell. Endocrinol. 30, 247266. Feige, J.J., Cochet, C., Pirollet, F. and Chambaz, E.M. (1983) Biochemistry 22, 1452-1459. Cochet, C., Feige, J. J. and Chambaz, E. M. (1983) Biochim. Biophys. Acta 743, 1-12. Hathaway, G. M. and Traugh, J. A. (1982) in Current Topics in Cellular Regulation (Horecker, B. and Stadtman, E. Eds) vol 21, pp 107-107, Academic Press, New York. Gonzatti-Haces, M. L. and Traugh, J. A. (1982) J. Biol. Chem. 257, 6642-6645. Walton, G.M. and Gill, G.N. (1983) J. Biol. Chem. 258, 4440-4446. Stetler, D.A. and Rose, K. M. (1983) Biochim. Biophys. Acta 739, 105113. Ackerman, P., Glover, C. V. C. and Osheroff, N. (1988) J. Biol. Chem. 263, 12653-12660. Caizergues-Ferrer, M., Belenguer, P., Lapeyre, B., Amalric, F., Wallace, M.O. and Olson, M. O. J. (1987) Biochemistry 26, 7876-7883. Liischer, B., Kuenzel, E. A., Krebs, E. g. and Eisenman, R. N. (1989) EMBO J. 8, 1111-1119. Glineur, C., Bailly, M. and Ghysdael, J. (1989) Oncogene 4, 101-108. Barbosa, M.S., Edmonds, C., Fisher, C,, Schiller, J.T., Lowry, D.R., Vousden, K.H. (1990) EMBO J. 9, 153-160. Gr~isser, F.A., Scheidtmann, K.H., Tuazon, P.T., Traugh, J.A. and Walter, G. (1988) Virology 165, 13-22. Krebs, E. G., Eisenman, R. N., Kuenzel, E. A., Litchfield, D. W., Lozeman, F. J., Ltischer, B. and Sommercorn, J. (1988) in Cold Spring Harbor Symposia on Quantitative Biology LIII, pp 77-84. Manak, J.R., de Bisschop, N., Kris, R.M., Prywes, R. (1990) Genes and development 4, 955-967. Meek, D.W., Simon, S. Kikkawa, U. and Eckhart, W. (1990) EMBO J. 9, 3253-3260. Carroll, D. and Marshak, D. R. (1989) J. Biol. Chem. 264, 7345-7348. Sommercorn, J., Mulligan, J. A., Lozeman, F. J. and Krebs, E.G. (1987) Proc. Natl. Aead. Sci. USA 84, 8834-8838. Sommercorn, J. and Krebs, E. G. (1987) J. Biol. Chem. 262, 38393843. Ackerman, P. and Osheroff, N. (1989) J. Biol. Chem. 264, 1195811965. 870
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23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ackerman, P., Glover, C. V. C. and Osheroff, N. (1990) Proc. Natl. Acad. Sci. USA 87, 821-825. Carpenter, G. and Cohen, S. (1990) J. Biol. Chem. 265, 7709-7712. Jones, O. W. and Berg, P. (1966) J. Mol. Biol. 22, 199-209. Riggs, A. D., Suzuki, H. and Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83. Rigby, P. W. J., Dieckman, M., Rhodes, C. and Berg, P. (1977) J. Mol. Biol. 113, 237-251. Laemmli, U. K. (1980) Nucleic Acid Res. 8, 1-5. Cochet, C., Feige, J. J., Pirollet, F., Keramidas, M. and Chambaz, E. M. (1982) Biochem. Pharmacol. 31, 1357-1361. Filhol, O., Cochet, C. and Chambaz, E. M. (1990) Biochemistry 29, 9928-9936. Feige, J. J., Pirollet, F., Cochet, C. and Chambaz, E. M. (1980) FEBS Lett. 121, 139-142. Pfaff, M. and Anderer, F. A. (1988) Biochim. Biophys. Acta 969, 100109. Belenguer, P., Baldin, V., Mathieu, C., Prats, H., Bensaid, M., Bouche, G. and Amalric, F. (1989) Nucl. Acid. Res. 17, 6625-6636. Glover, C. V. C. (1986) J. Biol. Chem. 261, 14349-14354. Job, D., Pirollet, F., Cochet, C. and Chambaz, E.M. (1989) FEBS Lett. 108, 508-512. Job, D., Cochet, C., Pirollet, F. and Chambaz, E.M. (1979) FEBS Lett. 98, 303-308. Mannermaa, R.M. and Oikarinen, J. (1989) Biochem. Biophys. Res. Comrnun. 162, 427-434.
871
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 862-871
December 31, 1990
DNA BINDING ACTIVITY OF CASEIN KINASE II Odile FILHOL, Claude COCHET & Edmond M. CHAMBAZ Unit6 INSERM 244, DBMS/BRCE, Centre d'Etudes Nucl6aires, 85X, F-38041 Grenoble Cedex, France Received October 31, 1990
Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations ; duplex ~ phage DNA exhibited at least two types of binding sites and the high affinity system (Kd _=_ 6 x 1013M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its o~ subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors. ~ 1990 Academic Press, Inc.
Casein kinase II (CKII) serine-threonine protein kinase various eucaryotic sources and two different subunits with an
is an ubiquitous enzyme belonging to the family (1). CKII has been purified from shown to be an oligomeric protein made of o~2132 stoichiometry (2). The ~ subunit has
been shown to bear the catalytic site whereas the B subunit is the target of a self-phosphorylation reaction and appeared to be required for optimal enzymatic activity (3, 4). CKII has been isolated either from soluble tissue fractions or from nuclear extracts in which it was termed Nil kinase. Distinctive properties of CKII include use of GTP as well as ATP as phosphate donor, activation by polyamines and inhibition by micromolar concentrations of heparin (5, 6). The biological functions and mechanisms of control of CKII in living cell remain to be fully understood. A number of potential cytoplasmic substrates have been characterized including enzymes of glycogen and 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
862
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lipid metabolism, as well as cytoskeleton proteins and factors involved in the protein synthesis machinery (1, 6, 7). On the other hand, a number of nuclear components including HMG proteins (8) RNA polymerase II (9), topoisomerase II (10), nucleolin (11), oncoproteins such as Myc, Myb, ErbA, E7 and SV40 proteins (12-16) and recently SRF and P53 proteins have been described as CKII substrates mostly following in vivo studies (17, 18). The possible implication of CKII in the regulation of nuclear activities is further suggested by recent reports showing that the enzyme may be activated in response to several mitogens such as serum (19), insulin (20, 21), insulin like growth factor I (21) and epidermal growth factor (20, 22, 23). CKH may thus be examined as a possible component in the transduction of the mitogenic signals from the plasma membrane to the cell nucleus (24). In this communication, we present evidence that purified casein kinase II selectively associates with double stranded DNA in vitro. This association involves high affinity binding sites and results in an apparent enzyme activation which may be explained by prevention of the kinase denaturation in vitro. In addition, CKII-DNA interaction was shown to specifically involve the o~ subunit of the kinase.
MATERIALS AND METHODS
Chemicals y32p-ATP and cz32p-dCTP (3,000 Ci/mmol) was from Amersham (UK). Nucleotides, polynucleotides, proteins, polyvinylpyrolidone, EDTA, were from Sigma Chemicals (St Louis, MO), while sucrose was purchased from Merck (Germany). Ficoll was from Pharmacia, and nitrocellulose filter disks were furnished by Millipore Co. Nick translation kit, calf thymus DNA, Lambda DNA and tRNA were from Boehringer (Germany).
Sucrose
density
gradient
analysis
Samples of casein kinase II in a final volume of 0.12 ml were analyzed by sedimentation in the absence or in the presence of calf thymus DNA, as indicated, in linear 5 - 25 % sucrose density gradients (4,4 ml) in 10 mM Tris HC1 pH 7.4 buffer containing 2.5 mM MgC12, 0.1 mM CaC12 and 0.15 M NaC1. Centrifugation was carried out at 200,000 g for 3.5 hours at 4°C in a 20.60 Kontron centrifuge. Fractions of 0.3 ml were collected and assayed for casein kinase II activity, as previously described (5). DNA was detected in the fractions by recording the optical density at 260 nm.
DNA binding assays DNA binding activity of casein kinase II was measured by the nitrocellulose filter disk assay (25, 26) using 32p labeled calf thymus DNA 863
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or Coliphage ~ DNA. Both DNA were previously labeled by nick translation (27) yielding a specific activity of (0.5 - 1) 108 cpmAtg DNA. Prior to the assay, the nitrocellulose filters (HA 45 from Millipore) were soaked in DNA binding buffer (10 mM Tris-HC1, pH 7.4), containing 0.1 mg/ml bovine serum albumin and 10 mM MgC12 for 30 min at 22°C. Binding reactions were run in 50 Ixl of the binding buffer in the presence of 0.5-2 ng of 32p labeled DNA and purified casein kinase II which was diluted from a stock solution in binding buffer at the very last moment. Reaction was initiated by the addition of the enzyme and carried out at 22°C for the indicated periods of time. It was stopped upon addition of 500 ~tl of chilled binding buffer immediatly followed by filtration of the reaction mixture through a nitrocellulose membrane. The membrane was washed twice with 3 ml of binding buffer, dried and counted for its radioactivity content in 5 ml of aquasol II scintillation fluid.
Western blotting and DNA binding activity of the casein kinase II s u b u n i t s The two subunits of casein kinase II (2 ~tg) were separated by SDS PAGE and then transfered onto nitrocellulose filters by the bidirectional diffusion method described by Laemmli (28). This transfer yielded two mirror images each containing equal amounts of proteins on each filter. One filter was treated by Coomassie blue staining and the other one was preincubated for 30 min at room temperature in 50 ml renaturation buffer (10 mM Tris HCI pH 7, containing 1 mM EDTA, 0.02 % BSA, 50 mM NaC1, 0.02 % polyvinylpyrrolidone, 0,02 % Ficoll). This buffer was removed and 20 ml of binding buffer containing 150 ng of 32p labeled DNA (3x106 cpm) was added. The filter was incubated under continuous shaking for one hour at 22°C and then washed three times for 30 min in 70 ml of binding buffer containing O.1 M NaC1. The dried filter was exposed to autoradiography using hyperfilm MP films from Amersham.
RESULTS
Interaction of casein kinase II with DNA in vitro We first performed sucrose density gradient analysis of casein kinase II previously incubated with calf thymus DNA. The experiment depicted in Figure 1 shows that in an isotonic ionic strength medium (0.15 M NaC1), the enzyme sedimented as a 13 S macromolecule, a value which is higher than expected with regard to its known molecular weight (140 kDa). Furthermore, the recovery of the enzyme activity following centrifugation was strikingly low. By contrast, when casein kinase II was run in the presence of DNA, it was observed that the enzyme co-sedimented with DNA and that the enzyme activity was recovered to nearly 100 %. (Figure 1). This observation strongly suggested an interaction between casein kinase II and DNA resulting in a complex in which the enzyme activity was 864
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
.c ~
/
A
12. v0 >-
B
E tO LO
/
> t--
o< 150, nl CO
5 1.5 0.8 >5 5 25
Ixg/ml Ixg/ml Ixg/ml tx g / m 1 ng/ml ng/ml
Samples of CK II (5ng) were incubated for 30 min with 10ng/ml of 32p labeled ~, DNA under standard conditions in the presence of different polynucleotides at a concentration that gives 50% of DNA-binding inhibition (IDs0). calculated to r e p r e s e n t a binding capacity of about 1 mol CKII per mol of DNA. Modulation
Several
of
agents
casein
have
activity. M a g n e s i u m
kinase
been
II
DNA
described
as
interaction.
modulators
of
casein
kinase
II
and p o l y a m i n e s are potent activators of CK II in vitro
(6, 3). As illustrated in F i g u r e 2 (panel B) submillimolar concentrations of spermine when
increased
assayed
the
interaction
between
casein
by the n i t r o c e l l u l o s e filter binding A
kinase
assay.
II
and
Similarly,
DNA MgC12
B
~!!~iii~i!i¸i~!~
i~ iii ll ii .....
Fi~,ure 3 . D N A binding activity of the casein kinase II subunits The two CK II subunits were separated by SDS-PAGE, and transfered onto two nitrocellulose filters by the bidirectional diffusion method. One filter was stained with Coomassie blue and the other incubated with 32p labeled DNA as described in Material and Methods. A : Coomassie blue stained filter. B : Autoradiography of the homologous filter incubated with 32p DNA.
867
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
stimulated the DNA binding activity of the enzyme at concentrations previously reported to optimally support casein kinase II activity as well as its nuclear uptake in vitro (29, 30). Heparin which is a well characterized selective inhibitor of casein kinase II (31, 6) inhibits binding of the enzyme to DNA (Figure 2, panel C). Identification of the casein kinase II subunit involved in DNA binding Since casein kinase II is an oligomeric protein made of two different ~ and B subunits, with an ot2B2 stoichiometry (2), it was of interest to examine whether one of the subunit may be selectively involved in the CK II-DNA interaction. The two subunits can be separated by SDS-PAGE (2, 5). Following electrophoresis, the polypeptides were transfered onto nitrocellulose filters. After a renaturation step, the filters were incubated with 32P-labeled DNA. Subsequent autoradiography revealed that only the subunit was able to bind DNA under these conditions (Figure 3). Although these experiments demonstrate that at least, part of the DNA binding affinity of casein kinase II resides in a domain of the o~ subunit, they do not exclude the possibility that the B subunit might participate in this process when the enzyme is under its native, oligomeric form.
DISCUSSION The ubiquitous casein kinase II has been found distributed both in the cytoplasmic and the nuclear compartments in a number of eucaryotic cells examined so far (1, 6). The observation that the kinase concentrates in the nuclear (30) or in the nucleolar (32, 33) compartments when the cells are stimulated to proliferate, together with a number of reported potential nuclear CKII protein targets (8-18), strongly suggests that the enzyme may be examined as a transducer in the mechanism of action of growth factors and the control of nuclear activities (24). The present report shows that purified CKII interacts with genomic DNA in vitro. This interaction resulted in an association which was clearly demonstrated by the co-sedimentation of a DNA-CKII complex upon centrifugation analysis. Purified CKII diluted in an isotonic medium (i.e. 0.15 M NaC1 b u f f e r ) h a s been shown to form aggregates in which the kinase is inactivated (34). It has been observed that higher salt concentration (i.e. 0.5 M NaC1) was required to prevent this aggregation and to preserve the kinase activity (35). In the presence of DNA, the kinase aggregation appeared to be prevented by its binding to the polynucleotide and CKII activity could be fully recovered when DNA concentration was optimal. 868
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Binding of CKII to DNA could be characterized in some detail ; kinetic analysis disclosed that duplex DNAs show a high affinity for CKII, exhibiting at least two types of binding sites. Interestingly, in the case of ~. phage DNA, the high affinity system was calculated to represent maximum binding capacity of about 1 mol CKII per mol DNA. This would suggest that the bacteriophage DNA may be an interesting simplified model to search for specific recognition sequences for CKII. This limited number of high affinity binding sites also strongly support the idea of a specific DNA-CKII interaction. This is in agreement with displacement experiments which showed that optimum CKII binding required double stranded DNA and that various polynucleotides including single stranded DNA were ineffective. These observations ruled out a CKII interaction involving only a non specific affinity for polyanions. By contrast, the polyanionic structure of heparin was able to inhibit the CKII-DNA interaction. However, heparin is a selective inhibitor of CKII activity and heparin-like structures have been shown to associate with purified CKII (36). The present data give an experimental support to a recent study in which homologies were observed between the catalytic domain of the CKII subunit and the putative D N A binding domain of nuclear factor I, on the basis of data bank aminoacid sequences (37). The present observations are of special interest in view of the current ideas concerning the possible role of CKII in the control of nuclear activities, especially in response to cell growth factors (24). The requirement of a double stranded DNA for CKII association and the saturable CKII binding capacity exhibited by the model genomic DNAs examined, suggest that CKII-DNA interaction may have a biological significance in vivo. This would be in line with previous observations showing that CKII is actively taken up by nuclear preparations in vitro to become strongly associated with chromatin structures (30). Such nuclear CKII association may involve both binding to DNA and to nuclear proteins, the later being possibly themselves DNA-binding proteins. It was found in the present study that polyamines induced an increase in the CKII-DNA association ; this effect should, at least partly, explain the previous observation that these polycations markedly stimulated the CKII uptake and its association with intact nuclei (30). The present observations are in line with the hypothesis t h a t casein kinase II may be examined as a possible component in the transduction of mitogenic signals from the cell membrane to the nucleus. It remains to be examined whether specific DNA recognition sequences are involved and whether this interaction may direct a specific CKII targeting in chromatin ultimately playing a role in the control of specific gene expression related to cell proliferation processes. 869
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Acknowledgments : This work was supported by the INSERM (U 244), the Commissariat ~t l'Energie Atomique (DBMS), the Fondation pour la Recherche M6dicale, the GEFLUC and the Ligue Nationale Fran~aise contre le Cancer. We are grateful to S. Lidy for typing the manuscript.
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