Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 945-952
October 31, 1991
POLYAMINE BINDING ACTIVITY OF CASEIN KINASE II Odile FILHOL, Claude COCHET, Thierry DELAGOUTTE and Edmond M. CHAMBAZ Unit~ INSERM 244, DBMS/BRCE, Centre d'Etudes Nucl6aires, 85X, F-38041 Grenoble Cedex, France Received September 18, 1991
Protein phosphorylation by the ubiquitous casein kinase II (CKII) is known to be sensitive to naturally occuring polyamines. Using isolated recombinant t~ and B subunits of the kinase, as well as the ct2B2 oligomeric enzyme, it is shown that (i) CKII binds [3H]-spermine with Kds in the micrGmolar range. (ii) The 13 subunit appears mostly responsible for this binding activity. (iii) The isolated t~ (catalytic) subunit is not activated by polyamines. (iv) The polyamine-dependent activation of the oligomeric CKII requires the B subunit, which appears as a regulatory component in the native kinase. These observations suggest that there may be a functional interaction between polyamines and CKII in living cells, especially in the response to cell growth factors and trophic hormones. © 1991 A c a d e m i c
Press,
Inc.
Polyamines are ubiquitous components which are required for normal cell growth (1). Although these polycations have been implicated in the regulation of major cellular functions, such as DNA, RNA and protein synthesis, their mechanism of action at the molecular level remain obscure. We have previously suggested that polyamines may express at least part of their biological action through an effect upon a multifunctional protein kinase, i.e. casein kinase II (CKII). This hypothesis was based on the major following observations : (i) The activity of CKII is markedly activated in vitro by polyamines, spermine being the most potent (2, 3) ; (ii) CKII tightly associates with nuclear preparations in vitro and this kinase nuclear uptake is markedly enhanced in the presence of polyamines (4) which also increased the binding of CKII to double stranded DNA (5) ; (iii) There exists a temporal relationship between the nuclear accumulation of
Abbreviations CKII : casein kinase II ; ACTH : adrenocorticotrophic hormone ; SDS : sodium dodecyl sulfate ; cDNA : complementary DNA.
945
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CKII and the increase in intracellular polyamine content in adrenocortical cells following exposure to their trophic hormone ACTH (6). Although an effect through the protein substrate conformation may contribute to the activation of CKII-dependent phosphorylations by polyamines (3), a direct interaction between these polycations and the e n z y m e itself has, to our knowledge, never been considered. Eukaryotie CKII is an oligomeric enzyme made of two different (or and B) subunits, with an tx2B2 stoichiometry (3, 7). We have recently expressed the isolated subunit cDNAs of the kinase in a baculovirus-directed-insect cell expression system. This system gives access to the isolated enzyme subunits as well as to a functional recombinant oligomeric enzyme (8). The present study addressed the question of a possible molecular interaction between polyamines and CKII and examined the contribution of each kinase subunit in this process. The results disclosed that among naturally occuring polyamines, CKII binds preferentially spermine with high affinity. In addition, the B subunit appears to play an essential role in this interaction. These observations support our w o r k i n g hypothesis suggesting that polyamines may be examined as intracellular messengers in the regulation of CKII activity. M A T E R I A L S AND M E T H O D S •
Materials
[3H]-spermine tetrahydrochloride (AS : 23.5 Ci/mmol) was purchased from New England Nuclear. Red-120 agarose, spermine, spermidine, cadaverine and polylysine were obtained from Sigma. Phenyl Sepharose was from Pharmacia. Spermine-agarose was from Pierce. Grace's insect cell culture m e d i u m was obtained from Gibco. The pEV55 Dmc~ and pEV55 Drab plasmids were a kind gift from CVC Glover (University of Georgia, USA). •
Methods
- R e c o m b i n a n t i s o l a t e d g. and B s u b u n i t s of C K I I were produced in Sf9 insect cells infected with baculo virus containing the full length eDNA of either CKII ot or CKII B subunit (8). Co-infection with both viruses yielded a recombinant enzyme with an 0t2132 stoichiometry. Active recombinant ct subunit and oligomeric kinase were purified to near homogeneity as in (8). The recombinant B subunit was purified through 3 chromatographic steps : the soluble Sf9 cell extract was loaded onto a phenyl sepharose column previously equilibrated with 10 mM Tris HC1, pH 7.5, 1 mM DTT, 1 % glycerol, (TDG buffer) containing 0 . 1 % Triton X-100 and 1 M NaC1. A 1 to 0 M linear NaC1 gradient in TDG buffer containing 0 . 1 % Triton X-100 was applied and the B subunit was detected in collected fractions by western blot assay. After dilution to a final concentration of 0.1 M NaC1, the active fractions were applied onto a spermine agarose column equilibrated in TDG buffer containing 0 . 1 % Triton X-100. A 0-1.2 M linear NaC1 gradient in the same buffer was applied. The last step used a Red-120 agarose 946
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
column, yielding a 13 subunit which was 90 % pure, as judged by coomassie blue staining. - B i n d i n g of s p e r m i n e was assayed using a gel filtration assay. Aliquots of recombinant proteins were incubated (5-15 min at 4°C) with 0.5 ~tM [3H]spermine (_= 106 cpm) and different concentrations of unlabeled polyamines in 80 ~tl of TDG buffer containing 1 mg/ml BSA. The mixtures were then rapidly centrifuged through small Sephadex G-50 superfine columns, at 4°C, according to (9). Bound [3H]-spermine was determined by radioactive counting of the excluded volume, after substraction of the blank values (usually less than 0.2 % of the input radioactivity) in the absence of recombinant proteins. - W e s t e r n blot a n a l y s i s were performed using an antiserum prepared against Drosophila casein kinase II (10) that recognized both the a and 13 subunits of the enzyme. RESULTS
1. Interaction of spermine with casein kinase II subunits The ability of CKII to bind [3H]-spermine was examined with the purified recombinant CKII a and 13 subunits and the recombinant oligomeric 0~2132 enzyme. As illustrated in Figure 1A, increasing amounts of the oligomeric form of the kinase were able to bind [3H]-spermine. When tested at the same concentrations, the isolated 13 subunit was also found to bind the polyamine, although with a lower efficiency than the oligomeric enzyme. On the other hand, the isolated 0c subunit which bears the catalytic site of the enzyme (11, 12) was unable to bind [3H]-spermine in a detectable fashion. We next examined the interaction of the CKII isolated subunits with immobilized spermine. Recombinant polypeptides were loaded on spermine-agarose and were eluted by increasing NaC1 concentrations. The experiments depicted in Figure 1B showed that the cx subunit was eluted at a relatively low salt concentration (0.4 M NaC1), whereas the isolated 13 subunit was eluted at 0.6 M NaC1. Under the same conditions, elution of the oligomeric CKII required 0.9 M salt concentrations. Since the behavior of the different polypeptides through this affinity system should reflect their relative affinities for the immobilized spermine, these observations confirm that CKII is a polyamine binding protein as well as its B subunit. By contrast, the tx subunit showed a low polyamine affinity only detectable with the chromatographic method. Interestingly, the oligomeric form of the enzyme exhibited a polyamine binding activity, higher than that exhibited by the isolated 13 subunit. 2. Kinetic p a r a m e t e r s of the spermine-CKII interaction Time-course experiments show that the binding reaction reached a plateau after 5 min of incubation at 4°C (not shown). When recombinant 947
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B
(~'
NaCl [hl]
200
97 68 43
/3 200 97 68
NaCl[~l
43 alpha
29
20 .beta
A ~"
18
18
.w
1 2~"
.w
(kOa
6-1
.
(kDa
/
6s
2
29
~ 0 i~J. ~" 0
~, 20
, .
. 40
recombinant subunits
Figure
1 .
. 60
.m
18 HW (kOa
,
80 (nM)
Interaction
of
spermine
with
the
isolated
recombinant subunits of casein kinase II. A. Increasing concentrations of recombinant ~ subunit ( R - I ) , B subunit ( e - e ) or recombinant oligomeric a2132 CKII (UI-UI) were incubated with 0.5 ~tM [3H]-spermine for 15 min at 4°C. Bound [3H]-spermine was determined by gel filtration, as described in Materials and Methods. B. Aliquots (2.4 Ixg protein) of recombinant a or 1~ subunits or oligomeric et21~2 casein kinase II were loaded on spermine agarose columns (100 ~tl). The columns were eluted stepwise by 0.3 ml of TDG buffer containing increasing concentrations of NaCI. Proteins in each fraction were precipitated with 10% TCA, analysed on a 12% SDS polyacrylamide gel and visualized by coomassie blue staining.
oligomeric CKII was incubated at 4°C with 0.5 ~tM [3H]-spermine in the presence of increasing concentrations of unlabeled spermine, a typical saturation curve was obtained (Figure 2). The binding sites of the enzyme were saturated at 200 ~tM spermine with an IDs0 of 50 ~M. A Scatchard plot analysis (13) of these disclosed at least two types of binding sites ( F i g u r e 2). A p p a r e n t d i s s o c i a t i o n c o n s t a n t s of 11.5 x 10-6M and 136 x 10-6M were calculated for the two binding systems, respectively. The corresponding binding capacities were 0.12 x 10-6M (1.7 sites/mol of CKII) and 0.77 x 10-6M (10.8 sites/mol of CKII), respectively. 3. Specificity of the polyamines CKII interaction Recombinant oligomeric CKII was incubated with [3H]-spermine in the presence of different unlabeled polyamines and bound [3H]-spermine was determined. As shown in Table I, at a 50 IxM concentration, spermine gave a 50 % displacement of bound [3H]-spermine whereas spermidine and cadaverine, at the same concentration, were ineffective. However, a synthetic polycationic compound, such as polylysine which is known to 948
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lO t,i,-X
E
o.
tl..
~4
0 "ID
t-. o
c
E Q.
?
12.
2
~
0
.~ 100
. . . . . 200 300
400
B (nM)
10~ 8
"1"
1'00
2'00
spermine (~M) Analysis of spermine binding to casein kinase II. Aliquots (71 nM) of recombinant oligomeric CKII were incubated for 15 min at 4°C with 0.5 txM [3H]-spermine and increasing concentrations of unlabeled spermine. Data were plotted according to Scatchard (inset). Figure
2.
stimulate CKII activity (7), was found to be a potent competitor of the [3H]-spermine-CKII interaction. 4 . E f f e c t of s p e r m i n e on m o n o m e r i c a n d o l i g o m e r i c recombinant CKII activity We have shown previously that the o~ subunit bears the catalytic site of the enzyme (11) and that the B subunit is required for optimal catalytic
Table I Effect of different unlabeled polyamines on the binding of [3H]-spermine to recombinant oligomeric casein kinase II UNLABELED
POLYAMINE
(50 ~M)
[3H]-SPERMINE
- NONE
11 5 11 13 5
- SPERMINE -
-
SPERMIDINE CADAVERINE
- POLYLYSINE
BOUND (CPM)
903 549 198 059 339
CKII aliquots (35 nM) of recombinant oligomeric CKII were incubated for 15 min at 4°C with 0.5 ~tM [3H]-spermine and different unlabeled naturally occurring polyamines or polylysine. Bound [3H]-spermine was determined by the gel filtration assay. 949
V o l . 180, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
~"
4000,
•~
3oo0,
2000,
J
lOOO,
o
0
-
0.0
•
0.2
-
i
-
0.4
=
-
0.6
,
0.8
-
,
1.0
SPERMINE [mM]
Fieure
3.
Effect of spermine on o l i g o m e r i c and m o n o m e r i c r e c o m b i n a n t casein kinase II activity.
Recombinant c~ subunit (B-B) or recombinant oligomeric c t 2 6 2 ( 0 - 0 ) CKII were assayed for protein kinase activity with topoisomerase II as substrate in the presence of 2 mM magnesium and increasing amounts of spermine. Phosphorylated proteins were analyzed by electrophoresis on a 7.5 % SDS polyacrylamide gel and 32p incorporation in topoisomerase II was measured by scintillation counting.
activity (12). Using isolated recombinant subunits, we examined the contribution of each subunit in the modulation of CKII activity by polyamines. The kinase activity of monomeric and oligomeric forms of CKII was assayed, using topoisomerase II as the protein substrate, in the presence of increasing concentrations of spermine. As shown in figure 3, p h o s p h o r y l a t i o n by CKII was stimulated 3 fold in the presence of spermine, this effect reaching a plateau for 200 ~tM spermine. By contrast, spermine at the same concentrations was without effect on the kinase activity of the isolated o~ subunit. Therefore, this experiment suggests that the polyamine modulates the oligomeric CKII activity, following its binding to the 6 subunit of the enzyme. DISCUSSION The catalytic activity of casein kinase II has been reported to be sensitive to submillimolar concentrations of polyamines (3, 14). The present study discloses that CKII is clearly a polyamine-binding protein. Among the major naturally occuring polyamines, the enzyme exhibits the best affinity for spermine, which is also the most potent as CKII activator (3). Under its native form, as an oligomeric protein with an ¢~21~2 subunit stoichiometry, CKII binds spermine to at least two types of binding sites with affinity constants in the micromolar range. An interesting observation came from binding studies using recombinant isolated a and B 950
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
subunits, as compared to the oligomeric form of the kinase. Spermine binding could be assayed only with the isolated g subunit, whereas no detectable polyamine remained bound to the ~ peptide, following the rapid gel filtration method employed to separate bound from free ligand. Although this non equilibrium method might contribute to this result, it is in line with the weak affinity of the ~ subunit for polyamine, as shown by its behavior on a spermine-coupled solid support. However, on identical molar basis, the spermine binding affinity of the oligomeric enzyme was higher than that of the isolated 13 subunit. This suggests that the g subunit conformation is different when isolated as compared to when it is integrated in the oligomeric o~21~2 structure, resulting in conformational adjustment and polyamine affinity increase. However, a contribution of the o~ subunit in the binding properties of the native enzyme is not excluded. The present study discloses that the activity of the isolated CKII ¢x subunit, which bears the catalytic site of the enzyme, was not sensitive to polyamine. The activation of CKII by spermine required the presence of the 13 subunit and the oligomeric structure of the kinase. This points to a possible key role of the g subunit in the regulation of CKII. The finding that CKII is a polyamine binding protein with a Kd in the micromolar range comes in support for a significant biological role of this interaction in living ceils, in which polyamines may reach millimolar concentrations (2, 15). In addition, the binding affinity of the different naturally occuring polyamines is well correlated with their relative potency as CKII activators (16). Intracellular p o l y a m i n e level is highly d e p e n d e n t upon growth factors and trophic hormones which have been shown to induce ornithine decarboxylase and cell polyamine uptake activation (2, 15). Polyamines increase the affinity of CKII for chromatine and double stranded DNA (4, 5) and this would be in line with the nuclear CKII accumulation observed under cell growth stimulation (4). Polyamines are known to bind to DNA and to promote chromatin condensation (17, 18). These polycations may act as bifunctional ligands with an affinity for both DNA and CKII and may thus modulate CKII-DNA interaction. The present findings support the idea of a functional link between polyamines and CKII in living cells, especially in the response to trophic hormone and growth stimuli.
Acknowledgments This work was supported by the INSERM (U244), the Commissariat l'Energie A t o m i q u e (DSV/DBMS), the Fondation pour la R e c h e r c h e M6dicale, the GEFLUC and the Ligue Nationale Fran~aise contre le Cancer.
951
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18.
Mamont, P. S., Bey, P. & Koch-Weser, J. (1980) In Polyamines in Biochemical Research. pp 147-183. John Wiley, New York. Feige, J. J., Cochet, C. & Chambaz, E. M. (1985) In Recent Progress in Polyamine Research (L. Selmeci, M. E. Brosnan, and N. Seiler, Eds) vol. 13, pp 181-190. Akadamiai Kiado, Budapest. Cochet, C. & Chambaz, E. M. (1983) Mol. Cell. Endocrinol. 30, 247-266. Filhol, O., Cochet, C. & Chambaz, E.M. (1990) Biochemistry 29, 99289936. Filhol, O., Cochet, C. & Chambaz, E. M. (1990) Biochem. Biophys. Res. Commun. 173, 862-871. Filhol, O., Loue-Mackenbach, P., Cochet, C. & Chambaz, E.M. (1991) Biochem. Biophys. Res. Commun., in press. Pinna, L.A. (1990) Biochim. Biophys. Acta, 1054, 267-284. Filhol, O., Cochet C., Wedegaertner, P., Gill, G.N. & Chambaz, E.M. (1991) Biochemistry, in press. Penefski, H.S. (1977) J. Biol. Chem. 252, 2891-2899. Padmanabha, R. & Glover, C.V.C. (1987) J. Biol. Chem. 262, 1829-1834. Feige, J.J., Cochet, C., Pirollet, F. & Chambaz, E.M. (1983) Biochemistry 22, 1452-1459. Cochet, C. & Chambaz, E.M. (1983) J. Biol. Chem. 258, 1403-1406. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Cochet, C., Job, D., Pirollet, F. & Chambaz, E. M. (1980) Endocrinology 106, 750-757. Heby, O. (1981) Differentiation 19, 1-20. Traugh, J.A., Lin, W.J., Takada-Axelrod, F. & Tuazon, P.T. (1990) Ir Advances in Second Messenger and Phosphoprotein Research. The Biology and-Medicine of Singal Transduction. (Y. Nishizuka, M. Endo, C. Tanaka, Eds) Vol. 24, Raven Press. Gosule, L.C. & Schellman, J.A. (1976) Nature 259, 333-335. Marton, L.J. & Feuerstein, B.G. (1986) Pharm. Res. 3, 311-317.
952
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 945-952
October 31, 1991
POLYAMINE BINDING ACTIVITY OF CASEIN KINASE II Odile FILHOL, Claude COCHET, Thierry DELAGOUTTE and Edmond M. CHAMBAZ Unit~ INSERM 244, DBMS/BRCE, Centre d'Etudes Nucl6aires, 85X, F-38041 Grenoble Cedex, France Received September 18, 1991
Protein phosphorylation by the ubiquitous casein kinase II (CKII) is known to be sensitive to naturally occuring polyamines. Using isolated recombinant t~ and B subunits of the kinase, as well as the ct2B2 oligomeric enzyme, it is shown that (i) CKII binds [3H]-spermine with Kds in the micrGmolar range. (ii) The 13 subunit appears mostly responsible for this binding activity. (iii) The isolated t~ (catalytic) subunit is not activated by polyamines. (iv) The polyamine-dependent activation of the oligomeric CKII requires the B subunit, which appears as a regulatory component in the native kinase. These observations suggest that there may be a functional interaction between polyamines and CKII in living cells, especially in the response to cell growth factors and trophic hormones. © 1991 A c a d e m i c
Press,
Inc.
Polyamines are ubiquitous components which are required for normal cell growth (1). Although these polycations have been implicated in the regulation of major cellular functions, such as DNA, RNA and protein synthesis, their mechanism of action at the molecular level remain obscure. We have previously suggested that polyamines may express at least part of their biological action through an effect upon a multifunctional protein kinase, i.e. casein kinase II (CKII). This hypothesis was based on the major following observations : (i) The activity of CKII is markedly activated in vitro by polyamines, spermine being the most potent (2, 3) ; (ii) CKII tightly associates with nuclear preparations in vitro and this kinase nuclear uptake is markedly enhanced in the presence of polyamines (4) which also increased the binding of CKII to double stranded DNA (5) ; (iii) There exists a temporal relationship between the nuclear accumulation of
Abbreviations CKII : casein kinase II ; ACTH : adrenocorticotrophic hormone ; SDS : sodium dodecyl sulfate ; cDNA : complementary DNA.
945
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CKII and the increase in intracellular polyamine content in adrenocortical cells following exposure to their trophic hormone ACTH (6). Although an effect through the protein substrate conformation may contribute to the activation of CKII-dependent phosphorylations by polyamines (3), a direct interaction between these polycations and the e n z y m e itself has, to our knowledge, never been considered. Eukaryotie CKII is an oligomeric enzyme made of two different (or and B) subunits, with an tx2B2 stoichiometry (3, 7). We have recently expressed the isolated subunit cDNAs of the kinase in a baculovirus-directed-insect cell expression system. This system gives access to the isolated enzyme subunits as well as to a functional recombinant oligomeric enzyme (8). The present study addressed the question of a possible molecular interaction between polyamines and CKII and examined the contribution of each kinase subunit in this process. The results disclosed that among naturally occuring polyamines, CKII binds preferentially spermine with high affinity. In addition, the B subunit appears to play an essential role in this interaction. These observations support our w o r k i n g hypothesis suggesting that polyamines may be examined as intracellular messengers in the regulation of CKII activity. M A T E R I A L S AND M E T H O D S •
Materials
[3H]-spermine tetrahydrochloride (AS : 23.5 Ci/mmol) was purchased from New England Nuclear. Red-120 agarose, spermine, spermidine, cadaverine and polylysine were obtained from Sigma. Phenyl Sepharose was from Pharmacia. Spermine-agarose was from Pierce. Grace's insect cell culture m e d i u m was obtained from Gibco. The pEV55 Dmc~ and pEV55 Drab plasmids were a kind gift from CVC Glover (University of Georgia, USA). •
Methods
- R e c o m b i n a n t i s o l a t e d g. and B s u b u n i t s of C K I I were produced in Sf9 insect cells infected with baculo virus containing the full length eDNA of either CKII ot or CKII B subunit (8). Co-infection with both viruses yielded a recombinant enzyme with an 0t2132 stoichiometry. Active recombinant ct subunit and oligomeric kinase were purified to near homogeneity as in (8). The recombinant B subunit was purified through 3 chromatographic steps : the soluble Sf9 cell extract was loaded onto a phenyl sepharose column previously equilibrated with 10 mM Tris HC1, pH 7.5, 1 mM DTT, 1 % glycerol, (TDG buffer) containing 0 . 1 % Triton X-100 and 1 M NaC1. A 1 to 0 M linear NaC1 gradient in TDG buffer containing 0 . 1 % Triton X-100 was applied and the B subunit was detected in collected fractions by western blot assay. After dilution to a final concentration of 0.1 M NaC1, the active fractions were applied onto a spermine agarose column equilibrated in TDG buffer containing 0 . 1 % Triton X-100. A 0-1.2 M linear NaC1 gradient in the same buffer was applied. The last step used a Red-120 agarose 946
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
column, yielding a 13 subunit which was 90 % pure, as judged by coomassie blue staining. - B i n d i n g of s p e r m i n e was assayed using a gel filtration assay. Aliquots of recombinant proteins were incubated (5-15 min at 4°C) with 0.5 ~tM [3H]spermine (_= 106 cpm) and different concentrations of unlabeled polyamines in 80 ~tl of TDG buffer containing 1 mg/ml BSA. The mixtures were then rapidly centrifuged through small Sephadex G-50 superfine columns, at 4°C, according to (9). Bound [3H]-spermine was determined by radioactive counting of the excluded volume, after substraction of the blank values (usually less than 0.2 % of the input radioactivity) in the absence of recombinant proteins. - W e s t e r n blot a n a l y s i s were performed using an antiserum prepared against Drosophila casein kinase II (10) that recognized both the a and 13 subunits of the enzyme. RESULTS
1. Interaction of spermine with casein kinase II subunits The ability of CKII to bind [3H]-spermine was examined with the purified recombinant CKII a and 13 subunits and the recombinant oligomeric 0~2132 enzyme. As illustrated in Figure 1A, increasing amounts of the oligomeric form of the kinase were able to bind [3H]-spermine. When tested at the same concentrations, the isolated 13 subunit was also found to bind the polyamine, although with a lower efficiency than the oligomeric enzyme. On the other hand, the isolated 0c subunit which bears the catalytic site of the enzyme (11, 12) was unable to bind [3H]-spermine in a detectable fashion. We next examined the interaction of the CKII isolated subunits with immobilized spermine. Recombinant polypeptides were loaded on spermine-agarose and were eluted by increasing NaC1 concentrations. The experiments depicted in Figure 1B showed that the cx subunit was eluted at a relatively low salt concentration (0.4 M NaC1), whereas the isolated 13 subunit was eluted at 0.6 M NaC1. Under the same conditions, elution of the oligomeric CKII required 0.9 M salt concentrations. Since the behavior of the different polypeptides through this affinity system should reflect their relative affinities for the immobilized spermine, these observations confirm that CKII is a polyamine binding protein as well as its B subunit. By contrast, the tx subunit showed a low polyamine affinity only detectable with the chromatographic method. Interestingly, the oligomeric form of the enzyme exhibited a polyamine binding activity, higher than that exhibited by the isolated 13 subunit. 2. Kinetic p a r a m e t e r s of the spermine-CKII interaction Time-course experiments show that the binding reaction reached a plateau after 5 min of incubation at 4°C (not shown). When recombinant 947
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B
(~'
NaCl [hl]
200
97 68 43
/3 200 97 68
NaCl[~l
43 alpha
29
20 .beta
A ~"
18
18
.w
1 2~"
.w
(kOa
6-1
.
(kDa
/
6s
2
29
~ 0 i~J. ~" 0
~, 20
, .
. 40
recombinant subunits
Figure
1 .
. 60
.m
18 HW (kOa
,
80 (nM)
Interaction
of
spermine
with
the
isolated
recombinant subunits of casein kinase II. A. Increasing concentrations of recombinant ~ subunit ( R - I ) , B subunit ( e - e ) or recombinant oligomeric a2132 CKII (UI-UI) were incubated with 0.5 ~tM [3H]-spermine for 15 min at 4°C. Bound [3H]-spermine was determined by gel filtration, as described in Materials and Methods. B. Aliquots (2.4 Ixg protein) of recombinant a or 1~ subunits or oligomeric et21~2 casein kinase II were loaded on spermine agarose columns (100 ~tl). The columns were eluted stepwise by 0.3 ml of TDG buffer containing increasing concentrations of NaCI. Proteins in each fraction were precipitated with 10% TCA, analysed on a 12% SDS polyacrylamide gel and visualized by coomassie blue staining.
oligomeric CKII was incubated at 4°C with 0.5 ~tM [3H]-spermine in the presence of increasing concentrations of unlabeled spermine, a typical saturation curve was obtained (Figure 2). The binding sites of the enzyme were saturated at 200 ~tM spermine with an IDs0 of 50 ~M. A Scatchard plot analysis (13) of these disclosed at least two types of binding sites ( F i g u r e 2). A p p a r e n t d i s s o c i a t i o n c o n s t a n t s of 11.5 x 10-6M and 136 x 10-6M were calculated for the two binding systems, respectively. The corresponding binding capacities were 0.12 x 10-6M (1.7 sites/mol of CKII) and 0.77 x 10-6M (10.8 sites/mol of CKII), respectively. 3. Specificity of the polyamines CKII interaction Recombinant oligomeric CKII was incubated with [3H]-spermine in the presence of different unlabeled polyamines and bound [3H]-spermine was determined. As shown in Table I, at a 50 IxM concentration, spermine gave a 50 % displacement of bound [3H]-spermine whereas spermidine and cadaverine, at the same concentration, were ineffective. However, a synthetic polycationic compound, such as polylysine which is known to 948
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lO t,i,-X
E
o.
tl..
~4
0 "ID
t-. o
c
E Q.
?
12.
2
~
0
.~ 100
. . . . . 200 300
400
B (nM)
10~ 8
"1"
1'00
2'00
spermine (~M) Analysis of spermine binding to casein kinase II. Aliquots (71 nM) of recombinant oligomeric CKII were incubated for 15 min at 4°C with 0.5 txM [3H]-spermine and increasing concentrations of unlabeled spermine. Data were plotted according to Scatchard (inset). Figure
2.
stimulate CKII activity (7), was found to be a potent competitor of the [3H]-spermine-CKII interaction. 4 . E f f e c t of s p e r m i n e on m o n o m e r i c a n d o l i g o m e r i c recombinant CKII activity We have shown previously that the o~ subunit bears the catalytic site of the enzyme (11) and that the B subunit is required for optimal catalytic
Table I Effect of different unlabeled polyamines on the binding of [3H]-spermine to recombinant oligomeric casein kinase II UNLABELED
POLYAMINE
(50 ~M)
[3H]-SPERMINE
- NONE
11 5 11 13 5
- SPERMINE -
-
SPERMIDINE CADAVERINE
- POLYLYSINE
BOUND (CPM)
903 549 198 059 339
CKII aliquots (35 nM) of recombinant oligomeric CKII were incubated for 15 min at 4°C with 0.5 ~tM [3H]-spermine and different unlabeled naturally occurring polyamines or polylysine. Bound [3H]-spermine was determined by the gel filtration assay. 949
V o l . 180, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
~"
4000,
•~
3oo0,
2000,
J
lOOO,
o
0
-
0.0
•
0.2
-
i
-
0.4
=
-
0.6
,
0.8
-
,
1.0
SPERMINE [mM]
Fieure
3.
Effect of spermine on o l i g o m e r i c and m o n o m e r i c r e c o m b i n a n t casein kinase II activity.
Recombinant c~ subunit (B-B) or recombinant oligomeric c t 2 6 2 ( 0 - 0 ) CKII were assayed for protein kinase activity with topoisomerase II as substrate in the presence of 2 mM magnesium and increasing amounts of spermine. Phosphorylated proteins were analyzed by electrophoresis on a 7.5 % SDS polyacrylamide gel and 32p incorporation in topoisomerase II was measured by scintillation counting.
activity (12). Using isolated recombinant subunits, we examined the contribution of each subunit in the modulation of CKII activity by polyamines. The kinase activity of monomeric and oligomeric forms of CKII was assayed, using topoisomerase II as the protein substrate, in the presence of increasing concentrations of spermine. As shown in figure 3, p h o s p h o r y l a t i o n by CKII was stimulated 3 fold in the presence of spermine, this effect reaching a plateau for 200 ~tM spermine. By contrast, spermine at the same concentrations was without effect on the kinase activity of the isolated o~ subunit. Therefore, this experiment suggests that the polyamine modulates the oligomeric CKII activity, following its binding to the 6 subunit of the enzyme. DISCUSSION The catalytic activity of casein kinase II has been reported to be sensitive to submillimolar concentrations of polyamines (3, 14). The present study discloses that CKII is clearly a polyamine-binding protein. Among the major naturally occuring polyamines, the enzyme exhibits the best affinity for spermine, which is also the most potent as CKII activator (3). Under its native form, as an oligomeric protein with an ¢~21~2 subunit stoichiometry, CKII binds spermine to at least two types of binding sites with affinity constants in the micromolar range. An interesting observation came from binding studies using recombinant isolated a and B 950
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
subunits, as compared to the oligomeric form of the kinase. Spermine binding could be assayed only with the isolated g subunit, whereas no detectable polyamine remained bound to the ~ peptide, following the rapid gel filtration method employed to separate bound from free ligand. Although this non equilibrium method might contribute to this result, it is in line with the weak affinity of the ~ subunit for polyamine, as shown by its behavior on a spermine-coupled solid support. However, on identical molar basis, the spermine binding affinity of the oligomeric enzyme was higher than that of the isolated 13 subunit. This suggests that the g subunit conformation is different when isolated as compared to when it is integrated in the oligomeric o~21~2 structure, resulting in conformational adjustment and polyamine affinity increase. However, a contribution of the o~ subunit in the binding properties of the native enzyme is not excluded. The present study discloses that the activity of the isolated CKII ¢x subunit, which bears the catalytic site of the enzyme, was not sensitive to polyamine. The activation of CKII by spermine required the presence of the 13 subunit and the oligomeric structure of the kinase. This points to a possible key role of the g subunit in the regulation of CKII. The finding that CKII is a polyamine binding protein with a Kd in the micromolar range comes in support for a significant biological role of this interaction in living ceils, in which polyamines may reach millimolar concentrations (2, 15). In addition, the binding affinity of the different naturally occuring polyamines is well correlated with their relative potency as CKII activators (16). Intracellular p o l y a m i n e level is highly d e p e n d e n t upon growth factors and trophic hormones which have been shown to induce ornithine decarboxylase and cell polyamine uptake activation (2, 15). Polyamines increase the affinity of CKII for chromatine and double stranded DNA (4, 5) and this would be in line with the nuclear CKII accumulation observed under cell growth stimulation (4). Polyamines are known to bind to DNA and to promote chromatin condensation (17, 18). These polycations may act as bifunctional ligands with an affinity for both DNA and CKII and may thus modulate CKII-DNA interaction. The present findings support the idea of a functional link between polyamines and CKII in living cells, especially in the response to trophic hormone and growth stimuli.
Acknowledgments This work was supported by the INSERM (U244), the Commissariat l'Energie A t o m i q u e (DSV/DBMS), the Fondation pour la R e c h e r c h e M6dicale, the GEFLUC and the Ligue Nationale Fran~aise contre le Cancer.
951
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18.
Mamont, P. S., Bey, P. & Koch-Weser, J. (1980) In Polyamines in Biochemical Research. pp 147-183. John Wiley, New York. Feige, J. J., Cochet, C. & Chambaz, E. M. (1985) In Recent Progress in Polyamine Research (L. Selmeci, M. E. Brosnan, and N. Seiler, Eds) vol. 13, pp 181-190. Akadamiai Kiado, Budapest. Cochet, C. & Chambaz, E. M. (1983) Mol. Cell. Endocrinol. 30, 247-266. Filhol, O., Cochet, C. & Chambaz, E.M. (1990) Biochemistry 29, 99289936. Filhol, O., Cochet, C. & Chambaz, E. M. (1990) Biochem. Biophys. Res. Commun. 173, 862-871. Filhol, O., Loue-Mackenbach, P., Cochet, C. & Chambaz, E.M. (1991) Biochem. Biophys. Res. Commun., in press. Pinna, L.A. (1990) Biochim. Biophys. Acta, 1054, 267-284. Filhol, O., Cochet C., Wedegaertner, P., Gill, G.N. & Chambaz, E.M. (1991) Biochemistry, in press. Penefski, H.S. (1977) J. Biol. Chem. 252, 2891-2899. Padmanabha, R. & Glover, C.V.C. (1987) J. Biol. Chem. 262, 1829-1834. Feige, J.J., Cochet, C., Pirollet, F. & Chambaz, E.M. (1983) Biochemistry 22, 1452-1459. Cochet, C. & Chambaz, E.M. (1983) J. Biol. Chem. 258, 1403-1406. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Cochet, C., Job, D., Pirollet, F. & Chambaz, E. M. (1980) Endocrinology 106, 750-757. Heby, O. (1981) Differentiation 19, 1-20. Traugh, J.A., Lin, W.J., Takada-Axelrod, F. & Tuazon, P.T. (1990) Ir Advances in Second Messenger and Phosphoprotein Research. The Biology and-Medicine of Singal Transduction. (Y. Nishizuka, M. Endo, C. Tanaka, Eds) Vol. 24, Raven Press. Gosule, L.C. & Schellman, J.A. (1976) Nature 259, 333-335. Marton, L.J. & Feuerstein, B.G. (1986) Pharm. Res. 3, 311-317.
952
