From the Cellular and Molecular Biochemistry Research Laboratory, Department of Laboratory Medicine and Pathology, University of Minnesota and the Department of Veterans Affairs Medical Center, Minneapolis, Minnesota 55417
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ABSTRACT |
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Protein kinase CK2 has been implicated in control of cell growth and proliferation. Since growth stimuli evoke its preferential association with chromatin and nuclear matrix, we examined the dynamics of CK2 in nucleosomes fractionated on the basis of their transcriptional activity in the rat prostate. In this model, androgens induce expression of androgen-dependent genes but inhibit the androgen-repressed genes, whereas absence of androgens has the reverse effect. The level of CK2 was higher in the active than in inactive nucleosomes from normal prostate. Differential alterations in the levels of CK2 activity in the transcriptionally active versus inactive nucleosomes were evoked by androgen deprivation or administration. Comparison of the distribution of CK2 in active and inactive nucleosomes under varying androgenic conditions showed that the relative CK2 activity intrinsic to the transcriptionally active nucleosomes remained fairly stable, concordant with gene activity specific to the androgenic status. However, CK2 associated with inactive nucleosomes declined to a minimal level on androgen deprivation but increased rapidly on androgen administration (reflecting expression of multiple androgen-dependent genes). We suggest a role for CK2 in promoting the conformational transition of inactive nucleosomes to the active form and in the function of transcriptionally active nucleosomes.
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INTRODUCTION |
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CK21 (formerly known as
casein kinase 2 or II) is a ubiquitous protein serine/threonine kinase
that has been implicated in multiple cellular functions, including
growth control and proliferation. The enzyme is a tetramer consisting
of ,
', and
subunits (42, 38, and 28 kDa, respectively) with
the possible
2
2,
'
2,
or
'
'
2 configuration. There appear to be several
substrates for CK2 in both the cytoplasm and the nucleus. Among the
putative nuclear substrates are RNA polymerases, topoisomerase II,
protein B23, nucleolin, SV40 large T antigen, certain protooncogene
products, and growth factors, as well as certain chromosomal nonhistone proteins (1-6). The involvement of CK2 in phosphorylation of these
growth-related proteins has provided additional support for its role in
growth control.
CK2 appears to be dynamically regulated with respect to its nuclear localization (7, 8) which is manifested by its preferential association with chromatin and nuclear matrix (7, 9, 10). The association of CK2 with these compartments is modulated in response to growth stimuli (7, 9-12) which is of interest since both chromatin and nuclear matrix play fundamental roles in genomic activity and cell proliferation. Nuclear matrix, the structural framework that represents residual components of the nuclear lamina-pore complex, nucleoli, and fibrogranular internal matrix, is believed to play a fundamental role in chromatin organization, regulation of gene activity, and cell proliferation (13-17). The transcriptional machinery resides in chromatin, and the nucleosome is its repeating subunit structure. As might be expected, nucleosomes appear to undergo dynamic conformational transition from inactive to active forms under specific conditions (for reviews see Refs. 18 and 19).
Recent studies have shown that transcriptionally active and inactive nucleosomes can be fractionated on the basis of their conformation, as reflected by the accessibility of thiol groups in the histone H3 (20, 21). This procedure has been successfully employed to fractionate nucleosomes from yeast, 3T3, HeLa S3, COLO 320, and rat liver cells (20, 21). Considering the dynamic association of CK2 with the nuclear matrix and chromatin (9, 11), we decided to examine the role CK2 plays in the nucleosomes in relation to their transcriptional activity by employing the experimental model of androgenic regulation of the prostate. Withdrawal of androgen from adult rats results in apoptosis in the prostate epithelial cells, whereas androgen administration to castrated rats evokes a regrowth of the gland with activation of genomic activity within 1-4 h (22-24). An interesting feature of this model is that during regression of the gland (and overall cessation of the androgen-dependent gene activity), certain genes called androgen-repressed genes (e.g. clusterin) are activated. On the other hand, androgenic stimulation evokes early expression of genes such as C3 and, over time, of a large number of prostatic androgen-dependent genes, whereas the androgen-repressed genes are suppressed (25-27). These markers can thus be used to differentiate the type of nucleosomes isolated from prostatic tissue under varied androgenic conditions. We have used this paradigm previously to demonstrate that CK2 is preferentially associated with the chromatin and nuclear matrix fractions isolated from purified nuclei and that this association is dynamically regulated in a spatiotemporal manner in response to altered androgenic status (7, 9, 11, 12). In the present work, we have documented that CK2 is associated to a greater extent with the transcriptionally active than the inactive nucleosomes. However, androgenic alterations in the transcriptional activity of the prostate evoke a differential regulation of CK2 associated with the active and inactive nucleosomes suggesting a possible role for CK2 in nucleosome organization and function.
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EXPERIMENTAL PROCEDURES |
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Materials
Animals--
Male Sprague-Dawley rats weighing 295-325 g
(Harlan Sprague-Dawley, Indianapolis, IN) were used as the source of
ventral prostate or liver. The animals were maintained under standard
conditions and were orchiectomized via the scrotal route under Metofane
anesthesia as described previously (9). For restoration of androgens, animals were injected with 5-DHT in sesame oil (1 mg/100 g of body
weight); control animals received an appropriate volume of the vehicle
alone.
Chemicals--
Synthetic dodecapeptide substrate
(Arg-Arg-Arg-Ala-Asp-Asp-Ser-Asp-Asp-Asp-Asp-Asp) for assay of CK2
activity was purchased from Peptide Technologies Corp. (Gaithersburg,
MD). Affi-Gel 501 was purchased from Bio-Rad. Micrococcal nuclease was
supplied by Worthington. Mouse monoclonal antibody raised against the
last 262 amino acids of the C-terminal end of the CK2- was from
Transduction Laboratories (Lexington, KY). All reagents were of the
highest purity available.
Methods
Preparation of Nucleosomes-- Nuclei were isolated from pooled ventral prostate tissue (15 prostate glands from normal, 15 from 2-day castrated, or 45 from 6-day castrated rats) or from liver as described previously (9-11) except that 5 mM sodium butyrate, 0.1 mM EPNP, and 0.5 mM PMSF were included throughout the preparative procedure. DNA content was assessed by measuring the A260 or by the Burton method (28), as appropriate. Nucleosomes were isolated from purified nuclei by adapting previously described procedures (20, 21). Briefly, purified nuclei were suspended at a concentration of 1 mg of DNA/ml in buffer A (consisting of 10 mM Tris-HCl, pH 7.4, 25 mM KCl, 25 mM NaCl, 0.5 mM CaCl2, 5 mM MgCl2, 5 mM sodium butyrate, 0.1 mM EPNP, and 0.5 mM PMSF). This suspension was warmed at 37 °C for 2 min followed by addition of micrococcal nuclease at a final concentration of 10 units/ml. The nuclease digestion was carried out for 5 min at 37 °C and was terminated by the addition of 0.2 M EGTA, pH 7.0, to a final concentration of 2 mM. These conditions were established so as to strictly ensure that 10-11% of the DNA was digested. After the tubes stood on ice for 10 min, the samples were centrifuged at 10,000 × g for 20 min. The supernatant phase containing the released nucleosomes was collected.
Fractionation of Active and Inactive Nucleosomes-- The supernatant fraction was treated with 0.2 M EDTA to a final concentration of 5 mM. The sample was then loaded onto an organomercurial agarose column (Affi-Gel 501) equilibrated with buffer B (10 mM Tris-HCl, pH 7.4, 25 mM KCl, 25 mM NaCl, 5 mM EDTA, 5 mM sodium butyrate, 0.1 mM EPNP, and 0.5 mM PMSF). To remove the unbound nucleosomes, the column was eluted at a flow rate of 20 ml/h with buffer B until A260 reached the base line. This fraction represented the transcriptionally inactive nucleosomes. The organomercurial-bound nucleosomes were then eluted with buffer C (buffer B with 0.5 M NaCl). Column fractions were monitored for A260 to identify the peak. Fractions corresponding to the peak were pooled. Finally, elution with buffer C containing 10 mM DTT was used to isolate the bulk of transcriptionally active nucleosomes. The pooled fractions from various peaks were concentrated with the aid of a Centricon 10 (Amicon, Inc., Beverly, MA) and then washed twice with 10 volumes of TMED buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, and 0.5 mM DTT, pH 7.9) containing 0.2 M NaCl. The final protein and DNA concentration were determined as described previously (28, 29).
Sephacryl S-300 Superfine (Amersham Pharmacia Biotech) column chromatography was carried out to determine if there was a contamination of free CK2 in the nucleosomal preparations. The resin was equilibrated in buffer B in a 0.9 × 12 cm column, and the nucleosomal sample was loaded in a volume of 200 µl in buffer B. The elution was carried out with buffer B at a flow rate of 8 ml/h, and 2-ml fractions were collected and analyzed for A260 and CK2 activity.Assay of CK2 Activity in Nucleosomal Preparations--
Assay of
CK2 activity in various nucleosomal preparations was carried out by
employing a synthetic dodecapeptide (30). The reaction medium consisted
of 30 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, 150 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 50 mM 2-glycerophosphate, 0.2 mM dodecapeptide substrate, 0.05 mM
[-32P]ATP (specific radioactivity 3 × 106 dpm/nmol of ATP). The reaction was started by the
addition of the enzyme source (e.g. various nucleosome
preparations). A paper-binding method was used to assay the
32P incorporated into the dodecapeptide substrate (9-11,
31). All assays of CK2 were carried out in triplicate over a time
course to ensure the linearity of the reaction. Each experiment was
repeated at least three times.
Identification of Active and Inactive Prostatic Nucleosomes with
C3 and Clusterin Probes--
To characterize the prostatic
transcriptionally active and inactive nucleosomes, the DNA in various
preparations was probed with cDNAs for C3 and clusterin genes, as
these genes are expressed in a testosterone-dependent or
testosterone-repressed manner, respectively. DNA was prepared from
nucleosomes by following standard purification procedures. Briefly,
nucleosomes were digested with 50 µg/ml RNase A (DNase-free) at
37 °C for 30 min. After SDS was added to a final concentration of
0.1% (v/v), the samples were digested with 100 µg/ml Proteinase K
(DNase-free) at 37 °C for 2 h and then extracted twice with
phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform.
The DNA was precipitated by the addition of 50% isopropyl alcohol, 2.0 M ammonium acetate (final concentrations) at 20 °C
overnight, and the pellets were washed with 70% ethanol and air-dried
prior to being dissolved in 100 µl of TE buffer (10 mM
Tris-HCl, pH 7.4, 1 mM EDTA). Genomic DNA was also isolated
by using a kit (Puregene, Minneapolis, MN). The DNA concentration was
measured by the Burton method (28). A sample of DNA (20 µg) was
slot-blotted onto Nytran membrane (Schleicher & Schuell) and used for
hybridization with C3 and clusterin probes. The C3 cDNA probe used
is a 150-base pair PstI fragment from pA34 (received from
Dr. Malcolm Parker, London), and the clusterin probe is a 1560-base
pair EcoRI fragment from pGEMB4 (received from Dr. Martin
Tenniswood through Dr. Mark Rosenberg, University of Minnesota,
Minneapolis). The probes were labeled with [
-32P]dATP
by the random priming method. The blotted membranes were incubated
overnight with the radiolabeled probes in a standard hybridization
buffer at room temperature, washed under high stringency conditions at
50 °C, and exposed on Kodak BioMax MR film for 12 days.
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RESULTS |
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Characteristics of Nucleosomes from Rat Ventral Prostatic
Tissue--
Fig. 1 shows the
chromatographic profile of the active and inactive nucleosomes isolated
from rat ventral prostate nuclei by utilizing the Affi-Gel 501 column
fractionation. The first peak (flow-through) represents the
transcriptionally inactive nucleosomes, whereas the second large peak
represents the transcriptionally active nucleosomes eluted with 10 mM DTT (Fig. 1). Unlike the previous experience (20, 21),
elution with 0.5 M NaCl yielded only a negligible peak and
was not investigated further. A somewhat larger peak was observed with
NaCl elution for liver nucleosomes (result not shown). The
inset shows analysis of the DNA pattern of the fractionated
nucleosomes as determined by electrophoretic separation of DNA on a
1.6% agarose gel followed by ethidium bromide staining. As expected
from previous observations (20, 21), the inactive and active
nucleosomes yielded a similar profile. Because our studies involved
analyses of the prostatic tissue from rats subjected to altered
androgenic status, we also established that isolation profiles of the
inactive and active nucleosomes were similar from animals castrated for
48 or 144 h, or 144 h and then treated with 5-DHT for
4 h (not shown).
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Association of Protein Kinase CK2 with the Nucleosomes-- In previous work, we have shown the translocation of CK2 to the chromatin of rat prostate in response to androgenic stimulus (9). We therefore decided to examine the nature of CK2 association with the transcriptionally inactive and active nucleosomes isolated from rat prostate. As expected, the amount of DNA in the inactive nucleosome fraction was 2-3 times greater than that in the active nucleosomes. Associated CK2 activity was detected in nucleosomes (inactive as well as active) isolated from normal prostate and liver (Table I), but a severalfold greater amount of CK2 was present in the transcriptionally active than in the inactive nucleosomes. The results are similar when calculated per unit of the DNA or protein in each nucleosome preparation.
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Protein Kinase CK2 in Nucleosomes from Prostatic Tissue of Androgen-deprived Rats-- On androgen deprivation, the CK2 activity in the inactive nucleosomes declined precipitously so that compared with the control values it was reduced by 94 and 98% at 48 and 144 h post-orchiectomy, respectively (Table II). The active nucleosomes under these conditions also showed a decline in associated CK2; however, this decline was of a relatively smaller magnitude, being on average 78 and 87% at 48 and 144 h post-orchiectomy, respectively. The persistence of CK2 in the transcriptionally active nucleosomes may reflect ongoing activity related to expression of androgen-repressed genes such as clusterin after androgen deprivation (25-27). The results were essentially the same when expressed per unit of protein or DNA (Table II).
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Protein Kinase CK2 in Nucleosomes from Prostatic Tissue of Androgen-treated Castrated Rats-- Administration of a single dose of androgen to previously castrated rats evokes an early (within 4 h) expression of certain genes such as C3 while shutting down the expression of testosterone-repressed genes such as clusterin. This response is followed within 8 h by the expression of bulk mRNA in a target tissue-dependent manner. We, therefore, examined the changes in CK2 associated with the inactive and active nucleosomes in response to early changes in transcriptional activity evoked by androgen within 4 h. The results in Table II show that there was a significant increase in CK2 association with the active and inactive nucleosomes within 4 h after testosterone administration to 144-h castrated rats. Interestingly, there was a hint of a greater rate of increase in CK2 associated with the inactive nucleosomes (Table II).
Immunoblot analysis of CK2 associated with inactive and active nucleosomes corresponding to the data shown in Table II is presented in Fig. 4. The immunoreactive CK2-
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DISCUSSION |
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Chromatin is the site of transcriptional activity in the nucleus. Expression of genes in a cell is a dynamic and highly regulated process that is constantly modulated by the influence of external and internal signals. Thus, it is reasonable that the subunit structure of chromatin called the nucleosome would be subject to dynamic conformational changes in accord with the transcriptional activity of the genome (e.g. see Refs. 17-19). Investigation of the mechanisms underlying the conformational changes that define the differences in the functional activities of the inactive and active nucleosomes is therefore of intense current interest. To this end, methods have recently been developed to fractionate the nucleosomes on the basis of their transcriptional activity (20, 21). Because CK2 has been implicated in the regulation of cell growth and proliferation, we have employed these methods to investigate the dynamics of the CK2 signal in the transcriptionally active and inactive nucleosomes from rat ventral prostate in which genomic activity can be modulated by altering the androgenic status of the animal. In our previous studies employing this experimental model, we documented that CK2 rapidly associates with chromatin and nuclear matrix in response to the androgenic growth stimulus (9-12). We have now demonstrated that differential association of CK2 occurs in the nucleosomes in normal cells such that it is present to a much greater extent in the transcriptionally active than the inactive nucleosomes. However, dynamic changes occur in the CK2 associated with these nucleosomes when transcriptional activity in the chromatin is altered, such as by changing androgen levels (i.e. by castration or administration of androgen to castrated rats). The observed dynamics of CK2 alterations in this experimental model must be considered in the context of the temporal androgenic response of prostatic epithelial cells, as discussed subsequently.
Androgen deprivation in the adult rat leads to a cascade of effects culminating in programmed death (apoptosis) of more than 80% of the prostatic epithelial cells within 5-7 days (27). However, the earliest response of the cells is the cessation of expression of a large variety of the androgen-dependent genes. Concomitantly, there is a specific rapid expression of androgen-repressed genes such as the clusterin gene (25-27). Thus, even during the tissue regression phase, it is possible to isolate both the transcriptionally active and inactive nucleosomes from chromatin. On the other hand, administration of androgen to 6-day castrated animals evokes regrowth of the prostate. The earliest events are an expression of androgen-dependent messages, including the C3 gene (within 4 h after androgen administration), followed by expression of bulk mRNA (i.e. expression of many genes) and protein synthesis. Under these conditions, the androgen-repressed genes are not expressed. In this case, the inactive nucleosomes would represent the fraction rich in sequences not being expressed (such as clusterin), whereas the active nucleosomes would represent the fraction expressing androgen-dependent genes such as C3.
A number of observations emerge from this study which suggest distinct roles for CK2 in the active and inactive nucleosomes. It appears that CK2 activity is high in the transcriptionally active nucleosomes compared with the inactive nucleosomes under the various tissue-specific changes in the transcriptional activity in the prostate. The proportionally high level of CK2 activity in transcriptionally active nucleosomes (compared with that in inactive nucleosomes) accords with a role in the control of gene activity. On androgen deprivation, CK2 associated with the active nucleosomes declines relatively slowly and remains proportionally high compared with that in the inactive nucleosomes. This finding suggests that CK2 association is required for the function of active nucleosomes.
Equally remarkable is the significant amount of CK2 activity in the transcriptionally inactive nucleosomes in normal prostate, and its dramatic modulation with altered growth conditions which suggests a distinct role for CK2 in this fraction. During the induction of changes in the prostate by androgen withdrawal, the level of CK2 in the inactive nucleosomes declines rapidly so that at 144 h, only a minimal amount is detectable in these nucleosomes. This loss of CK2 associated with inactive nucleosomes is commensurate with the cessation of androgen-dependent gene activity. However, on administration of androgen to castrated rats which initiates extensive transcriptional activity, the level of CK2 localized to the inactive nucleosomes increases at a rapid rate. An interpretation of this observation is that rapid association of CK2 with the inactive nucleosomes may be needed to evoke their transition from the inactive to active conformation. This is supported by the fact that during androgen deprivation most of the gene activity is shut down, precluding the need for a transition of inactive nucleosomes to the transcriptionally active conformation. On the other hand, androgenic stimulus evokes the expression of numerous genes in the prostate over a sustained period (e.g. see Ref. 32), thus necessitating the generation of transcriptionally active nucleosomes. The increasing association of CK2 observed in this experimental model thus hints at a possible role for CK2 in the conformational transition of the nucleosomes from transcriptionally inactive to transcriptionally active state.
Future work will determine the nature of protein substrates for CK2 in the active and inactive nucleosomes. However, CK2-mediated phosphorylation of proteins such as B23 and nucleolin which are known to be involved in the rRNA synthesis has been documented (33, 34). Also of interest are the observations on the association of CK2 with several growth-related nuclear proteins and transcription factors (35-41) which may participate in mediating the CK2 signal in the nucleus. The present work provides further evidence that translocation of CK2 to its sites of action may serve as a mechanism of its intracellular regulation.
In summary, these studies have demonstrated that CK2, a nuclear protein kinase signal that has been implicated in growth control, is differentially associated with transcriptionally active and inactive nucleosomes and that this association is dynamically regulated in response to altered transcriptional activity. Furthermore, it appears that association of CK2 with the transcriptionally inactive nucleosomes may participate in promoting their transition to the active conformation.
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FOOTNOTES |
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* This work was supported in part by U. S. Public Health Service Research Grant CA-15062 awarded by the National Cancer Institute, Department of Health and Human Services, and by the Medical Research Fund of the Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cellular and Molecular
Biochemistry Research Lab. (151), Veterans Affairs Medical Center, One
Veterans Dr., Minneapolis, MN 55417. Tel.: 612-725-2000 (ext. 2594);
Fax: 612-725-2093; Email: ahmedk{at}maroon.tc.umn.edu.
1
The abbreviations used are: CK2, formerly casein
kinase 2; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane;
5-DHT, 5
-dihydrotestosterone; PMSF, phenylmethylsulfonyl
fluoride; DTT, dithiothreitol.
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REFERENCES |
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