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(Received for publication, March 15, 1995; and in revised form, June 21, 1995) From the
Protein B23 is a nucleolar and nuclear matrix-associated
phosphoprotein that is involved in ribosome synthesis. Its expression
and phosphorylation in rat ventral prostate, an androgen target organ,
are profoundly influenced by androgens. Induction of programmed cell
death (apoptosis) in the prostatic epithelium by androgen deprivation
in the animal induces an early decline in protein B23 in the absence of
a corresponding loss of protein B23 mRNA. We have now demonstrated that
prostatic nuclei retain the ability to transcribe the B23 mRNA and that
a significant amount of this mRNA persists even after 7 days of
androgen deprivation when >80% of the prostatic epithelial cells
have undergone apoptosis. The B23 mRNA from these nuclei is also
translatable in vitro. However, the majority of the B23 mRNA
is associated with free and short-stretch polysomes, which may account
for the castration-induced decline in synthesis of protein B23 in
vivo. In addition, the mechanism of down-regulation of protein B23
in apoptotic prostatic cells appears to relate to two coordinate
signals, which include loss of phosphorylation of the protein as well
as the expression of a protease active toward dephosphorylated protein
B23, under these conditions.
Programmed cell death or apoptosis has been described in diverse
biological systems mediated by a variety of
signals(1, 2, 3) , and the response of the
prostatic glandular epithelium to androgen withdrawal is one of the
frequently studied models(3, 4) . Androgen withdrawal
via orchiectomy in the rat induces an energy-dependent cascade of
biochemical and morphological changes that lead to the death of 80% of
the secretory epithelium between days 2 and 6(4) .
Morphologically, the nucleolus dissolves(5) , and the chromatin
is condensed and fragmented to form the membrane-bound apoptotic bodies
that appear in appreciable numbers after 2 days of androgen
deprivation(6) . Biochemically, there is an increase in
intracellular calcium and enhancement of calcium/magnesium-dependent
endonuclease activity that reaches its maximum 4-5 days after
androgen withdrawal (7) . Apoptosis in the prostate is
associated with modulation of gene expression so that the expression of
some genes is repressed and that of others is enhanced. Among the
latter are c-fos, c-myc, heat shock
proteins(8) , glutathione S-transferase(9) ,
and TRPM-2/sulfated glycoproteins(10) . On the other hand, the
synthesis of ribosomes, especially their assembly into polysomes,
markedly declines after androgen deprivation (11, 12) . Also, prostatic ribosomes from animals
treated with 5 We
have been interested in the mechanisms underlying the decline in
prostatic rRNA synthesis and assembly after androgen deprivation.
Protein B23, a conserved phosphoprotein that is localized to the
granular and fibrillar regions of the nucleolus where rRNA synthesis
and assembly take place(13, 14) , seems to have
different functions at different stages of the cell cycle. It is
capable of binding nucleic acids and exhibits both helix-destabilizing (15) and ribonuclease activities that implicate the protein in
preribosomal RNA processing and transport(16, 17) .
Various observations suggest that protein B23 plays a role in DNA
synthesis (18, 19) and might also have a structural
role as one of the components of the perichromosomal layer that is
involved in chromosome organization in mitosis (20) and as one
of the nuclear matrix-associated proteins(21) . Protein B23 is
phosphorylated by protein kinase CK2 in interphase (22) and by
p34 In a previous report, we documented that expression and
phosphorylation of protein B23 started to decline at a modest rate in
the first 24 h after androgen withdrawal (26) and that by 48 h
post-castration, B23 was undetectable despite the presence of B23 mRNA
up to 7 days of androgen withdrawal(26) . These changes in
expression coincided with the decline in ribosome synthesis and the
morphological alterations associated with apoptosis, suggesting that
protein B23 might be involved in these processes(26) . In the
present work, we have explored the mechanisms by which the prostatic
glandular epithelium controls the expression of protein B23 during
apoptosis. Our data indicate that regulation of protein B23 expression
after androgen withdrawal is not at the transcriptional level. There
appears to be no new cis-acting element that would hinder B23
mRNA translation in vitro. This mRNA shows a differential
pattern of association with prostatic polysomes in response to androgen
deprivation, but this effect alone might not explain the specific and
abrupt decline in protein B23 expression observed after 48 h of
androgen deprivation. It appears that the primary means of regulation
of protein B23 expression after androgen withdrawal is its proteolytic
degradation and that the decline in protein B23 phosphorylation as well
as the expression (or release of inhibition) of a specific protease(s)
after androgen withdrawal may be the prerequisites for protein B23
degradation.
Figure 1:
In vitro transcription of B23
mRNA in prostatic nuclei. The nuclear run-on assay was carried out on
prostatic nuclei isolated from normal control and castrated rats as
described under ``Methods.'' Lane a, normal control
rats; lane b, 4-day castrated rats; lane c, 7-day
castrated rats. Relative densitometric values for B23 were 1.0 (lane a), 0.8 (lane b), and 0.5 (lane c),
and those for
Figure 2:
Effect of androgen deprivation on
steady-state level of B23 mRNA using Northern blot analysis. Total
prostatic RNA was electrophoresed, transferred to nylon membrane, and
probed with randomly labeled B23 cDNA as described under
``Methods.'' Lanesa-c correspond to
prostatic total RNA from intact normal and 4- and 7-day castrated rats,
respectively. Relative densitometric values were 1, 0.8, and 0.4,
respectively.
Figure 3:
Effect of androgen deprivation on B23 mRNA
association with prostatic polysomes. Prostatic cytosol was
fractionated on a sucrose gradient, and RNA from different fractions
was extracted as described under ``Methods.'' Each fraction
was examined for the presence of B23 mRNA,
Figure 4:
Effect of androgen deprivation on
translation of protein B23 in vitro. Prostatic poly(A) mRNA
was isolated from animals at the indicated times (days (d))
after androgen withdrawal and translated in vitro, followed by
immunoprecipitation of protein B23 using the specific antibody as
described under ``Methods.''
Figure 5:
Effect of androgen on proteolytic
degradation of purified protein B23 added to prostatic homogenate in vitro. Purified radioiodinated protein B23 was incubated,
in a final reaction volume of 100 µl, with 20 µl of prostatic
cellular homogenate (20%, w/v) from intact or from 4-day castrated
rats. After incubation for the indicated periods of time, the reaction
was stopped by the addition of gel electrophoresis sample buffer. The
material was subjected to SDS-PAGE as described under
``Methods.'' The dried gel was exposed to Kodak X-Omat film
overnight at -70 °C. The toppanel shows
protein B23 incubated with prostatic homogenate obtained from intact
rats. The bottompanel depicts protein B23 incubated
with prostatic homogenate prepared from 4-day castrated rats. The
control lane in both panels included the addition of protease
inhibitors as described under
``Methods.''
Figure 6:
Effects of protein B23 phosphorylation
status on its susceptibility to proteolytic degradation. A,
effect of dephosphorylation of protein B23 on its proteolytic
degradation. Purified radioiodinated protein B23 was treated with
potato acid phosphatase followed by alkaline phosphatase prior to
incubation with prostatic cellular homogenate prepared from intact or
4-day castrated rats as described for Fig. 5. In all panels,
heparin and poly(Glu,Tyr) (poly GT) were added as inhibitors
of protein kinase CK2. After incubation for the indicated periods of
time, the reaction was stopped by the addition of gel electrophoresis
sample buffer. The sample was subjected to SDS-PAGE, and the dried gel
was exposed to Kodak X-Omat film overnight at -70 °C. The toppanel represents dephosphorylated protein B23
incubated with prostatic homogenate obtained from intact rats. The middlepanel shows phosphorylated protein B23
incubated with prostatic homogenate prepared from 4-day castrated rats.
The bottompanel depicts dephosphorylated protein B23
incubated with prostatic homogenate prepared from 4-day castrated rats.
The control lane in all panels included the addition of the protease
inhibitors listed under ``Methods.'' B, effects of
various conditions on proteolytic degradation of protein B23 and actin
by prostatic homogenate from castrated rats. Lane a,
The nature of
the role of phosphorylation of protein B23 in its proteolytic
degradation in prostate tissue from castrated rats was further
examined. The result in Fig. 6B (lane b),
employing recombinant protein B23.1, shows that dephosphorylated
protein B23 under conditions that favor dephosphorylation (i.e. presence of excess P Protein B23 plays an integral role in ribosome synthesis.
Changes in rRNA synthesis and assembly are among the earliest responses
to androgen action in the prostate. Employing this paradigm, we
previously examined the changes in prostatic protein B23
phosphorylation and level in response to androgen withdrawal (i.e. during induction of programmed cell death) and administration (i.e. during prostatic epithelial regeneration)(26) .
These studies established that although protein B23 expression declines
to undetectable levels by 48 h after androgen deprivation, the
steady-state level of its mRNA does not change as rapidly, being
B23 mRNA in the prostate gland of
the intact animal exists mainly associated with long-stretch polysomes,
where it is probably being translated. We have now demonstrated that
after androgen withdrawal, B23 mRNA is mainly associated with either
free ribosomes or short-stretch polysomes. However, this effect is also
observed for The possibility of proteolytic degradation of
prostatic protein B23 as a result of androgen deprivation was examined.
The initial incubation of radioiodinated protein B23 with prostatic
homogenate obtained after androgen withdrawal did not yield significant
degradation products. However, prior incubation of protein B23 with
phosphatases led to the appearance of degradation products when
incubated with prostatic homogenate obtained from castrated animals,
but not when incubated with homogenates from intact animals. This
finding strongly indicates that dephosphorylation of protein B23 and
the expression (or release of inhibition) of protease(s) after androgen
withdrawal are both necessary for the observed decline in protein B23.
This mechanism might be restricted to a specific group of proteins that
includes B23 since, on androgen withdrawal, the expression of certain
proteins is enhanced and actin protein persists. This accords with our
observation that actin is not degraded under these conditions. Our data
also suggest that phosphorylation of protein B23 enhances its stability
and resistance to proteolytic degradation. The level of
phosphorylation of B23 correlates with cellular proliferative
activities and is enhanced at
mitosis(17, 18, 19, 20, 21, 22) .
Protein B23 is phosphorylated by protein kinase CK2(22) , and
CK2 is the rate-limiting factor for B23 phosphorylation in the prostate
gland after androgen deprivation(26) . Furthermore, both
protein B23 (21) and protein kinase CK2 (42) are
associated with the nuclear matrix, and phosphorylation of nuclear
matrix-associated protein B23 is directly affected by changes in
nuclear matrix-associated CK2 activity(43) . Upon androgen
deprivation, nuclear matrix-associated CK2 declines rapidly, which
affects the rate of phosphorylation of proteins intrinsic to that
fraction. On the other hand, after androgen administration to castrated
rats, nuclear matrix-associated CK2 is increased within 1
h(43) . The physiological significance of these dynamic changes
in the association of CK2 with the nuclear matrix is apparent in regard
to protein B23, where the decline in phosphorylation would enhance its
susceptibility to proteolytic degradation during programmed cell death.
On the other hand, the early availability of nuclear matrix-associated
CK2 activity after androgen administration might play a role in the
phosphorylation and stability of the newly translated protein B23,
which starts to appear within 4 h after androgen administration in the
cascade of events leading to epithelial regeneration(26) . The role of certain proteases in the induction of apoptosis has been
documented in several systems(44, 45) . Apoptosis
induced in thymocytes by staurosporine (a kinase inhibitor) could be
inhibited by a protease inhibitor, which suggests a role for both
kinases and proteases in this process(46) . Nuclear
matrix-associated proteins seem to be a preferential target for
proteolysis during apoptosis. Lamin A, lamin B, poly(ADP-ribose)
polymerase, and topoisomerases are all targets of proteolytic
degradation during apoptosis in many
systems(39, 47, 48) . As is the case for
protein B23, nucleolin is another nuclear matrix protein that is
localized to the nucleolus and involved in rRNA synthesis and has been
shown to be a preferential target of serine proteases during programmed
death induced in target cells by cytotoxic lymphocytes(49) .
Although the nature of the proteases responsible for degradation of
these nuclear matrix proteins, including protein B23, is still unknown,
a Ca In summary, we have
documented that cells undergoing apoptosis retain the ability to
transcribe B23 mRNA. The existing mRNA for this protein declines very
slowly on induction of apoptosis. It appears that the long-lived mRNA
is available for translation when the cells are stimulated to grow.
Disappearance of protein B23 from the cell appears to relate to its
proteolytic degradation, and phosphorylation of the protein (by protein
kinase CK2) plays a role in preventing its degradation. To our
knowledge, this is the first report to document the kinetics and
mechanism of protein B23 disposition in cells undergoing programmed
cell death.
Volume 270,
Number 36,
Issue of September 08, pp. 21009-21015, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dihydrotestosterone support a significantly higher
incorporation of radiolabeled amino acids into proteins than do
ribosomes isolated from castrated animal controls(11) . kinase during mitosis(23) . The
expression and phosphorylation of the protein are enhanced in different
cell types in response to mitogens, growth factors, and hormones,
including androgen in the prostate, suggesting that protein B23
constitutes a common signal required for cell
proliferation(23, 24, 25, 26, 27, 28) .
Materials
Animals
Male Sprague-Dawley rats weighing
295-325 g (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were
used as the source of ventral prostate tissue. The animals were
maintained under standard conditions and were orchiectomized via the
scrotal route under Metofane anesthesia as described
previously(26) .Chemicals
Heparin, phenylmethylsulfonyl fluoride,
poly(Glu,Tyr) (4:1), potato acid phosphatase, agarose-bound alkaline
phosphatase, and actin were purchased from Sigma. Rabbit anti-B23
antibody was the same as described previously(15) . DNase I
(RNase-free) and RNase A were purchased from Boehringer Mannheim. The
ribonuclease inhibitor RNasin and the rabbit reticulocyte lysate
(nuclease-free) were purchased from Promega. Proteinase K was purchased
from International Biotechnologies, Inc. (New Haven, CT).
Microcystin-LR was obtained from Life Technologies, Inc. All other
common reagents and chemicals were of the highest purity available.Methods
Nuclear Run-on Assay
Prostatic nuclei were
isolated from rat ventral prostate glands excised either from intact
animals or at different times after castration as described
earlier(26) . Equal amounts of nuclei, as assessed by DNA
assay(29) , were utilized for each time point examined. Nuclei
were suspended in 40% glycerol, 5 mM MgCl
, 0.1
mM EDTA, and 50 mM Tris-HCl, pH 8; shock-frozen; and
stored at -80 °C. The run-on transcription assay was
performed as described previously(30) . Before hybridization,
transcripts (in 100 µl) were partially hydrolyzed with 4 µl of
10 N NaOH for 10 min on ice, followed by neutralization with
20 µl of 2 M NaHPO
. The alkali
treatment reduces the length of transcripts to 50-600
nucleotides, the majority of chains being 200-300 nucleotides in
length, and leads to more uniformly labeled molecules by removing
unlabeled 5`-regions from transcripts that had already been initiated in vivo and then elongated in vitro(31) .
Excess amounts of denatured B23 cDNA (5 µg) as well
-actin
cDNA were immobilized on nylon filters with a dot-blot apparatus.
Filters were prehybridized for 30 min at 60 °C in prehybridization
buffer, followed by hybridization in hybridization buffer containing
1.5 10
cpm run-on transcripts for 2 days at 60
°C as described previously(26) . The density of different
signals was assessed using a two-dimensional Bio-Rad densitometer.Northern Analysis
Total RNA from 3 g of rat
ventral prostates was prepared as described previously(26) .
Total RNA (10 µg) was separated on a formaldehyde-agarose gel and
transferred to nylon filters(30) . The B23 probe was isolated
as described previously (26) and labeled using a random primed
DNA labeling kit (Boehringer Mannheim). Prehybridization,
hybridization, and autoradiography were as described
before(26) . The autoradiograms were quantitated by using the
two-dimensional Bio-Rad densitometer. The size of the mRNAs was
estimated by comparison with ethidium bromide-stained 28 S and 18 S
rRNAs.In Vitro Translation
Prostatic poly(A) RNA was
isolated from total RNA using the PolyATtract mRNA isolation system
from Promega. Translation was carried out using the rabbit reticulocyte
lysate system at 30 °C for 60 min. The reaction containing between
5 and 50 µCi of [S]methionine (>1000
Ci/mmol) was stopped by adding RNase A (50 µg/ml) in buffer
containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and
50 mM
-glycerophosphate. The mixture was incubated with
B23-specific antibody bound to activated Sepharose 4B beads using
cyanogen bromide as described previously(26, 32) . The
bound protein was washed four times with 10 mM Tris-HCl, pH
7.4, 100 mM NaCl, and 50 mM -glycerophosphate
and then eluted with buffer containing 10 mM Tris-HCl, pH 7.4,
4 M MgCl, and 50 mM -glycerophosphate, followed by two washes with the same
solution. Bovine serum albumin (500 µg) was added to the eluted
material as a carrier, and the mixture was precipitated with 15%
trichloroacetic acid, washed with absolute ethanol, and dried. The
dried sample was dissolved in -mercaptoethanol/SDS buffer, boiled
for 5 min, and subjected to SDS-PAGE. (
)The gel was dried
and exposed to Kodak X-Omat AR film for 4 days at -80 °C. The
density of the bands was quantitated using the two-dimensional Bio-Rad
densitometer.Polysome Gradients
Excised prostate glands were
immediately sliced and lysed in ice-cold extraction buffer containing
10 mM HEPES, pH 7.5, 3 mM MgCl, 40 mM KCl, 5% glycerol, 20 mM dithiothreitol, and 0.2% Nonidet
P-40 supplemented with 1000 units/ml RNasin, 150 µg/ml
cycloheximide, 1 mM phenylmethylsulfonyl fluoride, and 5 mg/ml
heparin. Mitochondria and nuclei were removed by centrifugation at
10,000 g for 10 min. Separation of polysomes from the
post-mitochondrial supernatant was carried out on linear 15-40%
sucrose gradients in extraction buffer centrifuged at 4 °C for 2 h
at 38,000
g as described previously(33) .
After centrifugation, gradients were separated into fractions and
extracted with phenol/chloroform, and the integrity of the
electrophoretically separated RNA in each fraction was assessed by
ethidium bromide staining. Subsequently, serial dilutions of each
fraction were applied to nylon filters using a slot-blot apparatus,
immobilized by heating at 80 °C for 2 h, and then hybridized with
randomly labeled B23.1 cDNA probe,
-actin probe, or 28 S probe as
described above. As a control, an aliquot of each fraction was treated
with RNase A before application to the nylon filter to ensure that any
observed signal was attributable to RNA in the polysome fraction.Radiolabeling of Protein B23
Prostatic nucleoli
were prepared from the isolated nuclei as described
previously(34) . All preparative solutions contained 50
mM -glycerophosphate as phosphatase inhibitor. Protein
B23 was purified from nucleoli using anti-B23 antibody coupled to
Sepharose 4B as described previously(32, 35) .
Iodination of purified protein B23 (from tissue or recombinant source)
was performed using IODO-BEADS (Pierce) and
NaI(36) . Radiolabeled protein B23 was dialyzed
in a 2000-fold volume of 10 mM Tris-HCl, pH 7.4, to get rid of
phenylmethylsulfonyl fluoride and leupeptin prior to the degradation
assay. The homogeneity of the protein was assessed using a silver stain
of SDS-PAGE and autoradiography.
B23 Proteolytic Degradation Assay
Rat prostate
glands were excised either from intact animals or at different times
after castration and were immediately homogenized in ice-cold buffer
containing 10 mM HEPES, pH 7.5, 3 mM MgCl, 40 mM KCl, 20 mM dithiothreitol, and 0.2% Nonidet P-40. The homogenate was used
within 15 min of excision of the gland. Dialyzed radiolabeled protein
B23 was incubated for different times with homogenates obtained from
intact and castrated animals. The reaction was stopped by adding sample
buffer containing 2% SDS and 5% -mercaptoethanol, and the mixture
was boiled for 5 min prior to SDS-PAGE. The gel was dried and exposed
to Kodak X-Omat film overnight. Protease inhibitors such as
phenylmethylsulfonyl fluoride (1 mM), leupeptin (20
µg/ml), aprotinin (25 µg/ml), and iodoacetamide (10
mM) were added as a control. When indicated, purified protein
B23 was treated with potato acid phosphatase bound to Sepharose 4B for
2 h at 37 °C in buffer containing 0.18 M
(NH)SO and 1 mM MgCl, pH 5.5, followed by dialysis in 3 µM triethanolamine, 0.3 M NaCl, 0.1 mM
MgCl, 0.01 mM ZnCl, pH 7.6, and
incubation with agarose-bound alkaline phosphatase for 2 h at 37 °C
prior to the degradation assay(37) .
Effect of Androgen Deprivation on B23 mRNA
Transcription
To determine if the continued high level of B23
mRNA after androgen deprivation was attributable to ongoing
transcription or to enhanced stability of the mRNA, the prostatic
nuclei obtained from intact and castrated rats were examined for their
ability to transcribe B23 mRNA in vitro. As determined by the
nuclear run-on assay, the nuclei retained the ability to transcribe B23
mRNA even at 7 days after androgen withdrawal (Fig. 1). The
decline in the ability to transcribe B23 mRNA observed after 7 days was
comparable to the decline observed in the cellular steady-state level
of B23 mRNA examined either by Northern blot analysis (Fig. 2)
or by slot-blot analysis (26) . The rate of decline in the
ability of prostatic nuclei to transcribe -actin after androgen
withdrawal was comparable to that observed for protein B23 (Fig. 1).
-actin were 1.0 (lane a), 0.7 (lane
b), and 0.4 (lane c).
Effect of Androgen on Distribution of B23 mRNA in
Prostatic Cytosol
Using a 15-40% sucrose gradient, a
different distribution of cytosolic B23 mRNA was observed in prostate
glands obtained from castrated animals than in prostates obtained from
intact animals (Fig. 3A). After androgen withdrawal,
the majority of the B23 mRNA existed in association either with free
ribosomes or with short stretches of polysomes (Fig. 3, A and B). The -actin mRNA displayed a pattern similar
to that of B23 mRNA (Fig. 3C), and this differential
distribution of both mRNAs was secondary to the general decline in 28 S
RNA-containing ribosomal subunits, as evidenced by the decrease in the
area under the curve after androgen withdrawal in Fig. 3D. To confirm that the signal obtained by the
radiolabeled probe is due to the interaction with RNA in the polysome
fractions, a sample of the various polysome fractions was preincubated
with RNase A. This treatment abolished the signals, indicating that the
probes are indeed recognizing the B23-specific mRNA in the polysome
fraction (Fig. 3A).
-actin mRNA, and 28 S
RNA-containing ribosomal particles by using the corresponding randomly
radiolabeled probes as described under ``Methods.'' The
amounts of total RNA loaded in each lane are indicated. The polysome
fractions from top to bottom of the slot blot represent the fractions
collected from top to bottom of the sucrose gradient, respectively. A, detection of B23 mRNA in different prostatic polysome
fractions isolated from intact rats (normal control) or from rats after
5 days (5d) of androgen deprivation. To ensure that the
observed signals were due to binding of the radiolabeled probes to RNA
in the polysome fractions, samples from different polysome fractions
were treated with RNase A prior to the hybridization step. B,
effect of androgen on the distribution of B23 mRNA in prostatic
polysomes. The relative density of different bands in A was
plotted against the sucrose concentration to facilitate analysis of the
autoradiogram in A. C, effect of androgen on the
distribution of -actin mRNA in prostatic polysomes. D,
effect of androgen on the distribution of 28 S ribosomal particles in
prostatic polysomes.
Effect of Androgen Deprivation on in Vitro Translation of
B23 mRNA
Steroid hormones can alter the mRNA initiation sites of
many genes(38) . To examine the possibility of the development
of cis-acting elements in B23 mRNA that would hinder its
translation upon androgen deprivation, an in vitro translation
assay was performed. Even at 6 days of androgen withdrawal, B23 mRNA
was capable of being translated in vitro (Fig. 4).
Effect of Androgen Deprivation on Protein B23
Degradation
Incubation of purified radioiodinated protein B23
with prostatic cellular homogenates from normal and castrated rats did
not indicate a significant differential degradation of the protein (Fig. 5, compare toppanel with bottompanel). However, we have previously shown that protein
B23 phosphorylation in vivo declines dramatically after 2 days
of androgen deprivation, suggesting that the extent of phosphorylation
may influence B23 degradation(26) . Therefore, purified
radiolabeled protein B23 was incubated first with acid phosphatase
followed by alkaline phosphatase and then with prostatic homogenate in
the presence of 20 µg/ml heparin and 2 mg/ml poly(Glu,Tyr) as
protein kinase CK2 inhibitors. Under these conditions, a significant
appearance of degradation products was observed only when
dephosphorylated protein B23 was incubated with prostatic homogenate
obtained from 4-day castrated rats (Fig. 6A, compare bottompanel with top and middlepanels). The appearance of the degradation products could
be minimized by the addition of the protease inhibitors described
under ``Methods.'' A Ca-dependent protease
is known to be associated with the nuclear matrix, where it might be
involved in the degradation of certain nuclear matrix
proteins(39, 40) . In accord with this, an addition of
5 mM CaCl
to the medium markedly enhanced the
appearance of degradation products (data not shown).
I-labeled protein B23 (recombinant, nonphosphorylated; 5
µg) was incubated for 60 min at room temperature in medium
consisting of 30 mM Tris-HCl, pH 7.45, 5 mM MgCl
, 1 mM dithiothreitol, 0.1 mM ATP, 40 mM -glycerophosphate, 2 µM Microcystin-LR, and 350 ng of CK2 in a final volume of 50 µl
(phosphorylation medium). Subsequently, 50 µl of control buffer
(0.32 M sucrose, 3 mM MgCl, 5 mM
-mercaptoethanol) were added, and incubation was continued for 4 h
at room temperature (control). Lane b, the conditions were the
same as described for lane a, except that phosphatase
inhibitors (ATP, -glycerophosphate, and Microcystin-LR) were
omitted, and 0.1 mM NaHPO was included
(to provide nonphosphorylation conditions). Instead of the control
buffer, 50 µl of 50% prostatic homogenate (from 4-day castrated
rats) prepared in the control buffer were added, along with inhibitors
of CK2 (50 µg/ml heparin, 1 mg/ml poly(Glu,Tyr)), to prevent any
phosphorylation of protein B23 catalyzed by CK2 during incubation with
the homogenate. Lane c, the conditions were the same as
described for lane a, except that prostatic homogenate was
added as described for lane b, but without inhibitors of CK2
to promote phosphorylation conditions for CK2. Lane d, 4.5
µg of I-labeled actin were incubated under
nonphosphorylating conditions as described above for 60 min in medium
consisting of 50 mM imidazole HCl, pH 6.3, 2 mM MgCl
, 2.5 units of alkaline phosphatase, and 2.5 units
of acid phosphatase, followed by the addition of control buffer plus
CK2 inhibitors and 2.5 mM CaCl, and incubation was
continued for 4 h as described above. Lane e, the conditions
were the same as described for lane d, except that the second
incubation (4 h) was carried out following the addition of prostatic
homogenate as described above. C, effect of treatment with CK2
on degradation of protein B23 at 30 min by prostatic homogenate from
4-day castrated rats. Lane a, 5 µg of I-labeled B23 were incubated under nonphosphorylating
conditions as described for B (lane b), except that
the second phase of incubation was reduced from 4 h to 30 min. Lane
b, the conditions were the same as described for B (lane c), except that the second incubation was for 30
min only as described above. In all panels, arrowheads indicate the position of protein B23. In addition, the position of
a 24-kDa marker is shown in B and C.
, CK2 inhibitors, phosphatases)
was almost completely degraded after 4 h of incubation with prostatic
homogenate from 4-day castrated rats. On the other hand, prior
incubation of protein B23 with CK2 under conditions that favor
phosphorylation (presence of excess ATP, phosphatase inhibitors)
protected a 22-kDa segment of protein B23 from being degraded under the
same conditions (Fig. 6B, lane c). Since these
results suggested that phosphorylation may influence the rate or
pattern of proteolytic degradation of protein B23, further experiments
were undertaken in which proteolytic degradation of protein B23 was
examined at 30 min of reaction time. The results in Fig. 6C (lane b) show that when protein B23 was treated with CK2 (i.e. phosphorylation conditions), some undegraded protein B23
was apparent in addition to the resistant 22-kDa segment that was also
observed after 4 h of incubation (e.g.Fig. 6B, lane c). It is noteworthy that
intact protein B23 was not detected when nonphosphorylated protein B23
was subjected to the same treatment (Fig. 6C, lane
a). Also, it appears that the 22-kDa band has undergone further
degradation under these conditions. However, it is possible that the
rate and pattern of degradation of protein B23 in vivo may
also be influenced by stoichiometry of phosphorylation and/or the
presence of other intrinsic kinases. The present observations on the
effects of the phosphorylation status of protein B23 on its proteolytic
degradation appear to be relatively specific since pretreatment of
actin with protein phosphatases under conditions that favor
dephosphorylation did not demonstrate a corresponding degradation of
actin (or any of the contaminating proteins in the sample) (Fig. 6B, lanes d and e). Thus, the
observed degradative changes in protein B23 are not due to the
treatment conditions, and furthermore, it appears that protein B23
degradation may be catalyzed by specific protease(s) that may act on a
relatively specific set of proteins.
60% of normal even after 7 days of castration (26) when
>80% of the prostatic cells have undergone apoptosis(4) .
The present results demonstrate that persistence of the B23 mRNA after
androgen deprivation is the result of ongoing transcription rather than
enhanced stability. The decline in the rate of in vitro transcription of protein B23 mRNA was comparable to that of
-actin, which suggests that these changes are general effects of
androgen withdrawal and cannot explain the observed specific rapid
decline in protein B23 expression.-actin mRNA. This similar association might be
attributed to the general reduction in rRNA synthesis, as evidenced by
the decline in 28 S RNA-containing ribosomal particles, and accords
with the decline in rRNA synthesis and assembly which is one of the
most dramatic effects of androgen withdrawal in the prostate
gland(11, 12) . The observed pattern of association of
B23 mRNA or actin mRNA with ribosomes after androgen withdrawal might
contribute to the decline in protein expression in both cases. However,
it cannot explain the aforementioned specific abrupt decline in protein
B23 expression (26) in that actin protein is detected despite
the fact that the majority of its mRNA is associated with free or
short-stretch polysomes. Furthermore, it is likely that this
differential pattern of association with polysomes might still be
compatible with some protein expression that is translated from the
mRNA associated with long-stretch polysomes. It is noteworthy that the
expression of many androgen-repressed genes is enhanced after androgen
withdrawal, which again suggests that protein expression can occur
despite the decline in the formation of long-stretch
polysomes(8, 9, 10) . Competition between
different mRNAs to associate with polysomes has been suggested as a
mechanism for translational control(41) . In the case of
protein B23 mRNA, there are no cis-acting elements that would
arise after androgen deprivation and hinder its translation, at least in vitro.-dependent protease exists in the nuclear lamina (39) and may be involved in this process(40) . It is
noteworthy that the addition of Ca
to the incubation
medium markedly enhances the degradation of protein B23. The relation
between the decline in protein B23 or its phosphorylation may have
certain implications for programmed cell death. An obvious effect of a
decline in protein B23 would be on ribosome assembly. Also, protein B23
has been suggested to have a structural role; the decline in protein
B23 and its charge might affect the overall tensional integrity of the
tissue matrix(50) , leading to altered gene expression
associated with programmed cell death.
)
We thank Alan T. Davis for help in various phases of
the experimental work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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