(Received for publication, December 7, 1994; and in revised form, April 8, 1995)
From the
Post-translational modification of a carboxyl-terminal negative
regulatory domain in vitro by either casein kinase II or
protein kinase C allosterically activates the latent sequence-specific
DNA binding function of p53. Reported here is a biochemical approach to
determine the types of signaling pathways and enzymes that are involved
in p53 activation in cells. Using a novel chromatographic method, we
have been able to separate three distinct biochemical forms of p53 that
have been synthesized in vivo; two are in an activated state,
and one is in a latent state for sequence-specific DNA binding. The two
activated forms of p53 appear to be controlled individually by either a
constitutive or a UV-inducible signaling pathway. p53 lacking the
COOH-terminal casein kinase II site (p53 p53 protein functions as a tumor suppressor and appears to be
normally involved in the cellular response to DNA damage (Vogelstein
and Kinzler, 1992; Lane, 1992). The induction of a G Regulation of
p53 protein function remains less well understood. p53 protein is
normally undetectable by immunohistochemical analysis in most tissue
cells (Hall et al., 1993; Hall and Lane, 1994). The stability
and activity of p53 is dramatically elevated in the nucleus of some
cells following DNA damage (Maltzman and Czyzyk, 1984; Kastan et
al., 1991; Lu and Lane, 1993; Zhan et al., 1993; Hall et al., 1993; Midgley et al., 1995) or during
spermatogenesis (Almon et al., 1993), but the gene products
regulating these pathways are not well defined. Extragenic factors
appear to control the stability of p53 as suggested by the observation
that cells derived from some patients with Bloom's syndrome fail
to induce the endogenous wild type p53 following DNA damage (Lu and
Lane, 1993), and some patients with an inherited predisposition to
cancer produce abnormally high levels of wild type p53 in normal cells
in the absence of exogenous damage (Barnes et al., 1992; Birch et al., 1994). Regulation of the biochemical function of
p53 through reversible phosphorylation is likely (Ullrich et
al., 1992a), as at least seven kinases have been shown to modify
p53, including casein kinase II (Meek et al., 1990), Cdc-2
(Bischoff et al., 1990; Addison et al., 1990), casein
kinase I-isozyme (Milne et al., 1992a), protein kinase C
(Baudier et al., 1992), DNA-activated protein kinase
(Lees-Miller et al., 1990), mitogen-activated protein kinase
(Milne et al., 1994), and JNK1 (Milne et al., 1995).
The regulatory significance of these modifications is only beginning to
be unraveled. Mutation of an NH Direct evidence linking the
activation of a kinase to the activation of p53 protein
sequence-specific DNA binding in a physiologically relevant system has
not been observed. However, a role for protein kinase C in the
activation of the DNA binding function of p53 in vivo is
compelling, based on two independent lines of evidence. The first
relies on the identification of biochemical forms of p53 synthesized in vivo which are modified post-translationally at the protein
kinase C site. Protein kinase C phosphorylation of p53 in vitro inhibits the binding of a monoclonal antibody (PAb421), whose
binding site overlaps the protein kinase C phosphorylation site in the
regulatory domain of p53 (Hupp and Lane, 1994a; Delphin and Baudier,
1994). Biochemical studies have also revealed that activation of the
specific DNA binding function of p53 in vivo is coincident
with post-translational modification at the protein kinase C site, as
indicated by lack of reactivity to PAb421 antibody (Hupp and Lane,
1994b; Delphin and Baudier, 1994). PAb421 non-reactivity is also
observed on p53 synthesized in vivo from inducible expression
vectors which lead to a growth arrest in glioblastoma cells (Ullrich et al., 1992b; Fiscella et al., 1994), suggesting
that an enzyme with a specificity for the protein kinase C site
modifies the induced form of p53 during growth arrest. The second
experimental evidence relies on the observation that the protein kinase
C activator phorbol 12-myristate 13-acetate can induce a
G Casein kinase II is a highly conserved
serine-threonine protein kinase that is constitutively expressed in all
eukaryotic cell types examined, including yeast, insects, and
vertebrates. The enzyme is a heterotetramer composed of two Although casein kinase
II has been implicated in the regulation of growth control based on its
phosphorylation of key proteins and enzymes, only a few examples exist
in which the kinase can modulate the biochemical activity of a protein,
including topoisomerase II (Ackerman et al., 1988),
topoisomerase I (Turman and Douvas, 1993), CREM (deGroot et
al., 1993), SRF (Manak et al., 1990; Marais et
al., 1992), DNA ligase I (Prigent et al., 1992), E-B
viral ZEBRA protein (Kolman et al., 1993), c-Jun (Lin et
al., 1991), Myc/Max (Bousset et al., 1993), and p53 (Hupp et al., 1992). The biochemical mechanism whereby casein kinase
II alters polypeptide structure in these situations is not defined. p53
is an allosterically regulated tetramer, which can be reversibly
activated by multi-site phosphorylation of its negative regulatory
domain (Hupp and Lane, 1994b). As the carboxyl-terminal casein kinase
II site of p53 is the only phosphorylation site on p53 that is highly
conserved at the primary amino acid level, understanding the mechanism
whereby casein kinase II activates p53 provides a unique opportunity to
study how phosphorylation affects allosteric regulation of a
DNA-binding protein. Reported here, using a eukaryotic cell line that
we now show can be used to produce correctly folded forms of both
latent and activated p53 and using a chromatographic method to separate
three forms of p53 produced in vivo, are data demonstrating
that (i) two distinct signaling pathways are involved in p53 activation
in the same cell line, (ii) the acidic casein kinase II site is
required for catalytic activation of p53 by casein kinase II in
vitro, and (iii) casein kinase II-independent activation of p53
can occur in vivo.
Figure 3:
Reactivity of latent and activated forms
of p53 to conformationally specific monoclonal antibodies. Increasing
amounts of activated p53 (A, 0.40 M eluate),
activated p53 (B, 0.48 M eluate), and latent p53 (C, 0.56 M eluate) were incubated in BPT buffer (5%
BSA, phosphate-buffered saline, and 0.1% Tween 20) at room temperature
for 2 h in ELISA wells precoated with the indicated monoclonal
antibodies. The amount of p53 bound to monoclonal antibody was detected
with polyclonal antibody CM-1 and swine-anti-rabbit IgG conjugated to
horseradish peroxidase as described (Harlow and Lane, 1988). C, latent p53 (0.56 M eluate) was serially diluted in
0.1 M carbonate buffer (pH 9.25) and used to coat ELISA wells
at 4 °C for 16 h. Following blocking with BPT buffer for 1 h at
room temperature, the indicated monoclonal antibody was diluted to 2
µg/ml and incubated at room temperature for 2 h. The amount of IgG
complexed to p53 was determined using goat anti-mouse IgG coupled to
horseradish peroxidase as described (Harlow and Lane,
1988).
Figure 5:
Reactivity of p53
Figure 1:
Domain structure of human p53. A, relative binding site of monoclonal antibodies specific for
p53. PAb1620 recognizes a conformationally sensitive epitope on
correctly folded forms of p53, whereas PAb240 recognizes unfolded or
denatured forms of p53 (Gannon et al., 1990). B,
amino acid sequences derived from the final 30 amino acids of human p53
(Soussi et al., 1990). Panel shows the site of phosphorylation
by casein kinase II (darkenedarrow; Bischoff et
al., 1992; Meek et al., 1990), the predicted sites of
protein kinase C phosphorylation (open-headed arrow; Baudier et al., 1992; Hupp and Lane, 1994a), the binding site of
PAb421 (Wade-Evans and Jenkins, 1985), the minimal binding site of DnaK
for peptides derived from the COOH terminus of p53 (Fourie et
al., 1994, T. R. Hupp and D. P. Lane, submitted for publication),
and the binding site of the inhibitory antibody ICA-9 (this study). The
relative location of the conserved basic amino acids(370-386)
containing the negative regulatory domain and the conserved acidic
amino acids enveloping the casein kinase II site(388-393) are as
indicated.
By
contrast to other cell lines, insect cells infected with recombinant
p53 baculovirus produce relatively large amounts of in vivo activated p53 (Hupp and Lane, 1994a, 1994b). As such, this cell
line provides an excellent model system with which to study the
signaling pathways that can activate the specific DNA binding function
of p53 in cells. Wild type human p53 expressed in insect cells can be
chromatographically separated into three forms using heparin-Sepharose
chromatography (Fig. 2). The discovery of a method to physically
separate different forms of p53 permits careful biochemical analysis of
different forms of the protein. The earliest fraction
(p53
Figure 2:
Chromatographic separation of three
biochemical forms of p53 expressed in baculovirus-infected insect cell
lines. Baculovirus vectors containing recombinant human p53 were
infected into Sf9 cells at 27 °C for 72 h as indicated under
``Experimental Procedures.'' Cells were harvested and lysed
by the addition of an equal volume of buffer containing protease and
phosphatase inhibitors (0.15 M phosphate-buffered saline (pH
7.2), 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM DTT, 1% Nonidet P-40, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 10 mM NaF). After an
incubation at 0 °C for 15 min, the sample was centrifuged at 14,000
rpm in minicentrifuge, and, after dilution in an equal volume of Buffer
B, the supernatant was directly loaded onto a heparin-Sepharose column
at a ratio of 0.15 mg of protein/ml of resin. Bound protein was eluted
with a 20 column volume linear gradient in Buffer B from 0.05 to 1 M KCl. p53 was assayed throughout the gradient by
immunoblotting with DO-1 (data not shown) or by sequence-specific DNA
binding with the indicated monoclonal antibodies. Representative peak
fractions are as indicated: 0.40 M eluate (lane1, no antibody; lane2, PAb421; lane3, DO-1), 0.48 M eluate (lane4, no antibody; lane5, PAb421; lane6, DO-1), 0.56 M eluate (lane7, no antibody; lane8, PAb421; lane9, DO-1). The darkenedarrow marks the position of p53
The latest
eluting fraction from the heparin matrix is in a latent state for
sequence-specific DNA binding (lane7) but is
allosterically activated in vitro after the binding of two
PAb421 molecules/p53 tetramer (lane8), as reported
previously (Hupp and Lane, 1994b). The NH
Is the latent state of wild type p53
induced by an unfolding of the conformationally sensitive core domain?
As p53 is an allosterically regulated, oligomeric protein, it is likely
that cooperative interactions between subunits are involved in
modulating the activity of the protein. An unresolved issue requires
determining the relationship between the unfolding of the
conformationally sensitive core DNA binding domain and allosteric
activation catalyzed by modification of the COOH-terminal regulatory
domain. Specifically, we address whether the latent form of p53
produced in insect cells, and purified by the regime we have reported
previously (Hupp and Lane, 1994b), is unfolded and, if so, whether
allosteric activation by modification of the COOH terminus in vivo induces a re-folding of the conformationally sensitive internal
domain. A two-site ELISA was used to probe the conformational states
of latent and in vivo activated p53 (Fig. 3). As
expected, both latent and activated forms of p53 bind to the
NH A control is
essential to demonstrate that the antibodies PAb1620 and PAb240 can be
used to discriminate between folded and unfolded forms of wild type
p53. Latent p53 was subject to denaturation by coating the pure protein
onto ELISA wells followed by detection with the indicated monoclonal
antibodies. Under these conditions in which the PAb1620 epitope is
destroyed, the latent protein becomes unfolded as defined by strong
reaction to PAb240 (Fig. 3D). These results establish
that p53 can be artificially unfolded in vitro and that PAb240
is functional in the assay described in this report. The absence of
PAb421 reactivity to unfolded p53 (Fig. 3D) may relate
to a concealment of the PAb421 epitope, as adsorption to ELISA wells
may occur through the charged carboxyl terminus harboring the PAb421
epitope.
Figure 4:
Regulation of p53-activation pathways in vivo. A, Sf9 cells were cultured in media
containing serum (5%) and after the addition of baculovirus expressing
wild type p53, cells were grown in serum-containing media for 42 h
prior to harvesting. B, Sf9 cells were cultured in media
containing serum (5%) and before the addition of baculovirus expressing
wild type p53, cells were incubated in media without serum for 5 h.
Cells were grown in serum-free media for 42 h prior to harvesting as
indicated in A. C, Sf9 cells were cultured in media
containing serum (5%) and before the addition of baculovirus expressing
wild type p53, cells were incubated in media without serum for 5 h.
After 36 h of protein production in serum-free media, cells were
UV-irradiated (60 J/m
When cells are deprived of serum, the production of
activated p53
To address these questions, the casein kinase II
phosphorylation site (PDSD) was first deleted from the COOH terminus of
p53 and the recombinant protein was characterized biochemically.
p53 We have previously reported the use of a
monoclonal antibody (ICA-9) that catalyzed the allosteric inhibition of in vitro or in vivo activated p53 (Hupp and Lane,
1994b), indicating that activation of p53 by phosphorylation is
reversible. The immunoreactivity of p53
Figure 6:
Casein kinase does not activate p53
Although activation of p53 by casein kinase II
requires ATP or GTP (Hupp et al., 1993), consistent with the
ability of casein kinase II to use GTP or ATP as phosphate donor
(Pinna, 1990), and activation of p53 is coincident with stoichiometric
phosphorylation by this kinase (Hupp et al., 1992), the actual
requirement of the acidic casein kinase II consensus motif in the COOH
terminus for casein kinase II-dependent p53 activation has not been
established. In addition, as the To investigate whether p53
Figure 7:
Activation of p53
Differences in the specific activity of p53 and p53
Figure 8:
ICA-9
does not destabilize PAb421-activated p53
Figure 9:
Casein kinase II-independent activation of
p53
Analysis of the varying
conformational forms of p53 has been accomplished using monoclonal
antibodies that recognize different forms of the protein (Fig. 1A). Using monoclonal antibodies specific for
folded (PAb1620) or unfolded (PAb240) forms of p53, it has been
established that oligomeric p53 is subject to conformational
flexibility in vivo (Gannon et al., 1990; Vojtesek et al., 1995) and in vitro (Milner and Medcalf,
1991). Strong evidence for cooperative interactions between subunits of
the oligomer has come from the observations that mutant p53 drives the
wild type p53 into the mutant conformation (i.e. wild type p53
becomes PAb1620 Using
insect cell expression systems, we report here on the use of a
chromatographic method to separate three biochemical forms of human p53
produced in vivo, which has greatly facilitated biochemical
characterization of latent and two activated forms of p53. The
different elution profile suggests that all three forms of p53 have
altered conformation and/or ionic properties, based on their
differential affinity for heparin. Using these purified biochemical
forms of p53, antibody-binding experiments have demonstrated that
allosteric activation does not coincide with alterations in the
conformationally flexible domain; both latent and activated forms of
p53 are reactive with PAb1620 and are non-reactive with PAb240. In
addition, as these results indicate that the latent form of p53 is
properly folded, allosteric activation by protein kinases, bacterial
Hsp70,( This information contrasts with interpretations proposed previously
on the regulation of p53 conformation and activity using p53
synthesized in crude reticulocyte lysates (Hainault and Milner, 1993).
In the latter report, active p53 is folded correctly
(PAb1620
One possibility is
that the acidic casein kinase II motif resembles the substrate DNA in
net charge and that this electrostatic interaction with the active site
inactivates p53 through a pseudo-substrate mechanism. This model would
then require local conformational changes to eject the casein kinase II
site from its binding pocket in the active site, thus permitting
sequence-specific DNA binding. Such a model has been proposed for many
protein kinases; pseudo-substrate domains interact with the active site
for some protein kinases and direct modification of the
pseudo-substrate domain or allosteric changes dissociate the negative
regulatory domain from its binding site and allow substrate access to
the active site. A second model explaining the inactivity of
unphosphorylated p53 requires that the basic region harbors the
negative regulatory domain and its interaction with a distinct, acidic
domain on the tetramer may trap p53 in the inactive conformation. Due
to the basic nature of the region, this would reduce the likelihood
that the negative regulatory domain mimics nucleic acid and resides
within the active site for sequence-specific DNA binding. Biochemical
studies of pure p53 lacking the acidic casein kinase II phosphorylation
site in the COOH-terminal regulatory domain of p53 have indicated that
deletion of this site does not activate p53, localizing the negative
regulatory domain to the basic stretch of amino acids (Fig. 1B). We favor, therefore, a model of p53 latency
that does not involve an interaction of the negative regulatory domain
within the active site for sequence-specific DNA binding, but to a
presently unmapped site on the tetramer.
A
recent study aimed at determining the affects of casein kinase
II-phosphorylation site mutation on the activity of p53 have concluded
that casein kinase II site does not contribute to wild type p53
activity in vitro or in vivo (Fiscella et
al., 1994). However, it is clear from studies described in our
report (Fig. 6) that the casein kinase II phosphorylation site
is essential for catalytic activation by casein kinase II in
vitro. These former conclusions (Fiscella et al., 1994)
overlook the possibility that other dominant activators of p53
predominate in different cells and that casein kinase II-dependent
regulation of p53 occurs only under strict circumstances. As the casein
kinase II phosphorylation site in the COOH-terminal regulatory domain
of p53 is the only phosphorylation sequence that is conserved at the
primary amino acid level in all species examined presently (Soussi et al., 1990), it is important to establish the physiological
conditions and a biological system in which casein kinase II-dependent
activation of p53 is detected in vivo. The biochemical
character of p53
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
4) was characterized
biochemically and used to determine the affects of deletion of the
casein kinase II motif on the production of the two activated forms of
p53 in vivo. As observed with full-length p53, the production
of two distinct chromatographic forms of activated p53
4 occurs in vivo, indicating that p53 activation can occur through a
casein kinase II-independent pathway and suggesting that two other
factors are involved in activation of p53 in vivo.
/S
growth arrest (Kastan et al., 1991; Kuerbitz et al.,
1992) or apoptosis (Yonish et al., 1991; Lowe et al.,
1993; Clarke et al., 1993; Merritt et al., 1994)
following radiation treatment of cells requires a wild type p53 gene,
as cells lacking functional p53 fail to respond normally to ionizing
radiation-induced growth inhibitory signals. The biochemical activity
of p53 required for tumor suppression, and presumably the cellular
response to DNA damage, involves the ability of the protein to bind to
DNA sequence-specifically (El-Deiry et al., 1992; Tokino et al., 1994) and function as a transcription factor (Kern et al., 1992; Funk et al., 1992; Scharer and Iggo,
1992; Pietenpol et al., 1994). Genes with binding sites for
p53 include the promoter regions of the creatine kinase gene (Weintraub et al., 1991; Zhao et al., 1994), the ribosomal gene
cluster (Zambetti et al., 1993), Rb (Osifchin et al.,
1994), cyclin G (Okamato and Beach, 1994), the p21
gene
(El-Diery et al., 1993), and the introns of the gadd45 (Kastan et al., 1992) and mdm-2 genes (Momand et al., 1992; Oliner et al., 1992; Barak and Oren,
1992). Of these genes, the mRNA levels of gadd45 (Kastan et al., 1992), mdm-2 (Barak et al., 1993),
and p21
genes (El-Deiry et al., 1994; Fiscella et al., 1994) are increased in a p53-dependent manner
post-irradiation, suggesting that p53 controls the transcription of
gene products with a direct role in growth control. In fact, p21 can
directly inhibit the initiation of SV40 viral DNA synthesis by
neutralizing proliferating cell nuclear antigen activity (Waga et
al., 1994), whereas inhibition of the initiation of chromosomal
DNA replication in the Xenopus system by p21 coincides with an
inhibition of Cdk2 cyclin-dependent kinases (Strausfeld et al., 1994). Although sequence-specific transcriptional activation
appears to represent an important role for p53, it has also been
established that p53 binds to TBP (Seto et al., 1992; Martin et al., 1993) and can inhibit transcription through
TATA-dependent (Mack et al., 1993) and TATA-independent
mechanisms (Hernandez, 1993; Ueba et al., 1994), presumably by
interactions with the basal transcriptional machinery.
-terminal DNA-activated
protein kinase phosphorylation site reduces the ability of p53 to
inhibit cell cycle progression (Fiscella et al., 1993).
Mutating the COOH-terminal casein kinase II phosphorylation site of
mouse p53 inactivates its growth suppressor activity in rodent cell
lines (Milne et al., 1992b). Direct evidence that
phosphorylation can control the activity of human p53 has been
supported by biochemical studies showing that two distinct protein
kinases (protein kinase C and casein kinase II) can activate the latent
sequence-specific DNA binding function of p53 in vitro (Hupp et al., 1992, Hupp and Lane, 1994a; Delphin and Baudier,
1994). These results suggest that p53 has evolved a functionally
relevant interaction with different enzymes and that distinct kinases
may activate p53 in vivo.
/S growth arrest of transformed cells harboring a
functional p53 allele (Skouv et al., 1994; Delphin and
Baudier, 1994), presumably by modification of p53 by protein kinase C.
Although these data provide evidence that protein kinase C is involved
in p53 activation in cells, it does not distinguish between the 12
isoforms of protein kinase C which exist, nor other enzymes that may
covalently modify serine residues at the canonical protein kinase C
site. In contrast to protein kinase C, a link between the induction of
a casein kinase II-dependent pathway and the activation of the
sequence-specific DNA binding function of p53 in vivo has not
been established.
and
two
subunits and can utilize GTP or ATP as a phosphate donor
(Pinna, 1990). The biochemical mechanism of casein kinase II regulation
is not well understood, as the kinase does not undergo reversible
inactivation/activation in response to growth factors and mitogens like
many second messenger-dependent protein kinases. However, biochemical
and physiological studies have revealed two points of potential
regulation. Enzymatic analysis has revealed that association of the
catalytic core
subunit with the
subunit can regulate the
activity of the kinase. The
subunit contains the binding site of
basic effectors like spermine and polylysine (Meggio et al.,
1994), the interaction with which stimulates substrate phosphorylation
by the
subunit. Although the
subunit stimulates the
activity of the
subunit for most substrates,
actually
inhibits kinase activity toward calmodulin (Meggio et al.,
1992). Casein kinase II associates with the nuclear matrix (Tawfic and
Ahmed, 1994), and nuclear fractions contain pools of free
and
free
subunit, which are differentially solubilized by NaCl in
vitro (Stigare et al., 1993). Physiological studies have
also revealed that the
subunit is degraded much more rapidly than
the
subunit (Luscher and Litchfield, 1994). Thus, if casein
kinase II activity is regulated, the most likely candidate at present
would be its
subunit; rate-limiting assembly of the
heterotetramer can dramatically modulate the specific activity of the
kinase. A series of recent reports has demonstrated that physiological
regulation can be observed under specific conditions; casein kinase II
activity is stimulated by mitogens (Sommercorn et al., 1987),
after heat shock (van Delft et al., 1993), in a cell
type-specific manner during development and embryogenesis (Mestres et al., 1994), and its activity is strikingly elevated in
human squamous cell carcinoma or in adenocarcinoma of the lung
(Daya-Makin et al., 1994) and up to 25-fold in prostatic
hyperplasia (Yenice et al., 1994).
Reagents, Enzymes, and Proteins
Recombinant p53
and monoclonal antibodies DO-1, PAb421, PAb1620, PAb240, and ICA-9 were
purified as described (Hupp and Lane, 1994b). Heparin-Sepharose, MonoQ
(SR 5/5), and Superose 12-gel filtration columns were obtained from
Pharmacia Biotech Inc. Phosphocellulose P-11 was obtained from Whatman.
The DnaK overproducing strain was obtained from Dr. Maciej Zylicz,
Gdansk, Poland. Mutagenesis kits were obtained from Amersham Corp.
Buffer B contains 15% glycerol, 25 mM HEPES (pH 7.6), and 0.1
mM EDTA. Insect cell growth media is Ex-cell 400 (Sera-Lab)
supplemented with 5% fetal calf serum.Purification of Casein Kinase II
Casein kinase II
from rabbit muscle was purified as described (Hupp et al.,
1992) with the following changes. Muscle from one rabbit was ground in
1 liter of homogenization buffer (4 mM EDTA, 2 mM DTT,()
1 mM benzamidine) and
centrifuged at 5000
g for 50 min. The soluble
supernatant containing 20 g of protein was batch adsorbed to 100 ml of
phosphocellulose resin for 2 h in Buffer F (10% glycerol, 20 mM HEPES (pH 7.6), 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and
0.05% Triton X-100) containing 50 mM KCl. The column was
washed with Buffer F containing 0.35 M KCl, and casein kinase
II activity was step eluted with Buffer F containing 1.2 M KCl
and 0.05% Triton X-100. After phosphocellulose, fractions containing
casein kinase II were applied to a heparin-Sepharose column and eluted
with a linear gradient from 0.05 to 1 M KCl in Buffer F
containing 0.05% Triton X-100. Active fractions were applied to a MonoQ
column and eluted with a linear KCl gradient in Buffer F containing
0.1% Triton X-100 from 0.1 M KCl to 0.66 M KCl.
Active casein kinase II was concentrated to 0.25 mg/ml using
Centricon-30, BSA was added to a final concentration of 1 mg/ml, and
kinase was stored frozen in liquid nitrogen. Casein kinase activity was
monitored during the purification by assaying for radioactive phosphate
incorporation into p53 and casein (10% glycerol, 1 mg/ml BSA, 25 mM HEPES (pH 7.6), 1 mM DTT, 0.05% Triton X-100, 0.1 M KCl, and 25 ng of p53 or 500 ng of casein) in the absence and
presence of PAb421 (200 ng). PAb421 binds near the casein kinase II
site of p53 and reduces the rate of phosphorylation, providing a method
to access casein kinase II dependence during purification (Hupp et
al., 1992).
Purification of DnaK
The Escherichia coli Hsp70 homologue (DnaK) was purified from an overproducing strain
by a modification of the published protocol (Hupp et al.,
1993). Cells were grown in LB media at 30 °C to an optical density
of 0.6, at which point an equal volume of prewarmed LB (55 °C) was
added and cells were grown for an additional 2 h at 42 °C. Cells
were pelleted by centrifugation and resuspended in 10% sucrose, 50
mM HEPES (pH 7.6), and the cell suspension was lysed by the
addition of KCl to 0.5 M, DTT to 1 mM, and lysozyme
to 0.5 mg/ml. After a 20-min incubation at 0 °C and centrifugation
for 10 min in a minicentrifuge, crude lysate was applied to a
DEAE-Sepharose column at a protein(mg):resin(ml) ratio of 10:1. Bound
protein was eluted and fractions containing DnaK, localized by
immunoblotting, were dialyzed against Buffer D (10 mM imidazole (pH 7.0), 10% sucrose, 10 mM MgCl,
0.1 mM EDTA, 20 mM KCl, and 1 mM DTT) and
applied to an ATP-agarose column (Sigma A-2682) in Buffer D at a
protein(mg):resin(ml) ratio of 5:1. After a wash in Buffer D containing
1.0 M KCl, and equilibration in Buffer D, DnaK was eluted from
the column using Buffer D containing 5 mM ATP. The fractions
of DnaK eluting from ATP-agarose were dialyzed overnight at 3 °C
against Buffer B containing 2 mM EDTA and 0.25 M KCl.
Immunological Methods
Protein immunoblotting was
performed as described (Harlow and Lane, 1988). ELISA was performed by
first coating plates with p53 or p534 at 2 µg/ml in sodium
carbonate buffer (pH 9.0) for 1 h at 22 °C and subsequently
developing the ELISA with monoclonal antibodies as indicated (Harlow
and Lane, 1988). Two-site ELISA was performed by first coating wells
with pure monoclonal antibody at 2 µg/ml in carbonate buffer (pH
9.0) at 4 °C for 16 h. After coating the non-reacted sites with BPT
buffer (5% BSA, phosphate-buffered saline, and 0.1% Tween 20), p53 was
detected with polyclonal serum CM-1 as indicated in Fig. 3legend.
Generation of ICA-9
A 20-amino acid synthetic
peptide derived from the COOH-terminal regulatory domain of p53 (40
µg; GQSTSRHKKLMFKTEPDSD) was coupled to 400 µg of keyhole
limpet hemocyanin and used as an antigen for the eventual generation of
hybridoma cell lines (Harlow and Lane, 1988) secreting antibodies
specific for p53. Detection of antibodies to p53 involved the use of
pure human p53 in an ELISA. The specificity of ICA-9 for the
COOH-terminal casein kinase II site was established by immunoblotting
with p53 lacking the casein kinase II site (Fig. 5) and by using
synthetic peptides in a competition ELISA assay.()
The antibody was classed as IgG
and purified for
biochemical studies using Protein A and gel filtration chromatography.
4 to ICA-9.
Full-length p53 (50 ng; lanes1, 3, and 5) or p53
4 (50 ng; lanes2, 4,
and 6) were immunoblotted, after SDS-polyacrylamide denaturing
gel electrophoresis, using the indicated antibodies: the
NH
-terminally specific antibody DO-1 (epitope from amino
acids 19-26, lanes1 and 2) and two
COOH-terminally specific antibodies, PAb421 (epitope from amino acids
372-381, lanes3 and 4) and ICA-9
(epitope from amino acids 388-393, lanes5 and 6).
Construction of p53
Single-stranded
pBSK vector DNA harboring the wild type human p53 gene (Midgley et
al., 1992) was used as a template for deletion mutagenesis
designed to remove the final four amino acids of p53. The resultant
allele was subcloned into the T7.7 expression vector for overproduction
in and purification from E. coli as indicated (Hupp and Lane,
1994b) or was subcloned into pVL1393 baculovirus expression vector and
used to isolate recombinant virus overproducing p534 Expression Vectors for Protein
Production in E. coli and in Sf9 Insect Cells
4 in Sf9 insect
cells as described by the manufacturer of insect cell expression
systems (Invitrogen).
Activation of p53
p53 (6-12 ng) was added to
10 µl of Activation buffer (10% glycerol, 1.0 mg/ml BSA, 0.05 M KCl, 0.1 mM EDTA, 5 mM DTT, 0.05% Triton X-100,
10 mM MgCl, 0.5 mM ATP (or 50 µM ATP when activated using protein kinases), and 25 mM HEPES (pH 7.6)), followed by incubation at 30 °C for 30 min
with 25 ng of PAb421, 50 ng of casein kinase II (MonoQ fraction; Hupp
and Lane, 1994) from rabbit muscle, or 2 µg of DnaK.
DNA Binding Assay Using Native Gel
Electrophoresis
Reactions containing activated p53 were placed
at 0 °C, and 10 µl of a DNA binding buffer (20% glycerol, 1.0
mg/ml BSA, 0.05 M KCl, 0.1 mM EDTA, 5 mM DTT, 0.05% Triton X-100, 10 mM MgCl, 0.5
mM ATP, 5 ng of radiolabeled consensus site oligonucleotide
(Hupp et al., 1992), and 100 ng of supercoiled pBluescript
competitor DNA) was added. After an incubation on ice for 15 min,
reaction products were separated on a 4% polyacrylamide gel (in 0.5
TB buffer and 0.1% Triton X-100) by electrophoresis at 200 V
for 3 h at 8 °C. Gels were dried prior to exposure to x-ray film.
Separation of Three Biochemical Forms of p53 Produced
in Vivo
Recombinant p53 assembles into predominantly latent
tetramers in bacterial or mammalian cell lines (Hupp et al.,
1992), suggesting that activation of p53 is rate-limiting in some cells
or that activated p53 is preferentially degraded. Activation of the
latent sequence-specific DNA binding function of p53 function in
vitro is catalyzed by protein kinase C, casein kinase II, or a
monoclonal antibody (PAb421, which binds to the protein kinase C site).
DnaK protein, whose binding site adventitiously overlaps with the
PAb421 binding site()
(Fourie et al.,
1994), also activates the latent DNA binding function of p53 (Hupp et al., 1992). These four proteins all target specific amino
acids in the COOH-terminal negative regulatory domain (Fig. 1B), suggesting that activation of p53 by these
diverse reagents occurs through a common biochemical mechanism.
), eluting at 0.40 M KCl, is active
for sequence-specific DNA binding (lane1), is
supershifted by the binding of two DO-1 antibodies/p53 tetramer (lane3), but is non-reactive with the
COOH-terminally specific antibody PAb421 (lane2).
The second active fraction (lane4,
p53
), eluting from the column at 0.48 M KCl, is also bound by two DO-1 molecules/p53 tetramer (lane6), but binds to only one PAb421 antibody/p53 tetramer (lane5). The enzymes which modify p53 at the COOH
terminus in vivo to give rise to these two active forms of p53
are not known. One activator may be protein kinase C-like, as protein
kinase C phosphorylation inhibits the binding of PAb421 to the COOH
terminus (Hupp and Lane, 1994a). Addressed below is whether casein
kinase II is a major activator of p53 in vivo.
DNA complexes bound to two
monoclonal antibodies, the open-headed arrow marks the
migration of p53
DNA complexes bound to one monoclonal antibody,
and the stem marks the migration of in vivo activated
p53.
-terminally
specific antibody DO-1 does not activate the latent form of wild type
p53 (lane9).
Analysis of the Conformationally Flexible Domain of
Latent and Activated Forms of p53 Produced in Recombinant Insect
Cells
p53 has a conformationally flexible central domain, which
is usually unfolded in point mutant proteins of the type found in human
tumor cells or in denatured wild type p53 (Bartek et al.,
1991; Gannon et al., 1990). Two conformationally specific
monoclonal antibodies can be used to access structure-function
relationships within the tetramer (Fig. 1A). Antibody
PAb1620 can bind only to the native conformation of human p53 and
recognizes a conformationally sensitive epitope that is destroyed by
denaturation. The other antibody, PAb240, recognizes a linear epitope
that defines an unfolded variant of p53; PAb240 binds to either mutant
p53 expressed in tumor cells or binds to denatured wild type p53
(Gannon et al., 1990).-terminally specific antibody DO-1 (Fig. 3, A-C). In addition, the latent form of p53 binds well to
PAb421, but activated p53
does not bind to PAb421,
possibly due to modification at the protein kinase C site. Strikingly,
both latent and activated forms of p53 react with PAb1620, but do not
bind PAb240 (Fig. 3, A-C), indicating that both
latent and activated forms of p53 are correctly folded and that the
insect cell expression system does not produce unfolded variants of
p53. Notably, these results clearly indicate that the latent character
of p53 does not arise from an unfolding of the internal DNA binding
domain as defined by strong reactivity to PAb240.
Two Distinct Enzymatic Pathways Can Activate p53 in
Cells
Do the two activated pools of p53 arise due to differences
in the stoichiometry of phosphorylation at the protein kinase C sites
by one enzyme, or are two distinct enzymes involved in p53 activation
in this system? In order to investigate whether extracellular signals
can modulate the production of the two activated forms of p53, we set
out to alter growth conditions of cells infected with human p53 in
order to subsequently assay for the production of the two activated
pools of p53. As observed previously, cells grown in media containing
serum produce both activated pools of p53 with altered immunoreactivity
to carboxyl-terminal monoclonal antibodies (Fig. 4A, lanes 1-8). p53 is not bound by
antibody PAb421 (lane3), and it is weakly
destabilized by stoichiometric amounts of ICA-9 (lane4), whereas p53
is bound to one
PAb421 antibody (lane7) and is potently inhibited by
stoichiometric amounts of antibody ICA-9 (lane8).
Thus, the two activated pools of p53 are distinguished based on the
differential reactivity of two different monoclonal antibodies to the
carboxyl-terminal regulatory site. These results suggest that two
distinct enzymes are involved in p53 activation in this cell-based
system.
) and were grown in media without
serum for another 6 h. Lysate from cells were applied to
heparin-Sepharose resin to resolve the two activated forms of p53 as
indicated in Fig. 2. Sequence-specific DNA binding assays were
performed without or with the indicated monoclonal antibodies (25 ng)
and p53 (25 ng of 0.4 M eluate (lanes1-4) or 0.48 M eluate (lanes
5-8)); lanes1 and 5, no
antibodies; lanes2 and 6, DO-1; lanes3 and 7, PAb421; lanes4 and 8, ICA-9. The migration of p53
DNA complexes without,
with one, or with two monoclonal antibodies bound/tetramer is indicated
by the arrowheads.
is suppressed (Fig. 4B, lanes 1-4), while UV
irradiation triggers the production of activated p53
in serum-deprived cells (Fig. 4C, lanes1-4). Activated p53
is not
affected by these manipulations (Fig. 4, A and B, lanes 5-8) and appears to be activated
through a signal-independent pathway. These results show for the first
time that two independent signaling pathways may activate p53 in the
same cell line, that this may occur at two sites in the COOH terminus,
and show a direct link between alterations in environmental stress and
activation of the sequence-specific DNA binding activity of p53.
Addressed below is whether casein kinase II is involved in one of the
enzymatic pathways involved in p53 activation in vivo.
Deletion of the Casein Kinase II Motif in the COOH
Terminus of p53
Characterization of wild type p53, purified by
conventional chromatography, has given important information on the
regulation of protein activity by a negative regulatory site. This
latter domain harbors two conserved, hydrophilic motifs; the basic
motif (amino acids 370-386) contains the binding site of the
activating antibody PAb421, E. coli Hsp70, and protein kinase
C, whereas the acidic domain comprises the conserved casein kinase II
site (Fig. 1B). Presumably one or both of these
COOH-terminal motif's contribute to p53 latency. One possible
model to explain p53 inactivity is that the acidic region of the
negative regulatory domain (amino acids 388-393) mimics nucleic
acid and this pseudo-substrate interaction directly inactivates p53 by
residing in the active site for sequence-specific DNA binding. This
hypothesis can be tested by deletion of this acidic casein kinase II
site. Addressed in this report are (i) the effects of deletion of the
final four amino acids of p53 (PDSD), containing the casein kinase II
site, on the latent character of the tetramer, (ii) whether this site
is required for casein kinase II-dependent and casein kinase
II-independent activation of p53 in vitro, and (iii) whether
casein kinase II is directly involved in the activation of p53 in
vivo.4 was purified to homogeneity after overproduction in
recombinant strains of E. coli or baculovirus infected insect
cells, using heparin-Sepharose, phosphocellulose, and gel filtration
chromatography. E. coli was first used to produce p53
4,
as bacteria can be used to generate large amount of latent tetramers in
an unphosphorylated state (Hupp and Lane, 1994b). Unphosphorylated
p53
4 had the same Stokes radius (65 Å) and sedimentation
coefficient (6.2 S) as full-length p53 (Hupp and Lane, 1994b), as
determined using gel filtration and velocity sedimentation, indicating
that deletion of this motif did not alter the tetrameric nature of p53
(data not shown).
4 to monoclonal antibody
ICA-9 (specific for the casein kinase II motif, Fig. 1B) was analyzed by immunoblotting to determine
whether this antibody epitope resides within the casein kinase II site.
Immunoreactivity of full-length p53 (Fig. 5) was observed to
monoclonal antibodies DO-1 (lane1), PAb421 (lane3), and ICA-9 (lane5). In contrast,
although p53
4 reacted with DO-1 (lane2) and
PAb421 (lane4), it failed to bind to ICA-9 in an
immunoblot (lane6). These results affirm that
deletion of the final four acidic amino acids removes essential
determinants of ICA-9 antibody binding.
p53
Deletion of 30 amino acids from the COOH-terminal regulatory
site constitutively activates p53 (Hupp et al., 1992), but
whether the casein kinase II site itself contributes to p53 latency is
not known. As observed with full-length p53 (Fig. 6, lane1), pure fractions of unphosphorylated p534 Is Not Activated by Casein Kinase
II
4 are
unable to bind to DNA sequence-specifically (Fig. 6, lane3), indicating that deletion of the casein kinase II
phosphorylation site does not activate the specific DNA binding
function of the protein. This result indicates that the negative
regulatory domain does not reside within the acidic casein kinase II
site and that this acidic motif does not promote a direct steric block
of DNA binding. In addition, these results clearly pinpoint the other
highly conserved hydrophilic motif, encompassed by the basic stretch of
amino acids from 370 to 386 (Fig. 1B), as the negative
regulatory domain.
4.
Purified fractions of p53 (25 ng; from left to right, lanes1 and 2) or p53
4 (25 ng, from leftto right, lanes3 and 4) derived from gel filtration, were incubated with casein
kinase II at 30 °C for 30 min in activation buffer (2 ng of kinase
purified from rabbit muscle, lanes2 and 4,
as indicated) and subsequent DNA binding activity was measured at 0
°C as indicated under ``Experimental Procedures.'' The
activated, tetrameric p53
DNA complex is indicated by the open-headed arrow.
subunit contains the binding
site of basic effectors, it is also possible that the mechanism of p53
activation by casein kinase II involves the binding of the
subunit to the basic stretch of amino acids within the PAb421 binding
site.
4 can be activated by casein
kinase II, catalytic amounts of kinase were added in the presence of
ATP under conditions in which wild type p53 can be activated in an ATP-
or GTP-dependent manner (Hupp et al., 1993). Although casein
kinase II from rabbit muscle activates effectively full-length latent
p53 (Fig. 6, compare lane2 and lane1), p53
4 is not activated by casein kinase II
(compare lane 4 and lane 3). These results do not
rule out a role for the
subunit in the catalytic activation of
p53 through direct binding to basic amino acids within the negative
regulatory domain, but they do highlight the requirement for the acidic
DSD motif. In addition, these results also do not rule out the
possibility that stoichiometric amounts of casein kinase II can lead to
p53 activation through stable binding to the carboxyl terminus in a
manner analogous to PAb421 or DnaK.
Activation of p53
Does the casein kinase II site of p53
actually play an essential role in the activation reaction? If so, then
p534 by Proteins That Target the
Negative Regulatory Domain
4 should be irreversibly inactive and not responsive to other
activating agents. Two well established activators of full-length p53,
which bind to sequences within the regulatory domain (Fig. 1B, PAb421 and DnaK), were used to examine the
ability of p53
4 to respond to alternate activation in
vitro. As observed with full-length p53 (Hupp et al.,
1992), both PAb421 and DnaK can also activate the latent function of
p53
4 (Fig. 7A, lanes 2-6 and 8-12, respectively). Thus, activation of latent p53 by
PAb421 or DnaK does not require the acidic casein kinase II site. In
addition, protein kinase C is also able to activate p53
4 in a
manner similar to wild type p53
indicating that loss of the
casein kinase II site does not block activation by a distinct kinase.
4 by PAb421 and
DnaK. A, pure fractions p53
4 were analyzed in a
sequence-specific DNA binding assay by dividing the reaction into two
stages; p53
4 (25 ng) was first incubated at 30 °C for 30 min
with either PAb421 (lanes 1-6; 0, 1.5, 3, 6, 12, and 24
ng of PAb421, respectively) or DnaK (lanes 7-12; 0,
0.25, 0.5, 1, 2, and 4 µg of DnaK, respectively) followed by
incubation with radiolabeled target DNA at 0 °C. Products of the
reaction were separated by native gel electrophoresis as indicated
under ``Experimental Procedures.'' The open-headed arrow marks the migration of PAb421-activated p53 tetramers bound to
target DNA and the position of DnaK-activated p53
DNA complexes
are bracketed. B, summary of the specific activity of p53 or
p53
4 when activated by either PAb421 or
DnaK.
4, when
activated by PAb421 or DnaK (Fig. 7B), indicate that
deletion of the casein kinase II motif does perturb the structure of
the tetramer in an undefined manner.
PAb421-activated p53
The biochemical affects of the binding of the allosteric
inhibitory antibody, ICA-9, to p534 Is Immune to Inhibition by
ICA-9
4 was examined. ICA-9 binds to
the conserved casein kinase II motif of full-length p53 (Fig. 5)
and destabilizes (i) p53 activated in vitro by kinase or
antibody PAb421 or (ii) in vivo activated p53 which is
purified from insect cell expression systems (Hupp and Lane, 1994b).
The ability of ICA-9 to inhibit p53 has been important to establish
that p53 activation by phosphorylation is a reversible event, and
establishes a mechanism in which the activity of the protein can be
reversibly regulated by allosteric means. Following activation of
latent p53 tetramers by PAb421 (Fig. 8, lane4), a titration of ICA-9 leads to the destabilization of
PAb421
p53
DNA complexes (lanes5 and 6). PAb421-activated p53
4
DNA complexes (lane1) are immune to inhibition by ICA-9 (lanes2 and 3), consistent with the absence of the
ICA-9 epitope on p53
4.
4. p53
4 (25 ng; lanes 1-3) or p53 (25 ng; lanes 4-6) was
activated by PAb421 (12 ng) and prebound to target DNA. Subsequently,
ICA-9 was titrated (lanes2 (50 ng), 3 (500
ng), 5 (50 ng), and 6 (500 ng)) and reaction products
were analyzed as indicated under ``Experimental
Procedures.''
Casein Kinase II-independent Activation of p53 in
Vivo
p534 baculovirus expression vectors were constructed
to determine whether the two biochemically active forms of p53 produced
in insect cells (Fig. 2) require the COOH-terminal casein kinase
II site. As used for wild type p53, lysates obtained from insect cells
expression p53
4 were subjected to chromatography on
heparin-Sepharose to separate distinct biochemical forms of p53
4.
Interestingly, the profile observed with p53
4 was identical to
that observed with p53 (Fig. 9): one active fraction eluting at
0.40 M KCl (lane1), which does not bind to
PAb421 (lane2, p53
); and a
second active fraction eluting at 0.48 M KCl (lane4), which binds to one PAb421/tetramer (lane5, p53
). As p53
4 can
be activated by alternate agents in vitro (Fig. 7),
these results suggest that casein kinase II is not one of the major
enzyme(s) responsible for p53
4 activation in this cell line, but
that two other factors may be catalyzing p53 activation in
vivo.
4 in vivo. p53
4 baculovirus expression vectors
were constructed and used to infect insect cells, as indicated for wild
type p53 (see ``Experimental Procedures''). After p53
4
production, cells were lysed and applied to a heparin column as
indicated for full-length p53 in Fig. 2. Bound protein was
eluted with a linear KCl gradient and p53 fractions (30 ng) were
analyzed in a DNA binding assay with and without the indicated
antibodies: lanes1 and 4, no antibody; lanes2 and 5, 100 ng of PAb421; lanes3 and 6, 100 ng of
DO-1.
Allosteric Activation and Conformational
Flexibility
Analysis of the regulation of the sequence-specific
DNA binding function of wild type p53 has shown that p53 assembles into
latent tetramers and post-translational modification of a COOH-terminal
negative regulatory domain in vitro by protein kinase C or
casein kinase II unmasks the cryptic site-specific DNA binding function
of the tetramer (Hupp et al., 1992; Hupp and Lane, 1994a). A
monoclonal antibody that binds near the protein kinase C site can mimic
the effects of kinases and activate p53 through a concerted transition
of subunits within the tetramer after the binding of two antibody
molecules/p53 tetramer (Hupp and Lane, 1994b). The induction of a
specific conformational change within the p53 tetramer after
post-translational modification of the regulatory site is only inferred
as p53 is converted from a low affinity to a high affinity DNA binding
form. Direct, physical evidence for conformational changes within the
tetramer during activation is lacking. To this end, we explored the
possibility that alterations in the conformationally flexible core
domain are a consequence of p53 activation./PAb240
; Milner and
Medcalf, 1991) and cosynthesis of inactive mutant and active wild type
p53 inactivates DNA binding by wild type p53 in vitro (Bargonetti et al., 1992; Halazonetis and Kandil, 1993).
These results indicate that the conformation of the internal core
domain can have a profound affect on tetramer activity. A major
question thus remains unresolved from the independent analysis of the
conformationally flexible domain of p53 and allosteric activation of
DNA binding. Does activation of latent p53 coincide with an alteration
in the conformationally flexible core DNA binding domain?
)
and monoclonal antibody PAb421 does not
therefore involve the re-folding of ``denatured'' p53.
/PAb240
), whereas p53
inactivated by zinc chelators becomes inactive and unfolded
(PAb1620
/PAb240
). The removal of
zinc ions presumably distorts and unfolds the core domain, exposing the
PAb240 epitope (Cho et al., 1994). Our data using purified
protein indicates that the correctly folded, latent form of p53
(PAb1620
/PAb240
) is biochemically
distinct from unfolded, inactive p53
(PAb1620
/PAb240
). This is further
substantiated by demonstrating that latent p53 can be unfolded in
vitro after its dilution into a destabilizing buffer and coating
onto a plastic surface, exposing the PAb240 epitope (Fig. 3D). These data indicate that allosteric
activation is not coupled to an alteration in refolding of the domain
recognized by PAb1620 and that a novel, as yet unidentified,
conformational change in p53 is involved in the interconversion between
latent and activated tetramers.
Acidic Amino Acids within the Casein Kinase II Site Do
Not Harbor the Negative Regulatory Domain
The tetrameric nature
of p53 and the existence of the casein kinase II motif (amino acids
388-393) within the presumed negative regulatory domain region
provide an excellent model system to study the affects of
phosphorylation on the allosteric regulation of an oligomeric protein.
Prior to these studies, it has not been clear whether the minimal
negative regulatory domain is contained within the basic or acidic
region within the COOH terminus. Knowing this information will aid in
the formation of models to explain the mechanism whereby
unphosphorylated p53 remains in the latent state.Casein Kinase II-independent Activation of p53 in
Vivo
The sequence-specific DNA binding activity of full-length
p53 is directly regulated by casein kinase II and protein kinase C in vitro (Hupp and Lane, 1994a). These results indicate that
the COOH-terminal regulatory site of p53 has evolved a functionally
relevant interaction with distinct regulatory enzymes and implies that
diverse signaling pathways may be involved in activating the tumor
suppressor function of p53, but the enzymes that specifically target
the COOH terminus in cells are not yet clear. The situation is likely
to prove very complex, as some cell lines appear to use casein kinase
II for activating the growth suppressor function of p53 (Milne et
al., 1992b), whereas other cell lines modify wild type p53
resulting in loss of the PAb421 epitope in the COOH-terminal regulatory
site (Ullrich et al., 1992b; Fiscella et al., 1994;
Hupp and Lane, 1994a; Delphin and Baudier, 1994), suggesting the
activation of a protein kinase C-like pathway. In addition, murine
cells may even overcome kinase-dependent pathways giving rise to an
alternatively spliced and activated form of p53, lacking the
COOH-terminal negative regulatory domain encoded by exon 11
(Kulesz-Martin et al., 1994; Wu et al., 1994).
Nevertheless, we show in this report for the first time that altering
the growth state of the cell can directly affect the activation of the
sequence-specific DNA binding activity of p53. In the Sf9 cell line we
are studying, one p53 activation pathway can be activated by UV
irradiation or suppressed by serum withdrawal, whereas the other
pathway is constitutive. In addition, the constitutively activated
pathway can be stimulated by phorbol esters, indicating
that distinct signaling events can channel information into the
regulatory site of p53 and activate its DNA binding function.
4 characterized in vitro facilitates
interpretations of experiments designed to explore the types of
activating factors which function in vivo. Catalytic
activation of p53
4 by casein kinase II is not possible in
vitro, confirming that this regulatory site mutation effectively
precludes casein kinase II-dependent activation of p53. Using the
combined strength of the insect cell line for the production of two
activated forms of p53 and the technical discovery that three
biochemical forms of p53 can be resolved chromatographically, we show
that the production of the two major activated forms of p53 occurs in vivo through a casein kinase II-independent pathway. This
is consistent with data showing that transcriptional activation or
sequence-specific DNA binding does not require the casein kinase II
phosphorylation site in one type of cell line (Fiscella et
al., 1994). However, we cannot rule out the possibility that
casein kinase II does activate p53 to a small extent in vivo,
since small amounts of casein kinase II-activated p53 might be
unresolved from the high levels of activated p53 produced through
casein kinase II-independent means. Clearly, the production of single
and double regulatory site mutations in p53 coupled with the use of the
eukaryotic expression system we are using to activate p53 in vivo and the chromatographic method used to resolve different forms of
p53 provides a powerful model system to identify signaling pathways
which can regulate the sequence-specific DNA binding activity of p53 in
cells.
We thank our colleagues in Dundee for assistance and
critical reading of the manuscript, particularly Kathryn Ball, Carol
Midgley, and Silke Hansen.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.