(Received for publication, September 30, 1994; and in revised form, December 16, 1994)
From the
The p53 tumor suppressor protein is a transcription factor with sequence-specific DNA binding activity that is thought to be important for the growth-inhibitory function of p53. DNA binding appears to require activation of a cryptic form of p53 by allosteric mechanisms involving a negative regulatory domain at the carboxyl terminus of p53. The latent form of p53, reactive to the carboxyl-terminal antibody PAb421, is produced in a variety of eukaryotic cells, suggesting that activation of p53 is an important rate-limiting step in vivo. In this report we provide evidence that phosphorylation of serine 378 within the carboxyl-terminal negative regulatory domain of the human p53 protein by protein kinase C correlates with loss of PAb421 reactivity and a concomitant activation of sequence-specific DNA binding. These effects are reversed by subsequent dephosphorylation of the protein kinase C-reactive site by protein phosphatases 1 (PP1) and 2A (PP2A), which restore the reactivity of p53 to PAb421 and regenerate the latent form of p53 lacking significant DNA binding activity. Thus, p53 is subject to both positive and negative regulation by reversible enzymatic modifications affecting the latent or active state of the protein, suggesting a possible mechanism for the regulation of its tumor suppressor function.
Inactivation or loss of p53 is a common event associated with the development of human cancers. Inactivation of p53, resulting from mutations within the p53 gene, or interaction of the p53 protein with viral and cellular oncogenes, is intimately associated with tumorigenesis(1, 2, 3, 4, 5) . At the cellular level, loss or inactivation of wild type p53 leads to deregulation of the cell cycle and DNA replication, inefficient DNA repair, loss of cellular apoptotic responses, selective growth advantage, and, consequently, tumor formation. Many studies now indicate that it acts as an important regulator in the cellular response to oxidative stress and DNA damage (6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Biochemical studies have suggested several potential mechanisms underlying p53-mediated growth suppression. Its ability to act as a transcription factor has been particularly well studied, both as a transactivator and repressor of gene expression. Transactivation by p53 is sequence-specific and correlates with its binding to DNA sequences that are similar to or identical with the recently reported consensus binding site(16, 17) . p53 can efficiently activate transcription from promoters bearing such sites, in vitro and in vivo(18, 19, 20, 21, 22, 23) . In contrast, suppression appears less selective, and p53 has been shown to suppress a variety of promoters containing TATA elements(20, 24, 25, 26, 27) , an effect that may involve binding of p53 to components of the basal transcription machinery, such as the TATA binding protein(26, 28, 29) . Most oncogenic mutants of p53 have lost their ability to function effectively as transcription regulators, suggesting that these activities of p53 may be critical to its tumor suppressor function. Alternatively, chimeric p53 proteins with foreign transactivation and/or dimerization domains inhibit cell growth, indicating that conservation of the central region of p53, mediating sequence-specific DNA binding, is sufficient and necessary to confer growth-suppressive function to such hybrid proteins(30) . This suggests that DNA binding and activation of p53 target genes is an important step in p53-mediated tumor suppression.
The DNA binding domain of p53 is located in the core region of the molecule(31, 32, 33, 34) . Regulation of DNA binding activity is mediated by the dimerization and tetramerization domains within the carboxyl terminus of p53 and by a regulatory domain within the carboxyl-terminal 30 amino acids that has been implicated in negative autoregulation of sequence-specific DNA binding(35, 45) . Cellular p53 is phosphorylated at amino- and carboxyl-terminal sites(36) , suggesting that kinases and phosphatases may be involved in the regulation of p53 tumor suppressor function. A role for serine 389, a target site for casein kinase II, in murine p53-mediated growth suppression has been proposed(46) . Phosphorylation of the carboxyl terminus of human p53 by casein kinase II has been shown to unmask the cryptic DNA binding activity of bacterial expressed p53(35) , indicating that p53 is subject to positive regulation by post-translational modification and raising the possibility that such activation may be important for p53 growth suppressor function. In contrast, substitution of Ser-389 in murine p53 with aspartic acid has been reported to have no apparent effect on the DNA binding activity of in vitro translated p53 protein (47) and raised the question of whether phosphorylation of serine 389 by casein kinase II is important for murine p53 growth suppressor function via a mechanism which, however, is ancillary to sequence-specific DNA binding(47) . More detailed studies appear required to clarify this issue.
Most recently, murine p53 has been shown to be phosphorylated by protein kinase C(37) . However, the site of phosphorylation and the functional consequences of such a modification are not known. Potential consensus target sites are located within the carboxyl-terminal 30 amino acids. Studying potential modifications at the carboxyl terminus of p53 are of particular interest, since loss of p53 reactivity to the carboxyl-terminal antibody PAb421 occurs in vivo upon growth arrest of glioblastoma cells as a result of overexpression of p53 (38) and in resting lymphocytes(39) . These findings suggest a correlation between loss of PAb421 reactivity and the growth suppressor function of p53.
In this report we show that protein
kinase C targets a serine(s) in the negative regulatory domain within
the carboxyl-terminal basic region of the human p53 protein.
Phosphorylation of this site is associated with the unmasking of
cryptic p53, and, strikingly, loss of PAb421 reactivity occurs
concurrently with activation of sequence-specific DNA binding. These
effects are reversible, and dephosphorylation by protein phosphatases 1
(PP1) ()or 2A (PP2A) restores PAb421 reactivity and
regenerates the latent form of p53. Thus, p53 is subject to both
positive and negative regulation by reversible enzymatic modifications
affecting the latent or active state of the protein, suggesting a
possible mechanism for the regulation of its tumor suppressor function.
Figure 1:
Protein kinase C
phosphorylates p53. A, SDS-PAGE and silver stain analysis of
purified p53 proteins (lane 1, His-p53; lane 2, p53; lane 3, His-p53). B, phosphorylation of purified
human p53 proteins produced in bacteria: His-p53 (a) or in Sf9
insect cells: p53 (b) and His-p53 (c). Reactions were
incubated for 15 min at 30 °C with the indicated dose of protein
kinase C (nanograms/reaction) or for the indicated period of time (in
minutes) with 10 ng of PKC/reaction. Maximum levels of P
incorporation (in moles of phosphate/mol of p53) measured were:
bacterial p53, 0.9; baculovirus p53, 0.7; baculovirus His-p53,
0.65.
Figure 3:
Protein kinase C and protein phosphatases
1 (PP1) and 2A (PP2A) modulate p53 reactivity to PAb421. A,
PP1 and PP2A dephosphorylate PKC-P-labeled baculovirus
His-p53. Proteins were analyzed by SDS-PAGE and autoradiography. B, effects of PKCmediated phosphorylation and subsequent
dephosphorylation by protein phosphatases PP1 and PP2A, on the
immunoreactivity of baculovirus-produced p53 to antibodies PAb421 and
PAb1801 (lanes 1-4), as determined by Western blot
analysis. Lanes 5 and 6, effects of phosphatases on
untreated p53 protein.
At a molar ratio of p53/PKC of 250,
more than 80% of the total incorporated phosphate is transferred within
3 min of the start of the reaction, indicating that p53 is an effective
PKC substrate (Fig. 1B, panel c). PKC
phosphorylation of unphosphorylated p53 produced in bacteria results in
the incorporation of up to 0.9 mol of phosphate per mol of p53 and a
ratio of
0.7 mol of phosphate per mol of p53 was calculated for
p53 produced in insect cells. Similarly, incorporation of
0.65 mol
of phosphate/mol of p53 was measured for the purified baculovirus
His-p53 fusion protein.
The presumed consensus target site for PKC
phosphorylation in p53 is located at the carboxyl terminus and
encompasses the PAb421 epitope (37) . Several observations are
consistent with a serine(s) in this region being targeted by PKC.
Initial studies using monoclonal antibody DO1-immunoprecipitated in
vitro translated full-length p53, or carboxyl-terminal deletion
mutants of the p53 protein (p53(1-389) and p53(1-347)), as
substrates, indicated that phosphorylation occurs within the
carboxyl-terminal 46 amino acids (relative P
incorporation: wild type p53, 1.0; p53(1-389), 1.0; p53(1-147), <0.1. Equal amounts of p53
protein were used, as determined by Western blot analysis, not shown).
Subsequently, purified p53 protein was used. Phosphorylation of
purified baculovirus-p53 is inhibited by prior inclusion of the
monoclonal antibody PAb421, which targets the carboxyl terminus of p53
(residues 370-378), but not PAb1801, which targets the amino
terminus of p53 or PAb1620 (Fig. 2A). Phosphorylation
of purified p53 is also inhibited by co-incubation with a PAb421
epitope containing peptide (residues 369-382,
NH
-LKTKKGQSTSRHKKCOOH) which is also effectively
phosphorylated (Fig. 2A, lane 3), directly
implicating this region as the potential target for phosphorylation by
PKC (Fig. 2, A and B). In contrast, other
carboxyl-terminal peptides covering residues 340-357,
369-382, and 389-393 (includes the casein kinase II site)
do not compete as PKC substrates. Further analysis of the targeted
region indicates that peptide NH
-TSRHKKL-COOH (residues
377-383) is still effectively phosphorylated by PKC (Fig. 2B). Site-specific mutations, including changes
in residues 377 and 378 from threonine or serine to glycine,
respectively, indicate that it is serine 378 that is phosphorylated (Fig. 2B), consistent with phosphoamino acid analysis
of the phospho-PAb421 peptide which demonstrates that a serine is
phosphorylated (Fig. 2C). Another p53 synthetic peptide
(residues 372-379) encompassing serine 376 and containing glycine
at positions 377 and 378, NH
-KKGQSGGR, was reproducibly not
phosphorylated by PKC (data not shown). Importantly, as evidenced by
phospho-p53 phosphoamino acid analysis, PKC also phosphorylates wild
type p53 at a serine (Fig. 2C). Altogether, these
findings strongly suggest that serine 378 within the PAb421 epitope of
p53 is the predominant site phosphorylated by PKC.
Figure 2:
Protein kinase C targets the PAb421
epitope in p53. A, phosphorylation of purified baculovirus p53
(-PKC, lane 1; +PKC, lane 2) in the
presence of carboxyl-terminal p53 peptides, including the competing
p53-PAb421 epitope peptide
NH-LKTKKGQSTSRHKK-COOH(369-382) or the
carboxyl-terminal antibody PAb421 and the amino-terminal antibody
PAb1801. Proteins were analyzed by SDS-PAGE and autoradiography. B, phosphorylation of p53-421 (lane 1) or truncated
p53-421 peptide (TSRHKKL, residues 377-383, lane 2) and
corresponding mutants (substitutions: lane 3, Ser-378
Gly; lane 4, Thr-377
Gly). Other peptides: lane
5, GPDSD (residues 389-393); lane 6,
MFRELNEALELKDAQAGK. Equal amounts of labeled peptides were spotted onto
phosphocellulose paper, washed with 1% H
PO
, and
detected by autoradiography. C, identification of PKC as a p53
serine kinase. Phosphoamino acid analysis of PKC-labeled baculovirus
His-p53 and p53-421 peptide is shown. TLC migration of amino acid
standards indicates the nature of labeled residue. O,
origin.
Equal amounts of protein kinase C-treated p53 protein were resolved by SDS-PAGE and subsequently subjected to immunoblot analysis using as probes either antibody PAb421 or PAb1801. Strikingly, phosphorylation of p53 by protein kinase C reduces the reactivity of p53 to PAb421 but not to PAb1801, a monoclonal antibody that targets the amino terminus of p53 (Fig. 3B, lane 1 versus 2). That such a change in PAb421 reactivity is detected using denatured and immobilized p53 indicates that the specific loss in the ability of p53 to interact with PAb421 antibody can be attributed to a direct steric effect of the added phosphate. Prior incubation of the PKC-treated p53 protein with phosphatases PP1 or PP2A effectively restores subsequent PAb421 reactivity (Fig. 3B, lanes 3 and 4). In contrast, treatment of non-PKC-treated purified p53 protein with PP1 or PP2A does not significantly increase the levels of PAb421-reactive p53 species (Fig. 3B, lane 5 versus lanes 6 and 7), suggesting that the major form of the isolated protein is nonphosphorylated at the PKC-reactive site. This is consistent with the observation that comparable levels of phosphate are incorporated into bacterial and baculovirus p53 proteins (Fig. 1).
Fig. 4A shows that purified
p53, pretreated with PP1, binds DNA predominantly in the form of two or
three complexes (similar to the non-PP1-treated p53); a fast migrating
complex consisting of a p53 tetramer bound to DNA (p53, see lane 2) and slower migrating complexes consisting of
oligomeric forms of p53 bound to DNA (p53
, lane 2, e.g. associated tetramers?), consistent with previous reports (e.g.(42) ). Inclusion of PAb421 leads to supershift
of the p53
complex and an apparent disruption of oligomeric
p53 complexes. In addition, PAb421 produces a pronounced activation of
cryptic p53, as indicated by the increase in the levels of a
PAb421-activated p53-DNA complex, p53
/Ab (lanes
3-9, see also, for example, Fig. 4C, lane 3), as previously reported for p53 protein produced in
bacteria(35) .
Figure 4:
Regulation of the DNA binding function of
p53. A, left side, DNA binding activity of purified
baculovirus His-p53 protein and activation of latent p53 by PAb421
(0-100 ng) (lanes 2-9). Weak formation of one
intermediate is detected. Right side, activation by PAb1801
(1.5-100 ng) (lanes 10-16) and dosedependent
formation of Ab-p53-DNA intermediates. B, activation of p53
DNA binding by protein kinase C. Purified baculovirus His-p53 was
incubated with PKC (0, 0.1, 1, 5, 10 ng of PKC/reaction, lanes
2-11) and tested for DNA binding in the absence (lanes
2-6) or presence (lanes 7-11) of PAb421 (100
ng/reaction). C, inhibition of p53 DNA binding by protein
phosphatases. Purified baculovirus His-p53 was treated with PKC (lanes 8-16) and subsequently further incubated in the
absence (lanes 8-10) or presence (lanes
11-16) of phosphatases PP1 and PP2. D, effect of
protein phosphatases on DNA binding of untreated baculovirus His-p53 in
the absence (lanes 1-3) or presence (lanes
4-6) of PAb421. p53, p53
tetramer; p53
, p53 oligomer; Ab,
antibody; Ab
, Ab
, Ab
,
predicted number of antibody molecules in pAb1801-p53-DNA complex. p53
protein was analyzed by electromobility gel shift assay as described
under ``Experimental Procedures.'' Lanes labeled as probe contain labeled DNA probe
only.
Interestingly, throughout a PAb421 titration (Fig. 4A, lanes 3-9), the
p53/Ab complex represents the apparent single predominant
PAb421-p53-DNA complex that is formed. Only weak formation of a faster
migrating apparent intermediate is detected. This suggests that the
activated protein-DNA complex has a defined ratio of p53, DNA, and
antibody molecules.
Fig. 4A (lanes
10-16) shows the effects of adding PAb1801 on the formation
of p53-DNA intermediates. In contrast to the effects observed with
PAb421, addition of PAb1801 does not activate latent p53, but instead
supershifts the p53 and p53
complexes and
produces the formation of multiple p53-DNA intermediates. Only one
intermediate (p53
/Ab
) is formed upon
supershift of the p53
complex with PAb1801. This is
consistent with the PAb1801-saturated complex containing two bivalent
molecules of antibody bound per p53 tetramer
(p53
/Ab
). Three intermediate complexes are
formed upon supershift of the oligomeric p53-DNA complex
p53
, consistent with it representing two associated p53
tetramers and the PAb1801-saturated complex having two molecules of
bivalent antibody bound per p53 tetramer
(p53
/Ab
). The findings that PAb1801, unlike
PAb421, does not prevent or disrupt the formation of oligomeric p53
bound to DNA is consistent with oligomerization being mediated by the
basic carboxyl terminus of p53(44) . Comparison of the
migration of the PAb1801-induced p53
/Ab
complex with that of the predominantly formed PAb421-p53-DNA
complex, is consistent with two molecules of bivalent PAb421 being
bound to p53 in the activated complex p53
/Ab. This is in
agreement with the recent findings by Hupp and Lane(48) ,
reported after submission of this manuscript, and is further supported
by experiments using PKC-activated p53, as described below.
PKC effectively activates DNA
binding of either the tetrameric or oligomeric forms of p53, although
the increase in the levels of p53 tetramer bound to DNA is more
pronounced (Fig. 4, B and C). This activation
is dose-dependent (Fig. 4B); however, it does not
exactly mirror the profile of the dose-dependent phosphorylation of p53
by PKC (see Fig. 1). This could reflect the requirement of a
minimum number of p53 molecules in a p53 tetramer to be phosphorylated
in order for the activation to occur, i.e. activation of DNA
binding is not directly proportional to phosphorylation and may be
regulated in a concerted manner, similar to activation of p53 by PAb421
which requires binding of more than one molecule of antibody. Addition
of PAb421 (lanes 7-11), reveals a parallel decrease in
the supershifted p53/Ab
complex, as well as an
increase in the appearance of one novel PAb421-p53-DNA complex
(p53
/Ab
). The formation of this novel complex
can be explained by the partial phosphorylation of a p53 tetramer and
the resulting loss in PAb421 reactivity. The apparent formation of only
one such intermediate complex is consistent with one molecule of
bivalent antibody binding to the partially phosphorylated p53 tetramer
and two molecules of PAb421 bound to nonphosphorylated p53, as
previously suggested by experiments described above. The absence of the
significant formation of the p53
/Ab
complex
upon addition of PAb421 to untreated p53 protein (lane 7, see
also Fig. 4C, lane 3, and Fig. 4D) is further consistent with the p53 protein
produced in insect cells not being significantly phosphorylated at the
PKC reactive site. Phosphorylation at this site would be expected to
reduce PAb421 reactivity.
Additional observations can be made from
multiple experiments of this type: the p53/Ab
complex can be generated from distinct phosphorylated p53
species. In most binding reactions, a significant fraction of the novel
PAb421-p53 complex (p53
/Ab
) appears to derive
from the PKC-activated p53-DNA complex (p53
), which is
partially supershifted by PAb421 (see Fig. 4C, lane
8 versus 9). However, a significant fraction of the novel
PAb421-p53-DNA complex may also be generated by activation of a
partially phosphorylated, but latent and PAb421-responsive p53 protein
(similar to nonphosphorylated p53). This is indicated by the increase
in p53
/Ab
complex formation in the absence of
a comparable loss of supershiftable p53
(see Fig. 4, B and C). These results indicate that a partially
phosphorylated p53 tetramer can be active in DNA binding, but that
activation only occurs when more than one molecule of p53 in a p53
tetramer is phosphorylated. Alternatively, partial phosphorylation and
binding of one molecule of pAb421 may induce a concerted transition to
the activated state. The apparent existence of distinct phosphorylated
forms of p53 tetramers with differing DNA binding activities is likely
to reflect a difference in the degree of phosphorylation of such
complexes.
We show in this report that protein kinase C, and the protein phosphatases PP1 and PP2A, can modulate the negative autoregulatory function of the carboxyl terminus of p53 and, consequently, the sequence-specific DNA binding function of p53.
Taken together, several observations point to serine 378 being the predominantly targeted residue in human p53 involved in such regulation. 1) Phosphoamino acid analysis demonstrates that protein kinase C phosphorylates p53 and the p53-421 peptide at a serine(s). 2) Phosphorylation of p53 results in a direct, conformation independent, loss in PAb421 reactivity. 3) The p53-421 peptide competes effectively with p53 for phosphorylation by protein kinase C. 4) Peptide deletion and site-specific mutation analyses show that serine 378 is phosphorylated.
PKC-mediated phosphorylation of p53 can be associated with the formation of multiple distinct p53 tetramer species that are either latent or active in DNA binding. These are: 1) activated PAb421-negative p53, 2) activated PAb421-reactive p53, 3) latent PAb421-reactive p53, which appear to reflect differences in the degree of phosphorylation of a p53 tetramer. Existence of a PKC-activated/PAb421-reactive form of p53 indicates that partial phosphorylation of a p53 tetramer can be sufficient to activate DNA binding. Phosphorylation of more than one p53 molecule per tetramer is, however, required for the concerted transition to an activated state. Such modulation of the sequence-specific DNA binding function of p53 represents an apparent novel mechanism for transcription factor activation by PKC.
Such regulation of p53 is of particular interest
in light of recent findings that loss of PAb421 reactivity of p53
occurs in vivo upon growth arrest of glioblastoma cells as a
result of overexpression of p53(38) , in resting
lymphocytes(39) , and apoptotic cells, ()implicating
the PAb421-negative form of p53 as a possible active tumor suppressor
form of p53. Phosphorylation of human p53 by casein kinase II does not
generate a PAb421-negative form of p53 in vitro. (
)Similarly, mutation of serine 389 in murine p53 to
aspartic acid does not change pAb421 reactivity(47) . In
contrast, results presented here suggest that PKC and protein
phosphatases PP1 or PP2A, or cellular enzymes with the specificity of
these, may provide a mechanism to regulate the transition of p53 from a
latent (PAb421-reactive) to active (PAb421-negative) (or vice versa)
DNA binding transcription factor. The monoclonal antibody PAb421 could
be used as a molecular probe to distinguish various forms of p53,
monitor potential post-translational modification in vivo, and
establish the functional significance of modification of the
PKC-reactive site in the activation of cellular p53. Which type of
protein kinase might be a functionally relevant regulator of p53 is not
yet known, and identification of a presumed active kinase will
constitute an area of considerable interest.