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Volume 270, Number 10, Issue of March 10, 1995 pp. 5405-5411
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Sequence-specific DNA Binding Function of p53 by Protein Kinase C and Protein Phosphatases (*)

(Received for publication, September 30, 1994; and in revised form, December 16, 1994)

Ivone Takenaka ,  Francine Morin ,  Bernd R. Seizinger ,  Nikolai Kley (§)

From the Department of Molecular Genetics and Cell Biology, Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.

INTRODUCTION

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) (^1)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.

EXPERIMENTAL PROCEDURES

Purification of Human p53 Proteins

Human WTp53 or His-human WTp53-fusion proteins were generated by cloning of p53 cDNAs into the pBlueBac III or pBlueBacHis vectors (Invitrogen), respectively, and subsequent selection of recombinant baculoviruses. 48 h postinfection, extracts of infected Sf9 insect cells were prepared by sonication in buffer A (50 mM Tris-HCl, pH 7.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and protease inhibitors aprotinin, pepstatin A, and leupeptin at 5 µg/ml each). Lysates were centrifuged at 20,000 rpm for 30 min, 4 °C. Supernatant was chromatographed through a Q-Sepharose fast flow FPLC column (Pharmacia Biotech Inc.) and eluted with a linear salt gradient in a buffer containing 25 mM Tris-HCl, pH 7.5, 50-500 mM NaCl, 1 mM EDTA, and 1 mM DTT. Immunoblot analysis revealed the fractions containing p53. The Q-Sepharose p53 eluate, and, in certain cases, the cell lysate directly, was bound to an affinity column of Protein A-Sepharose beads, coupled to the monoclonal antibody PAb421, in a buffer containing 150 mM NaCl, 15% glycerol, 40 mM HEPES, pH 7.5, 0.2 mM EDTA, 3 mM EGTA, 1 mM DTT, 0.2 mM Na(2)S(2)O(5), protease inhibitors (pepstatin A, aprotinin, and leupeptin) at 5 µ/ml each, and 0.5% Nonidet P-40. Bound p53 was eluted with a synthetic peptide encompassing the PAb421 epitope (NH(2)LKTKKGQSTSRHKK-COOH) at 0.1 mg/ml in the same buffer. Fractions containing p53 were pooled, separated from the peptide by chromatography through a gel filtration column (G-50), and dialyzed against 200 mM NaCl, 50% glycerol, 40 mM HEPES, pH 7.5, 0.2 mM EDTA, 1 mM EGTA, and 1 mM DTT, and protease inhibitors at 5 µg/ml, as above. p53 protein was >95% pure as judged by SDS-PAGE and silver staining, whether or not the initial Q-Sepharose step was introduced. Thus, lysates were directly added to the PAb421 column in subsequent purifications. Baculovirus His-p53 fusion protein, or bacterial His-p53 protein generated by expression of p53 from a recombinant pET19 (Novagen) expression vector (kindly provided by N. Horikoshi, R. Zandomeni, and R. Weinmann), was purified by nickel affinity chromatography, essentially as described previously(32) . p53 proteins were >95% pure as judged by SDS-PAGE and silver staining.

Phosphorylation of p53

Purified p53 protein was phosphorylated in a reaction mixture (20 µl) containing: reaction buffer PK (25 mM Tris-HCl, pH 7.0, 2 mM MgCl(2), 0.1 mM ATP containing [-P]ATP, 0.8 mM CaCl(2), and 10 mM phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidylserine, Triton X-100 mixed micelles). Where indicated, peptides at 1 mM final concentration or monoclonal antibodies (100 ng) were present. Reactions were incubated at 30 °C with purified (>97%) protein kinase C (a mixture of alpha, beta, and isoforms; UBI). Where indicated, PKC peptide inhibitor was added, and the reaction was incubated for an additional 15 min at 30 °C, in the absence or presence of purified protein phosphatases PP1 and PP2A (UBI; 0.02 unit/reaction). Products were subsequently separated by electrophoresis in 15% SDS-polyacrylamide gels. Reactions containing peptides were separated through a step gradient polyacrylamide gel (up to 20%). Radioactivity incorporated into p53 was subsequently determined and expressed as moles of phosphate incorporated per mol of p53 protein.

Phosphoamino Acid Analysis

PAb421 peptide or purified His-p53 (2 µg) were phosphorylated with PKC, resolved by 10% or 25% SDS-PAGE, and transferred to Immobilon membranes. Phosphoamino acid analysis was performed essentially as described previously(40) . Labeled peptide or p53 was hydrolyzed with 6 N HCl for 1 h at 110 °C. Dried hydrolysates were resuspended in running buffer (pH 2.5) (5.9% glacial acetic acid, 0.8% formic acid (88%), 0.3% pyridine, and 0.3 mM EDTA) containing phosphoserine, phosphothreonine, and phosphotyrosine standards. P-Amino acids were resolved by phosphocellulose-TLC using running buffer (pH 2.5)(41) .

Activation of p53, Immunoblot Analysis, and Electromobility Gel Shift Assays

In these experiments, the consequence of phosphorylation and dephosphorylation on the reactivity of p53 with monoclonal antibodies PAb421 and PAb1801, and the DNA binding activity of p53, were determined for one and the same p53 protein sample.

Phosphorylation-Dephosphorylation

p53 protein (1 µg) was incubated in 10 µl of kinase reaction buffer PK (see above), supplemented with 1 mM ATP, in the presence or absence of protein kinase C (0-20 ng) at 30 °C for the indicated time periods (0-20 min) or 30 min in all subsequent experiments. Reactions were stopped by the addition of a peptide PKC inhibitor (Life Technologies, Inc.), and reaction volume was brought to 15 µl by the addition of phosphatase reaction buffer (10% glycerol, 40 mM Tris-HCl, pH 7.0, 0.1 mM CaCl(2), 40 mM MgCl(2)). Reactions were further incubated at 30 °C for 30 min in the presence or absence of phosphatases (purified PP1 (0.02 unit) or PP2A (0.02 unit, UBI)). Subsequently, 15 µl of ice cold Buffer BB (20% glycerol, 50 mM KCl, 1 mg/ml bovine serum albumin, 0.1% Triton X-100, 5 mM DTT) were added (30 µl final), and samples were put on ice. Separate aliquots were then analyzed by immunoblotting (10 µl) or tested for DNA binding activity by electromobility gel shift assay (2 µl).

Dephosphorylation

p53 protein (1 µg) was incubated in 10 µl of phosphatase reaction buffer in the presence or absence of purified PP1 or PP2A (0.02 unit, UBI), for 30 min at 30 °C. Ice cold buffer BB was added to the reaction (10 µl), and an aliquot (2 µl) of the samples was tested for DNA binding activity.

Immunoblot Analysis

Equal amounts of p53 protein (10 µl of reaction sample) were resolved by SDS-PAGE and analyzed by immunoblotting, using as probes the monoclonal antibodies PAb421 or PAb1801.

DNA Binding

Approximately 60 ng of p53 protein (2 µl of reaction sample) were incubated in a final reaction volume of 20 µl containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 1 mg/ml bovine serum albumin, 0.1% Triton X-100, 210 ng of poly(dIbulletdC) competitor, and 0.2 ng of P-labeled double-stranded p53 DNA recognition sequence 5`-GGACATGCCCGGGCATGTC-3`(17) . Reactions were incubated for 15 min at room temperature, and DNA-protein complexes were resolved on a 4.5% polyacrylamide gel containing 0.5 times TBE (1 times TBE = 50 mM Tris borate, pH 8.3, 1 mM EDTA). Mutant oligonucleotide used for competition studies to confirm specificity of binding was altered in the second consensus sequence box to 5`-GGACATGCCCGGGCTTTTC-3`.

In Vitro Translation and Immunoprecipitations

In vitro translated proteins were expressed using SP6 transcribed RNA (0.1-1 µg) and rabbit reticulocyte lysate. Plasmid proSp53, encoding full-length wild type p53, was used as a template for in vitro transcription of full-length p53, and truncated versions of p53 were generated using DraII (p53 1-389) and StuI (p53 1-347) linearized p53 recombinant plasmids. Proteins were immunoprecipitated in a buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 0.1 mM ZnCl(2), 0.5 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, using immobilized monoclonal antibody DO1 (agarose conjugate, Santa Cruz Biotechnology). Monoclonal antibodies PAb1801 and PAb421 (Oncogene Science) and polyclonal antibody CM1 (Novocastra Laboratories) were used for Western blotting.

RESULTS

Phosphorylation of p53 by Protein Kinase C

Human p53 protein produced in Sf9 insect cells or bacteria was purified to near homogeneity (Fig. 1A) and used in phosphorylation studies. Fig. 1B shows that p53 protein from either source is an effective substrate for protein kinase C (panels a and b) that is rapidly modified upon addition of the enzyme (panel c). Phosphorylation was reduced in the absence of Ca and cofactors (data not shown). However, consistently, a low level of P-labeled p53 was detected even in the absence of added PKC (Fig. 1B, 0 ng PKC, panels a and b), when baculovirus produced p53, as opposed to bacterially produced p53, was used. This observation is similar to results previously reported for purified baculovirus-mouse p53 protein (37) and could be due to partial co-purification of a p53-associated kinase activity from insect cells. If so, however, the site of phosphorylation would be different from the site targeted by protein kinase C, as suggested by its relative insensitivity to protein phosphatases (see below, Fig. 3A).

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(2)-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(2)-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(2)-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(2)-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(3)PO(4), 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.


Protein Phosphatases PP1 and PP2A Dephosphorylate the PKC-reactive Site in p53

Concurrently to the analysis of the effects of PKC, we tested the effects of two protein phosphatases: protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Both enzymes target the serine in p53 which is phosphorylated by PKC; as shown in Fig. 3A, either purified phosphatase effectively dephosphorylates PKC-labeled phospho-p53 (lane 2 versus lanes 4 and 6). Neither phosphatase, however, significantly decreased the level of P incorporation associated with the incubation of p53 with labeled ATP in the absence of added protein kinase C (lane 1 versus lanes 3 and 5), an effect possibly mediated by a co-purified kinase activity other than protein kinase C.

Protein Kinase C Phosphorylation of p53 Reduces PAb421 Reactivity

Since phosphorylation of p53 by PKC appears to occur within the PAb421 epitope, we tested as to whether this would affect the reactivity of p53 to PAb421. In light of the findings that the PAb421 epitope is targeted by PKC, we continued to use His-p53 protein purified by nickel affinity chromatography, rather than p53 purified by PAb421 affinity chromatography.

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).

Regulation of the DNA Binding Function of p53 by Monoclonal Antibody PAb421

Previous reports suggested that bacterial, baculovirus, or in vitro translated p53 protein exists in tetrameric form and binds DNA predominantly as a tetramer or in forms of higher molecular weight complexes(34, 42, 43, 48) . Activation of DNA binding by allosteric mechanisms involves the carboxyl terminus of p53 and can be triggered by the binding of the carboxyl-terminal antibody PAb421(35) . First, we wished to determine how many molecules of monoclonal antibody PAb421 per presumed p53 tetramer are present in activated p53-DNA complexes, which would allow us to interpret the nature of possible PKC-modified phospho-p53-DNA complexes.

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^t/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^t and p53^o complexes and produces the formation of multiple p53-DNA intermediates. Only one intermediate (p53^t/Ab) is formed upon supershift of the p53^t complex with PAb1801. This is consistent with the PAb1801-saturated complex containing two bivalent molecules of antibody bound per p53 tetramer (p53^t/Ab). Three intermediate complexes are formed upon supershift of the oligomeric p53-DNA complex p53^o, consistent with it representing two associated p53 tetramers and the PAb1801-saturated complex having two molecules of bivalent antibody bound per p53 tetramer (p53^o/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^t/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^t/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.

Activation of the Latent DNA Binding Function of p53 by Protein Kinase C

In the light of recent findings implicating the carboxyl-terminal 30 (35) or carboxyl-terminal 18 residues (45) , (^2)in the basic region of the p53 molecule as a negative autoregulatory domain, and, having established that PKC specifically phosphorylates p53 in this region, we tested as to whether phosphorylation and dephosphorylation of p53 are associated with a change in sequence-specific DNA binding activity. Purified p53 protein was subjected to phosphorylation and/or dephosphorylation reactions as performed for antibody reactivity studies (see Fig. 3) and subsequently assayed for DNA binding.

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^t/Ab complex, as well as an increase in the appearance of one novel PAb421-p53-DNA complex (p53^t/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^t/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^t/Ab complex can be generated from distinct phosphorylated p53 species. In most binding reactions, a significant fraction of the novel PAb421-p53 complex (p53^t/Ab) appears to derive from the PKC-activated p53-DNA complex (p53^t), 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^t/Ab complex formation in the absence of a comparable loss of supershiftable p53^t (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.

Inhibition of the DNA Binding Function of p53 by Protein Phosphatases

PKC-induced activation of DNA binding and loss of PAb421 reactivity are reversible upon subsequent treatment with protein phosphatases 1 and 2A. Remarkably, dephosphorylation of p53 is associated with a decrease in DNA binding, and the latent, PAb421-responsive form of p53 is restored (Fig. 4C, lanes 11-16). Thus, once activated, p53 does not remain activated unless it is phosphorylated. In contrast, incubation of untreated p53 with PP1 and PP2A has no significant effect on p53 binding to DNA (Fig. 4D, lanes 2 and 3), demonstrating that inhibition of DNA binding by dephosphorylation is specific to the PKC-activated form of p53. This is consistent with the absence of a significant increase in p53 immunoreactivity to PAb421 upon treatment with phosphatases (see Fig. 3) and the lack of a significant formation of PAb421-p53-DNA intermediates upon addition of PAb421 to untreated p53 (Fig. 4, C and D), which also indicate that the untreated purified p53 protein is not significantly phosphorylated at the PKC reactive site in Sf9 insect cells.

DISCUSSION

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, (^3)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. (^3)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.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Molecular Genetics and Cell Biology, Room K2119A, Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000. Tel.: 609-252-3276; Fax: 609-252-3307.

(^1)
The abbreviations used are: PP, protein phosphatase; PKC, protein kinase C; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. Chung and N. Kley, unpublished observations.

(^4)
F. Morin and N. Kley, unpublished observation.

(^3)
S. Velasco-Miguel and N. Kley, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. L. V. Crawford for providing us with plasmid proSp53, Dr. S. Velasco-Miguel for providing His-p53 baculovirus stock, and Dr. R. Weinmann for critical reading of the manuscript.

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