©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Distinct Signaling Pathways Activate the Latent DNA Binding Function of p53 in a Casein Kinase II-independent Manner (*)

(Received for publication, December 7, 1994; and in revised form, April 8, 1995)

Ted R. Hupp David P. Lane (§)

From the Cancer Research Campaign Laboratories, Department of Biochemistry, Dundee University, Dundee DD1 4HN, Scotland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (p534) 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 p534 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.


INTRODUCTION

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/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.

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-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.

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/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.

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 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).

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.


EXPERIMENTAL PROCEDURES

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.


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).



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.


Figure 5: Reactivity of p534 to ICA-9. Full-length p53 (50 ng; lanes1, 3, and 5) or p534 (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 p534 Expression Vectors for Protein Production in E. coli and in Sf9 Insect Cells

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 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.


RESULTS

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.


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), 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.


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 p53DNA complexes bound to two monoclonal antibodies, the open-headed arrow marks the migration of p53DNA complexes bound to one monoclonal antibody, and the stem marks the migration of in vivo activated 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-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).

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-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.

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.

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.


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) 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 p53DNA complexes without, with one, or with two monoclonal antibodies bound/tetramer is indicated by the arrowheads.



When cells are deprived of serum, the production of activated p53 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.

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. p534 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 p534, as bacteria can be used to generate large amount of latent tetramers in an unphosphorylated state (Hupp and Lane, 1994b). Unphosphorylated p534 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).

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 p534 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 p534 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.

p534 Is Not Activated by Casein Kinase II

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 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.


Figure 6: Casein kinase does not activate p534. Purified fractions of p53 (25 ng; from left to right, lanes1 and 2) or p534 (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 p53DNA complex is indicated by the open-headed arrow.



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 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.

To investigate whether p534 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), p534 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 p534 by Proteins That Target the Negative Regulatory Domain

Does the casein kinase II site of p53 actually play an essential role in the activation reaction? If so, then p534 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 p534 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 p534 (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 p534 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.


Figure 7: Activation of p534 by PAb421 and DnaK. A, pure fractions p534 were analyzed in a sequence-specific DNA binding assay by dividing the reaction into two stages; p534 (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 p53DNA complexes are bracketed. B, summary of the specific activity of p53 or p534 when activated by either PAb421 or DnaK.



Differences in the specific activity of p53 and p534, 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 p534 Is Immune to Inhibition by ICA-9

The biochemical affects of the binding of the allosteric inhibitory antibody, ICA-9, to p534 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 PAb421p53DNA complexes (lanes5 and 6). PAb421-activated p534DNA complexes (lane1) are immune to inhibition by ICA-9 (lanes2 and 3), consistent with the absence of the ICA-9 epitope on p534.


Figure 8: ICA-9 does not destabilize PAb421-activated p534. p534 (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 p534 were subjected to chromatography on heparin-Sepharose to separate distinct biochemical forms of p534. Interestingly, the profile observed with p534 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 p534 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 p534 activation in this cell line, but that two other factors may be catalyzing p53 activation in vivo.


Figure 9: Casein kinase II-independent activation of p534 in vivo. p534 baculovirus expression vectors were constructed and used to infect insect cells, as indicated for wild type p53 (see ``Experimental Procedures''). After p534 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.




DISCUSSION

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.

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/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?

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,() and monoclonal antibody PAb421 does not therefore involve the re-folding of ``denatured'' p53.

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/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.

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.

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.

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 p534 characterized in vitro facilitates interpretations of experiments designed to explore the types of activating factors which function in vivo. Catalytic activation of p534 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.


FOOTNOTES

*
This work is funded by the Cancer Research Campaign. 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.

§
Gibb fellow of the Cancer Research Campaign and Howard Hughes International Scholar. To whom correspondence should be addressed. Tel.: 01382-223181 (ext. 4806); Fax: 01382-224117.

The abbreviations used are: DTT, dithiothreitol; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

T. Hupp and D. Lane, unpublished observations.

T. Hupp and D. Lane, unpublished observations.

S. Hansen and D. Lane, unpublished observations.


ACKNOWLEDGEMENTS

We thank our colleagues in Dundee for assistance and critical reading of the manuscript, particularly Kathryn Ball, Carol Midgley, and Silke Hansen.


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