C-terminal Regulatory Domain Of P53 Research Articles

Abstract P3-10-03: Exploiting p53-dependent functional duality of estrogen receptor-beta to repurpose tamoxifen for triple negative breast cancer therapy

Abstract Whether estrogen receptor beta (ERβ/ESR2) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are generally high in triple-negative breast cancer (TNBC). Reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is a major driver of these cancers. We tested the hypothesis that WT versus mutant status of p53 will have an important role in determining the duality of ERβ functions. We showed that ERβ directly binds to TP53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 to be essential for the interaction. The highly sensitive in situ proximity ligation assay (PLA) showed that ERβ is capable of interacting with both wild type (WT) and mutant p53 in breast cancer cells and tumor tissues. ERβ and p53 antibodies validated for specificity were used for all experiments. Combination of proliferation, cell cycle, and apoptosis assays, RNAi technology, quantitative ChIP (qChIP), and real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of WTp53, whereas it is anti-proliferative in the context of mutant p53 in multiple breast cancer cell lines. The p53-dependent diametrically opposite functions of ERβ were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR) that differ only in the presence of WT versus mutant p53. A major gain-of-function of mutant p53 is its ability to bind and inactivate tumor suppressor p73. We show that ERβ binds and sequesters mutant p53 from mutant p53−p73 complex leading to reactivation of p73. Consistent with these observations, immunohistochemistry (IHC) in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing mutant p53 along with high levels of ERβ were of smaller size and lower stage. Complementing these findings, our analysis of the subgroup of the basal-like/TNBC tumors expressing mutant p53 (but not WTp53) in the METABRIC dataset showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had significantly better breast cancer-specific survival. The finding that ERβ–mutant p53 combination prognosticates survival in TNBC is important as to date there are no effective prognostic markers for TNBC and suggest the potential for using ERβ−mutant p53 combination in stratification of TNBC into therapeutically actionable subgroups.Furthermore, our findings provide a mechanistic explanation for the functional duality of ERβ and the controversy over its pro-versus anti-tumorigenic role.Surprisingly, Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells leading to increased transcription of anti-proliferation genes and knockdown experiments showed that the effect on transcription was dependent on both p73 and ERβ. Importantly, when combined with doxorubicin (Adriamycin) Tam decreased several fold the IC50 of doxorubicin resulting in increased apoptosis. The combination was more effective in inhibiting TNBC xenograft tumor growth in vivo compared to either treatment alone. Significant clinical implications of these findings include the potential for treating patients with doxorubicin at much lower dose than what is currently used in the management of TNBC, thereby reducing its major cumulative dose side effects. Importantly, although at present Tam is not indicated for TNBC, our data suggest the possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on ERβ and p53 status. If validated in a clinical trial, our findings could lead to a therapy that is fundamentally better in terms of effectiveness, cost and time needed to reach the TNBC patients. Citation Format: Gokul M Das, Utpal K Mukhopadhyay, Chetan C Oturkar, Christina Adams, Nadi Wickramasekera, Sanjay Bansal, Rajesh Medisetty, Austin Miller, Wendy M Swetzig, Laxmi Silwal-Pandit, Anne-Lise Borresen-Dale, Creighton J Creighton, Jun Hyoung Park, Santhi D Konduri, Alka Mukhopadhyay, Alexander Caradori, Benny Abraham Kaipparettu. Exploiting p53-dependent functional duality of estrogen receptor-beta to repurpose tamoxifen for triple negative breast cancer therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-03.

Abstract 3748: p53 status as a determinant of functional duality of estrogen receptor beta in breast cancer: Therapeutic implications

Abstract Whether estrogen receptor beta (ERβ) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are high in ERα negative cancers including triple-negative breast cancer (TNBCs). Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 (mut-p53) and it is the most predominant driver in these cancers. We tested the hypothesis that p53 status in breast cancer will have an important role in determining the duality of ERβ functions. We have shown that ERβ directly binds to p53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the domains of both proteins that are required for the ERβ-p53 interaction. The DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 are essential for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERβ-p53 interaction in situ in breast cancer cells expressing either wild type (wt)- or mut-p53. ERβ and p53 antibodies validated for specificity were used. In multiple cell lines, a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of wt-p53, whereas it is anti-proliferative in the context of mut-p53. The results were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR technology) that differ only in the presence of wt-versus mut-p53. ERβ binds and sequesters mut-p53 from mut-p53−p73 complex leading to reactivation of tumor suppressor p73. Consistent with these data, combination of immunohistochemistry (IHC) and PLA in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing wt-p53/ high ERβ had worse prognosis, both in terms of overall survival (OS) and progression-free survival (PFS). On the contrary, tumors expressing mut-p53/ high ERβ were of smaller size and stage. These findings were complemented by data from our analysis of the mut-p53 subgroup of the basal-like tumors in the METABRIC dataset which showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had better prognosis. Surprisingly, 4-Hyroxy Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells and combination of doxorubicin (Adriamycin) and Tam decreased the IC50 of doxorubicin by 10 fold leading to increased apoptosis. These data have significant clinical implications in targeting ERβ and mutant p53 signaling pathways for therapeutic purposes especially in ERα negative cancers such as TNBC. Importantly, although at present Tam is not standard of care for TNBC, our data suggest a possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on p53 status. Citation Format: Gokul M. Das, Utpal K. Mukhopadhyay, Christina Adams, Nadi Wickramasekera, Rajesh Medisetty, Sanjay Bansal, Laxmi Silwal-Pandit, Anne-Lise Borresen-Dale, Austin Miller, Wendy M. Sweizig, Angela Omilian, Wiam Bshara, Alka Mukhopadhyay. p53 status as a determinant of functional duality of estrogen receptor beta in breast cancer: Therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3748.

Abstract 3465: p53 status as a determinant of estrogen receptor beta function in breast cancer

Abstract While the importance of estrogen receptor alpha (ERα) in breast cancer is well documented, role of estrogen receptor beta (ERβ) still remains elusive. Whether ERβ is an oncogenic or a tumor suppressor protein in breast cancer remains controversial. ERβ levels are high in ERα negative cancers including triple-negative breast cancer (TNBCs). Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 (mut-p53) and it is the most predominant driver mutant in these cancers. Importantly, in addition to losing tumor suppressor activity, mut-p53 gains oncogenic functions, and therefore, has a major role in driving the cancer process. We tested the hypothesis that p53 status in breast cancer will have an important role in determining function of ERβ. We report that ERβ directly binds to p53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the domains of both proteins that are required for the ERβ-p53 interaction. The DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 are essential for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERβ-p53 interaction in situ in breast cancer cells expressing either wt- or mut-p53. Surprisingly, we found that ERβ has opposite functions depending on the wt/mut status of p53. A combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR) showed that in the context of the wt-p53, ERβ is pro-proliferative, whereas in the context of mut-p53, ERβ is anti-proliferative. ERβ binds wt-p53 and inhibits its transcriptional function. On the other hand, ERβ binds and sequesters mut-p53 from mut-p53−p73 complex leading to reactivation of tumor suppressor p73. Consistent with findings in cell culture models, combination of immunohistochemistry (IHC) and PLA in TNBC patient tissue microarray (TMA) representing 130 tumors followed by correlative analysis of linked patient tumor characteristics obtained from RPCI clinical data network showed that p53 status is an important determinant of ERβ function. For example, ERβ along with mut-p53 has negative effect on tumor growth, whereas ERβ in the wt-p53 context has opposite effect. Our finding that ERβ elicits opposite functions dependent on p53 status provides an important insight into the mechanism underlying the context-dependent function of ERβ and provides an explanation to the controversial reports on pro- versus anti-tumorigenic role of ERβ. These data would have significant clinical implications in targeting ERβ and mutant p53 signaling pathways for diagnostic, prognostic, and therapeutic purposes especially in ERα negative cancers such as triple negative breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Gokul M. Das, Utpal K. Mukhopadhyay, Sanjay Bansal, Nadi Wickramasekera, Rajesh Medisetty, Wendy M. Swetzig, Austin Miller, Jianmin Wang, Chetan Oturkar, Alka Mukhopadhyay, Santhi Konduri. p53 status as a determinant of estrogen receptor beta function in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2015-3465

Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression.

Strategies for p53 Reactivation in Human Sarcoma

Emerging strategies in cancer therapeutics link the genomic mutational and proteomic landscape, allowing intelligent reasoning on target selection. In this issue of Cancer Cell, Piccinin and colleagues use this approach to demonstrate that the mesenchymal protein Twist1 inhibits p53, providing a novel target for reactivation of p53 in human sarcoma.

Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a Kd of 1.95 ± 0.19 μM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM–p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices α1 and α2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM–p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM–p53 interaction are discussed.

Estrogen Receptor-α Binds p53 Tumor Suppressor Protein Directly and Represses Its Function

Estrogen receptor-alpha (ERalpha) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERalpha and p53. Ionizing radiation together with ERalpha knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the pro-proliferative role of ERalpha.

Calcium-Binding Properties of Wild-Type and EF-Hand Mutants of S100B in the Presence and Absence of a Peptide Derived from the C-Terminal Negative Regulatory Domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.

The Cell Cycle-regulated Protein Human GTSE-1 Controls DNA Damage-induced Apoptosis by Affecting p53 Function

GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.

Metastasis-Inducing Mts1/S100A4 Protein Binds to Tumor Suppressor p53 Protein

This study for the first time demonstrates a physical and functional interaction between the Ca2+-binding protein Mts1/S100A4 and tumor suppressor p53 protein. Using different in vitro and in vivo approaches, we have found that Mts1 can bind to the C-terminal regulatory domain of p53. The Mts1 binding to p53 promotes activation of the reporter gene transcription in vivo. A modulation of the p53 target gene (p21/WAF,bax,mdm-2, and thrombospondin-1) expression was observed upon Mts1 induction in the cells expressing the wild-type p53. These results suggest that the ability of Mts1 to enhance p53-dependent apoptosis of tumor cells leads to the decrease/disappearance of the tumor cells expressing the wild-type p53. Thus, Mts1 promotes selection of more aggressive, metastatic phenotype during tumor progression.

Tumor Suppressor p53 Protein Is a New Target for the Metastasis-associated Mts1/S100A4 Protein: FUNCTIONAL CONSEQUENCES OF THEIR INTERACTION

A physical and functional interaction between the Ca(2+)-binding protein Mts1 (S100A4) and the tumor suppressor p53 protein is shown here for the first time. We demonstrate that Mts1 binds to the extreme end of the C-terminal regulatory domain of p53 by several in vitro and in vivo approaches: co-immunoprecipitation, affinity chromatography, and far Western blot analysis. The Mts1 protein in vitro inhibits phosphorylation of the full-length p53 and its C-terminal peptide by protein kinase C but not by casein kinase II. The Mts1 binding to p53 interferes with the DNA binding activity of p53 in vitro and reporter gene transactivation in vivo, and this has a regulatory function. A differential modulation of the p53 target gene (p21/WAF, bax, thrombospondin-1, and mdm-2) transcription was observed upon Mts1 induction in tet-inducible cell lines expressing wild type p53. Mts1 cooperates with wild type p53 in apoptosis induction. Our data imply that the ability of Mts1 to enhance p53-dependent apoptosis might accelerate the loss of wild type p53 function in tumors. In this way, Mts1 can contribute to the development of a more aggressive phenotype during tumor progression.

The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A

Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B 1-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B 1-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.

The Ca(2+)-dependent interaction of S100B(beta beta) with a peptide derived from p53.

S100B(beta beta) was found to interact with the tumor suppressor protein, p53, and inhibit its PKC-dependent phosphorylation and tetramer formation [Baudier, J., Delphin, C., Grunwald, D., Khochbin, S., and Lawrence, J. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11627-11631]. Since PKC-dependent phosphorylation at the C-terminus of p53 is known to effect transcription and p53 tetramer formation [Sakaguchi, K., Sakamoto, H., Lewis, M. S., Anderson, C. W., Erickson, J. W., Appella, E., and Xie, D. (1997) Biochemistry 36, 10117-10124], we examined the interaction of S100B(beta beta) with a peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). In this paper, we report that S100B(beta beta) binds to the p53 peptide (CaK3 < or = 23.5 +/- 6.6 microM) in a Ca(2+)-dependent manner, and that the presence of the p53 peptide was found to increase the binding affinity of Ca2+ to S100B(beta beta) by 3-fold using EPR and PRR methods, whereas the peptide had no effect on Zn2+ binding to S100B(beta beta). Fluorescence and NMR spectroscopy experiments show that the p53 peptide binds to a region of S100B(beta beta) that probably includes residues in the "hinge" (S41, L44, E45, E46, I47), C-terminal loop (A83, C84, H85, E86, F87, F88), and helix 3 (V52, V53, V56, T59). Together these data support the notion that S100B(beta beta) inhibits PKC-dependent phosphorylation by binding directly to the C-terminus of p53.

Regulation of the specific DNA binding function of p53

The DNA binding activity of p53 is required for its tumor suppressor function; we show here that this activity is cryptic but can be activated by cellular factors acting on a C-terminal regulatory domain of p53. A gel mobility shift assay demonstrated that recombinant wild-type human p53 binds DNA sequence specifically only weakly, but a monoclonal antibody binding near the C terminus activated the cryptic DNA binding activity stoichiometrically. p53 DNA binding could be activated by a C-terminal deletion of p53, mild proteolysis of full-length p53, E. coli dnaK (which disrupts protein-protein complexes), or casein kinase II (and coincident phosphorylation of a C-terminal site on p53). Activation of p53 DNA binding may be critical in regulation of its ability to arrest cell growth and thus its tumor suppressor function.