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Biomedical subjects

Carol Prives

Publications and source records attributed to Carol Prives.

At least 19 recordsLinked to original sources
PubMed2006

The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity.

Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein-protein interactions with the E2.

Adenosine Triphosphate↗
PubMed2006

p53 and p73 display common and distinct requirements for sequence specific binding to DNA.

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73beta proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73beta than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73beta but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73beta than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Delta30) demonstrates the same sequence discrimination as does p73beta. In vivo, both p53 and p73beta exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.

Base Sequence↗
PubMed2006

p53-Dependent p21 mRNA elongation is impaired when DNA replication is stalled.

We have previously reported that when DNA replication is blocked in some human cell lines, p53 is impaired in its ability to induce a subset of its key target genes, including p21(WAF1/CIP1). Here, we investigated the reason for this impairment by comparing the effects of two agents, hydroxyurea (HU), which arrests cells in early S phase and impairs induction of p21, and daunorubicin, which causes a G(2) block and leads to robust activation of p21 by p53. HU treatment was shown to inhibit p21 mRNA transcription rather than alter its mRNA stability. Nevertheless, chromatin immunoprecipitation assays revealed that HU impacts neither p53 binding nor acetylation of histones H3 and H4 within the p21 promoter. Furthermore, recruitment of the TFIID/TATA-binding protein complex and the large subunit of RNA polymerase II (RNA Pol II) are equivalent after HU and daunorubicin treatments. Relative to daunorubicin treatment, however, transcription elongation of the p21 gene is significantly impaired in cells treated with HU, as evidenced by reduced occupancy of RNA Pol II at regions downstream of the start site. Likewise, in the p21 downstream region after administration of HU, there is less of a specifically phosphorylated form of RNA Pol II (Pol II-C-terminal domain serine 2P) which occurs only when the polymerase is elongating RNA. We propose that while the DNA replication checkpoint is unlikely to regulate the assembly of a p21 promoter initiation complex, it signals to one or more factors involved in the process of transcriptional elongation.

Acetylation↗
PubMed2006

Mutational analysis of the p53 core domain L1 loop.

The p53 tumor suppressor gene acquires missense mutations in over 50% of human cancers, and most of these mutations occur within the central core DNA binding domain. One structurally defined region of the core, the L1 loop (residues 112-124), is a mutational "cold spot" in which relatively few tumor-derived mutations have been identified. To further understand the L1 loop, we subjected this region to both alanine- and arginine-scanning mutagenesis and tested mutants for DNA binding in vitro. Select mutants were then analyzed for transactivation and cell cycle analysis in either transiently transfected cells or cells stably expressing wild-type and mutant proteins at regulatable physiological levels. We focused most extensively on two p53 L1 loop mutants, T123A and K120A. The T123A mutant p53 displayed significantly better DNA binding in vitro as well as stronger transactivation and apoptotic activity in vivo than wild-type p53, particularly toward its pro-apoptotic target AIP1. By contrast, K120A mutant p53, although capable of strong binding in vitro and wild-type levels of transactivation and apoptosis when transfected into cells, showed impaired activity when expressed at normal cellular levels. Our experiments indicate a weaker affinity for DNA in vivo by K120A p53 as the main reason for its defects in transactivation and apoptosis. Overall, our findings demonstrate an important, yet highly modular role for the L1 loop in the recognition of specific DNA sequences, target transactivation, and apoptotic signaling by p53.

Alanine↗
PubMed2006

Excess HDM2 impacts cell cycle and apoptosis and has a selective effect on p53-dependent transcription.

Mutational inactivation of p53 is only one of the ways that tumors lose p53 function. An alternate route is through overexpression of HDM2, the negative regulator of p53. To further understand how excess HDM2 regulates p53-mediated functions, we generated H1299 cell clones that constitutively express both ectopic HDM2 and tetracycline-regulated inducible p53. We found that over a range of p53 concentrations constitutively expressed HDM2 did not affect the levels of p53 protein. Nevertheless, cells with excess HDM2 displayed numerous changes in their response to p53. After DNA damage, such cells had both increased p53-mediated G2 arrest and reduced cell death. They also showed selective impairment of p53 target gene induction in that some p53 targets were unaffected whereas others were markedly less well induced in the presence of extra HDM2 protein. We also found that excess HDM2 was correlated with reduced p53 acetylation but did not affect p53 association with target promoters in vivo. Indeed, there was no significant difference in the amount of HDM2 associated with p53 at target promoters that differed in their expression depending on the presence of extra HDM2. Thus, HDM2 can selectively down-regulate the transcription function of p53 without either degrading p53 or affecting the interaction of p53 with target promoters.

Apoptosis↗
PubMed2006

Mutant p53 gain of function: the NF-Y connection.

The molecular mechanisms underlying mutant p53 gain of function are becoming increasingly complex. In this issue of Cancer Cell, Di Agostino et al. identify the heterotrimeric transcription factor NF-Y as an interacting partner of mutant p53. They show that mutant p53/NF-Y complexes bind to NF-Y target promoters and recruit p300 in response to DNA damage, resulting in aberrant transactivation of NF-Y target genes and cell cycle deregulation. These data thereby implicate transcriptional activation by mutant p53 as a key mechanism responsible for its oncogenic activity.

CCAAT-Binding Factor↗
PubMed2005

The continuing saga of p53--more sleepless nights ahead.

Despite previous assumptions that the tumor suppressor protein p53 exists primarily and functionally as a single species, recent papers document the existence and unexpected properties of numerous p53 isoforms in cells. The complexity of p53 continues to unfold.

Alternative Splicing↗
PubMed2005

P53 and prognosis: new insights and further complexity.

Despite the massive amount of knowledge that has accumulated about p53, there is still much to learn about its role in tumor suppression. New papers now provide evidence that levels of Mdm2 in humans or presence of mutant forms of p53 in mice can have profound impacts on survival and tumorigenesis. These findings may lead to new diagnostic and therapeutic approaches to treating cancer patients.

Animals↗
PubMed2005

The S phase checkpoint: when the crowd meets at the fork.

Accumulation of unrepaired DNA lesions is the biggest threat to genomic stability. DNA damage checkpoints create windows of time that allow the cell to repair assaults on DNA in each phase of the cell cycle. When DNA lesions arise in S phase, however, the checkpoint machinery must work to coordinate DNA replication and repair processes. In fact some upstream components of the DNA damage checkpoint play parallel roles in maintaining the continuity of DNA replication and signaling to downstream components.

Adaptor Proteins, Signal Transducing↗
PubMed2005

DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation.

Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.

14-3-3 Proteins↗
PubMed2005

Inhibition of TFII-I-dependent cell cycle regulation by p53.

The multifunctional transcription factor TFII-I is tyrosine phosphorylated in response to extracellular growth signals and transcriptionally activates growth-promoting genes. However, whether activation of TFII-I also directly affects the cell cycle profile is unknown. Here we show that under normal growth conditions, TFII-I is recruited to the cyclin D1 promoter and transcriptionally activates this gene. Most strikingly, upon cell cycle arrest resulting from genotoxic stress and p53 activation, TFII-I is ubiquitinated and targeted for proteasomal degradation in a p53- and ATM (ataxia telangiectasia mutated)-dependent manner. Consistent with a direct role of TFII-I in cell cycle regulation and cellular proliferation, stable and ectopic expression of wild-type TFII-I increases cyclin D1 levels, resulting in accelerated entry to and exit from S phase, and overcomes p53-mediated cell cycle arrest, despite radiation. We further show that the transcriptional regulation of cyclin D1 and cell cycle control by TFII-I are dependent on its tyrosine phosphorylation at positions 248 and 611, sites required for its growth signal-mediated transcriptional activity. Taken together, our data define TFII-I as a growth signal-dependent transcriptional activator that is critical for cell cycle control and proliferation and further reveal that genotoxic stress-induced degradation of TFII-I results in cell cycle arrest.

Animals↗
PubMed2004

p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2.

The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. We report here that in several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. We demonstrate as well that E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance.

Apoptosis↗
PubMed2004

p53 linear diffusion along DNA requires its C terminus.

In cells, sequence-specific transcription factors must search through an entire genome to find their target sites in promoters. Such sites may be identified by using one-dimensional (linear diffusion) and/or three-dimensional (association/dissociation) mechanisms. We show here that wild-type p53 possesses the ability to linearly diffuse on DNA. p53 lacking its C terminus is incapable of such sliding along DNA, while the isolated C terminus of p53 is even more effective than the full-length protein at one-dimensional linear diffusion. Importantly, neither acetylation-mimicking mutations nor phosphorylation of residues within the C terminus stimulates linear diffusion by p53. Supporting these in vitro observations, we found that C-terminally deleted p53 (p53Delta30) expressed at physiological levels is deficient in binding to and transactivating downstream promoters in vivo. Therefore, our data show that the C terminus is a positive regulator of DNA binding in vivo and in vitro, and indicate that the mechanism may involve linear diffusion.

Acetylation↗
PubMed2004

Regulation of p53 activity through lysine methylation.

p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.

Amino Acid Sequence↗
PubMed2004

p53 in the cytoplasm: a question of overkill?

The tumor suppressor p53 regulates cellular responses to stress by serving in the nucleus to regulate transcription of genes involved in processes including cell cycle arrest, DNA repair, and apoptosis. Nevertheless, recent papers show that p53 may have a separate cytoplasmic role in directly regulating the Bax-dependent mitochondrial pathway to cell death.

Animals↗
PubMed2004

The linchpin? Pin1 meets p73.

A recent paper shows that the peptidyl-prolyl isomerase Pin1 conformationally alters p73, promoting its acetylation by p300 in a c-Abl dependent manner. Given previous findings with p53, Pin1 may represent a common mediator linking proapoptotic cooperative activity of the p53 family members.

Acetyltransferases↗
PubMed2003

p73 Expression in human normal and tumor tissues: loss of p73alpha expression is associated with tumor progression in bladder cancer.

PURPOSE: To characterize the expression profile of p73 in human normal tissues by immunohistochemistry (IHC) and to analyze the correlation between p73 expression and bladder cancer progression. EXPERIMENTAL DESIGN: CJDp73 was characterized for p73alpha detection in Western blot and IHC through its application to isoform-transfected 293 cells. Normal tissues were analyzed by IHC with the CJDp73 antiserum. Transitional cell carcinoma (TCC)-derived cell lines were subjected to reverse transcription-PCR and Western blot. TCC tissue microarrays were analyzed for p73alpha expression by IHC, and the results were statistically analyzed. RESULTS: p73 immunostaining was nuclear and restricted to epithelial cells of certain organs such as squamous epithelium of the epidermis and transitional epithelium of the bladder. The expression was also observed in certain specialized glandular epithelia such as acinar cells of breast and parotid gland. Four of seven TCC-derived cell lines had low to undetectable p73alpha protein levels. We found undetectable or low p73alpha expression in 104 of 154 (68%) TCC cases, this phenotype being more frequently observed in invasive tumors when compared with superficial lesions. This association was statistically significant (P < 0.0001). We also observed a significant association between p53, p63, and p73alpha alterations with bladder cancer progression (P < 0.0001). CONCLUSIONS: p73alpha plays a tumor suppressor role in bladder cancer, and its inactivation occurs through an epigenetic mechanism, most probably involving protein degradation.

Cell Line↗