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C Prives

Publications and source records attributed to C Prives.

At least 55 records · Page 3Linked to original sources
PubMed1998

High mobility group protein-1 (HMG-1) is a unique activator of p53.

The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. We report the identification of a factor from HeLa nuclear extracts that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. We demonstrate that recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro and also that HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53Delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. Finally, using transient transfection assays we show that HMG-1 can increase p53 and p53Delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures, and these data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA.

DNA↗
PubMed1998

The 400 kDa subunit of the PCAF histone acetylase complex belongs to the ATM superfamily.

PCAF histone acetylase is found in a complex with more than 20 associated polypeptides. Here we report cloning and characterization of the 400 kDa PCAF-associated factor referred to as PAF400. PAF400 is almost identical to TRRAP, which binds to c-Myc and E2F, and has significant sequence similarities to the ATM superfamily including FRAP, ATM, ATR, and the catalytic subunit of DNA-PK. Remarkably, PAF400 and FRAP share sequence similarity in broad regions that cover 80% of the entire PAF400 sequence. However, unlike the other members of the ATM superfamily, PAF400 is not a protein kinase as judged from the lack of kinase motif and autophosphorylation activity. We discuss the possibility that PAF400 may play a role in signaling of DNA damage to p53 by stimulation of p53 acetylation.

Acetyltransferases↗
PubMed1998

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication.

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells were observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb. G2 arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1997

DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.

DNA-damaging agents signal to p53 through as yet unidentified posttranscriptional mechanisms. Here we show that phosphorylation of human p53 at serine 15 occurs after DNA damage and that this leads to reduced interaction of p53 with its negative regulator, the oncoprotein MDM2, in vivo and in vitro. Furthermore, using purified DNA-dependent protein kinase (DNA-PK), we demonstrate that phosphorylation of p53 at serines 15 and 37 impairs the ability of MDM2 to inhibit p53-dependent transactivation. We present evidence that these effects are most likely due to a conformational change induced upon phosphorylation of p53. Our studies provide a plausible mechanism by which the induction of p53 can be modulated by DNA-PK (or other protein kinases with similar specificity) in response to DNA damage.

Binding Sites↗
PubMed1997

Identification of redox/repair protein Ref-1 as a potent activator of p53.

p53 can be isolated from cells in a form that is inert for binding to DNA but that can be stimulated dramatically by phosphorylation, antibody binding, or short single strands of DNA. This suggests that upon genotoxic stress, cells can convert latent p53 to one that is active for DNA binding. Surprisingly, we observed that latent p53 is as effective in activating transcription in vitro as is active p53. We found that HeLa nuclear extracts can stimulate DNA binding by latent p53 and have purified from them a p53-stimulating protein that we have determined to be the product of the Ref-1 gene. Interestingly, Ref-1 is a dual function protein that can both regulate the redox state of a number of proteins and function as a DNA repair (A/P) endonuclease. We observed that oxidized forms of full-length and carboxy-terminally truncated p53 (p53 delta30), which are inactive for DNA binding, are both stimulated by the Ref-1 protein. However, in the presence of reducing agent, Ref-1 is an extremely potent stimulator of full-length p53 but not p53 delta30. These and additional data indicate that Ref-1 protein stimulates p53 by both redox-dependent and -independent means and imply a key role for it in p53 regulation. Importantly, we have also determined that Ref-1 can stimulate p53 transactivation in vivo. This is the first example of a noncovalent protein modifier of p53 function identified in cells.

Carbon-Oxygen Lyases↗
PubMed1997

Cyclin-dependent kinase regulation of the replication functions of polyomavirus large T antigen.

The amino-terminal portion of polyomavirus (Py) large T antigen (T Ag) contains two phosphorylation sites, at T187 and T278, which are potential substrates for cyclin-dependent kinases (CDKs). Our experiments were designed to test whether either or both of these sites are involved in the origin DNA (ori DNA) replication function of Py T Ag. Mutations were generated in Py T Ag whereby either or both threonines were replaced with alanine, generating T187A, T278A, and double-mutants (DM [T187A T278A]) mutant T Ags. We found that the Py ori DNA replication functions of T278A and DM, but not T187A, mutant T Ags were abolished both in vivo and in vitro. Consistent with this finding, it was shown that the ori DNA binding and unwinding activities of mutant T278A Py T Ag were greatly impaired. Moreover, whereas wild-type Py T Ag is an efficient substrate for phosphorylation by cyclin A-CDK2 and cyclin B-cdc2 complexes, it is phosphorylated poorly by a cyclin E-CDK2 complex. In contrast to mutant T187A, which behaved similarly to the wild-type protein, T278A was only weakly phosphorylated by cyclin B-cdc2. These data thus suggest that T278 is an important site on Py T Ag for phosphorylation by CDKs and that loss of this site leads to its various defects in mediating ori DNA replication. S- and G2-phase-specific CDKs, but not a G1-specific CDK, can phosphorylate wild-type T Ag, which suggests yet another reason why DNA tumor viruses require actively cycling host cells.

Antigens, Polyomavirus Transforming↗
PubMed1997

p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

Amino Acid Sequence↗
PubMed1997

Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage.

Amino Acid Sequence↗
PubMed1997

p53 represses Sp1 DNA binding and HIV-LTR directed transcription.

The HIV-LTR region contains binding sites for, and is regulated by, a number of transcription factors including Sp1 and NF-kB. The wild-type p53 tumor suppressor protein represses transcription from the HIV-LTR promoter while oncogenic mutant forms of p53 stimulate expression from the HIV-LTR. We have shown previously that wild-type p53 is a site specific DNA binding protein that binds to a region of the SV40 virus which contains GC-box DNA binding sites for the ubiquitously expressed transcription factor Sp1. In this study using DNase I footprinting, we have shown that purified p53 is able to protect the Sp1 binding sites and the adjacent NF-kB site of the HIV-LTR. Furthermore we have demonstrated that when p53 and Sp1 are mixed together both proteins change each other's interaction with DNA. Interestingly, we noted that oncogenic mutant p53 is also able to change the interaction of Sp1 with DNA. We confirmed p53 dependent repression of HIV-LTR driven transcription by comparing the expression from an HIV-LTR reporter construct in the presence and absence of p53. EMSA of an oligonucleotide sequence derived from the HIV-LTR sequence demonstrated a slight decrease in Sp1 DNA binding activity with nuclear extract derived from the cell line expressing a high level of wild-type p53. These data suggest that the influence of p53 on the transcription of promoters with Sp1 binding sites may be partially due to a change in the DNA binding ability of Sp1.

Animals↗
PubMed1996

Cell-cycle related regulation of poly(A) polymerase by phosphorylation.

The poly(A) tail found on almost all eukaryotic messenger RNAs is important in enhancing translation initiation and determining mRNA stability. Control of poly(A)-tail synthesis thus has the potential to be a key regulatory step in gene expression and is indeed known to be important during early development in many organisms. To study a possible basis for such regulation, we examined phosphorylation of poly(A) polymerase (PAP) by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor, MPF). We show here that PAP can be phosphorylated in vivo and in vitro by MPF. Consistent with this, PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell-cycle when MPF is known to be active. We show further that hyperphosphorylation by MPF dramatically reduces the activity of purified PAP, and that PAP isolated from mitotic HeLa cells is similarly inhibited by hyperphosphorylation. This repression probably contributes to the well established reductions in poly(A)+ RNA and/or protein synthesis known to occur in M-phase cells.

Animals↗
PubMed1996

Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein.

In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters.

Animals↗
PubMed1996

Regulation of mutant p53 temperature-sensitive DNA binding.

We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 --> Ala, Arg-175 --> His, Arg-248 --> Trp, Arg-249 --> Ser, and Arg-273 --> His). While all mutants were defective for binding to DNA at 37 ;C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33 ;C), and several mutants activated transcription from a p53-responsive promoter at 26 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37 degrees C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.

Amino Acid Sequence↗
PubMed1996

p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.

It is well established that induction of the p53 tumor suppressor protein in cells can lead to either cell cycle arrest or apoptosis. To further understand features of p53 that contribute to these cell responses several p53-null Saos2 and H1299 cell lines were generated that express wild-type or mutant forms of p53, or the cyclin-dependent kinase inhibitor p21/WAF1, under a tetracycline-regulated promoter. Our results show that the cellular level of p53 can dictate the response of the cell such that lower levels of p53 result in arrest whereas higher levels result in apoptosis; nevertheless, DNA damage can heighten the apoptotic response to p53 without altering the protein level of p53 in cells. We also demonstrate that arrest and apoptosis are two genetically separable functions of p53 because a transcriptionally incompetent p53 can induce apoptosis but not arrest, whereas induction of p21/WAF1, which is a major transcriptional target of p53, can induce arrest but not apoptosis. Finally, we show that a full apoptotic response to p53 requires both its amino and carboxyl terminus, and our data suggest that there is synergism between transcription-dependent and -independent functions of p53 in apoptosis. Thus, there are multiple independent cellular responses to p53 that together may account for the extraordinarily high frequency of p53 mutations in diverse types of human tumors. The implications of these results are discussed and a model is proposed.

Apoptosis↗
PubMed1996

p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A.

We and others previously showed that cyclin G is a transcriptional target of the p53 tumor suppressor protein. However, cellular proteins which might form a complex with cyclin G have not yet been identified. To gain insight into the biological role of cyclin G, we used the yeast two-hybrid screen and isolated two mouse cDNAs encoding cyclin G-interacting proteins. Interestingly, both positive cDNAs encoded B' regulatory subunits of protein phosphatase 2A (PP2A). One clone encodes B'alpha, while the other clone codes for a new member of the B' family, B'beta. B'beta is 70% identical to other members of the B' family. B'alpha associated both in vitro and in vivo with cyclin G but not with the other mammalian cyclins. Furthermore, cyclin G formed a complex with B'alpha only after induction of p53 in p53 temperature-sensitive cell lines. These results indicate that cyclin G forms a specific complex with the B' subunit of PP2A and that complex formation is regulated by p53. Potential roles for the cyclin G-B' complex in p53-mediated pathways are discussed.

Alternative Splicing↗
PubMed1996

Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo.

The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.

Animals↗