Biomedical subjects

Carol Prives

Publications and source records attributed to Carol Prives.

31 records · Page 2Linked to original sources

PubMed2003

Decreased p21 levels are required for efficient restart of DNA synthesis after S phase block.

The cyclin-dependent kinase inhibitor p21, a major transcriptional target of the tumor suppressor p53, plays a critical role in cell cycle arrest in G1 and G2 after DNA damage. It was previously shown that in some human cell lines when S phase is arrested, p53 is transcriptionally impaired such that some p53 targets including p21 are only weakly induced. We show here that during S phase arrest proteasome-mediated turnover of p21 is significantly increased in a manner that is independent of p53. It is well established that p21 can interact both with cyclin-dependent kinase complexes and with proliferating cell nuclear antigen (PCNA). Interestingly, the scant amount of p21 detected during S phase block cannot fully saturate cyclin A-cyclin-dependent kinase 2 complexes and does not interact detectably with PCNA. Importantly, DNA elongation assays in isolated nuclei show that the C terminus of p21 containing the PCNA-binding domain effectively blocks this process. This implies that p21 down-regulation could be an essential requirement for efficient restart of DNA synthesis. In line with this, only cells expressing low levels of p21 immediately progress through the cell cycle upon release from S phase arrest, whereas the remaining few high p21 producing cells move much more slowly through S. Thus, p21 down-regulation is multiply determined and is required for the reversibility of the arrest in S phase.

Blotting, Northern↗

PubMed2003

Replication of damaged DNA in vitro is blocked by p53.

The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of gamma-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from gamma-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains.

Animals↗

PubMed2003

Cyclin-dependent kinases phosphorylate p73 at threonine 86 in a cell cycle-dependent manner and negatively regulate p73.

p73 transcription factors are members of the p53 family and participate in developmental processes and DNA damage response. p73 expression was shown to be regulated during the cell cycle, suggesting that p73 might play a role in cell growth and might be a target for cyclin-dependent kinases. Consistent with this hypothesis, we discovered that p73 interacts physically with various cyclins (A, B, D, and E). Furthermore, cyclin A/CDK1/2, cyclin B/CDK1/2, and cyclin E/CDK2 complexes can phosphorylate multiple p73 isoforms in vitro at threonine 86. A specific antibody directed against phosphorylated Thr86 showed that this site is phosphorylated in vivo and that such phosphorylation is regulated in a cell cycle-dependent manner. Thr86 phosphorylation is induced during S phase and is maximal in the G2/M phase. Accordingly inhibitors of cell growth, such as p16 and serum starvation, reduce Thr86 phosphorylation. Finally, we found that cyclin-dependent kinase (CDK)-dependent Thr86 phosphorylation represses the ability of p73 to induce endogenous p21 expression. Our results demonstrate that p73 proteins are targets of CDK complexes and that phosphorylation on Thr86 by CDKs regulates p73 functions.

Animals↗

PubMed2003

Questioning the role of checkpoint kinase 2 in the p53 DNA damage response.

Cdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by gamma irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity.

Cell Cycle Proteins↗

PubMed2003

Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization.

The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.

Active Transport, Cell Nucleus↗

PubMed2003

p73alpha regulation by Chk1 in response to DNA damage.

The checkpoint kinase 1 (Chk1) is an essential component of the DNA damage checkpoint. Previous studies have demonstrated an indispensable role for the p53-related transcription factor p73alpha in DNA damage-induced apoptosis. Here, we provide evidence that p73alpha is a target of Chk1. We found that endogenous p73alpha is serine phosphorylated by endogenous Chk1 upon DNA damage, which is a mechanism required for the apoptotic-inducing function of p73alpha. Consistent with this, we discovered that endogenous p73alpha interacts with Chk1 and is phosphorylated by Chk1 at serine 47 in vitro and in vivo. In contrast, Chk2 does not phosphorylate p73alpha in vitro. Moreover, mutation of serine 47 abolishes both Chk1-dependent phosphorylation of p73alpha upon DNA damage in vivo and the ability of Chk1 to upregulate the transactivation capacity of p73alpha. Our data indicate a novel biochemical pathway through which the p73alpha proapoptotic function requires DNA damage-triggered p73alpha phosphorylation by Chk1.

Animals↗

PubMed2002

Deconstruction of p53 functions and regulation.

The 11th international p53 workshop was held in Barcelona, Spain, May 15-18, 2002. The p53 workshop was organized by Carlos Cordón-Cardo (Memorial Sloan Kettering, New York). The workshop included 46 oral presentations and 206 posters.

Acetylation↗

PubMed2002

Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation.

We have purified and characterized human Chk2 both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity.

Cell Cycle Proteins↗

PubMed2002

The proline-rich domain of p53 is required for cooperation with anti-neoplastic agents to promote apoptosis of tumor cells.

In some cell types either DNA damage or p53 expression leads to minimal cell death, while combining the two leads to a strong apoptotic response. To further understand features of p53 that contribute to this increased cell death we used clones of H1299 cells that express wild-type or several mutant forms of p53 under a tetracycline-regulated promoter. In these cells the induction of wild-type p53 leads to significant apoptosis only when combined with exposure to a number of chemotherapeutic agents. A common target of p53, p21, is itself not sufficient to cause apoptosis in the presence of these chemotherapeutic compounds. Many agents also effectively increase cell death when a transcriptionally-defective p53, p53([gln22ser23]), is induced, although a dramatic exception is treatment with 5-FU, which strongly cooperates with wild-type but not p53([gln22ser23]). Our results with 5-FU thus show that genetically separable functions of p53 are involved in its ability to respond to DNA-damaging agents to induce apoptosis. Notably as well, deleting the C-terminal 30 amino acids of p53 does not affect this cooperative effect with DNA-damaging agents. By contrast, a p53 mutant lacking the PXXP-domain between residues 60-90, while at least partially transcriptionally-competent, cannot be rendered apoptotic by any compounds that we tested. Thus the PXXP domain provides an essential component of the ability of p53 to respond to DNA-damaging agents to cause cell death.

Antineoplastic Agents↗

PubMed2002

Cyclin G recruits PP2A to dephosphorylate Mdm2.

The function of cyclin G, a commonly induced p53 target, has remained elusive. We show that cyclin G forms a quaternary complex in vivo and in vitro with enzymatically active phosphatase 2A (PP2A) holoenzymes containing B' subunits. Interestingly, cyclin G also binds in vivo and in vitro to Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate Mdm2 at T216. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at T216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Hdm2 at S166. Thus, our data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to regulate both Mdm2 and p53.

Animals↗

PubMed2002

p53 leans on its siblings.

Despite the common assumption that p53 by itself can induce apoptosis, results of a recent study implicate the homologous genes p63 and p73 in p53-mediated programmed cell death.

Animals↗

PubMed2002

Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein.

The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.

Animals↗

PubMedDate not supplied

The Chk2 protein kinase.

Checkpoint kinase 2 (Chk2) is a multifunctional enzyme whose functions are central to the induction of cell cycle arrest and apoptosis by DNA damage. Insight into Chk2 has derived from multiple approaches. Biochemical studies have addressed Chk2 structure, domain organization and regulation by phosphorylation. Extensive work has been done to identify factors that recognize and respond to DNA damage in order to activate Chk2. In turn a number of substrates and targets of Chk2 have been identified that play roles in the checkpoint response. The roles and regulation of Chk2 have been elucidated by studies in model genetic systems extending from worms and flies to mice and humans. The relationship of Chk2 to human cancer studies is developing rapidly with increasing evidence that Chk2 plays a role in tumor suppression.

Animals↗