Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop

Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.     

Essential role played by the C-terminal domain of glycoprotein I in envelopment of varicella-zoster virus in the trans-Golgi network: interactions of glycoproteins with tegument

Varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN). Here we report that glycoprotein I (gI) is required within the TGN for VZV envelopment. Enveloping membranous TGN cisternae were microscopically identified in cells infected with intact VZV. These sacs curved around, and ultimately enclosed, nucleocapsids. Tegument coated the concave face of these sacs, which formed the viral envelope, but the convex surface was tegument-free. TGN cisternae of cells infected with VZV mutants lacking gI (gI(Delta)) or its C (gI(DeltaC))- or N-terminal (gI(DeltaN))-terminal domains were uniformly tegument coated and adhered to one another, forming bizarre membranous stacks. Viral envelopment was compromised, and no virions were delivered to post-Golgi structures. The TGN was not gI-immunoreactive in cells infected with the gI(Delta) or gI(DeltaN) mutants, but it was in cells infected with gI(DeltaC) (because the ectodomains of gI and gE interact). The presence in the TGN of gI lacking a C-terminal domain, therefore, was not sufficient to maintain enveloping cisternae. In cells infected with intact VZV or with gI(Delta), gI(DeltaN), or gI(DeltaC) mutants, ORF10p immunoreactivity was concentrated on the cytosolic face of TGN membranes, suggesting that it interacts with the cytosolic domains of glycoproteins. Because of the gE-gI interaction, cotransfected cells that expressed gE or gI were able to target truncated forms of the other to the TGN. Our data suggest that the C-terminal domain of gI is required to segregate viral and cellular proteins in enveloping TGN cisternae.     

p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C-terminal acetylation

The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2.     

Recognition of cisplatin-damaged DNA by p53 protein: critical role of the p53 C-terminal domain

It was shown previously that the p53 protein can recognize DNA modified with antitumor agent cisplatin (cisPt-DNA). Here, we studied p53 binding to the cisPt-DNA using p53 deletion mutants and via modulation of the p53-DNA binding by changes of the protein redox state. Isolated p53 C-terminal domain (CTD) bound to the cisPt-DNA with a significantly higher affinity than to the unmodified DNA. On the other hand, p53 constructs involving the core domain but lacking the C-terminal DNA binding site (CTDBS) exhibited only small binding preference for the cisPt-DNA. Oxidation of cysteine residues within the CD of posttranslationally unmodified full length p53 did not affect its ability to recognize cisPt-DNA. Blocking of the p53 CTDBS by a monoclonal antibody Bp53-10.1 resulted in abolishment of the isolated CTD binding to the cisPt-DNA. Our results demonstrate a crucial role of the basic region of the p53 CTD (aa 363-382) in the cisPt-DNA recognition.     

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.     

Reciprocal interference between the sequence-specific core and nonspecific C-terminal DNA binding domains of p53: implications for regulation.

The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.     

p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis.     

Stable formation of mutated p53 multimers in a Chinese hamster cell line causes defective p53 nuclear localization and abrogates its residual function

We have previously described a methotrexate-resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm-2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C-terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced beta-tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function.     

hGTSE-1 expression stimulates cytoplasmic localization of p53

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.     

Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability.

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.     

Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain

A synthetic 22-mer peptide (peptide 46) derived from the p53 C-terminal domain can restore the growth suppressor function of mutant p53 proteins in human tumor cells (G. Selivanova et al., Nat. Med. 3:632-638, 1997). Here we demonstrate that peptide 46 binds mutant p53. Peptide 46 binding sites were found within both the core and C-terminal domains of p53. Lys residues within the peptide were critical for both p53 activation and core domain binding. The sequence-specific DNA binding of isolated tumor-derived mutant p53 core domains was restored by a C-terminal polypeptide. Our results indicate that C-terminal peptide binding to the core domain activates p53 through displacement of the negative regulatory C-terminal domain. Furthermore, stabilization of the core domain structure and/or establishment of novel DNA contacts may contribute to the reactivation of mutant p53. These findings should facilitate the design of p53-reactivating drugs for cancer therapy.     

The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth.

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.     

Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.