The Role Of P53 In Cell Apoptosis

Bad (Bcl-2 antagonist of cell death), a member of the Bcl-2 protein family, is a positive regulator of cell death and is localized in the cytoplasm under physiological conditions. Like other BH3-only proteins, Bad selectively displaces Bax to heterodimerize with Bcl-2 or Bcl-xL while promoting apoptosis. The apoptotic activity of Bad is determined largely by its phosphorylation status at serine 112, Serl36, and Serl55. In unstressed cells, Bad is hyperphosphorylated by several protein kinases, including protein kinase A and protein kinase B (AKT), and as a result is held onto by 14-3-3. However, in response to apoptotic stimuli, Bad is rapidly dephosphorylated and migrates to the mitochondria, where itinduces cell death.The p53 tumor suppressor is a pivotal mediator of cell cycle arrest and apoptosis in response to diverse stress stimuli. It transactivates various target genes, including those for p21waf1, Bax, p53DINP1, Bid, Puma, and many other proteins. However, a steady input of evidence for the transcription-independent function of p53 in apoptosis has been accumulating recently. Although a detailed mechanism has not yet been determined, recent data have revealed that upon an apoptotic stimulus, p53 translocates into mitochondria to interact directly with Bcl-2 or Bcl-xL to displace and activate proapoptotic Bax signaling. Similarly, disruption of the association complex between Bak/Mcl-1 or Bak/Bcl-2 by p53 is also related to p53-dependent but transcription- independent apoptosis.Although the regulation of several Bcl-2 family molecules, including Puma, Noxa, Bax, and Bid, by p53 has been studied intensively, the interplay between Bad (Bcl-2 antagonist of cell death) and p53 has not yet been reported thus far. Here, we report that p53 activates Bad transcription and expression through binding to a short conserved sequence located approximately 6.6 kb upstream of the translation start point. We also demonstrate that Bad physically interacts with cytoplasmic p53, thereby preventing p53 from entering the nucleus and resulting in reduced transcription of Bad. Moreover, Bad is able to direct p53 to the mitochondria and forms a p53/Bad complex at the mitochondria. Two lines of evidences support this hypothesis: first, when mitochondria purified from p53-deficient H1299 cells are incubated with p53 and either wild-type (wt) Bad or mutant Bad (this mutant binds p53 yet is unable to migrate to mitochondria), p53 can be detected only in mitochondria incubated with wt Bad and not in those incubated with mutant Bad; second, knockdown of Bad expression reduces mitochondrial localization of p53. The mitochondrial p53/Bad complex promotes apoptosis via activation and oligomerization of Bak. Elimination of Bad expression by RNA interference notably attenuates apoptosis induced by etoposide. Hence, our collective data provide the first evidence that Bad plays dual roles in both p53 transcription-dependent and -independent pathways.In summary, we have identified a p53-responsive element located approximately 6.6 kb upstream of the ATG translational start codon of the Bad gene and have shown that p53 transcriptionally activates Bad expression through binding to this region. We provide evidence that Bad is part of a negative feedback loop control system serving to maintain the equilibrium level of Bad by reducing p53 nuclear entry. Excess Bad binds to p53 within thecytosol, preventing p53 from entering the nucleus for further transcription of bad. Moreover, we demonstrate that Bad is able to direct p53 to the mitochondria and facilitates the intrinsic mitochondrion-mediated apoptosis through activation and oligomerization of Bak. Siva1, the CD27-binding protein, plays an important role in promoting both intrinsic and extrinsic cell death.Ectopically expressed Sival binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis. Siva-1 and its alternative splice form Siva-2, which lacks the death domain homology region (DDHR), mediates apoptosis in T-lymphocytes via a caspase-dependent mitochondrial pathway. Moreover, Siva1 was reported to be involved in TCR-mediated AICD (activation-induced cell death) with implications in peripheral tolerance, T-cell homeostasis and cancer through its inhibitory effect on NF-κB activity. Jacobs SBR and coworker have also shown that Siva1 is essential for p53-dependent apoptosis in cerebella granule neurons. Together, all the data reported for Siva1 thus far have demonstrated its proapoptotic function in stressed cells. Little is known about the function(s) of Siva1/2 besides apoptosis induction and the exact function of Siva1/2 in unstressed cells.Tumor suppressor p53, a key regulator of cell-cycle control, apoptosis, and genomic stability, transactivates stress response genes with various functions, including p21waf1 (7), Bax (8), Bad (9), Hmd2 (10-12), Siva (13) and many others. Some target gene products, such as Hdm2 and Bad can bind to p53, creating a negative feedback loop for down-regulating p53 level or activity. Normally, in unstressed cells, level of p53 is tightly regulated and is usually kept at a minimum by many factors among which Hdm2 is the central player. Accumulating data reveals that many cellular factors are involved in modulating Hdm2-mediated p53 ubiquitination and degradation, such as PACT and Daxx. PACT (p53-associated cellular protein-testes derived, also known as P2P-R, RBBP6) can directly interact with Hdm2 to facilitate p53- Hdm2 binding, thus increases ubiquitination and degradation of p53 (14). Similarly, Daxx, a p53 binding protein, stabilizes Hdm2 via stabilizing Hausp (herpesvirus-associated ubiquitin-specific protease, a deubiquitinating enzyme) to enhance the intrinsic E3 activity of Hdm2 towards p53, preventing p53 activation in unstressed cells (15). In addition, p53 also affects other cellular functions, including cell migration and tumorigenesis. It has been reported that in the absence of stress, p53 promotes cell motility by modulating intracellular signaling pathways, rather than by stimulating the production of secreted mitogenic factors. Consistently, the motility of cells lacking of p53 has also been reported to be deceased (16). Nonetheless, some contradictory results were also reported, Gadea G et. al showed that loss of p53 promotes RhoA-ROCK-dependent cell migration and invasion in 3D matrices (17).The tumor suppressor p53 induces potent anti-proliferative responses in stressed cells, and this ability of p53 is restrained in unstressed cells due to rapid ubiquitination and degradation mediated by Hdm2. Expression of Hdm2 is induced by p53, thereby establishing feedback inhibition. It is not completely understood how the p53-Hdm2 interaction is regulated and what other protein(s) is involved in the feedback inhibition of p53.Here we show that the p53-Hdm2 interaction in unstressed cells is promoted by Siva1, which, like Hdm2, is the product of a p53 target gene. Siva1 binds to both p53 and Hdm2 through distinct regions and enhances Hdm2-mediated p53 ubiquintion and degradation. In addition, Siva1 blocks p53-mediate gene expression, apoptosis, and cell migration. The function of Siva1 appears to be related to its ability to form a homo-dimer as the dimerization-defective splicing variant Siva2 fails to destabilize p53. In a xenograft mouse model, overexpression of Siva1 inhibits p53-mediated tumor suppression, while down-regulation of Siva1 increases the response. Upon DNA damage, the interactions of Siva1 with both p53 and Hdm2 are diminished. These results identify Siva1 as an important adaptor promoting p53 degradation via Hdm2.In summary, Siva1 may be part of the negative feedback loop that inhibits p53 activity at the end of a stress response.