The results in this study establish a fundamental role for ZBP-89 in the regulation of cell proliferation. Elevated expression of ZBP-89 induced growth arrest and apoptosis. However, the S-phase inhibition observed with ZBP-89 overexpression was abolished in a p53 null cell line. This result confirmed that the growth arrest mediated by ZBP-89 was p53 dependent whereas ZBP-89-mediated apoptosis was p53 independent. Elevated levels of ZBP-89 stabilize the p53 protein through direct physical contact, which potentiates the transcriptional activation of both a synthetic p53-responsive reporter and its endogenous target, the p21waf1
gene. The potentiation was p53 dependent since ZBP-89 could not activate the promoter in the absence of the p53 protein. Similarly, our prior study showed that ZBP-89 activation of the p21waf1
promoter is butyrate dependent in a p53-deficient cell line and that ZBP-89 exerts no transcriptional regulation on the p21waf1
promoter alone (5
). ZBP-89 binds directly to the p21waf1
), but, in the absence of p53, ZBP-89 requires recruitment of a histone acetylase and inhibition of deacetylase activity (5
). Thus both p53 and p21waf1
appear to be downstream targets of ZBP-89. Accumulation of p53 is normally transient due to the induction of the p53 inhibitor MDM2 or decreased p14ARF activity. This raised the possibility that ZBP-89 might affect p53 protein levels by modulating p14ARF or MDM2 expression. However, both the MDM2-p53 interaction and p14ARF levels remained unperturbed despite elevated levels of ZBP-89. Thus ZBP-89 was able to overcome the tendency for p53 to be rapidly degraded by binding to a site distal to the MDM2 binding domain and formation of the p14ARF-MDM1-p53 trimeric complex, thereby preventing p53 nuclear export.
In transiently cotransfected HCT 116 p53 null cells, ZBP-89 caused an approximately five- to eightfold increase in cotransfected p53 protein, whereas the transcriptional activity of transfected p53 alone was potentiated only approximately twofold. The discrepancy between protein levels and activity is consistent with another report showing that stabilized p53 is not able to totally regain its transcriptional activation due to the formation of a trimeric complex containing MDM2, p53, and the retinoblastoma protein (23
). Further, the accumulation of p53 mediated by ZBP-89 may not directly translate to transcriptional activity due to the inhibitory effects of MDM2 suppressing the overall level of transcription.
A major mechanism that results in p53 stabilization involves activation of tumor suppressor protein p14ARF (mouse p19ARF) (37
). An increase in the activity of p14ARF promotes sequestration and degradation of MDM2, thereby reducing its activity (51
). p14ARF binds directly to MDM2 in a region distinct from the p53 binding domain and therefore does not disrupt the interaction between p53 and MDM2. The tumor suppressor protein BRCA1 (45
) and oncogene products, such as c-Abl, c-Myc, Ras, and E1A (10
), stabilize p53 through activation of p14ARF. For BRCA1, overexpression induces p14ARF expression, which in turn inhibits MDM2 (45
). p14ARF is required for the BRCA1 effect since overexpression of this tumor suppressor protein in p14ARF-deficient cells failed to induce accumulation of wild-type p53 (45
). BRCA1 binds to the C terminus of wild-type p53 and stabilizes the protein through direct binding. While BRCA1 induces accumulation of wild-type p53, its overexpression failed to induce accumulation of mutant p53. These studies were carried out with a prostate cell line (DU-145) which has a double point mutation of p53 (14
). Since BRCA1 and ZBP-89 both bind to the C terminus of p53 and preferentially stabilize wild-type over mutant forms of p53, we considered the possibility that ZBP-89 might also stabilize p53 in a p14ARF-dependent manner. However, the time course of serum starvation and lack of correlation with p14ARF protein expression diminished the likelihood that this tumor suppressor is required for the ZBP-89-induced stability of p53.
Most of the p53 mutations that occur in cancer are located in the DNA binding domain and effectively block its transactivating activity. p53 mutations also prevent MDM2 from targeting the protein for degradation, allowing mutant forms of p53 to accumulate in the cell (6
). Elevated mutant forms of p53 are thought to have a deleterious effect on p53 function by dimerizing with the remaining normal p53 proteins in the cell (7
). We found that a single p53 mutation, R273H, prevented ZBP-89 from inducing accumulation of the mutant p53 protein. Thus it appears so far that the effect of ZBP-89 on the p53 protein is specific for the wild-type form. This result may have relevance in cancers that tend to accumulate wild-type rather than mutant p53 despite activation of oncogenes or other cell stresses (13
). Cancer cells that accumulate wild-type p53 tend to undergo apoptosis and are more susceptible to radiotherapy and chemotherapy (33
). Therefore, the studies described here may further our understanding of how wild-type p53 might accumulate in transformed cells.
p53 may accumulate in cells due to mechanisms that interfere with MDM2 binding or activity. This may be accomplished by phosphorylation of the p53 N-terminal domain, subsequently blocking MDM2 binding and activity (2
). It is not clear how the ZBP-89–p53–MDM2 trimeric complex protects p53 from MDM2-mediated degradation. However, the heterokaryon assay clearly demonstrated that elevated levels of ZBP-89 prevent p53 nuclear export. The knowledge that ZBP-89 binds preferentially to the middle (DNA binding) and C-terminal domains of p53 and not to the N-terminal domain, recognized by the MDM2-p14ARF complex, suggests that an alternative mechanism is employed to stabilize p53. It has been shown recently that both the DNA binding domain and extreme C terminus of p53 are necessary for MDM2-mediated degradation (2
). Partial deletions or mutations of the p53 C terminus interrupt MDM2-directed degradation (29
). A recent study shows that p53 C-terminal lysine residues are the main sites of MDM2-mediated ubiquitin ligation, which targets p53 for proteasome degradation (41
). Modifications of the p53 C terminus, including phosphorylation (24
) and acetylation (4
), enhance the transcriptional activity of p53. Acetylation of p53 at these C-terminal lysines prevents nuclear ubiquitination (35
). Further, histone acetylase coactivator p300 binds the N-terminal domain of ZBP-89 and the C-terminal domain of p53 (5
). Thus, ZBP-89 may protect p53 from MDM2-mediated degradation by sterically masking the sites on p53 that confer sensitivity to degradation or by recruiting p300 to modify p53 through increased acetylation. This hypothesis would explain why the cellular MDM2 protein levels are not directly affected by ZBP-89 overexpression. Collectively, the results reported here reveal a novel function of ZBP-89 that supports its physiological role in growth regulation through a p53-dependent mechanism.