If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.
). In the late phase of the stress response, TBP-associated factor 1 (TAF1), the largest subunit of the basal transcription factor TFIID, phosphorylates p53 at Thr-55 and dissociates p53 from the p21 promoter by elevating MDM2 binding affinity of p53 (
). In addition, some unknown transcription-related factors may also be involved in p53 regulation, and identification of a novel regulator of the p53-MDM2 interplay is critical for understanding the mechanisms underlying p53 dynamics.
TLP is a member of the TBP family and is also named TBP-related factor 2 (TRF2) and TBP-like 1 (TBPL1) (
). However, little is known about the most fundamental question of how TLP regulates p53 target genes or p53 itself.
Here we aimed to investigate the role of TLP in p53 regulation and present evidence that TLP is a new regulatory factor of the p53-MDM2 interplay. In the genotoxic stress response, TLP promotes p53-driven apoptosis and senescence by mediating persistent p53 activation. TLP binds to the p53 TAD and inhibits MDM2 recruitment to p53, which results in suppression of p53 ubiquitination. We also aimed to real time chasing of p53 nuclear export and show that TLP is essential for suppressing MDM2-driven nuclear export of p53. Moreover, a cervical cancer-derived TLP mutant has little p53 binding ability and does not suppress cell growth. Taken together, our findings indicate that TLP disrupts the p53-MDM2 interaction and mediates long-lasting p53 activation in response to genotoxic stress.
Recent studies have proposed that the concentration and duration of activated p53 and its target genes determine the cell fate decision such as cell cycle arrest, apoptosis, and senescence (
). In the present study we showed that TLP stabilizes the p53 protein, thereby enhancing its function. We demonstrated that the TLP function is evident in the late phase of a high dose of UV exposure and is important to mediate the induction of apoptosis and senescence. This indicates that TLP is required for persistent p53 activation and direction of the cell fate decision. We further showed that TLP disrupts the p53-MDM2 interaction and demonstrated that TLP prevented p53 degradation by interfering with MDM2-mediated ubiquitination and nuclear export of p53. Moreover, we found that the expression of TLP is increased at the protein level in the late phase of UV irradiation, suggesting that elevated TLP binds to p53 and releases p53 from the MDM2-mediated negative control. In contrast, the MDM2 function toward p53 becomes stronger when the levels of TLP in cells are low. Notably, MDM2 is one of the targets of p53 and thus is down-regulated by TLP knockdown. Although this result seems to cause p53 stabilization, TLP depletion decreased p53 levels. This situation is because the turnover of p53 became faster in TLP-depleted cells. These phenomena suggest that TLP maintains the protein levels of p53 and MDM2 in cells. Our findings indicate that TLP acts as an MDM2 blocker in the p53-MDM2 negative feedback loop and establishes a novel mechanism for long-lasting p53 activation to direct appropriate cell fate decision (Fig. 7).
TBP recognizes the TATA element in promoters of many RNA polymerase II-driven genes, enabling it to constitute the basic transcriptional machinery on those promoters (
). TLP binds to some general transcription factors, such as TFIIA, and interferes with the TBP-TFIIA interaction. The TLP-bound TFIIA precursor is protected from Taspase1-mediated cleavage, which leads to inhibition of TBP-mediated transcription (
). Perhaps one role of TLP may be as a blocker of protein-protein interactions. We note that TLP depletion in HeLa cells increased p53 mRNA levels, although p53 protein levels were down-regulated in such a condition. This phenomenon seems to be governed by p53-independent TLP function, suggesting that TLP has multiple roles in both p53-dependent and -independent manners.
The interplay between p53 and transcription-related factors in stress response is highly complicated. It is basically unknown whether basal transcription factors exert their positive or negative effects on p53 function. TFIIH, a multiprotein complex involved in both transcription and DNA repair, and TBP, both, activate p53-driven transcription (
). TAF1-mediated phosphorylation of p53 increases p53-MDM2 interaction and releases p53 from the p21 promoter in the late phase of UV irradiation. In this study we confirmed that p53 is actually recruited to the promoters of its target genes. However, TLP is not significantly recruited to promoters of representative p53 target genes. These results suggest that TLP affects p53 activity through a mechanism distinct from association of transcription machineries on a promoter. Hence, our findings may help to resolve the mechanisms underlying p53 dynamics and linking basal transcription factors to p53 activation.
The nuclear export of p53 has also been widely discussed because a number of cancers show cytoplasmic localization of p53 (
). It has been speculated that MDM2 induces a conformational change of p53 to expose the nuclear export signal at the C-terminal domain of p53. MDM2 then mediates p53 monoubiquitination and promotes its nuclear export (
). We established a method for chasing real-time nuclear export of p53 using Dronpa and showed that TLP depletion increases the nuclear export of p53. However, TLP depletion does not fully export p53 to the cytoplasm, and the remaining p53 still activates transcription. These results suggest that TLP depletion is not sufficient for abolishing p53 activity. Because many factors cooperatively and rapidly regulate the p53 level in normal cells (
). In these cancer cells several altered cellular processes are likely to suppress p53 function. In this study we used HCT116 and HeLa cells. Interestingly, TLP-elevated p53 stability was clearly observed in HeLa cells. Human papillomavirus E6 protein in HeLa cell leads p53 to degradation (
), and because of these vulnerable conditions, TLP function for p53 may be strengthened. Furthermore, we found that multiple TLP mutations in human cancers were mapped in the p53 binding region of TLP. We used a TLP mutant, D99H, which is found in wild-type p53-expressing cervical cancer. This mutant had a weak p53 binding ability and exhibited a defect in suppression of cell growth. These findings are consistent with the notion that wild-type p53-expressing cancer cells have some p53-suppressive factors. Consequently, TLP is a novel regulator of the p53-MDM2 interplay that provides cells with sensitivity to genotoxic stress by mediating long-lasting p53 activation.
R. M., N. A., K. U., T. E., and T. T. conceived and designed the experiments. R. M., H. Tamashiro, K. T., H. S., S. S., and W. K. performed the experiments. R. M. and H. Takahashi analyzed the data. R. M., H. S., and T. T. contributed reagents, materials, and analysis tools. R. M. and T. T. wrote the paper.