Bombyx mori p53 requires multiple domains for transcription-dependent apoptosis induction

The transcription factor p53 is widely conserved among vertebrates and invertebrates and regulates various stress responses, including apoptosis. p53 proteins identified in the lepidopteran insects Bombyx mori (Bm-p53) and Spodoptera frugiperda exhibit proapoptotic activity through transient expression; however, the mechanism of apoptosis induction by lepidopteran p53 remains unclear, particularly regarding the domains required and the role of transcriptional activity. Here, we investigated the domains required for Bm-p53 activity using transient expression assays with domain/region deletion mutants. Bm-p53 contains three domains-TAD (transcriptional activation domain), DBD (DNA-binding domain), and NLS (nuclear localization signal)-which are conserved in mammalian and Drosophila p53s. However, Bm-p53 lacks an oligomerization domain, which is conserved in the C-terminal region of mammalian and Drosophila p53s. We found that all examined domains/region-TAD, DBD, NLS, and the C-terminal region (CR)-are crucial for the full proapoptotic activity of Bm-p53. Additionally, Bm-p53 can activate transcription from a promoter containing human p53-binding sites. This activity is completely lost in cells expressing Bm-p53 with deletions in the DBD, NLS, and CR, whereas TAD deletion results in a modest reduction. Both the proapoptotic and transcriptional activities were suppressed in Bm-p53 with point mutations corresponding to human p53 mutational hotspots. Collectively, our findings demonstrate that Bm-p53 induces apoptosis primarily through transcriptional activation of downstream genes, requiring nuclear localization via the NLS and DNA binding via the DBD, and supported by the TAD and CR. Notably, transcription-independent mechanisms may also contribute to apoptosis induction, indicating a more complex regulatory model of Bm-p53 function.