Negative regulation of p53 family protein p73 by transcription factor E2F-1

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We have found for the first time that E2F-1 has an ability to promote the ubiquitin-dependent proteolytic degradation of p73 in COS7 and SAOS-2 cells. Similar results were also obtained in H1299, U2OS, H4 and A549 cells. Deletion analysis revealed that the transactivation function of E2F-1 is required for the degradation of p73. We also demonstrated that a novel HECT-type E3 ubiquitin protein ligase termed NEDL2 interacts with the PY motif of p73, and thereby inducing its ubiquitination. Unexpectedly, NEDL2-mediated ubiquitination of p73 resulted in an incre ase in its half-life, and enhanced its transcriptional activity. These observations strongly suggest that there exists a non-proteolytic regulatory function of the ubiquitination. By using a yeast-based two-hybrid screening, we have identified RanBPM as a binding partner of p73. RanBPM bound to the extreme COOH-terminal region of p73, and increased its stability by inhibiting the ubiquitination levels of p73. It is likely that the COOH-terminal Lys residues could be target(s) for ubiquitination. In addition, we have found that U-box-type E3/E4 ubiquitin protein ligase termed UFD2a induces the proteasome-dependent proteolytic degradation of p73 through the physical interaction with p73. Of note, UFD2a-mediated ubiquitination of p73 was not detected under our experimental conditions. Thus, our present results indicate that p73 is regulated not only by the ubiquitination-dependent degradation pathway but also by the functional interaction with UFD2a in a ubiquitination-independent manner.