5D). After UVC treatment, in the more sensible cells lines (RKO and HepG2), a robust caspase-3 activity increase was observed at 5 and 8 hours, respectively, in the presence of H(3KR)V5 mutant, in comparison to HuR-V5. Similar results were

obtained in MLP29 and SAMe-D cells at 16 and 36 hours after UVC treatment (Fig. 5D). These data highlight the proapoptotic phenotype associated with the H(3KR)V5 mutant and therefore to the lack of HuR NEDDylation. To further explore the mechanism by which HuR gets NEDDylated, we examined the interaction between Mdm2 and HuR by IP. Mdm2 interacts with both WT and H(K326R)V5 mutant (Fig. 6A), Also, HuR-V5 was cotransfected with Mdm2 WT and Mdm2 mutants (nuclear localization signal [NLS] and C464A). NLS, a Mdm2 mutant characterized by its exclusively cytoplasmic localization,29, 30 produced buy Galunisertib the same stabilization of HuR, compared to Mdm2 WT (Fig. 6B), suggesting that Mdm2-mediated HuR NEDDylation PLX-4720 chemical structure takes place in the cytoplasm. The C464A Mdm2 mutant, residue required for Mdm2 function as an E3 Ub ligase,27 had the same effect as WT MdM2 (Fig. 6B). In summary, these data indicate that Mdm2 interacts with HuR in the cytoplasm and participates in its stabilization independently of Mdm2 Ub ligase activity.

HuR is, predominantly, a nuclear protein.31 Using immunofluorescence, we observed that HuR-V5 expression was mostly nuclear, similar to the endogenous protein, whereas HuR mutants

had a more diffuse expression, with a predominantly cytoplasmic 上海皓元医药股份有限公司 localization in the case of H(K326R)V5 (Fig. 6C, upper panel). These results were confirmed by western blotting analysis (Fig. 6C, lower panel). Interestingly, the cysteine protease (NEDP1), which specifically removes NEDD8 molecules from conjugated substrates, reduced, by approximately 50%, the nuclear localization of HuR-V5, having no effect on cytoplasmic content. These data emphasize the role of NEDDylation in HuR nuclear localization (Fig. 6D). Given that HuR plays a central role in the post-transcriptional regulation of many critical proteins involved in fundamental processes in tumorigenesis (e.g., cell cycle, apoptosis and survival, proliferation, proangiogenesis, etc.), we analyzed the mechanisms regulating HuR overexpression in HCC and colon cancer. It was previously reported that in gastric cancers, HuR is transcriptionally up-regulated via the NFκB-PI3K axis.8 In liver cells, we reported that HuR expression levels increased proportionately to their transformation status.22 Here, we observed a strong correlation in the levels of HuR and Mdm2 during the transformation from primary hepatocytes to hepatoma, in colon cancer cells, and, more important, in a cohort of human metastatic colon cancer and HCC samples. We report that HuR is a NEDDylation substrate, and that Mdm2 mediates this NEDDylation by acting as an E3 NEDD8 ligase in the cytoplasm.