2B,C). We studied HuR ablation in terms of cell response in MLP29, SAMe-D, RKO, and the human hepatoma cell line, HepG2. HuR silencing induced a strong activation of caspase-3 and a lower percentage of cells entering into S phase in those cell lines (Fig. 2D). These data suggest that HuR ablation promotes apoptosis and cell-cycle arrest. Furthermore, Mdm2 silencing in MLP29, hepatoma, and RKO cells led to a significant

decrease in HuR levels and its target, cyclin A (Fig. 2E). In addition, in RKO cells, the ablation of Mdm2 increased by 9.7 times the activity of caspase-3 activity (Fig. 2F, left graphics), whereas no changes in this apoptotic response was detected in MLP29 and SAMe-D cells 48 hours after transfection (data not shown). However, silencing of Mdm2 for a longer selleck inhibitor period (72 hours) rendered a significant increase of caspase-3 activity and cell-cycle arrest in those cell lines (Fig. 2F). All these data suggest a cross-talk between AG-014699 supplier HuR and Mdm2, where at least part of the oncogenic features linked to Mdm2 could be attributed to HuR functionality. Mdm2 acts as an E3 Ub ligase and can regulate its own stability through autoubiquitination, targeting itself for proteasomal degradation.27 Importantly, Mdm2 exhibits E3 NEDD8 ligase activity, promoting p53 stability.14

Because we found that Mdm2 could regulate HuR levels (Fig. 2E), we examined whether this occurred through NEDDylation. To this end, we overexpressed Mdm2 and His6-NEDD8 in the MLP29 cell line. IP of HuR revealed the appearance of high molecular bands with HuR medchemexpress immunoreactivity in the presence of either Mdm2 or NEDD8 (Fig. 3A). To gain

insight about HuR NEDDylation, MLP29 cells were transfected with a plasmid expressing HuR-V5, along with His6-tagged NEDD8, and the proteins conjugated to His6-NEDD8 were purified as described previously.28 We found that HuR NEDDylation, in the presence of His6-NEDD8, was increased by Mdm2, suggesting that Mdm2 regulates HuR NEDDylation (Fig. 3B). To examine the influence of NEDDylation on HuR stabilization, we used siRNA to knock down NEDD8 expression. Sequential transfections to silence NEDD8 completely destabilized HuR-V5, even in the presence of Mdm2 (Fig. 3C). To show the importance of Mdm2-mediated NEDD8 conjugation in HuR stabilization, we cotransfected HuR and Mdm2 with the cysteine protease (NEDP1), which removes NEDD8 molecules from conjugated substrates.13 Whereas Mdm2 alone induced the accumulation of HuR-V5, coexpression with NEDP1 clearly decreased the amount of V5 expressed (Fig. 3D). In agreement with this, the expression of NEDP1 induced the destabilization of endogenous HuR in MLP29 cells and RKO cells (Fig. 3E). Ub-mediated proteolysis of HuR by stress signals has been previously reported to regulate its stability.9 We thus examined whether HuR ubiquitination following a stress signal, such as UVC, would affect NEDDylated HuR-V5.