Based on these results, we propose that HuR and Mdm2 expression cooperatively influences tumor cell growth by controlling the expression of cell-cycle regulators. Interestingly, it has been previously reported that Mdm2 mRNA level is stabilized by HuR, establishing a regulatory network among these targets either at protein and mRNA levels and highlighting the tight balance that controls both.32 The Ub-proteasome pathway has been implicated in HuR levels and function.9, 33 Interestingly, several NEDDylated proteins seem to be either substrates or components of ubiquitin E3s, revealing an intriguing relationship between
ubiquitination and NEDDylation. NEDDylated-HuR-V5 shifts to ubiquitinated conjugation after UV treatment, which is accompanied by a decrease in HuR-V5 levels. In addition, endogenous HuR was also a target for NEDDylation, and Mdm2 was demonstrated to increase its stabilization as a result of
a robust enrichment in this post-translational modification. Analysis of single amino acid substitutions in the RRM3 and C-terminal region of HuR demonstrated that three evolutionarily conserved lysines, K283, K313, and, particularly, K326, affected the stability of HuR. The three KR mutants were less stable than the control HuR-V5, but their intrinsic ability to associate with target mRNAs (e.g., PTMA or cyclin D1) were not affected, although the triple mutant, H(3KR)V5, showed a clear proapoptotic phenotype. NEDD8 silencing drastically reduces levels of HuR, and NEDDylation experiments showed that H(K326R)V5 is deficient for NEDD8 conjugation and
this corresponds to increased destabilization of the mutant. Florfenicol Also, H(K326R)V5 showed increased ubiquitination in the presence of Mdm2 and Ub, suggesting that suppression of NEDDylation renders HuR more susceptible to ubiquitination. HuR is localized mainly in the nucleus and translocates to the cytoplasm in response to specific stimuli. It was previously reported that NEDD8 plays a role in the correct nuclear localization of L11, protecting it from degradation.29 We observed that NEDP1 transfection lowered the nuclear content of HuR-V5 and that the mutant, H(K326R)V5, was mostly cytoplasmic. HuR export to the cytoplasm is associated with a reduction of this protein as a result of proteasomal degradation after UVC exposure (Supporting Fig. 5B). K313R and K326R mutants were degraded by the proteasome in the cytoplasm, because proteasome inhibition by MG132 treatment induced an accumulation of K313 and K326 HuR mutants (Supporting Fig. 5A). Also, the Mdm2 NLS mutant, which is localized exclusively in the cytoplasm, and Mdm2, bearing a Adriamycin mw mutation in C464, a residue involved in Mdm2 ubiquitination, were able to promote HuR stabilization.