Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. We previously found that the mRNA binding protein, ELAV 1 (HuR), is upregulated in primary brain tumors and stabilizes growth factor mRNAs such as VEGF and IL-8. To better understand the role of HuR in brain tumor growth, we altered levels of HuR in glioma cells by shRNA or ectopic expression and measured tumor cell phenotype using in vitro and in vivo models. In HuR-silenced cells, we found a significant decrease in anchorage-independent growth and cell proliferation with a concomitant induction of apoptosis. Using an intracranial tumor model with primary glioblastoma cells, HuR silencing produced a significant decrease in tumor volume. In contrast, overexpression of HuR produced in vitro chemoresistance to standard glioma therapies. Since bcl-2 is abundantly expressed in glioma and associated with tumor growth and survival, we determined the impact of HuR on its regulation as a molecular validation to the cellular and animal studies. Using UV crosslinking and RNA immunoprecipitation, we show that HuR bound to the 3' untranslated region of all bcl-2 family members. Silencing of HuR led to transcript destabilization and reduced protein expression. Polysome profiling indicated loss of HuR from the translational apparatus. In summary, these findings reveal a HuR-dependent mechanism for cancer cell survival and sensitivity to chemotherapeutic drugs suggesting that HuR should be considered as a new therapeutic target.

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.Precise and timely subcellular localization of proteins is essential for their biological functions, and conversely, the control of protein localization provides the cells with a convenient way to regulate their functions. Posttranslational modifications of proteins play critical roles in these aspects. For instance, three of the most important outcomes after protein phosphorylation are typically the changes in protein localization, stability, or activity (32, 38). Eukaryotic DNA replication is strictly controlled by mechanisms that regulate the cell cycle to ensure both genetic inheritance and stability. DNA replication is initiated at a precise time when the cellular replication machinery is ready, and then the genome is replicated for a single round in each cell cycle. Cell cycle entry and progression are regulated by cyclin/cyclin-dependent kinases (CDKs) that phosphorylate key regulatory proteins (5,59,65,74). At the G 1 /Sphase transition, cyclin/CDK complexes phosphorylate the components of the cellular DNA prereplication complex (pre-RC), including origin recognition complex, Cdc6, Cdt1, and MCM2-7, enabling the initiation of replication and subsequently preventing reduplication in the same cell cycle (5, 6, 54). The specific mechanisms vary for different components in different organisms. For example, for the replicative DNA helicase complex composed of the MCM2-7 subunits (71), phosphorylation inhibits its activity in humans and mice (29,30). In budding yeast, phosphorylation leads t...

The ataxia mutation (ax J ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The ax J gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the ax J mice. Transgenic rescue of the ax J mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the ax J mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of ax J mice. However, the ax J -rescued mice still displayed the Purkinje cell axonal swellings that are seen in the ax J mice, indicating that this cerebellar alteration is not the primary cause of the ax J movement disorders. These results show that the motor defects observed in the ax J mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the ax J mice.