Myeov2 alters L11 subnuclear localization without nucleolar disruption.

<p>(<b>A</b>) HeLa cells were transfected with Myc-L11, together with either Flag-Myeov2 or Flag- Myeov2ΔFD plasmids, and subjected to immunocytochemistry. Arrows and arrowheads indicate Myeov2-expressing and control cells, respectively. Scale bar, 20 µm. (<b>B</b>) Quantification of the data in (A). Subnuclear localization of Myc-L11 in Flag-Myeov2-positive cells was examined. Cells that express L11 in nucleous only (yellow), both in nucleolus and nucleoplasm (blue) and nucleoplasm only (pink), were counted. N denotes the number of cells counted. (<b>C</b>) Co-immunoprecipitation of Flag-Myeov2 or Flag-Myeov2ΔFD with HA-L11 in HEK293T cells. Cell lysates were immunprecipiated with anti-Flag antibodies and analyzed using both Flag and HA antibodies. (<b>D</b>) HeLa cells transfected with EGFP-NPM and HA-Myeov2 were treated with 10 nM ActD for indicated time, and subjected to immunocytochemistry. Expression of Myeov2 did not induce nucleolar disruption in the absence of ActD (top panels). Nuclear disruption was observed in Myeov2-expressing cells at 1 hr of ActD treatment (middle panels). 4 hrs of ActD treatment resulted in nucleolar disruption irrespective of Myeov2 expression (lower panels). Scale bar, 20 µm.</p

Model for the role of Myeov2 in nucleus.

<p>(<b>A</b>) Most of L11 is neddylated and localized in nucleolus. In response to nucleolar stress, L11 is deneddylated and relocates to nucleoplasm, leading to activation of p53. (<b>B</b>) Myeov2 blocks L11 neddylation by their interaction and suppresses translocation of L11 into nucleolus, and make cells sensitive to stresses.</p

Expression of Myeov2 maintains L11 in a lower neddylation level in cells.

<p>(<b>A</b>) HEK293 cells were transfected with HA-Nedd8 and either Flag-Myeov2 or Flag-Myeov2ΔFD. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (<b>B</b>) HEK293T cells were transfected with either Flag-Myeov2 or Flag- Myeov2ΔFD and then treated with 10 µM MG132 for 1 hour prior to harvest. Cell lysates were subjected to immunoblot analyses. Arrowhead indicates putative Myeov2 target protein. (<b>C, D and E</b>) HEK293T cells were transfected as indicated. Neddylated proteins were precipitated from denatured cell lysates using Talon metal affinity resin and immunoblotted with indicated antibodies. Arrowheads indicate neddylated L11.</p

Myeloma Overexpressed 2 (Myeov2) Regulates L11 Subnuclear Localization through Nedd8 Modification

<div><p>Nucleolus is a dynamic structure that controls biogenesis of ribosomal RNA and senses cellular stresses. Nucleolus contains a number of proteins including ribosomal proteins that conduct cellular stresses to downstream signaling such as p53 pathway. Recently, it has been reported that modification by a ubiquitin-like molecule, Nedd8, regulates subnuclear localization of ribosomal protein L11. Most of L11 is normally localized and neddylated in nucleolus. However, cellular stress triggers deneddylation and redistribution of L11, and subsequent activation of p53. Although Nedd8 modification is thought to be important for L11 localization, the mechanism of how neddylation of L11 is regulated remains largely unknown. Here, we show that Myeloma overexpressed 2 (Myeov2) controls L11 localization through down-regulation of Nedd8 modification. Expression of Myeov2 reduced neddylation of proteins including L11. We also found that Myeov2 associates with L11 and withholds L11 in nucleoplasm. Although Myeov2 interacted with a Nedd8 deconjugation enzyme COP9 signalosome, L11 deneddylation was mediated by another deneddylase Nedp1, independently of Myeov2. Finally, p53 transcriptional activity is upregulated by Myeov2 expression. These data demonstrate that Myeov2 hampers L11 neddylation through their interactions and confines L11 to nucleoplasm to modulate nucleolar integrity. Our findings provide a novel link between oncogenic stress and p53 pathway and may shed light on the protective mechanism against cancer.</p></div

Myeov2 associates with COP9 signalosome.

<p>(<b>A</b>) Protein identified by mass spectrometry of Myeov2 immunoprecipitants. The colored squares show how often a protein was identified in multiple experiments. (<b>B</b>) Co-immunoprecipitation of HA-Myeov2 and Flag-CSN5 in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using Flag and HA antibodies. (<b>C</b>) Co-immunoprecipitation of Flag-Myeov2 and endogenous CSN5 in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using Flag and CSN5 antibodies. (<b>D</b>) Schematic structure of Myeov2 deletion mutants. (<b>E</b>) Co-immunoprecipitation of CSN5, Cul1, and Cul3 with Flag-Myeov2 in HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using indicated antibodies.</p