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Fig. 4. Nucleoplasmic nucleostemin competes with L23 for MDM2 binding. (A1) Triple coimmunoprecipitation of nucleostemin (HA), MDM2 (Myc) and L23 (Flag) by the indicated antibodies (IP) showed that L23 coexpression reduces the interaction between nucleostemin and MDM2. Double coimmunoprecipitation shows that L23 interacts with MDM2 (A2) but not with nucleostemin (A3). (B) The ability of nucleostemin to bind MDM2 in the presence of L23 overexpression is significantly increased by the combined mutations of dB and G256V [dB(256)]. (C) The dB(256) mutant is capable of competing with L23 for MDM2 binding in the triple coimmunoprecipitation experiments. MDM2 protein complexes (top panel) immunoprecipitated from lysates (bottom panel) containing the same amount of L23 and increasing amounts of dB(256). (D) Both nucleostemin (HA) and L23 (Flag) reside primarily in the nucleolus under normal growth conditions (Ctrl). When exposed to ADR (2 µM), actinomycin D (ActD, 0.05 µg/ml) and MPA (40 µM) for 4 hours, L23 is redistributed to the nucleoplasm, as is nucleostemin, but to a lesser extent. Distribution of nucleostemin and L23 is shown in different ADR-treated cells because of the autofluorescent property of ADR. Scale bar: 10 µm. (E) Compared with mock-treated cells, ADR and MPA significantly increased the binding between nucleostemin and MDM2. ActD had a lesser effect. (F) Proteins were extracted from cells coexpressing HA-tagged nucleostemin, Myc-tagged MDM2 and Flag-tagged L5, L11 or L23, and immunoprecipitated by anti-Myc antibody. Western blots showing that L23 competes with nucleostemin for MDM2 binding better than L5 and L11.
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