Figure 10. Functional interaction between HEY1 and RPL11.

(A) Wild-type HEY1, but not the mutant HEY1-S68D, prevents MDM2-mediated p53 degradation. H1229 cells were transfected, where indicated, with expression vectors for p53 (10 ng), MDM2 (1 μg), RPL11 (1 μg), HEY1 (1 μg) or HEY1-S68D (1 μg). Twenty-four hours after transfection cells were harvested for immunoblot analysis with the specified antibodies. (B) RPL11 cooperates with HEY1 to prevent MDM2-mediated p53 degradation. H1229 cells were transfected, where indicated, with expression vectors for p53 (10 ng), MDM2 (1 μg), RPL11 (0.5 μg) or HEY1 (0.5 μg). Twenty-four hours after transfection cells were harvested for immunoblot analysis with the specified antibodies. (C) RPL11 cooperates synergistically with HEY1 to enhance p53 transcriptional activity. U2OS cells were transfected with 100 ng of PIG3-Luc in the presence or absence of 100 ng of expression vector for HEY1, 50 ng of expression vector for RPL11 or both together. After transfection, cells were incubated 24 h. Subsequently cell lysates were assayed using a dual luciferase reporter system. Normalized values are expressed relative to the activity of the reporter in the absence of HEY1. The results shown represent the averages of results of four independent experiments assayed in duplicate + S.D. (D) Simulation of HEY1 phosphorylation at residue S68 inhibits its interaction with MDM2. Whole-cell extracts from U2OS cells previously transfected with expression vector for MDM2 were incubated with GST fusion proteins of HEY1 or HEY1-S68D coupled with Sepharose beads. The associated proteins were detected by western blotting using anti-MDM2 antibody. (E) Simulation of HEY1 phosphorylation at residue S68 inhibits its interaction with RPL11. Whole-cell extracts from U2OS cells previously transfected with expression vector for Flag-tagged RPL11 were incubated with GST fusion proteins of HEY1 or HEY1-S68D coupled with Sepharose beads. The associated proteins were detected by western blotting using anti-Flag antibody. (F) MDM2 decreases the level of ectopically expressed HEY1. U2OS cells were transfected with expression vectors for MDM2 (2 μg) and Flag-tagged HEY1 (2 μg) or HEY1-S68D (2 μg). Twenty-four hours after transfection cells were harvested for immunoblot analysis with the specified antibodies. (G) Effects of Nutlin-3 on MDM2-mediated degradation of HEY1 or p53. H1229 cells were transfected, where indicated, with expression vectors for HEY1 (1 μg), p53 (10 ng) and MDM2 (1 μg). Eighteen hours after transfection cells were washed and incubated with vehicle or 10 μM Nutlin-3 for an additional 24 h. Subsequently, cells were harvested for immunoblot analysis with the specified antibodies.