Abstract
Mdm2 is a member of the RING finger family of ubiquitin ligases and is best known for its role in targeting the tumor suppressor p53 for ubiquitination and degradation. Mdm2 can bind to itself and to the structurally related protein MdmX, and these interactions involve the RING finger domain of Mdm2 and MdmX, respectively. In this study, we performed a mutational analysis of the RING finger domain of Mdm2, and we identified several amino acid residues that are important for Mdm2 to exert its ubiquitin ligase function. Mutation of some of these residues interfered with the Mdm2-Mdm2 interaction indicating that a homomeric complex represents the active form of Mdm2. Mutation of other residues did not detectably affect the ability of Mdm2 to interact with itself but reduced the ability of Mdm2 to interact with UbcH5. Remarkably, MdmX efficiently rescued the ubiquitin ligase activity of the latter Mdm2 mutants in vitro and within cells. Because the interaction of Mdm2 with MdmX is more stable than the Mdm2-Mdm2 interaction, this suggests that Mdm2-MdmX complexes play a prominent role in p53 ubiquitination in vivo. Furthermore, we show that, similar to Mdm2, the Mdm2-MdmX complex has Nedd8 ligase activity and that all mutations that affect the ubiquitin ligase activity of Mdm2 also affect its Nedd8 ligase activity. From a mechanistic perspective, this suggests that the actual function of Mdm2 and Mdm2-MdmX, respectively, in p53 ubiquitination and in p53 neddylation is similar for both processes.
Highlights
RING finger domains and U-box domains adopt similar structural folds providing an interface for the interaction with their cognate E2 ubiquitin-conjugating enzymes (4 –9)
HECT domain E3s have a similar modular structure, but in contrast to RING finger or U-box E3s, HECT domain E3s appear to play a direct catalytic role in the covalent attachment of ubiquitin to substrate proteins (10 –12)
The results obtained indicate that the ability of Mdm2 to form homomeric complexes is required for both its ubiquitin ligase and its Nedd8 ligase activity. We show that both the ubiquitin ligase and the Nedd8 ligase activity of some of the Mdm2 mutants are efficiently rescued by MdmX in vitro and within cells
Summary
Cell Lines and Plasmids—H1299 cells and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum. Bacterial expression constructs for glutathione S-transferase (GST) fusion proteins of wild-type Hdm and HdmX were described previously [29]. The expression constructs used in transient transfection experiments (Fig. 4) encoding wild-type Mdm (pCocMdm2), wildtype p53, His-tagged ubiquitin, and HA-tagged HdmX were described previously [29, 33, 34]. Ubiquitination, Neddylation, and Degradation Assays—For in vitro ubiquitination experiments, wild-type Mdm, Hdm, and HdmX and the various Mdm and Hdm mutants were expressed as GST fusion proteins in Escherichia coli DH5␣. For ubiquitination of p53 within cells, one 6-cm plate of H1299 cells or HEK293T cells was transfected with expression constructs encoding p53 (200 ng), His-tagged ubiquitin (1 g), Mdm or the respective Mdm mutants (200 ng), and HdmX (600 ng). Growth on uracil-deficient plates requires stronger interaction of the respective proteins than growth on adenine-deficient plates, whereas growth on histidine-deficient plates requires the least efficient interaction [37]
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