The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity
2006; Springer Nature; Volume: 26; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601465
ISSN1460-2075
AutoresMasha V. Poyurovsky, Christina Priest, Alex Kentsis, Katherine L. B. Borden, Zhen‐Qiang Pan, Nikola P. Pavletich, Carol Prives,
Tópico(s)RNA modifications and cancer
ResumoArticle14 December 2006free access The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity Masha V Poyurovsky Masha V Poyurovsky Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Christina Priest Christina Priest Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Alex Kentsis Alex Kentsis Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Katherine LB Borden Katherine LB Borden Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Zhen-Qiang Pan Zhen-Qiang Pan Department of Oncological Sciences, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Nikola Pavletich Nikola Pavletich Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Carol Prives Corresponding Author Carol Prives Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Masha V Poyurovsky Masha V Poyurovsky Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Christina Priest Christina Priest Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Alex Kentsis Alex Kentsis Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Katherine LB Borden Katherine LB Borden Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Zhen-Qiang Pan Zhen-Qiang Pan Department of Oncological Sciences, Mount Sinai School of Medicine, New York University, New York, NY, USA Search for more papers by this author Nikola Pavletich Nikola Pavletich Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Carol Prives Corresponding Author Carol Prives Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Author Information Masha V Poyurovsky1, Christina Priest1, Alex Kentsis2, Katherine LB Borden2, Zhen-Qiang Pan3, Nikola Pavletich4 and Carol Prives 1 1Department of Biological Sciences, Columbia University, New York, NY, USA 2Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, NY, USA 3Department of Oncological Sciences, Mount Sinai School of Medicine, New York University, New York, NY, USA 4Memorial Sloan-Kettering Cancer Center, New York, NY, USA *Corresponding author. Department of Biological Sciences, Columbia University, 816 Fairchild Building, New York, NY 10027, USA. Tel.: +1 212 854 2557; Fax: +1 212 865 8246; E-mail: [email protected] The EMBO Journal (2007)26:90-101https://doi.org/10.1038/sj.emboj.7601465 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein–protein interactions with the E2. Introduction Human Mdm2 is a highly conserved homologue of the product of the murine double minute (mdm2) gene. A multidomain nuclear phosphoprotein whose principal function in normal tissue is inhibition of the p53 tumor suppressor protein (Prives, 1998; Harris and Levine, 2005; Poyurovsky and Prives, 2006), Mdm2 controls p53 through two distinct mechanisms, by directly binding and masking the N-terminal transactivation domain of p53 (Momand et al, 1992) and by promoting proteasomal degradation of p53 (Haupt et al, 1997; Kubbutat et al, 1997). Mdm2 is a RING-type E3 ubiquitin ligase that primarily regulates its own levels and the levels of p53 in cells through proteasome-mediated degradation (Fang et al, 2000; Honda and Yasuda, 2000). In response to various stress signals, the Mdm2–p53 interaction is rapidly uncoupled through multiple mechanisms, including phosphorylation, acetylation, redistribution of protein complexes and changes in subcellular localization (Prives and Hall, 1999; Moll and Petrenko, 2003). RING-type E3s constitute the largest number of the RING-containing proteins. Approximately 50 of the 200 known RING proteins can be found in discrete subcellular structures that are visible by confocal microscopy (Saurin et al, 1996). The integrity of these structures depends on proper RING domain organization. For example, the RING domain of PML is required for the formation of PML nuclear bodies (Melnick and Licht, 1999). These observations, along with the fact that several RING domain proteins oligomerize in solution, have led to the hypothesis that RING domains serve as scaffolds for forming multi-protein complexes (Bailin et al, 1999; Borden, 2000; Brzovic et al, 2003). This view is supported by the fact that several unrelated RING domains self-assemble in vitro into supramolecular structures capable of scaffolding multiple interacting proteins on their surface and amplifying the specific activities of biochemical reactions (Kentsis et al, 2002a, 2002b). The Mdm2 RING domain is atypical when compared to canonical RING motifs both with respect to the spacing of Zn2+ coordinating cysteines and its multiple functions in cells (Boddy et al, 1994). First, the Mdm2 RING is essential for proper E3 ubiquitin ligase activity and mutation of any of the Zn2+ coordinating residues renders the protein inactive (Lai et al, 1998; Fang et al, 2000). Second, the Mdm2 RING domain was shown to bind to 5S RNA (Elenbaas et al, 1996). Third, this region of Mdm2 contains a cryptic nucleolar localization signal revealed upon protein interactions with p14ARF (Lohrum et al, 2000). Fourth, we found that the Mdm2 RING domain binds nucleotides with a strong preference for ATP and although such binding does not contribute to its E3 ubiquitin ligase, it is important for sub-nuclear translocation of Mdm2 from the nucleoplasm to the nucleolus (Poyurovsky et al, 2003). Finally, the lysine residues within the RING domain of Mdm2 have been shown to be substrates for CBP/p300-mediated acetylation leading to inhibition of the ubiquitin ligase activity (Wang et al, 2004). Collectively, these data suggest multiple important functions and modes of regulation of the RING domain of Mdm2, although it is unclear how these activities are carried out by a single domain. Here we demonstrate that the purified RING domain of Mdm2 can exist as two species in solution: as a monomer and as a spontaneously assembling structured supramolecular complex. Both species are capable of ATP binding and E3 ubiquitin ligase activity. Remarkably, the C-terminus of Mdm2 is required both for assembly and E3 activity, while being dispensable for nucleotide binding. We propose that intramolecular interactions made by the extreme C-terminus stabilize the RING structure, thereby providing a binding platform for E2. These data elucidate a previously undescribed aspect of the Mdm2 RING domain. Results Purification and behavior of Mdm2 RING domain constructs We set out to purify and characterize the Mdm2 RING domain (residues 400–491). The purification scheme for the fusion protein as well as the untagged Mdm2 RING domain is outlined in Figure 1A and B. While yields of the fusion protein were quite large (20 mg/l in Escherichia coli, 100 mg/l from insect cells) and displayed minimal protease sensitivity, indicative of being well folded, analytical gel filtration revealed that this construct displayed polydispersity both as a fusion protein and upon cleavage from the GST fusion partner. A proportion of the GST-RING protein showed clear monodispersity in solution, however, and could be purified away from the polydisperse fraction through two consecutive gel filtration steps (Figure 1C). Furthermore, the behavior of the fusion protein during purification was virtually indistinguishable from the cleaved Mdm2 construct. Protein fractions from the second gel filtration step were 99% pure, as determined by SDS–PAGE (Figure 1D). Figure 1.Purification of the RING domain of Mdm2. (A, B) Purification of Mdm2 protein. Flow-chart representations of the purification schemes for the RING domain of Mdm2 with (B) and without (A) a GST tag. (C) Mdm2 can be purified to homogeneity by size-exclusion chromatography. Mdm2 RING domain sample was prepared as outlined in (A). Upper panel: Superdex-200 gel-filtration absorbance profiles of Mdm2400−491 RING (#I); lower panel: peak fractions of around 16 ml were collected and subjected to a second round of gel filtration (#II). (D) Purified Mdm2 RING domain is a single species. Fractions eluted from the second Superdex-200 gel-filtration column (#II) were subjected to 12% SDS–PAGE gel and stained with Coomassie blue. (E) Analytical gel-filtration and static light-scattering analysis of the Mdm2 RING monomer fraction. The GST-Mdm2 peak fractions from the second gel filtration (#II) were injected onto an analytical gel-filtration column at 200 μM concentration and the effluent was monitored by refractive index (bottom trace, arrow) and 90° static light-scattering (top trace) detectors. Calculations from the Debye plot estimate a molecular mass of 36.1 kDa for the protein peak of the elution profile. Download figure Download PowerPoint The peak fractions of the monodisperse form of the GST-Mdm2 RING domain were analyzed by static light-scattering immediately after purification and following storage at −80°C and a thaw cycle (Figure 1E). The mass-proportional refractive index trace showed that more than 95% of the protein migrated as a single peak, indicating the presence of only one species in solution. The predicted molecular weight of the fusion protein is 36 kDa and light-scattering measurements estimated the molecular weight of the protein as ∼38 kDa, suggesting that the predominant protein species in this, peak, Mdm2 was a monomer. It is difficult to estimate the exact size and stoichiometry of the large molecular weight RING fractions owing to the heterogeneity of this material and the limitations of the gel-filtration column resolution. Mdm2 forms higher order structured oligomers in solution Several RING domain proteins, including PML, BRCA1, BARD1, KAP-1 and arenaviral protein Z were shown to self-assemble into approximately 50 nm spherical structures in vitro, termed 'bodies', dependent on Zn2+ coordination (Kentsis et al, 2002a, 2002b). The ability of Mdm2 to form bodies in vitro was assessed by negative staining electron microscopy (EM). Both the Mdm2 RING domain (Figure 2A, left panel) and the GST-Mdm2 RING (Figure 2A, middle panel) formed spherical structures similar in size to those previously described for arenaviral protein Z (Z) and BRCA1 RINGs. The mean diameter of the observed structures was about 50 nm; the slight physical heterogeneity is likely due to the differential grid adsorption or the potential influence of the fusion partner on the appearance of the body. Observed heterogeneity could also be attributed to the low (femtomolar) protein concentrations required for single-particle EM measurements that are well below the Kd calculated for association of Z bodies (Kentsis et al, 2002b). As expected, and consistent with previous results, the bodies were only observed only in the higher molecular weight fractions of Mdm2, whereas none were associated with the monomer (Figure 2A, right panel). Comparison of the elution profile of the oligomerized material with that of gel filtration standards allowed us to estimate a molecular weight range associated with this peak. Mdm2 RING oligomers eluted as a broad peak from 100 to 600 kDa. Taking into account the migration profile of the monomer, we estimate that the elution range of the body fractions could be produced by complexes of 2–16 subunits (Figure 2B). Previous studies on whether RING supramolecular assemblies are biochemically functional entities have revealed that the BRCA1/BARD1 hetero-oligomeric assembly is not only able to support ubiquitin conjugation and polymerization, but is also substantially more active than the unassembled heterodimer (Kentsis et al, 2002a). Identification of supramolecular assembly mediated by the RING domain of Mdm2 posed interesting questions, such as which regions of the RING domain are involved in the interaction and is there functional significance of these higher order Mdm2 oligomers. Figure 2.Supramolecular assembly of the Mdm2 RING domain. (A) The RING domain of Mdm2 self-assembles into spherical bodies. Peak fractions of the void and monomeric forms of Mdm2 RING and GST-Mdm2 RING protein, respectively, were subjected to single-particle EM at a nominal magnification of × 100 000 or × 80 000, using uranyl acetate counterstain. (B) Mdm2 gel-filtration profile juxtaposed with elution of sizing standards. Right panel: Superdex-200 gel-filtration elution profiles of gel filtration standards; left panel: Mdm2 RING domain sample from Figure 1C. Download figure Download PowerPoint The C-terminal five amino acids of Mdm2 are required for body assembly To identify the regions of the RING necessary for formation of the observed higher order oligomers, we focused on the hydrophobic C-terminus of Mdm2, as such regions are frequently implicated in protein–protein interactions. We deleted the last five or seven residues of the RING domain to generate two new recombinant proteins GST-Mdm2 (400–486) and GST-Mdm2 (400–484). The fusion proteins were then subjected to analytical gel-filtration chromatography to determine their oligomeric state (Figure 3A and B). Unexpectedly, deletion of as few as five amino acids from the C-terminus of Mdm2 led to a striking change in the gel-filtration profile of the protein. Specifically, both proteins exhibited only one elution peak consistent in size with the Mdm2 RING monomer, and the oligomeric peak was virtually absent from the preparations. The solution behavior of the Mdm2-ΔC7 RING domain remained unchanged following removal of the fusion protein, and the resulting elution profile was indistinguishable from that of the monomer fraction of the Mdm2 RING (Figure 3C and D). These data indicate that the extreme C-terminus of Mdm2 is directly involved in Mdm2 body assembly. Figure 3.Deletion of C-terminal residues alters the behavior of the Mdm2 RING domain in solution. (A) Deletion of amino acids 485–491 and 487–491 prevents body formation. SD-200 gel-filtration profile of GST-Mdm2 (400–484) (left panel) and GST-Mdm2 (400–486) (right panel). (B) GST-ΔC7-Mdm2-RING and GST-ΔC5-Mdm2-RING elute as single species on gel filtration. Identical peak fractions from GST-ΔC7-Mdm2-RING elution (top panel) and GST-ΔC5-Mdm2-RING (lower panel) were resolved on 12% SDS–PAGE and visualized by Coomassie blue staining. (C) Removal of GST fusion protein did not alter the solution behavior of ΔC7-Mdm2-RING domain. Gel-filtration profile of the ΔC7-Mdm2-RING domain prepared according to the procedure in Figure 1A. (D) Mdm2 RING and ΔC7-Mdm2-RING elute in the same volume from an SD-200 column. Peak fractions from ΔC7-Mdm2-RING elution (top panel) and Mdm2 RING (lower panel) were resolved on 12% SDS–PAGE and visualized by Coomassie blue staining. (E) Wild-type and C-terminally deleted forms of Mdm2 RING bind similar amounts of Zn2+. Inductively coupled plasma-mass spectromety (ICP-MS) metal analysis of the void, monomer and Mdm2-ΔC7 RING fractions as well as purification buffer was performed under standard analytical conditions. The concentration of the protein preparations were ∼5 μM in a volume of 2 ml each in buffer containing 50 mM bis-tris-propane (BTP), pH 6.8, 350 mM NaCl and 1 mM DTT. (F) C-terminally deleted Mdm2 and Mdm2 bodies retain nucleotide binding ability. Monomeric and oligomeric forms of wild-type GST-Mdm2400−491 prepared as in Figure 1B and Mdm2-ΔC7 protein at indicated amounts were incubated with ATP-γ32P for 10 min at 30°C, filtered through nitrocellulose and washed extensively with 25 mM Hepes buffer. ATP-bound proteins were quantified using a scintillation counter. Download figure Download PowerPoint Metal and ATP binding analysis of C-terminally deleted Mdm2 RING domains To further study the anomalous behavior of the intact RING domain construct, we used inductively coupled plasma-mass spectrometry (ICP-MS) to determine the metal content of the void and monomer fractions of Mdm2 and Mdm2 RING with the last seven residues deleted (Mdm2-ΔC7) (Figure 3E). Metal analysis confirmed the presence of stoichiometric amounts of Zn2+ in all protein preparations, demonstrating that these proteins are capable of zinc coordination and that the anomalous gel-filtration profiles of our constructs are due to other determinants. The metal content was also roughly equivalent between the GST fusion proteins and those purified away from GST, demonstrating that zinc was specifically associated with the RING domain. We also compared the nucleotide binding activity of the monomeric and oligomerized fractions of Mdm2, as well as the C-terminally deleted RING domain. All three Mdm2 preparations bound ATP to a similar extent (Figure 3F), and nucleotide binding by these preparations was lost following heat and guanidinium denaturation (data not shown). Thus, proper folding is required for nucleotide binding and these three forms of Mdm2 are likely to possess functional tertiary structure. Ubiquitin ligase activity of different Mdm2 species The ability of the Mdm2 body fraction to function as an E3 ubiquitin ligase was examined, as well as whether forced monomerization via the deletion of the C-terminus would have any effect on Mdm2 function. To this end, we purified a version of His-tagged ubiquitin with a PKA kinase site at the N-terminus, which allowed us to label it with 32P, along with His-UbcH5c, the E2 shown to function with Mdm2 in vivo (Tan et al, 1999). The relative activities of body and monomer versions of the Mdm2 RING domain were then analyzed by performing ubiquitination assays in vitro, followed by resolution of the reaction products by SDS–PAGE and their visualization by autoradiography (Figure 4A). Figure 4.Mdm2 monomers, oligomers and C-terminally deleted Mdm2 display differential ubiquitin ligase activities. (A) Mdm2 RING bodies and monomers exhibit similar E3 activity at saturating amounts of E2 protein. Ubiquitin ligation assays were performed as indicated in Materials and methods. Reaction mixtures contained Mdm2 monomer and body (1.5 μg) per reaction, ∼300 pmol 32P-labeled ubiquitin and a 50-fold concentration range of the E2, as indicated. Following incubation at 37°C, reactions were terminated and resolved by 8% SDS–PAGE, followed by autoradiography. (B) Mdm2 bodies exhibit lower dependence on E2 in ubiquitination reactions in vitro. Reactions were performed as in (A) in the presence of 7, 15, 20 and 40 ng purified UbcH5c protein. Reactions were resolved by 8% SDS–PAGE and visualized by autoradiography. (C) Deletion of the RING C-terminus abrogates Mdm2 E3 activity. The ubiquitin ligation assay was performed as in (A). Mixtures containing UbcH5c protein (500 ng) ∼300 pmol of 32P-labeled ubiquitin and purified Mdm2 monomer, body and Mdm2-ΔC7 proteins (1, 3, 5 and 7 μg) were incubated as in (A) and then resolved by 8% SDS–PAGE and visualized by autoradiography. (D) Mdm2 RING species used in the ubiquitin polymerization reactions. A Coomassie blue-stained 12% SDS–PAGE gel was used to visualize 1, 3, 5 and 7 μg of Mdm2 monomer, oligomer and Mdm2-ΔC7 RING proteins. Download figure Download PowerPoint Both monomeric and body fractions of Mdm2 RING domain showed activity in the polymerization assay over a broad range of E2 concentrations. We observed that the monomer was relatively more active over a range of E2 protein between 50 and 500 ng. However, monomers showed no detectable activity at 10 ng, whereas the Mdm2 body still exhibited some activity in the presence of only 10 ng of UbcH5c (Figure 4A, lanes 3 and 9; see Supplementary Figure 1 for a longer exposure). Based on this observation, we performed the ubiquitin polymerization experiment in the presence of limiting amounts of UbcH5c. Here, with sub-saturating amounts of UbcH5c, at every point, Mdm2 oligomers showed higher activity (Figure 4B). The ability of the Mdm2 bodies to participate in E2-dependent ubiquitin ligation along with the fact that they coordinate Zn2+ and bind ATP further supports our assumption that we have isolated properly folded yet structurally distinct Mdm2 proteins. Note that at higher saturating E2 concentrations, monomers exhibited somewhat higher activity than the bodies, possibly owing to the heterogeneity of the body fraction (Figures 4B and 6A). All subsequent in vitro ubiquitination reactions were performed in the presence of saturating amounts (500 ng) of UbcH5c. Figure 5.The C-terminus of MdmX can substitute for the Mdm2 C-terminus in ubiquitin polymerization with a conserved phenylalanine residue being essential for this activity. (A) MdmX is not able to catalyze ubiquitin conjugation. Ubiquitin ligation assays were performed as in Figure 4 and described in Materials and methods. Equivalent aliquots of reaction mixtures with UbcH5c protein (500 ng), ∼300 pmol of 32P-labeled ubiquitin and purified Mdm2 monomer, body and MdmX RING domains (at the indicated amounts) were resolved by 8% SDS–PAGE and visualized by autoradiography. (B) The C-terminal residues of the Mdm2 RING domain are necessary but not essential for E3 activity. Mdm2 and MdmX C-terminal swap proteins; Mdm2XC7 (Mdm2-Δ7 fused to the last seven residues of MdmX) and MdmX2C7 (MdmX-ΔC7 fused to the last seven residues of Mdm2) were generated and purified as described in Materials and methods. Ubiquitin ligation assays were as in (A) with the above proteins at the indicated amounts. In lanes 11–14, 3 and 5 μg curves of each protein were used. (C) A phenylalanine residue is the one common residue within the last five amino acids of Mdm2 and MdmX. Graphic representation of the C-terminal swap constructs used in (B) and (C), in which F490 of Mdm2 and F488 are circled as the single conserved residues. (D) Substitution of F490 for Q leads to altered behavior in solution of the Mdm2 RING domain. Comparison of the Superdex-200 elution profiles of wild-type Mdm2 RING domain and F490Q Mdm2 RING domain. (E) MdmX enhances E3 activity of the C-terminal mutant F490Q Mdm2. Ubiquitin ligation assays were performed as indicated in (A) with purified GST-Mdm2 RING (3 and 5 μg), GST-MdmX RING (1, 3 and 5 μg) and either no additional proteins, or Mdm2-F490Q (F490Q; 5 μg) or Mdm2-ΔC7 (ΔC7; 5 μg) proteins. Equivalent aliquots of each mixture were resolved by 8% SDS–PAGE and visualized by autoradiography (top panel). Reactions were quantitated by phosphorimaging and graphed as fold increase in activity over the no E3 control (lower panel). Download figure Download PowerPoint Far less predictable were our results with the C-terminally deleted Mdm2. Remarkably, loss of the last seven residues completely abrogated Mdm2 E3 activity under our conditions (Figure 4C). Thus, deletion of the hydrophobic tail led to the loss of the oligomeric fraction as well as Mdm2 E3 activity. We surmise that when E2 amounts are limiting (a situation likely to arise in the cell when there are many more E3s than E2s), Mdm2 bodies are more active E3 ligases than Mdm2 monomers. Further, the C-terminus plays an important role in the E3 ligase activity of the monomer and the body. The last seven amino acids of MdmX are able to substitute for the Mdm2 C-terminus Having established the requirement for an intact C-terminus in Mdm2 E3 activity, we set out to determine the potential function of this region in ubiquitin polymerization. To this end, we took advantage of the fact that MdmX, a closely related protein, possesses a homologous C-terminal RING domain but is devoid of E3 activity (Marine and Jochemsen, 2005). Initially, we confirmed this observation in our in vitro system with the purified MdmX RING domain and, as expected, did not observe E3 activity from the MdmX RING (Figure 5A). Figure 6.Deletion of the last seven amino acids of Mdm2 or mutation of F490 inhibits autodegradation and p53 degradation in vivo. (A) Mdm2-ΔC7 and F490Q are deficient in self- and p53 degradation in vivo. Increasing amounts (1, 2 and 4 μg) of plasmids encoding Myc-tagged full-length Mdm2, Mdm2-F490Q or Mdm2-ΔC7 were transiently expressed in H1299 cells along with HA-tagged p53 (350 ng) in the presence of a GFP plasmid (150 ng) to normalize transfection efficiency. Cells were harvested 36 h after transfection and soluble proteins were resolved by 10% SDS–PAGE, followed by Western blotting with anti-HA antibody for p53, mixture of Smp14 and 2A10 antibodies for Mdm2 and anti-actin and anti-GFP antibodies for their respective targets. (B) Mdm2-ΔC7 is stable in vivo. Myc-tagged full-length Mdm2 and Mdm2-ΔC7 (4 μg) were transiently coexpressed in H1299 cells in the presence of GFP. At 36 h after transfection, cells were treated with 25 μg/ml cyclohexamide and harvested at the indicated time points. Mdm2, actin and GFP were detected as in (A). (C) Proteasome inhibition has little effect on the levels of Mdm2-ΔC7. Myc-tagged full length Mdm2 and Mdm2-ΔC7 (4 μg) were transiently co-expressed in H1299 cells in the presence of GFP. At 24 h after transfection, cells were treated with 50 μM LLnL and harvested at the indicated time points. Mdm2, actin and GFP were detected as in (A). Note that the upper band (arrow) is the ectopically expressed Myc-Mdm2. Longer exposure of the Mdm2 Western blot allows for the visualization of higher molecular weight forms of the proteins (predicted ubiquitin conjugates) only in lanes with wild-type Mdm2. Download figure Download PowerPoint Primary sequence comparison between the Mdm2 and MdmX C-termini revealed that both contain several hydrophobic residues, although there is only limited similarity between their last five amino acids. We performed a swap experiment where the last seven amino acids of Mdm2 and MdmX RING domains were exchanged (generating Mdm2-XC7 and MdmX-2C7) and the resulting proteins were purified and assayed for the E3 activity (Figure 5B and Supplementary Figure 2). Substitution of the last seven residues of MdmX for those of Mdm2 did not impart ubiquitin ligase function on the MdmX RING domain. Thus, other parts of the Mdm2 RING must also be required for its E3 ligase function and, reciprocally, they must be absent from the MdmX RING domain. Importantly, however, the C-terminal seven amino acids of MdmX were able to substitute for the last seven residues of Mdm2, resulting in a fully functional chimeric protein (Mdm2-XC7) (Figure 5B). By contrast, mixing the full-length MdmX-2C7 and Mdm2-Δ7 RING did not rescue Mdm2-Δ7 RING ligase activity. Further, the MdmX-2C7 RING had no effect on the activity of Mdm2-XC7. Nevertheless, the fact that Mdm2-XC7 is fully functional as an E3 ligase suggests that residues within the C-termini of Mdm2 and MdmX that are essential for the E3 ligase activity of Mdm2 must be conserved between them. As gel-filtration profiles of the Mdm2-ΔC5 and Mdm2-ΔC7 constructs were similarly monomeric, it is likely that one (or more) key determinant(s) of Mdm2 activity must be contained in the last five residues (Figure 3A and B). Phenylalanine is the only common residue within the last five amino acids of the two C-termini (F490 of Mdm2; F488 of MdmX) and so we focused on this residue in subsequent mutational analysis (Figure 5C). Mutation of F490 diminishes oligomerization and E3 activity of Mdm2 We generated an F490Q Mdm2 RING mutant and, following bacterial expression and purification, subjected the resulting protein to gel-filtration chromatography. Consistent with our original observation that Mdm2 body formation is mediated by the C-terminus, the predominant F490Q species detected was the monomer, although some heterogenous large molecular weight material was also present, in contrast to the completely monomeric C-terminally deleted Mdm2 RINGs (Figure 5D). Purified F490Q monomers were as inert as inactive Mdm2-ΔC7 (Supplementary Figure 3). By contrast, F490Q protein before gel filtration displayed a modest amount of E3 activity (Figure 5E, compare lanes 1 and 7). Our observation about the necessity o
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