1. Volume 12, Issue 4, Pages 875-887 (October 2003)
Nucleotide Binding by the MDM2 RING Domain Facilitates Arf-Independent MDM2 Nucleolar Localization 
Masha V. Poyurovsky, Xavier Jacq, Charles Ma, Orit Karni-Schmidt, Peter J. Parker, Martin Chalfie, James L. Manley, Carol Prives 
Molecular Cell 
Volume 12, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
Mdm2 Preferentially Binds Adenine Base-Containing Nucleotides through the P Loop Motive in Its RING Domain
(A) Mdm2 RING domain contains a Walker A motif. Sequence alignment of nucleotide binding motifs in Mdm2 family members. The conserved nucleotide binding motif (Walker A sequence or P loop) is highlighted.
(B) ATP binding by purified Mdm2. Extensively purified full-length Mdm2 bound to SMP14-Sepharose was incubated with 10 nM ATP and 5 μCi ATP-γ32P for 10 min at 30°C and then filtered through nitrocellulose filters prior to counting by liquid scintillation.
(C) Nucleotide specificity of Mdm2. Following incubation of Mdm2 with ATP, increasing concentrations of unlabeled competitor nucleotides as indicated were added to reaction mixtures. The ATP-γ32P-bound fraction was analyzed as in (B). The graph represents bound fraction after incubation with the indicated nucleotides.
(D) Dissociation constants of wild-type and mutant Mdm2 proteins. Nitrocellulose filter binding mixtures containing 10 nM ATP and 5 μCi ATP-γ32P were incubated with GST-Mdm wild-type and mutant (G448S and K454A) proteins in the presence of increasing amounts of unlabeled ATP. KD values were calculated according to the Michaelis-Menten equation for first order kinetics.
(E) The Mdm2 RING finger is sufficient for nucleotide binding. GST or GST-Mdm (GST-RING) (10, 50, and 100 ng) incubated with ATP-γ32P, filtered through nitrocellulose followed by counting by liquid scintillation.
(F) Decreased nucleotide binding by P loop mutant forms of Mdm GST, wild-type Mdm2, and mutant G448S, T454C, K455A, GST-RING proteins (0.25, 0.5, 1.5, 2 μg) were incubated with 10 nM ATP and 5 μCi ATP-γ32P for 10 min at 30°C, filtered through nitrocellulose, and counted by liquid scintillation.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Nucleotide Binding Causes a Conformational Change at the C Terminus of Mdm2
Full-length his-Mdm2 protein (100 ng) was incubated with 0, 1, 5, 25, and 100 ng of trypsin for 15 min at room temperature. Mixtures either lacked nucleotide or contained 5 mM ATP or ADP as indicated. Reactions were terminated by the addition of SDS-PAGE loading buffer and subsequently resolved on an 8% SDS-PAGE gel and subjected to immunoblotting with antibodies to Mdm2 protein. Monoclonals 4B11 and 2A10 recognize the C terminus of Mdm2, and monoclonal 3F8 recognizes a region within the N-terminal portion of Mdm2.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
P Loop Mutations Have Differential Effects on Mdm2 and p53 In Vivo
(A) P loop mutants vary in their stability in transfected cells. U20S cells were transfected with either empty vector (3 μg) or wild-type or mutant Flag-Mdm2-expressing plasmids (1, 2, and 3 μg). Cotransfected GFP (300 ng) and actin were used for transfection and loading controls. Cells were harvested 40 hr after transfection, and 40 μg of total cell extract in each case was used for immunoblot analysis with anti-Flag, anti-actin, and anti-GFP antibodies as indicated.
(B) Proteasome-mediated degradation of wild-type and P loop Mdm2 mutants. H1299 cells transfected as in (A) were subjected to treatment with LLnL (50 μM) 24 hr posttransfection. Cells were harvested at the indicated times after treatment, and 40 μg of total extract was used for immunoblot analysis with antibodies as in (A).
(C) P loop Mdm2 mutants differ in their E3 ubiquitin ligase activity. H1299 cells were cotransfected with constructs expressing HA-ubiquitin (10 μg) and Flag-Mdm2 (24 μg). After 40 hr cell lysates (1 mg) were prepared and immunoprecipitated with an anti-Flag antibody, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-HA antibody 12CA5 (top panel). The membrane was than stripped and reprobed with a mixture of anti-Mdm2 monoclonal antibodies: SMP14 and 2A10 (second panel from top). Prior to the immunoprecipitation aliquots of total cell extracts (5% input) were taken, and the relative amounts of input Mdm2 were determined by immunoblotting with anti-Flag antibody (top panel marked Input Western).
(D) P loop mutants are differentially able to degrade p53. U20S cells were cotransfected with either empty vector (3 μg) or wild-type or mutant Flag-Mdm2-expressing plasmids (1, 2, and 3 μg) and HA-p53 (350 ng). Cotransfected GFP (300 ng) was used to control for transfection efficiency. Cells were harvested 40 hr after transfection, and 40 μg of cell extract was used for immunoblotting with anti-Flag, anti-HA, anti-GFP, and anti-p21 antibodies as indicated. Numerical representation of the relative protein levels was obtained by densitometry analysis of the immunoblot where initial level of p53 was taken as 1, and the amount of wild-type Mdm2 in the 3 μg DNA transfection was taken as 1.
(E) Quantitation of the relative p53 downregulation by Mdm2 and the P loop mutants. Scion Image program was used to quantitate relative amounts of Mdm2 and p53 on the immunoblot. The graph is representative of [(initial p53 level/p53 in the presence of Mdm2)/amount of Mdm2 in each transfection], fold change in p53 per amount of Mdm2.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
The RING Domain of Mdm2 Alters the Stability of GFP in C. elegans
(A) The RING domain of Mdm2 reduces the number of cells expressing GFP from the unc-4 promoter. Animals expressing GFP, the wild-type Mdm2 RING domain fused to GFP, and the K454A Mdm2 RING domain fused to GFP were synchronized by bleaching gravid adults and observing the remaining newly hatched larvae. The number of GFP-expressing cells (regardless of intensity) was counted at the indicated times (n = 8). Each numbered line represents data from a different stable line. Note that the number of cells expressing GFP alone increases as animals age. The number of cells decreases when the RING domain is included.
(B) Examples of 48 hr posthatching adults. Adults expressing GFP alone (top panels), the wild-type Mdm2 RING domain fused to GFP (middle panels), and the K454A Mdm2 RING domain fused to GFP (bottom panels).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
P Loop Mutants Are Defective in p14ARF-Independent Nucleolar Localization
(A) Actinomycin D mediated nucleolar localization of Mdm2. H1299 cells were transfected with constructs expressing wild-type Flag-Mdm2 or the P loop mutants (3 μg). Twenty-four hours after transfection cells were treated with 50 nM ActD, and 18 hr after treatment cells were fixed, and Mdm2 and B23 proteins were visualized by immunofluorescence using M2 (anti-Flag antibody) and a polyclonal goat anti-B23 antibody. B23 was visualized using Alexa Fluor 488-conjugated secondary antibody (green), and Mdm2 protein was visualized using Alexa Fluor 594-conjugated secondary antibody (red). Nuclei of cells were visualized by DAPI staining.
(B) Nucleolar localization of wild-type and P loop mutant forms of Mdm2. Graphic representation of the fold change in nucleolar localization of exogenous Mdm2 protein after actinomycin D treatment. Total number of transfected cell counted was ∼1000 for each data point. Three independent transfection experiments were performed.
(C) The RING domain of Mdm2 alters the subcellular localization in the C. elegans hypodermis. Cells expressed GFP (top panel), the wild-type Mdm2 RING domain fused to GFP (middle panel), and the K454A Mdm2 RING domain fused to GFP (bottom panel).
(D) p14ARF facilitates nucleolar localization of Mdm2. U2OS cells transfected with Myc-p14ARF and Flag-Mdm2 expressing constructs as in (A) were fixed and stained with anti-Flag or anti-Myc antibodies and visualized using an Alexa Fluor 488-conjugated secondary antibody (green). The nuclei and nucleoli of cells were visualized by phase-contrast microscopy.
(E) Myc-p14ARF nucleolar localization of Mdm2 is independent of nucleotide binding. Cells were transfected with constructs expressing wild-type or mutant forms of Flag-Mdm2 (3 μg) with or without Myc-p14ARF (1 μg) as indicated in (A) and (B). The graph represents the amount of cells expressing Flag-Mdm2 proteins exhibiting nucleolar localization. For each experiment at least 300 transfected cells were counted; the graph is representative of two independent experiments.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
P Loop Mutants Are Defective in Nucleolar Localization In Vitro
(A) Nucleolar localization of Mdm2 RING domain in vitro. GST-Mdm as well as the RING domain mutants (4 μg) or GST alone (4 μg) were added to Triton X-100 buffer to permeabilize U2OS cells, and after 1 min cells were fixed and stained as described in the Experimental Procedures. Permeabilized cells were costained with anti-GST antibody to ensure equivalent reactivity (visualized using Alexa Fluor 488-conjugated secondary antibody [green]) and anti-B23 protein (visualized using Alexa Fluor 594-conjugated secondary antibody [red]). Nuclei of cells were visualized by DAPI staining.
(B) Graphic representation of nucleolar localization of GST-Mdm and P loop mutants in vitro. The graph represents three independent experiments where at least 300 cells were counted for each slide.
(C) Wild-type and mutant GST-RING Mdm2 proteins. GST-Mdm and the indicated mutants (3, 6, and 9 ng) were resolved by SDS-PAGE and then immunoblotted with anti-GST antibody.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 ATP-Bound Mdm2 Localizes to the Nucleolus
(A) Specific labeling of Mdm2 protein with FSBA. Mdm2 or BSA (100 ng) was incubated with FSBA (1 mM) in the presence or absence of ATP (1 mM) as indicated and described in the Experimental Procedures. Mixtures were incubated at 30°C for 10 or 40 min. The mixtures were resolved by SDS-PAGE, and FSBA labeling was determined by immunoblotting with anti-FSBA and anti-Mdm2 antibodies.
(B) FSBA labeled Mdm2 localizes to the nucleolus in vitro. U2OS cells were incubated with the indicated proteins and then fixed as in Figure 6, followed by staining with anti-GST or anti-FSBA antibodies. Nucleoli of the cells were visualized by phase-contrast microscopy. GST and FSBA antibodies were detected by Alexa Fluor 488-conjugated anti-mouse secondary antibody.
(C) Nucleotide-bound Mdm2 preferentially localizes to the nucleolus. Wild-type GST-RING Mdm2 protein either incubated or not with FSBA was added in permeabilization buffer to U2OS cells as in (B). Cells were fixed and stained with either anti-GST (left two bars) or anti-FSBA (right bar), and then the proportion of cells with nucleolar staining in each case was quantitated. The graph represents two independent experiments in which 500 to 1000 cells were counted per data point.
Molecular Cell  , DOI: ( /S (03) )