Nucleotide Binding by the MDM2 RING Domain Facilitates Arf-Independent MDM2 Nucleolar Localization

Molecular Cell, Vol. 12, 875–887, October, 2003, Copyright 2003 by Cell Press

Nucleotide Binding by the MDM2 RING Domain Facilitates Arf-Independent MDM2 Nucleolar Localization Masha V. Poyurovsky,1,3 Xavier Jacq,1,3,4 Charles Ma,1 Orit Karni-Schmidt,1 Peter J. Parker,2 Martin Chalfie,1 James L. Manley,1 and Carol Prives1,* 1 Department of Biological Sciences Columbia University New York, New York 10027 2 Protein Phosphorylation Laboratory Cancer Research UK London Research Institute Lincoln’s Inn Fields Laboratories 44 Lincoln’s Inn Fields London WC2A 3PX United Kindgom

Summary The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14ARF-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.

or Mdm2, or through the p14ARF protein (the alternative product of the INK4a locus) (Prives and Hall, 1999). To date, about 200 proteins have been identified with RING domains (Borden, 2000). A subgroup of these proteins (e.g., BRCA1, Cbl, TRAF2, and Mdm2) possesses E3 ubiquitin ligase activity, which is responsible for target specificity of the ubiquitination machinery. Mdm2 has a Cis3-His-Cis4 RING domain in which two molecules of zinc are coordinated by four pairs of amino acids in a “crossbrace” configuration (Lai et al., 1998). Mutations in the zinc-coordinating residues diminish the E3 ligase activity of the Mdm2 RING domain, supporting the idea that proper tertiary structure is essential for RING domain function (Fang et al., 2000; Lai et al., 1998). The Mdm2 RING domain has other described functions in addition to E3 ubiquitin ligase activity. Elenbaas et al. reported that the Mdm2 RING domain binds RNA, with residue G448 being required for this activity (Elenbaas et al., 1996). A cryptic nucleolar localization signal was identified within the RING domain (residues 466– 473), which is thought to be exposed as a consequence of binding between Mdm2 and p14ARF, and which is essential for Mdm2 nucleolar localization (Lohrum et al., 2000). Mdm2 has a nonclassical RING domain due to a longer stretch of amino acids (8 residues, compared to the canonical 2–3 residues) between the fourth (H452) and fifth (C461) ligand-coordinating residues (Fang et al., 2000; Lai et al., 1998). Here we identify and characterize a nucleotide binding motif located within these residues and present evidence that this motif plays a role in p14ARF-independent nucleolar localization of Mdm2. Our data indicate that nucleotide binding serves to negatively regulate the E3 ubiquitin ligase activity of Mdm2 by facilitating its redistribution within nuclear subcompartments. Results

Introduction The murine and human double minute (mdm2) genes encode highly conserved multidomain nuclear phosphoproteins that play essential roles in negatively regulating the p53 tumor suppressor protein (Prives, 1998). Mdm2 controls p53 through inhibition of p53 transcriptional activity direct binding of the N-terminal transactivation domain of p53 (Prives, 1998) and promotion of p53 degradation (Haupt et al., 1997; Kubbutat et al., 1997). Mdm2 is a RING domain-containing E3 ubiquitin ligase that actively downregulates its own levels as well as levels of p53 in cells through proteasome-mediated degradation (Fang et al., 2000; Honda and Yasuda, 2000). Mdm2 activity toward p53 can be negatively regulated by at least two mechanisms: phosphorylation of either p53 *Correspondence: [email protected] 3 These authors contributed equally to this work 4 Present address: Hybrigenics, SA 3/5 Impasse Reille, 75014 Paris, France.

Identification of ATP Binding Motif in the RING Domain of Mdm2 The Mdm2 RING domain contains a consensus Walker A or P loop motif, a common feature of ATP/GTP binding proteins (Walker et al., 1982). The P loop consensus sequence (GxxxxGK (T/S) in Mdm2 consists of G(448)CIVHGKT(455). It is present in all Mdm2 homologs as well as MdmX, an Mdm2 family member (Figure 1A). Among other known RING E3 ubiquitin ligase proteins, the Mdm2 RING domain is unique in possessing this motif. To determine whether Mdm2 can bind nucleotide(s), full-length His-tagged human Mdm2 expressed in E. coli was purified (see Supplemental Figure S1A at http:// www.molecule.org/cgi/content/full/12/4/875/DC1; lane marked Input Mdm2). Sepharose beads containing the Mdm2-specific monoclonal antibody SMP14 with or without purified Mdm2 were tested for nucleotide binding. Beads containing Mdm2 but not controls were shown to bind ␥-32P ATP (Figure 1B). Mdm2 was unable

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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 ␮Ci ATP-␥32P 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-␥32P-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 ␮Ci ATP-␥32P were incubated with GST-Mdm2410-491 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-Mdm2410-491 (GST-RING) (10, 50, and 100 ng) incubated with ATP␥32P, filtered through nitrocellulose followed by counting by liquid scintillation. (F) Decreased nucleotide binding by P loop mutant forms of Mdm2410-491. GST, wild-type Mdm2, and mutant G448S, T454C, K455A, GST-RING proteins (0.25, 0.5, 1.5, 2 ␮g) were incubated with 10 nM ATP and 5 ␮Ci ATP-␥32P for 10 min at 30⬚C, filtered through nitrocellulose, and counted by liquid scintillation.

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to hydrolyze ATP (data not shown). Binding to ␥-32P ATP was examined in the presence of various unlabeled nucleotide competitors (Figure 1C). Only adenine basecontaining nucleotides competed efficiently with labeled ATP for binding to Mdm2. This rules out nonspecific charge effects imparted by the phosphate group of the nucleotide. To determine whether the Mdm2 RING domain mediates ATP binding and whether the P loop is required, we made single amino acid substitutions in 3 key P loop residues (G448S, K454A, and T455C) in a GST-tagged RING domain construct (GST-RING410-491). Wild-type GSTRING domain bound both ␥-32P ATP (Figure 1E) and ␣-32P ATP (Supplemental Figure S1B), ruling out the possibility that binding reflected activity of a contaminating kinase. Notably, all of the mutant proteins exhibited lower affinity for ATP than did their wild-type counterpart over a 10fold concentration range (Figure 1F and Supplemental Figure S1B). Point mutations in the Walker A motif increased the KD for ATP by about 10-fold (13.5 ␮M for the wild-type protein to 150 ␮M and 177 ␮M for G448S and K445A, respectively; Figure 1D). These results establish the specificity of the Mdm2-ATP interaction and demonstrate the requirement of an intact P loop for optimal nucleotide binding. ATP Binding Causes a Conformational Change in the Mdm2 C Terminus We tested whether a conformational change could be detected in Mdm2 upon ATP binding as evidenced by altered susceptibility to brief trypsin proteolysis (Figure 2). Two C-terminal-specific Mdm2 antibodies, 4B11 and 2A10, revealed marked effects of ATP on partial digestion of Mdm2 by trypsin (Figure 2, top two panels). ADP caused somewhat greater susceptibility to digestion, as observed by increased reduction in the full-length input protein. By contrast, no significant change in the tryptic digestion pattern was observed when the products of the digest were visualized with the N-terminal antibody 3F8 (Figure 2, bottom panel). We postulate that ATP/ ADP binding imparts a conformational change on Mdm2 that is specific to the C-terminal portion of the protein. P Loop Mutants Exhibit Differential Stability and E3 Activity In Vivo To investigate in vivo functions of ATP binding by Mdm2, Flag-tagged versions of wild-type and P loop mutant forms of full-length Mdm2 were introduced into human tumor-derived cell lines (Figure 3). The T455C and G448S mutant proteins accumulated to far higher levels in U2OS cells than did the wild-type protein (Figure 3A). By contrast, the K454A mutant accumulated to slightly lower levels than wild-type Mdm2 (Figure 3A). Similar results were obtained in H1299, 293, HCT116, and LNCaP cells (data not shown). When H1299 cells were treated with the proteasome inhibitor LLnL, all Mdm2 variants accumulated to similar levels (Figure 3B). Furthermore, time course experiments using cycloheximide to block new protein synthesis showed that T455C and G448S have an increased half-life as opposed to K454A, which exhibited an increase in turnover compared to wild-type Mdm2 (data not shown). Thus, variation in the protein

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.

stability of these Mdm2 variants explains the differences in their steady-state levels in cells. To test the ubiquitin ligase activity of wild-type and P loop mutant forms of Mdm2, HA-tagged ubiquitin and Flag-tagged Mdm2 variants were expressed in H1299 cells. Ubiquitin conjugates were visualized by probing with an anti-HA antibody (Figure 3C). As expected, the two Mdm2 variants with elevated protein levels (T455C and G448S) were substantially underubiquitinated compared to wild-type Mdm2, while the K454A mutant showed levels of ubiquitination similar to that of wildtype Mdm2.

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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 ␮g) or wild-type or mutant Flag-Mdm2-expressing plasmids (1, 2, and 3 ␮g). Cotransfected GFP (300 ng) and actin were used for transfection and loading controls. Cells were harvested 40 hr after transfection, and 40 ␮g of total cell extract in each case was used for immunoblot analysis with anti-Flag, antiactin, 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 ␮M) 24 hr posttransfection. Cells were harvested at the indicated times after treatment, and 40 ␮g 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 ␮g) and Flag-Mdm2 (24 ␮g). 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 ␮g) or wild-type or mutant

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The ability of the mutant Mdm2 proteins to target p53 for degradation was examined. In line with their relative E3 activities, T455C and G448S mutants were unable to decrease p53 protein levels. Conversely, wild-type Mdm2 and the K454A mutant markedly decreased the level of cotransfected p53 (Figures 3D and 3E). In fact, on a per mole basis the K454A Mdm2 mutant displayed significantly greater ability (approximately 3-fold more activity) to reduce p53 levels than did wild-type Mdm2 (Figure 3E). Interestingly, all three mutants were capable of repressing p53 transcriptional activity, as evidenced by the decrease in the levels of p21 protein (Figure 3D). Mutation of P loop residues did not affect the ability of Mdm2 to interact with p53 (data not shown). The lack of a consistent phenotype between the three mutants indicates that ATP binding is not directly involved in modulating the ubiquitin ligase activity of Mdm2. Wild-Type and K454A Mutant Mdm2 RING Domains Vary in Stability in C. elegans We next tested the ability of wild-type Mdm2 RING domain to influence protein stability in a living organism, the worm, C. elegans. Utilizing green fluorescent protein (GFP), this system allowed visualization of protein accumulation in vivo and simultaneously permitted us to determine whether Mdm2 RING domain function is evolutionarily conserved. To this end, we examined Mdm2 RING domain-GFP fusion proteins expressed from the unc-4 promoter. The unc-4 gene is expressed in several ventral cord motor neurons: the DA cells in the embryo, the VA cells in first and second larval stages (L1 and L2), and the VC cells in L4 larvae and young adults. The number of ventral cord motor neurons expressing detectable GFP from the unc-4 promoter increases as animals develop, with adults having many more fluorescent cells than accounted for by the VC cells (Figure 4). The prolonged fluorescence could be due to GFP stability or continued gene expression. However, the number of GFP-expressing cells in older animals is reduced when the minimal RING domain of wild-type Mdm2 is fused to GFP (Figure 4). This result suggests that increased degradation of GFP has occurred and indicates that the expression of GFP alone in adults is a consequence of its stability. Importantly, in agreement with our results in mammalian cells, the K454A mutant domain reduced accumulation of GFP even further (Figure 4). These results support the likelihood that the K454A mutant is more active as an E3 ubiquitin ligase than the wild-type Mdm2. ATP Binding Mutants Are Defective in p14ARF-Independent Nucleolar Localization in Human Cells and in C. elegans To characterize potential differences in subcellular localization between the wild-type Mdm2 and the P loop

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).

mutants, we examined two stimuli known to cause nucleolar translocation of Mdm2: treatment with actinomycin D (ActD) and coexpression of p14ARF. ActD treatment of U2OS cell, at levels subinhibitory to transcriptional activity, was shown to cause relocalization of Mdm2 to

Flag-Mdm2-expressing plasmids (1, 2, and 3 ␮g) 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 ␮g of cell extract was used for immunoblotting with anti-Flag, anti-HA, antiGFP, 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 ␮g 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.

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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 ␮g). 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 ⵑ1000 for each data point. Three independent transfection experiments were performed.

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the nucleolus in an p14ARF-independent manner (Ashcroft et al., 2000). We performed similar experiments, in our case using H1299 cells (Figure 5). In the absence of ActD, Mdm2 and all of the P loop mutants exhibited a primarily nucleoplasmic staining pattern (Figure 5A, upper panel). After drug treatment, however, Mdm2 colocalized with B23 (nucleophosmin), a nucleolar marker (Finch et al., 1995) (Figure 5A, lower panel). Note that, using these conditions, nucleolar localization of wildtype Mdm2 was seen in only a relatively small subset (15%) of transfected cells. Perhaps the association of Mdm2 with the nucleolus is transient and at a given time only a fraction of the protein may exhibit such localization. Due to the fact that some nucleolar staining could be observed in a few cells even in the absence of drug treatment, results are presented as fold change in nucleolar localization. Remarkably, each of the nucleotide binding-deficient mutant Mdm2 proteins exhibited a significant reduction in its nucleolar localization. These results indicate that nucleotide binding facilitates Mdm2 nucleolar localization. The Mdm2 RING domain also localizes to the nucleolus in C. elegans. Since the ventral motor neurons in adult worms do not have easily distinguished nucleoli, Mdm2-GFP fusion proteins were expressed in the C. elegans hypodermis (the syncytial epidermis) using the promoter of the dpy-7 gene (Gilleard et al., 1997). The hypodermal nuclei are quite large and have prominent nucleoli. When GFP alone was expressed in the hypodermis under these conditions, it was excluded from the nucleolus (Figure 5C, top panels). However, when GFP was fused to the RING domain of wild-type Mdm2, fluorescence was localized to both the nucleoplasm and the nucleolus (Figure 5C, middle panels). Thus, the Mdm2 RING domain can localize to nucleoli of C. elegans hypodermal cells. Strikingly, GFP fused to the Mdm2 (K454A) RING domain localized to the nucleus but was at least partially excluded from the nucleolus (Figure 5B, bottom panels). The above data suggested that nucleotide binding might function to expose the cryptic nucleolar localization signal in Mdm2, similar to the effect of p14ARF binding (Lohrum et al., 2000). We therefore tested the effects of p14ARF on nucleolar localization of wild-type and P loop mutant forms of Mdm2 in U2OS cells (which lack endogenous p14ARF). As previously reported (Weber et al., 1999), ectopically expressed p14ARF was extensively localized to the nucleolus (Figure 5D, top panel). Consistent with a previous report (Xirodimas et al., 2001a), cotransfected p14ARF led to elevated levels of Mdm2 (Supplemental Figure S2). In parallel experiments, p14ARF also stabilized cotransfected p53 (data not shown). When introduced alone, Mdm2 was almost exclusively

nucleoplasmic, while upon coexpression with p14ARF nucleolar localization was observed in a fraction of transfected cells (Figure 5D, lower two panels). Importantly, both wild-type and P loop mutant forms of Mdm2 were localized to the nucleolus to similar extents (graph in Figure 5E). These data suggest that p14ARF binding and nucleotide binding are functionally independent. P Loop Mutants Are Defective in Nucleolar Localization In Vitro To gain more direct information about the role of nucleotide binding on RING domain-mediated Mdm2 nucleolar localization, we developed an in vitro localization assay (see Experimental Procedures). For this we used U2OS cells to ensure no contribution to the nucleolar localization from endogenous p14ARF. Equivalent amounts of purified wild-type or mutant GST-Mdm2 RING domain proteins were used (Figure 6C). Detergent-mediated cell membrane permeabilization allowed entrance of purified proteins into the cells, which could be detected within 1 min (Figure 6A). Nucleolar localization of GSTMdm2 proteins was confirmed by costaining with antibody against B23. GST- RING Mdm2 localization in vitro was more efficient than in ActD-treated cells in vivo (see above), with 25%–40% of all cells displaying either exclusive nucleolar staining or combined nucleolar and nucleoplasmic staining. In parallel samples, GST alone demonstrated no nucleolar localization (Figures 6A and 6B). A previously characterized RING domain mutant in which the nucleolar localization signal residues have been mutated (Nols-Mdm2; Lohrum et al., 2000) exhibited a strictly nucleoplasmic pattern (Figure 6B). Here too the P loop mutants were all deficient in nucleolar localization by at least a factor of two when compared to wild-type protein (Figure 6B). Collectively, our results imply that nucleotide binding facilitated movement of Mdm2 to the nucleolus requires its nucleolar localization signal yet is p14ARF independent. Nucleolar Mdm2 Is Bound to Nucleotide To extend the above observations, we utilized an ATP analog, fluorylsulfonylbenzoyladenosine (FSBA), which crosslinks specifically to the lysine residues within the P loop of ATP binding proteins (Tohgo et al., 1997). Detection of FSBA-bound Mdm2 was facilitated by the use of an anti-FSBA antibody previously employed to identify proteins crosslinked with FSBA by immunoblotting (Parker, 1993). The anti-FSBA antibody detected FSBA-labeled Mdm2 but not BSA, and excess ATP greatly reduced the amount of bound FSBA (Figure 7A). FSBA and the anti-FSBA antibody allowed us to visualize nucleotide-bound protein in the in vitro localization assay (Figure 7B). Wild-type GST-Mdm2-RING, GST

(C) The RING domain of Mdm2 alters the subcellular localization in the C. elegans hypodermis. Cells expressed GFP (top panel), the wildtype 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 wildtype or mutant forms of Flag-Mdm2 (3 ␮g) with or without Myc-p14ARF (1 ␮g) 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.

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Figure 6. P Loop Mutants Are Defective in Nucleolar Localization In Vitro (A) Nucleolar localization of Mdm2 RING domain in vitro. GST-Mdm2410-491 as well as the RING domain mutants (4 ␮g) or GST alone (4 ␮g) 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 488conjugated 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-Mdm2410-491 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-Mdm2410-491 and the indicated mutants (3, 6, and 9 ng) were resolved by SDS-PAGE and then immunoblotted with anti-GST antibody.

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alone, and Nols-Mdm2-RING were treated with FSBA, and each was added in the permeabilization buffer to U2OS cells and visualized using both anti-GST and antiFSBA antibodies. Anti-GST antibody staining of FSBAtreated Mdm2-RING showed significantly increased nucleolar localization (65%) over that observed with the same antibody in the absence of FSBA (ⵑ40%), suggesting that FSBA binding directly stimulates Mdm2 nucleolar localization (Figure 7C). In fact, saturating amounts of ATP (4–10 mM) also stimulated Mdm2-RING nucleolar localization in vitro (data not shown). Indeed, when identical parallel samples were visualized with FSBA antibody, ⵑ90% of cells showed strong nucleolar staining, and in general there was less nucleoplasmic staining, strongly indicating that nucleotide-bound Mdm2 is preferentially localized to the nucleolus (Figures 7B and 7C). No nucleolar localization of either GST or Nols-Mdm2-RING was observed with anti-GST antibody, and no anti-FSBA staining of GST alone was detected. Moreover, while FSBA interacted with the nucleolar localization signal defective mutant Nols-RING, no nucleolar staining of this variant could be detected (Figure 7B). When similar experiments were performed with the K454A mutant, no FSBA could be detected as the lysine residue in the P loop is essential for the crosslinking reaction (data not shown). This further confirmed both the specificity of the crosslinking reaction and the lack of nonspecific nucleolar staining associated with the anti-FSBA antibody. Experiments with FSBA thus support our hypothesis that efficient nucleolar localization requires ATP binding and an intact nucleolar localization signal. Discussion We demonstrate here that Mdm2 binds specifically to adenine base-containing nucleotides, and this is mediated by the P loop in its RING domain. Our data show that binding of Mdm2 to nucleotide facilitates its nucleolar localization in vivo and in vitro. We postulate that nucleolar localization is caused by a nucleotide-mediated conformational change in the RING domain. We identify a single P loop mutant K454A, which retains its E3 ubiquitin ligase activity while exhibiting a defect in nucleolar localization both in mammalian cells and in C. elegans. Proteins capable of binding tri- and diphosphate nucleotides have been well characterized. Regulatory proteins such as Ras and SV40 large T antigen (Maegley et al., 1996; Weiner and Bradley, 1991) and many others contain a P loop motif, which forms extensive interactions with the ␣ and ␤ phosphates of the nucleotide (Walker et al., 1982). The KD of Mdm2 for binding ATP (13.5 ␮M) falls within the fairly broad range of values for other known nucleotide binding proteins. The KD for the heterotrimeric G proteins is in the range of .1–1 ␮M, while small GTP binding proteins of the Ras superfamily exhibit 10–100 nM KD values (Malinski et al., 1996). A far higher dissociation constant has been reported for the Na,K-ATPase, which can only weakly bind ATP in the absence of K⫹ and has a KD of ⵑ200–400 ␮M (Hilge et al., 2003). Our data showing that nucleotide binding causes a detectable conformation change in Mdm2 is

consistent with previous observations. SV40 large T antigen undergoes allosteric activation of hexamer formation upon ATP binding (Borowiec et al., 1990) while E. coli DnaA protein undergoes a profound conformational change upon ATP binding that facilitates its interaction with DnaB and also renders it resistant to heat denaturation (Sekimizu et al., 1987). ATP binding also induces RecA filaments assembly on single-stranded DNA (Lusetti and Cox, 2002). A common feature of these examples is the facilitation by nucleotide binding of proteinprotein interactions. It is thus tempting to speculate that Mdm2 nucleotide binding may also affect its interactions with itself or with other proteins. The interaction between neuronal cell adhesion molecule (NCAM), which promotes axonal outgrowth, and fibroblast growth factor receptor 1 (FGFR1) provides a possibly relevant example of ATP regulating proteinprotein interactions (Kiselyov et al., 2003). The extracellular region of NCAM containing a P loop binds ATP; however, under the conditions of axonal extension its ATP binding region is obscured by FGFR1 binding. Once synaptic contact is formed, ATP release may uncouple the NCAM-FGFR1 complex and stop axonal growth. Similarly, depending on RING domain binding factors, the Mdm2 nucleotide binding motif may be masked, causing Mdm2 to remain in a nucleotide free form, despite the high concentration of intracellular ATP. Alternately, in the presence of a different subset of interacting proteins, Mdm2 may exist in the ATP- or ADP-bound form. Identification of Mdm2 RING domain binding proteins will allow insight into these possibilities. Nucleolar localization of Mdm2 is facilitated by ATP binding. Although targeting of a molecule to the nucleolus often results from its direct or indirect interaction with rDNA or its transcripts (Carmo-Fonseca et al., 2000), a variety of proteins with no apparent function in ribosome assembly have been shown to localize to this nuclear compartment (Pederson, 1998). Nucleolar proteins have been shown to function in nucleotide modification of several small RNAs, to play a role in the biosynthesis of the signal recognition particle, and to function in the sequestration and release of proteins involved in gene silencing, senescence, and cell division (Pederson, 1998). Growth regulatory proteins shown to localize to the nucleolus include basic and acidic fibroblast growth factor, angiogenin, parathyroid hormone-related peptide, the Werner syndrome gene product, and the oncogene c-myb-associated protein p160 (Pederson, 1998). Nucleotide binding regulates nucleolar localization of several proteins. B23 shuttles from the cytoplasm to the nucleolus in a GTP binding-dependent fashion (Finch et al., 1995). Recently, a nucleolar protein, nucleostemin, was identified that contains both a P loop and a GTP binding motif. While direct nucleotide binding has not been established, deletion of these motifs disrupts the interaction between nucleostemin and the nucleolus (Tsai and McKay, 2002). Indeed, a recent proteomics analysis of nucleolar components predicts that ⵑ24% of all nucleolar proteins bind nucleotides or nucleic acid (Andersen et al., 2002). The nucleolus may serve as a staging area for nuclear export. Ribosomal components are transferred from the nucleolus by the CRM1 pathway (Ho et al., 2000), which also shuttles p53 and Mdm2 to the cytoplasm (Geyer et

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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

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al., 2000). Both p53 and Mdm2 have been reported to be degraded in the nucleus under certain conditions (Shirangi et al., 2002; Xirodimas et al., 2001b), so nucleolar localization may serve as a sorter between nuclear activity, nuclear degradation, or cytoplasmic degradation of these proteins. Nucleolar localization and inactivation of Mdm2 could be a consequence of its interaction with the p14ARF tumor suppressor (Weber et al., 1999). Nevertheless, p14ARF can stimulate p53 activity in the absence of nucleolar sequestration (Llanos et al., 2001) so it is not necessary to assume that such relocalization is obligatorily linked to p14ARF function. Although the exact function of Mdm2 nucleolar localization remains to be elucidated, independent evidence supports the idea that nucleolar sequestration of Mdm2 downregulates its activity toward p53 (Ashcroft et al., 2000; Weber et al., 1999; Zhang and Xiong, 1999). We nevertheless cannot rule out the possibility that nucleolar localization of Mdm2 serves a purpose that is independent of its regulation of p53. Whatever, the function of Mdm2 nucleolar localization, our data provide a mechanism for facilitating this translocation that is likely independent of p14ARF. Uncoupling of Mdm2 nucleolar localization and E3 activity by mutational analysis has been experimentally challenging (Fang et al., 2000; Xirodimas et al., 2001a). In fact, our G448S and T455C mutants and the previously constructed Nols-Mdm2 mutant are all defective in both p53 degradation and nucleolar localization. By contrast, the K454A mutant Mdm2 functions better than wild-type Mdm2 in self- and p53 degradation and yet is at least partially defective in nucleolar localization. K454A Mdm2 thus provides evidence that these functions are separable. Remarkably, the Mdm2 RING domain is both functional as an E3 ubiquitin ligase and can localize to nucleoli in C. elegans, leading us to infer that, while there are no reported Mdm2 or p14ARF homologs in worms, the pathways directing Mdm2 ubiquitination and ATPregulated nucleolar localization are evolutionarily conserved. These observations, coupled with the fact that all Mdm2 and MdmX proteins from all species identified have conserved P loop motifs and nucleolar localization signals within their RING domains, highlight the potential significance of nucleolar localization in Mdm2 function. Although we found that nucleotide binding control of nucleolar localization is lost in the presence of overexpressed p14ARF, we cannot conclude that this is the case with endogenous levels of p14ARF. In fact, since the K454A mutant showed a defect in nucleolar localization in H1299 cells, which express endogenous p14ARF, p14ARF effects are likely not dominant over nucleotide binding effects under normal conditions. A recent publication describing Mdm2 interaction with the ribosomal L11 protein further strengthens the idea

that nucleolar localization can serve as a negative regulator of Mdm2 activity (Lohrum et al., 2003). Mdm2-L11 interaction leads to translocation of Mdm2 to the nucleolus under certain conditions, and this interaction is enhanced following ActD treatment. L11-induced nucleolar translocation of Mdm2 leads to increased p53 activity, further establishing the importance of nucleolar localization of Mdm2 as a negative regulator of its activity. Although evidence for extensive and multifaceted regulation of p53 continues to accumulate (Prives and Hall, 1999), much is still unknown about the mechanisms for regulating Mdm2. Understanding the reasons for and regulation of Mdm2 nucleolar localization could not only potentiate therapeutic approaches but also allows for a more lucid view of cellular response to stress. Although full understanding of how nucleotide binding has an impact upon Mdm2 function awaits further study, our results identify an important mechanism for regulating Mdm2 subnuclear localization. Experimental Procedures Plasmids and Antibodies Human wild-type Mdm2 cDNA was unidirectionally cloned with the addition of two N-terminal Flag tags, between the EcoRI and BamHI sites of pCDNA3. GST-Mdm2 RING deletion constructs aa 410–491 were unidirectionally cloned between the BamH1 and EcoR1 sites of the pGEX-2T vector using two primers for fragment amplification (5⬘- GCGGATCCGTGAAAGAGTTTGAAGAAACC-3⬘ and 5⬘-GCGAAT TCCTAGGGGAAATAAGTTAGCAC-3⬘) and used as a backbone for site-directed mutagenesis (QuikChange, Stratagene) to prepare substitution mutants K454A, T455C, and G448S. The integrity of the resulting constructs was verified by sequencing. Pc53-HA vector expresses full-length HA-p53 cDNA from the CMV promoter in pCDNA3. The PCMV-HA-ubiquitin expression construct was obtained from Dr. D. Bohmann. A nucleolar localization defective mutant construct, PcHdm-Nols in pCMV-neo plasmid, and a construct expressing ARF, pcDNA3-myc-ARF were kind gifts of Dr. K. Vousden and Dr. Y. Xiong, respectively. Mouse anti-FSBA antibody was a generous gift from Dr. Peter Parker (ICRF). Monoclonal antibodies against Flag, Myc, and HA epitopes were purchased from Sigma (M2), Santa Cruz Biotechnology (9E10), and BabCo (HA11). Goat anti-mouse Alexa Fluor 488/ 594-conjugated antibody and donkey anti-goat-conjugated antibody were from Molecular Probes. Goat anti-B23 antibody was from Santa Cruz. 4B11 (epitope: amino acids 383–491) and 2A10 (epitope: amino acids 294–339) and 3F8 (epitope located approximately between amino acids 122 and 204) monoclonal antibodies against human Mdm2 were gifts of Dr. A. Levine. Protein Purification Purification of recombinant His-6⫻ tagged Mdm2 protein was performed as previously described (Shieh et al., 2000). GST fusion proteins were expressed in BL21 (DE3) cells. After induction for 3 hr at 16⬚C with 0.1 mM IPTG, soluble proteins were extracted by sonication in lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% aprotinin, 1 mM DTT, 0.5 mM PMSF). The soluble protein fraction was incubated with glutathione-Sepharose beads

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.

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(Pharmacia) at 4⬚C for 1 hr, and the bound protein was eluted with reduced glutathione. Purified GST-Mdm2 proteins were then dialyzed against a buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 20% glycerol. ATP Binding Experiments Mdm2 protein (50 or 100 ng) was incubated with ␥-32P ATP for 10 min at 30⬚C in ATP binding solution (40 mM creatine-phosphate, 1 mM MgCl2, 15 mM NaCl, 0.5 mM DTT, 10 ␮g of BSA). Reaction mixtures were filtered through 25 mm nitrocellulose filters (Protran, Schleicher a Schuell, Keene, NH) under vacuum. Filters were washed three times with 5 ml of 25 mM HEPES buffer (pH 7.9), dried, and counted by liquid scintillation. For the nucleotide preference experiment 100 ng of recombinant Mdm2 protein was first incubated with ␥-32P ATP for 10 min at 30⬚C; then increasing concentrations of unlabeled competitor nucleotides were added, and the mixtures were incubated for an additional 10 min at 30⬚C before filtration. Partial Trypsin Digestion Recombinant full-length his-Mdm2 protein (100 ng) was preincubated for 10 min at 30⬚C in 10 mM MgCl2, 50 mM Tris (pH 8.0). Following preincubation, 0, 1, 5, 25, and 100 ng of trypsin was added to the mixtures, which were then incubated at room temperature for an additional 15 min. Then, SDS-PAGE loading buffer was added, and the mixtures were subjected to 8% SDS-PAGE gel and immunoblotting with antibodies that recognize different regions on Mdm2 protein. Cell Lines, Transfections, and Immunoblotting H1299 (human non-small-cell lung carcinoma), HCT116 (human colorectal cancer), U2OS (osteosarcoma), and 293T (adenovirus-transformed human embryo kidney) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). Transfections were performed using Lipofectamine 2000 Reagent (Invitrogen) in accordance with the manufacturer’s protocols. For ubiquitination experiments cells were plated in 10 cm dishes and transfected with 20 ␮g Flag-Mdm2 and 10 ␮g HA-ubiquitin. Cells were harvested 40 hr posttransfection. Western blotting was performed as previously described (Gottifredi et al., 2001). For immunoprecipitations, 1 mg of soluble protein was incubated with anti-Flag antibody at 4⬚C for 4 hr before Protein A/G beads (25 ␮l) were added, and binding was allowed to proceed for an additional 2 hr. The beads were washed twice with lysis buffer following two additional washes with low salt buffer (10 mM Tris [pH 8.0], 140 mM Nacl). Finally, 30 ␮l of protein sample buffer was added to the beads, and the beads were boiled for 5 min at 100⬚C, followed by immunoblot analysis with the indicated antibodies. Membrane stripping was performed following ECL Protocol (Amersham Bioscience). Worm Transformation and Gene Expression C. elegans were grown at 25⬚C as described (Brenner, 1974). unc-4 promoter constructs were generated by inserting the sequence GATCC after the start codon to create a BamHI site and cloning the resulting 2.5 kb HindIII-BamHI fragment containing the unc-4 promoter (Miller and Niemeyer, 1995) into the GFP vector pPD95.75 (A. Fire, personal communication) to produce vector TU#686. GFP fluorescence was observed using Zeiss Axioskop 2 microscope with suggested filter. Photos were taken with a digital Spot camera. A KpnI DNA fragment corresponding to aa 437–478 in Mdm2 containing the K454A mutation was produced by PCR and cloned into the KpnI site in TU#686 to produce TU#703 (this insertion places the Mdm2 sequence N-terminal to GFP). The K454A mutation was reverted back to wild-type sequence using the QuikChange kit from Stratagene to produce TU#702. A similar series of GFP fusions driven from the dpy-7 promoter (Gilleard et al., 1997) was obtained by exchanging a HINDIII-BamHI fragment of TU#686, TU#702, and TU#703 to produce TU#704, TU#705, and TU#706, respectively. Transformation of C. elegans was performed as described (Mello and Fire, 1995) using pRF6 (containing a domain rol-6 mutation) into wild-type animals (N2) for the unc-4 promoter constructs and PJM23 (containing the wild-type lin-15 gene) into lin-15 (n76s) animals (provided by P. Sternberg).

Immunofluorescence Cells fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. After fixation, the cells were permeabilized in PBS containing 0.2% Triton X-100 for 5 min. Cells were blocked in PBS containing 5% bovine serum albumin at room temperature for 30 min prior to incubation for 2 hr with primary antibodies, followed by 1 hr incubation with the indicated secondary antibodies. Nuclei were visualized by DAPI (4⬘,6⬘-diamidino-2-phenylindole) (Sigma) staining. For visualization of the nucleolus, immunostaining with a goat anti-B23 antibody was performed either in the presence or absence of anti-MDM2 antibodies. In Vitro Localization Experiments U2OS cells were plated on cover slips in 35 mm dishes and grown to 70% confluence. The cells were washed two times with PBS and permeabilized with 200 ␮l of 10% Triton X-100 in PBS. Where indicated, the Triton permeabilization solution contained purified recombinant proteins (500 ng to 4 ␮g) for 1 min, after which the Triton containing solution was aspirated. Residual cellular structures were then crosslinked using 4% paraformaldehyde in PBS for 20 min at room temperature. Localization of the recombinant proteins in permeabilized cells was determined by immunofluorescence. FSBA Labeling All recombinant proteins used for fluorosulfonyladenosine (FSBA) labeling were first buffer exchanged into the labeling reaction buffer (20 mM Tris [pH 7.5], 10% glycerol, 10 mM MgCl2, 0.2% Triton X-100) to remove dithiothreitol (DTT) from the buffer, which would otherwise react with FSBA and quench the reaction. Labeling reactions were performed according to the published protocol (Parker, 1993). For in vitro localization experiments, the reactions were terminated by buffer exchange. Using three consecutive rounds of buffer exchange using Sephadex G25 minispin columns (Amersham Bioscience) into PBS allowed for the removal of 90% of unbound nucleotides. Acknowledgments We are grateful to Vanesa Gottifredi for suggesting that we look at nucleolar localization of wild-type and mutant Mdm2 and Tingting Zhang for suggesting that GFP-Mdm2 RING fusion proteins might be less stable than GFP. We thank Kristine McKinney and Jinwoo Ahn for helpful discussions throughout this work, Ella Freulich for expert technical assistance, Nico U. Dosenbach for help with the initial part of this study, and Karen Vousden for sharing results and reagents. This work was supported by CA87497 (C.P.), GM37971 (J.L.M.), and GM30997 (M.C.). X.J. was supported by EMBO and Human Frontier Science awards. Received: February 2, 2003 Revised: August 4, 2003 Accepted: September 17, 2003 Published: October 23, 2003 References Andersen, J.S., Lyon, C.E., Fox, A.H., Leung, A.K., Lam, Y.W., Steen, H., Mann, M., and Lamond, A.I. (2002). Directed proteomic analysis of the human nucleolus. Curr. Biol. 12, 1–11. Ashcroft, M., Taya, Y., and Vousden, K.H. (2000). Stress signals utilize multiple pathways to stabilize p53. Mol. Cell. Biol. 20, 3224– 3233. Borden, K.L. (2000). RING domains: master builders of molecular scaffolds? J. Mol. Biol. 295, 1103–1112. Borowiec, J.A., Dean, F.B., Bullock, P.A., and Hurwitz, J. (1990). Binding and unwinding—how T antigen engages the SV40 origin of DNA replication. Cell 60, 181–184. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Carmo-Fonseca, M., Mendes-Soares, L., and Campos, I. (2000). To be or not to be in the nucleolus. Nat. Cell Biol. 2, E107–E112. Elenbaas, B., Dobbelstein, M., Roth, J., Shenk, T., and Levine, A.J.

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(1996). The MDM2 oncoprotein binds specifically to RNA through its RING finger domain. Mol. Med. 2, 439–451.

Prives, C. (1998). Signaling to p53: breaking the MDM2-p53 circuit. Cell 95, 5–8.

Fang, S., Jensen, J.P., Ludwig, R.L., Vousden, K.H., and Weissman, A.M. (2000). Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275, 8945–8951.

Prives, C., and Hall, P.A. (1999). The p53 pathway. J. Pathol. 187, 112–126.

Finch, R.A., Chang, D.C., and Chan, P.K. (1995). GTP gamma S restores nucleophosmin (NPM) localization to nucleoli of GTPdepleted HeLa cells. Mol. Cell. Biochem. 146, 171–178. Geyer, R.K., Yu, Z.K., and Maki, C.G. (2000). The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat. Cell Biol. 2, 569–573. Gilleard, J.S., Barry, J.D., and Johnstone, I.L. (1997). cis regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Mol. Cell. Biol. 17, 2301–2311. Gottifredi, V., Karni-Schmidt, O., Shieh, S.S., and Prives, C. (2001). p53 down-regulates CHK1 through p21 and the retinoblastoma protein. Mol. Cell. Biol. 21, 1066–1076. Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299. Hilge, M., Siegal, G., Vuister, G.W., Guntert, P., Gloor, S.M., and Abrahams, J.P. (2003). ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase. Nat. Struct. Biol. 10, 468–474. Ho, J.H., Kallstrom, G., and Johnson, A.W. (2000). Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151, 1057–1066. Honda, R., and Yasuda, H. (2000). Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase. Oncogene 19, 1473–1476. Kiselyov, V.V., Skladchikova, G., Hinsby, A.M., Jensen, P.H., Kulahin, N., Soroka, V., Pedersen, N., Tsetlin, V., Poulsen, F.M., Berezin, V., and Bock, E. (2003). Structural basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP. Structure 11, 691–701. Kubbutat, M.H., Jones, S.N., and Vousden, K.H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299–303. Lai, Z., Freedman, D.A., Levine, A.J., and McLendon, G.L. (1998). Metal and RNA binding properties of the hdm2 RING finger domain. Biochemistry 37, 17005–17015. Llanos, S., Clark, P.A., Rowe, J., and Peters, G. (2001). Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat. Cell Biol. 3, 445–452. Lohrum, M.A., Ashcroft, M., Kubbutat, M.H., and Vousden, K.H. (2000). Identification of a cryptic nucleolar-localization signal in MDM2. Nat. Cell Biol. 2, 179–181. Lohrum, M.A., Ludwig, R.L., Kubbutat, M.H., Hanlon, M., and Vousden, K.H. (2003). Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell 3, 577–587. Lusetti, S.L., and Cox, M.M. (2002). The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu. Rev. Biochem. 71, 71–100. Maegley, K.A., Admiraal, S.J., and Herschlag, D. (1996). Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. Proc. Natl. Acad. Sci. USA 93, 8160–8166. Malinski, J.A., Zera, E.M., Angleson, J.K., and Wensel, T.G. (1996). High affinity interactions of GTPgammaS with the heterotrimeric G protein, transducin. Evidence at high and low protein concentrations. J. Biol. Chem. 271, 12919–12924. Mello, C., and Fire, A. (1995). DNA transformation. Methods Cell Biol. 48, 451–482. Miller, D.M., III, and Niemeyer, C.J. (1995). Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. Development 121, 2877–2886. Parker, P.J. (1993). Antibodies to fluorylsulfonylbenzoyladenosine permit identification of protein kinases. FEBS Lett. 334, 347–350. Pederson, T. (1998). Growth factors in the nucleolus? J. Cell Biol. 143, 279–281.

Sekimizu, K., Bramhill, D., and Kornberg, A. (1987). ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell 50, 259–265. Shieh, S.Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000). The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289–300. Shirangi, T.R., Zaika, A., and Moll, U.M. (2002). Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage. FASEB J. 16, 420–422. Tohgo, A., Munakata, H., Takasawa, S., Nata, K., Akiyama, T., Hayashi, N., and Okamoto, H. (1997). Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase. J. Biol. Chem. 272, 3879–3882. Tsai, R.Y., and McKay, R.D. (2002). A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16, 2991–3003. Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951. Weber, J.D., Taylor, L.J., Roussel, M.F., Sherr, C.J., and Bar-Sagi, D. (1999). Nucleolar Arf sequesters Mdm2 and activates p53. Nat. Cell Biol. 1, 20–26. Weiner, B.M., and Bradley, M.K. (1991). Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen. J. Virol. 65, 4973–4984. Xirodimas, D., Saville, M.K., Edling, C., Lane, D.P., and Lain, S. (2001a). Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo. Oncogene 20, 4972–4983. Xirodimas, D.P., Stephen, C.W., and Lane, D.P. (2001b). Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2mediated degradation of p53. Exp. Cell Res. 270, 66–77. Zhang, Y., and Xiong, Y. (1999). Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol. Cell 3, 579–591.