Download The RING Domain of Mdm2 Can Inhibit Cell

[CANCER RESEARCH 62, 1222–1230, February 15, 2002]
The RING Domain of Mdm2 Can Inhibit Cell Proliferation1
Jinjun Dang, Mei-Ling Kuo, Christine M. Eischen,2 Lilia Stepanova, Charles J. Sherr, and Martine F. Roussel3
Departments of Tumor Cell Biology [J. D., M-L. K., L. S., C. J. S., M. F. R.], Biochemistry [C. M. E.], and Howard Hughes Medical Institute [L. S., C. J. S.], St. Jude Children’s
Research Hospital, Memphis, Tennessee 38105
ABSTRACT
Mdm2 is a p53-inducible phosphoprotein that negatively regulates p53
by binding to it and promoting its ubiquitin-mediated degradation. Alternatively spliced variants of Mdm2 have been isolated from human and
mouse tumors, but their roles in tumorigenesis, if any, remain elusive. We
cloned six alternatively spliced variants of Mdm2 from E␮-Myc-induced
mouse lymphomas, all of which lacked the NH2-terminal p53-binding
domain but conserved the remainder of the Mdm2 protein. Enforced
expression of full-length Mdm2 in primary mouse embryo fibroblasts or
bone marrow-derived, interleukin 7-dependent pre-B cells accelerated
their proliferation, whereas unexpectedly, overexpression of truncated
Mdm2 isoforms inhibited their growth. Truncated variants were active as
inhibitors whether they localized predominantly to the nucleus or cytoplasm. Despite the absence of the p53-binding domain, growth inhibition
remained strictly p53 dependent (but not p19Arf dependent) and could be
overcome by full-length Mdm2. The intact RING finger domain at the
Mdm2 COOH terminus (amino acids 399 – 489) was necessary and sufficient for growth inhibition by truncated Mdm2 proteins and could physically interact with either the RING finger domain or central acidic region
of full-length Mdm2. However, such interactions do not inhibit Mdm2 E3
ubiquitin ligase activity in vitro using p53 as a substrate. Expression of
growth-inhibitory Mdm2 isoforms in tumors remains an enigma.
INTRODUCTION
Mdm2 was discovered as an amplified gene on murine doubleminute chromosomes in a spontaneously transformed 3T3 cell line
(1). Subsequent analysis demonstrated that Mdm2 (Hdm2 in human)
is overexpressed in 5–10% of human tumors (2, 3), and that its
expression is not only able to immortalize primary rodent embryonic
fibroblasts but also to transform them in cooperation with activated
Ras (4, 5). Embryos lacking Mdm2 die in utero, but lethality is
rescued in a p53-null genetic background, indicating that an essential
function of Mdm2 is to negatively regulate p53 activity (6, 7). Oncogenic properties of Mdm2 are conferred, at least in part, by its
ability to inactivate p53. Similar to inactivating p53 mutations, overexpression of Mdm2 also induces chromosome instability, inappropriate centrosome duplication, and changes in ploidy (8).
Mdm2 binding to the p53 NH2 terminus antagonizes p53 transcriptional activity (9 –11), inhibiting p53 acetylation and transactivation
by interfering with p300/CBP (12, 13). Mdm2 also functions as an E3
ligase to ubiquitinate p53 (14 –16) and to enforce its export from the
nucleus to the cytoplasm, where it is degraded in proteasomes (17–
19). It is unlikely that Mdm2 E3 ubiquitin ligase activity alone is
sufficient to trigger p53 proteolysis, because Mdm2 mono-ubiquitinates p53 at multiple sites but does not catalyze addition of polyuReceived 9/5/01; accepted 12/14/01.
The costs of publication of this article were defrayed in part by the payment of page
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1
This work was supported in part by NIH Grants CA-71907 (to M. F. R.), Cancer
Center Core Grant CA-21765, and by the American Lebanese Syrian Associated Charities
of St. Jude Children’s Research Hospital. C. J. S. is an investigator of the Howard Hughes
Medical Institute.
2
Present address: Eppley Cancer Institute, University of Nebraska Medical Center,
986805 Nebraska Medical Center, Omaha, NE 68198-6805.
3
To whom requests for reprints should be addressed, at Department of Tumor Cell
Biology, DTRT 5006C, Mail Stop 350, St. Jude Children’s Research Hospital, 332 North
Lauderdale, Memphis, TN 38105. Phone: (901) 495-3481/3597; Fax: (901) 495-2381;
E-mail: [email protected]
biquitin chains that are necessary for recognition by the proteasome
(20). One possibility is that mono-ubiquitination of p53 is required to
expose a nuclear export signal, and that p53 polyubiquitination and
degradation then proceed in the cytoplasm (21–23). Because Mdm2 is
a direct transcriptional target of p53, its expression acts in a negative
feedback loop to terminate the p53 response (24). However, Mdm2 is
itself subject to positive regulation through Ras signaling (25) and to
negative control by ATM-mediated phosphorylation (26) and through
direct binding of the ARF tumor suppressor protein (27–29). Apart
from p53, ARF, and CBP/p300, Mdm2 has been found to directly
associate with the retinoblastoma protein, E2F1, Numb, MTBP, p73,
and ribosomal protein L5 (30, 31). Therefore, it is unlikely that p53 is
the only physiological target of Mdm2. A homologue of Mdm2,
Mdm4 (MdmX), although not a target of p53 transcriptional regulation, can also negatively regulate p53-mediated transcription (32–35).
The recent demonstration that loss of Mdm4 in the mouse germ-line,
like the disruption of Mdm2, results in embryonic lethality that is
rescued on a p53-null background has led to the conclusion that
Mdm2 and Mdm4 regulate p53 via different pathways (36).
The integrity of the COOH-terminal RING finger domain of Mdm2
is necessary for both its E3 ubiquitin protein ligase activity (15,
37–39) and RNA-binding activity (40). Mutation of cysteine 464 to
alanine disrupts the integrity of the RING finger and abolishes Hdm2mediated p53 ubiquitination and nuclear export (14, 21, 22, 37).
Mdm2 also mediates its own ubiquitination in a RING finger-dependent manner (Refs. 16 and 37), and Lysine 444 might be important for
Mdm2 E3 ligase activity.5
A number of alternatively spliced variants of Mdm2 have been identified and isolated from both human and rodent tumor cells (41– 49).
Expression of alternatively spliced Mdm2 transcripts correlates with
high-grade malignancy in human ovarian tumors, bladder carcinomas,
astrocytic tumors, and breast cancer (42, 43, 49), irrespective of their p53
status. Of the characterized Mdm2 variants, most sustained deletions of
the p53-binding domain. Some Mdm2 isoforms were reported to transform NIH-3T3 cells (42). In E␮-Myc transgenic mice, many of the B-cell
lymphomas that arise sustain either p53 or Arf loss of function, with or
without overexpression of Mdm2 (50). Some lymphomas also expressed
variant Mdm2 isoforms, which coexisted with full-length Mdm2, irrespective of whether the tumors sustained Arf deletions or p53 mutations.
This prompted us to clone and characterize these variants and to examine
their role in tumorigenesis.
MATERIALS AND METHODS
Isolation of Variant Mdm2 cDNAs. Full-length and alternatively spliced
Mdm2 mRNAs were amplified by RT-PCR4 (ProSTAR First-Strand RT-PCR
kit; Stratagene), using 1 ␮g of total RNA isolated from E␮-Myc-induced
lymphomas (Table 1; Ref. 50). Primers were chosen from the noncoding
region of the published Mdm2 cDNA sequence [bp 169, sense (⫹), 5⬘CCATCGATCACCGCGCTTCTCCTGCGGCC-3⬘ and bp 1702, antisense
(⫺) 5⬘-ATCGATATAAAATTCTATTTTTGTGAGCAGGTC-3⬘], including a
ClaI site (underlined; Ref. 4). The amplified cDNA fragments were cloned into
4
The abbreviations used are: RT-PCR, reverse transcription-PCR; HA, hemagglutinin;
WT, wild type; MEF, mouse embryo fibroblast; FBS, fetal bovine serum; IL, interleukin;
NLS, nuclear localization sequence; NES, nuclear export sequence; IRES, internal ribosomal entry site; GFP, green fluorescent protein; Ubc, ubiquitin conjugating.
5
H. Yasuda, personal communication.
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Table 1 Expression of Mdm2 spliced variants, p53, and ARF in
E␮-Myc-induced tumors
Mdm2
a
Tumor
Full length
Variant
p53
protein
ARF
protein
CR71
CR135
CR156
CR203
CR246
CR325
UDa
UD
⫹
⫹
UD
⫹
V6
V2, V5
V2, V3, V4
V2, V3, V5
V1
V3
WT
WT
WT
Mutant
Mutant
WT
UD
⫹
⫹
⫹
⫹
⫹
UD, undetectable; ⫹, protein present.
the pGEM-T Easy vector (Promega), and their nucleotide sequences were
determined. Mammalian expression vectors were constructed by subcloning
the cDNAs into pSR␣MSV-tkneo (generously provided by Drs. Charles Sawyers and Owen Witte UCLA, CA; Ref. 51) or MSCV-IRES-GFP retroviral
vectors at an EcoRI site located immediately 3⬘ to the long terminal repeat
(52).
Generation of Mdm2 Mutants and Epitope-tagged UbcH5. Full-length
Mdm2 in pGEM-T Easy or in the pSR␣MSV-tkneo vector was used to
generate deletion and point mutants by site-directed mutagenesis (QuickChange, Site-Directed Mutagenesis kit; Stratagene). The sense primers including EcoRI and BamHI sites (underlined) were used to construct Mdm2 deletion
mutants M1 (amino acids 198 – 489), M2 (amino acids 299 – 489), M3 (amino
acids 399 – 489), and M4 (amino acids 399 – 463). Primers were as follows:
M1, 198⫹, 5⬘-CGGAATTCGGATCCACCATGTGCAGCGGCGGCACGAGCAGC-3⬘; M2, 299⫹, 5⬘-CAGAATTCGGATCCACCATGGACTATTGGAAGTGTACCTCATGC-3⬘; M3, 399⫹, 5⬘-CGGAATTCGGATCCACCATGTCCAGCAGCATTGTTTATAGCAGC-3⬘; the antisense primers were
for M4, 463- 5⬘- CGGAATTCTATGCACACGTGAAACATGACATGAG-3⬘
(EcoRI site underlined), and for all constructs except M4, 1702 (⫺), 5⬘ATCGATATAAAATTCTATTTTTGTGAGCAGGTC-3⬘ (ClaI site underlined). Mutations were introduced into the RING domain, substituting arginine
for lysine 444 (M5) and alanine for cysteine 462 (M6), using the following
primers: M5, 5⬘-ATCTGCCAGGGGCGGCCTAGAAATGGCTGCATTGTTCACG-3⬘ (mutation underlined); M6, 5⬘-CACCTCATGTCATGTTTCACGGCTGCAAAGAAGCTAAAAAAA-3⬘. Constructs containing a HA tag or
FLAG-tag were created by PCR by fusion of the HA or FLAG sequence to the
5⬘ end of the amplified Mdm2 cDNA sequences. Primers used to generate
HA-Mdm2, HA-⌬464 – 489, and HA-M3 were as follows: HA, 2⫹, 5⬘-GGGATCCAGCCATGGGTTACCCATACGACGTCCCAGACTACGCTACCTGCAATACCAACATGTCTGTGTCTAC-3⬘; HA, 399⫹, 5⬘-GGATCCAGCCATGGGTTACCCATACGACGTCCCAGACTACGCTACCTCCAGCAGCATTGTTTATAGCAGC-3⬘ (BamHI sites underlined); antisense primers
1702(⫺) and 463(⫺) were the same as those listed above. Primers to generate
FLAG-M3, FLAG-M4, FLAG-M5, and FLAG-M6 were as follows: FLAG
amino acid, 399⫹, 5⬘-GACCATGGACTACAAGGACGACGATGACAAGTCCAGCAGCATTGTTTATAGCAGC-3⬘ (NcoI site underlined); antisense bp
1702(⫺) and amino acid 463(⫺) were as described above.
A FLAG-tagged UbcH5 construct was generated by PCR using human
UbcH5 cDNA and the following primers: FLAG-H5–1⫹, 5⬘-GACCATGGACTACAAGGACGACGATGACAAGGCGCTGAAGAGGATTCAGAAAGAA-3⬘ (NcoI site underlined); H5(⫺), 5⬘-GGATCCTTACATTGAATATTTCTGAGTCCATTC-3⬘ (BamHI site underlined). The PCR product was
subcloned into pGEM-T Easy, excised with EcoRI, and subcloned into the
pVL-1393 baculovirus expression vector (PharMingen). Baculovirus constructs containing full-length Mdm2 and mutants were generated by excision
with EcoRI of the Mdm2 inserts from pGEM-T Easy or the MSCV-IRES-GFP
vector and subcloning them into the EcoRI site of pVL1393.
Cell Culture and Viral Vector Production. WT, p53-null, Arf-null or
Cip1-null MEFs were isolated from 13.5 day midgestation mouse embryos as
described previously (53) and cultured at early passages in DMEM containing
10% FBS, 2 mM glutamine, 0.1 mM nonessential amino acids, 55 mM 2mercaptoethanol, and 10 ␮g/ml gentamicin, in 8% CO2 humidified incubators.
Primary pre-B cells were derived from mouse bone marrow harvested from
femurs and tibias of WT animals, expanded, and maintained on feeders of
NIH3T3 cells expressing human IL-7 (T220-29) or in liquid culture with
recombinant human IL-7 in RPMI 1640 containing 5% FBS, 2 mM glutamine,
55 mM ␤-mercaptoethanol, penicillin/streptomycin, in 10% CO2 humidified
incubators, as described previously (50, 54). NIH3T3 cells stably expressing a
zinc-inducible p19Arf protein (pMTCB6-HA-Arf) were generated by transfection of a mammalian expression vector (pMTCB6) in which the HA-tagged Arf
cDNA was cloned downstream of the sheep metallothionein promoter (MT1).
Cells were maintained in DMEM containing 10% FBS, 2 mM glutamine,
penicillin/streptomycin, and 400 ␮g/ml of G418. Production of high titer
ecotropic viruses in 293T cells and infections of MEFs (53) and pre-B cells
(50, 54) were carried out as described. Spodoptera frugiperda (Sf9) cells were
grown at 24°C in Grace’s medium (Life Technologies, Inc.) supplemented
with 5% FBS and infected with baculoviruses as described previously (54).
Protein Extraction, Immunoprecipitation, and Immunoblotting. MEFs
were trypsinized, and after two washes with PBS, were lysed in ice-cold
Tween 20 lysis buffer [50 mM HEPES (pH 7.5), 200 mM NaCl, 1 mM EDTA,
0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.4 unit of aprotinin/ml,
1 mM NaF, 10 mM ␤-glycerophosphate, and 0.1 mM sodium orthovanadate]
and left on ice for 1 h. After sonication at 4°C (7 s ⫻ 2), cellular debris was
removed by centrifugation in a microcentrifuge at 14,000 rpm for 15 min at
4°C. Proteins (200 ␮g/lane) were electrophoretically separated on denaturing
polyacrylamide gels containing SDS and transferred onto membranes (Osmonics, Westborough, MA). Membranes were immunoblotted with affinity-purified rabbit polyclonal antibodies to mouse p19Arf (55), a monoclonal antibody
directed to mouse Mdm2 (2A10) generously provided by Dr. Arnold Levine
(Rockefeller University, New York, NY); a monoclonal antibody to the HA
epitope (generously provided by Dr. Albert Reynolds, Vanderbilt University,
Nashville, TN); a monoclonal antibody (9E10) recognizing the NH2-terminal
epitope of c-Myc (56); or with commercial antibodies directed to mouse p53
(Ab-7; Oncogene Research Products), mouse p21Cip1 (F-5; Santa Cruz Biotechnology, Santa Cruz, CA), Mdm2 (SMP14; Santa Cruz Biotechnology), or
FLAG (anti-FLAG, M2; Sigma Chemical Co.). Sequential immunoprecipitation and immunoblotting were performed as described (57).
Immunofluorescence. Procedures were described in detail previously (18).
Cells (3 ⫻ 104) plated on coverslips were fixed and permeabilized in cold
acetone:methanol (1:1, v/v) for 15 min at ⫺20°C. Coverslips were air dried,
blocked with 10% FBS in PBS, and stained with the 2A10 antibody to Mdm2,
followed by a goat antimouse antibody conjugated with fluorescein (Amersham). Nuclei were visualized by 4⬘,6-diamidino-2-phenylindole staining.
In Vitro Ubiquitination Assay. The ubiquitination assay was based on
protocols published previously (14, 16, 58). p53 was ubiquitinated in 50 ␮l of
total volume reactions containing Sf9 lysates containing E1 (10 ␮g of lysate
protein), FLAG-UbcH5 (10 ␮g of lysate protein), Mdm2 (10 ␮g of lysate
protein), and p53 (10 ␮g of lysate protein) in 100 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 0.6 mM DTT plus 15 ␮M ubiquitin (Sigma Chemical Co.), 2 mM ATP,
10 mM ␤-glycerol phosphate, 5 ␮g/ml aprotinin, and 5 ␮g/ml ubiquitinaldehyde (Boston Biochemical). Reactions were allowed to proceed at 25°C
for 2 h. Products were resolved on 7.5% denaturing polyacrylamide gels,
transferred to membranes, and immunoblotted with anti-p53 antibodies (Ab-7)
visualized by ECL (Amersham). A His6-tagged, NH2-terminal p19Arf peptide
(N37) expressed in Escherichia coli (57) was purified on a nickel (Ni2⫹)
column according to the manufacturer’s instructions (Qiagen) and added (10
␮g) as an Mdm2 inhibitor. Mdm2 mutants V4, FLAG-M3, or FLAG-M4 from
Sf9 lysates were also added as indicated.
RESULTS
Characterization of Alternatively Spliced Mdm2 Variants.
RNA was extracted from E␮-Myc-induced B-cell tumors, the p53 and
p19Arf status of which had been determined previously (Ref. 50; Table
1). cDNAs encoding various Mdm2 isoforms were amplified from
total RNA by RT-PCR and subcloned, and their nucleotide sequences
were determined. This yielded six alternatively spliced Mdm2 variants
(designated V1 to V6) in addition to full-length Mdm2 cDNA (Fig.
1A). Single or multiple Mdm2 isoforms were expressed in different
B-cell tumors, regardless of whether full-length Mdm2 was overexpressed or not and without any obvious correlation with WT or mutant
p53 status (Table 1). Alignment of cDNA sequences of all variants
with that of Mdm2 predicted that five of the six Mdm2 isoforms (V1
to V5) would not encode portions of the p53-binding domain (mapped
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
Fig. 1. Structure, expression, and binding characteristics of Mdm2 variants isolated from E␮-Myc-induced B-cell mouse lymphomas. A, schematic structure of Mdm2 variants
isolated from E␮-Myc-induced B-cell lymphomas. p53 binding (amino acids 19 –102), p19Arf-binding (amino acids 210 –304), and RING finger (RD; amino acids 436 – 476) domains
are designated by unshaded boxes. NLSs, NESs, and sequences required for nucleolar localization (NrLS) are shown as black boxes. Arrows, predicted translation ATG start codons.
Deleted sequences within Mdm2 variants are shown as lines. Cellular localization of full-length Mdm2 and each variant was determined by indirect immunofluorescence with a
monoclonal antibody against Mdm2 (2A10). ND, undetermined. B, primary MEFs at early passage were infected with a control retrovirus vector (Lane 1), or with vectors expressing
full-length Mdm2 (Lane 2), or Mdm2 variants (V1 to V6, Lanes 3– 8). Cells were lysed 72 h after infection, and proteins were detected by direct immunoblotting (IB) with a monoclonal
antibody to Mdm2 (2A10). Right, protein markers (in thousands). C and D, Sf9 cells were coinfected with baculoviruses encoding p53 (C) or p19Arf (D) together with full-length Mdm2
(Lanes 1–3), Mdm2 variant V2 (Lanes 4 – 6), or Mdm2 variant V4 (Lanes 7–9). Cell lysates were precipitated with normal rabbit serum (NRS; C, Lanes 1, 4, and 7), a monoclonal
antibody against a Myc epitope (9E10; D, Lanes 1, 4, and 7), a monoclonal antibody to Mdm2 (2A10; Lanes 2, 5, and 8), or antibodies against p53 (Ab-1) or p19Arf (Lanes 3, 6, and
9). Proteins electrophoretically resolved on denaturing gels were blotted with monoclonal antibody to Mdm2, 2A10. IB, immunoblot.
within the first 120 NH2-terminal amino acids) because of deletions of
exons 3, 5, or 4 – 8. The one exception was the V6 isoform that lacked
sequences encoded by exon 8 alone (residues 134 –165). Variants V2
to V5 retained the entire COOH-terminal portion of Mdm2 from
amino acids 166 to 489, including nuclear localization (NLS) and
nuclear export (NES) signals located between amino acids 178 to 195,
the p19Arf-binding domain (amino acids 210 to 304), zinc finger
(amino acids 303 to 320), and RING finger domain (amino acids
436 – 476; Fig. 1A). Isoform V1 contained an additional in-frame
deletion of amino acids 231–240.
To characterize the proteins encoded by each Mdm2 isoform in
mammalian cells, the cDNAs were subcloned into retroviral expression vectors, packaged into virions, and used to infect primary, earlypassage MEF strains. Protein expression was monitored by direct
immunoblotting with a monoclonal antibody to Mdm2 (2A10). Fulllength Mdm2 encoded the expected Mr 90,000 protein (Fig. 1B, Lane
2), which, although larger than that predicted from its sequence,
undergoes posttranslational modifications (phosphorylation and
sumoylation) in mammalian cells. Mdm2 variants V1 and V6 encoded
little or no protein (Fig. 1B, Lanes 3 and 8, respectively), whereas all
others encoded protein levels comparable with that of WT Mdm2
(Lanes 4 –7). Similar relative levels of protein expression were observed after transcription and translation of the variant cDNAs in vitro
(data not shown), implying that V1 and V6 were poorly translated.
Mdm2 isoforms V3 and V5 encoded Mr 55,000 proteins that were
predicted to initiate at codon 198 (Fig. 1A). Therefore, these variants
lacked the NLS and NES and, in agreement, immunofluorescence
analysis (data not shown) revealed that both proteins were predominantly expressed in the cytoplasm (summarized in Fig. 1A). Deletion
of exon 5 in V1 changes its reading frame, also forcing internal
initiation at methionine 198 and probably accounting for the low level
of protein detected (Fig. 1B, Lane 3). In addition, the downstream
in-frame deletion resulted in production of a cytoplasmic polypeptide
smaller than the V3 and V5 isoforms (Fig. 1B, Lane 3). V6 lacked
exon 8, again resulting in disruption of its reading frame, but unlike
V1, its expression was undetectable. V2 lacked residues specified by
exon 3 and encoded a Mr 75,000 protein, the translation of which was
likely initiated from methionine 50 (Fig. 1B, Lane 4). This isoform is
identical to that previously identified as p76Mdm2 by others (59). The
V4 protein (Fig. 1B, Lane 6) initiates at methionine 1, but it contains
a large in-frame deletion from amino acids 11 to 155, as well as
deletion of serine 207. As predicted, both the V2 and V4 isoforms
were predominantly localized to the nucleus of infected MEFs (Fig.
1A). Of interest, although endogenous Mdm2 expression was not
detected in MEFs infected with the naked vector (Fig. 1B, Lane 1), we
observed a modest but consistent increase in its expression in cells
expressing the truncated Mdm2 variants (Fig. 1B, Lanes 3–7, and see
below).
Nucleotide sequence analysis predicted that all Mdm2 variants
would be unable to bind p53 but would still interact with p19Arf. To
confirm this, insect Sf9 cells were coinfected with baculoviruses
encoding either WT Mdm2 or variants V2 or V4, together with
baculoviruses encoding WT p53 (Fig. 1C) or p19Arf (Fig. 1D). Lysates of infected cells were then either precipitated with control
antibodies (NRS or 9E10), monoclonal antibodies to Mdm2 (2A10) or
p53 (Ab-1), or antibodies to the mouse p19Arf COOH terminus, and
proteins were separated on denaturing gels were immunoblotted with
antibody to Mdm2. As expected, the full-length Mdm2 protein coprecipitated with either p53 or p19Arf (Fig. 1, C and D, Lanes 1–3). In
contrast, Mdm2 variants V2 and V4 were unable to form complexes
with p53 but retained the ability to bind p19Arf (Fig. 1, C and D, Lanes
4 –9).
Overexpression of Truncated Mdm2 Isoforms Inhibits Cell
Proliferation. Because the Mdm2 variants were expressed in B-cell
tumors, we suspected that they might compete with WT Mdm2 for
p19Arf binding, facilitating the ability of endogenous Mdm2 to antagonize p53, and thereby accelerating cell proliferation. To test this, we
used retroviral vectors to introduce either full-length Mdm2 or variant
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
Fig. 2. Overexpression of Mdm2 variants inhibits cell growth. Primary early-passage,
WT MEFs (passage 4; A), WT bone marrow-derived pre-B cells (B) or primary MEFs
from mice lacking p53 (C, p53⫺/⫺), Cip1 (D, p21⫺/⫺), or Arf (E, Arf⫺/⫺) were infected
with retroviruses expressing Mdm2 full-length (wt, f) and variants (V2, ⫻; V4, Œ; V5,
E) or an empty vector MSCV-IRES-GFP (Vector, F) and seeded at 2 ⫻ 104 in 60-mm
diameter culture dishes 72–96 h after infection. Growth rates were determined by counting
cell numbers from triplicate cultures every day for 8 days (MEFs) or every other day for
12 days (pre-B cells). F, primary WT MEFs were infected with retroviruses expressing
full-length Mdm2 (WT, Lane 2), Mdm2 variant (V4, Lane 3), or empty vector (Vect, Lane
1) and lysed 72 h after infection. Proteins were separated on denaturing gels and blotted
with antibodies against Mdm2, p53, p21Cip1, and p19Arf as indicated.
isoforms into an engineered NIH-3T3 cell line in which p19Arf expression can be conditionally up-regulated by addition of zinc to the
culture medium. Two days after infection, p19Arf expression was
induced by addition of 100 ␮M zinc, and cell proliferation was
monitored using long-term colony assays. Contrary to our expectations, WT Mdm2 bypassed Arf-induced growth arrest, whereas the
variants did not (data not shown), inconsistent with the idea that
truncated Mdm2 variants antagonize p19Arf function in this manner.
Others reported that Mdm2 variants isolated from human tumor
cells were able to transform NIH-3T3 cells (42). Although the NIH3T3 cell line used in our studies lacks the Ink4a/Arf locus (55) and is
readily transformed by oncogenic Ras, enforced expression of variants
V2, V4, or V5 in these cells did not transform them but instead
decreased their growth rate in a manner similar to that seen with
primary cell strains (data not shown, but see below).
We next tested the effects of the enforced expression of each Mdm2
variant on the proliferation of both primary MEFs and mouse bone
marrow-derived pre-B cells. First, early-passage mouse primary
MEFs (Fig. 2A) were infected with retroviruses encoding full-length
Mdm2, variants V2, V4, or V5, or the empty control vector. The
MSCV-IRES-GFP vector carries the gene encoding GFP in cis, which
was used as an internal control to monitor infection efficiency. Three
to 4 days after infection, GFP-positive cells were seeded at 2 ⫻ 104
per dish, and proliferation was monitored by counting cells (triplicate
plates/day) for 8 days thereafter. MEFs infected with full-length
Mdm2 proliferated considerably more rapidly than cells infected with
the empty vector (Fig. 2A), became smaller in size (data not shown),
and arrested at confluence by day 8. These results contrast directly
with those reported by others who concluded that Hdm2 overexpression inhibited the proliferation of NIH-3T3 cells (60). In contrast,
MEFs infected with variants V2, V4, and V5 grew at a much reduced
rate and eventually stopped proliferating before becoming confluent
(Fig. 2A). Both nuclear (V2 and V4) and cytoplasmic (V5) Mdm2
variants had inhibitory effects on cell proliferation. Growth-arrested
MEFs remained viable for at least 4 weeks in culture and were
morphologically flat and enlarged (data not shown). Although the rate
of growth inhibition by each variant differed, V4 showed the strongest
effect and was chosen for subsequent experiments.
To confirm that growth inhibition was not limited to fibroblasts,
primary bone marrow-derived, IL-7-dependent pre-B cells were infected with the control vector or with those encoding WT Mdm2 or
V4 (Fig. 2B). Although full-length Mdm2 accelerated pre-B cell
proliferation, expression of V4 not only inhibited pre-B cell growth
but triggered apoptosis. Therefore, rather than promoting cell division
as we initially supposed, tumor-derived Mdm2 variants hampered the
proliferation of established NIH-3T3 cells, as well as that of primary
MEFs and pre-B cells.
Mdm2 Variants Inhibit Cell Proliferation in a p53-dependent
Manner. Expression of Mdm2 variants in a subset of E␮-Mycinduced tumors that retained WT p53, Mdm2, and Arf prompted us to
determine whether their ability to inhibit growth might depend on p53,
p19Arf, or p21Cip1, the latter a direct transcriptional target of p53. We
expressed either full-length Mdm2 or V4 in early-passage MEFs
derived from mice lacking p53, both p53 and Mdm2, Cip1, or Arf and
tested their effects on cell proliferation. The rates of proliferation of
p53-null MEFs (Fig. 2C) or of those lacking both p53 and Mdm2 (data
not shown) were unaffected by introduction of either full-length
Mdm2 or V4. Similar results were obtained with the V2 and V5
isoforms in these cells (data not shown). Therefore, although truncated Mdm2 variants cannot interact with p53 directly, their ability to
inhibit cell proliferation was still p53 dependent. The effect of V4 was
partially compromised in Cip1-null and Arf-null MEFs (compare
effects of V4 in Fig. 2, D and E, versus A), indicating that although the
absence of p21Cip1 or p19Arf facilitates faster cell proliferation, neither
is strictly required for V4-mediated inhibition.
The strict dependency on p53 and partial contributions of p21Cip1
and p19Arf for growth inhibition suggested that the variant Mdm2
isoforms might affect the expression of p53, p19Arf, Mdm2, and
p21Cip1 in infected cells. Indeed, p76Mdm2 (V2) was found previously
to antagonize the function of WT Mdm2, increasing the levels and
activity of p53 (59). In agreement, we had observed modest increases
in endogenous Mdm2 levels in cells coexpressing the truncated Mdm2
variants (Fig. 1B). Three days after infection with the control vector or
those encoding Mdm2 or V4, early-passage, WT MEFs were lysed,
and levels of p53, p21Cip1, and p19Arf were determined by immunoblotting (Fig. 2F). The very low levels of p53 expressed in primary
MEFs were not detectably changed in cells infected with WT Mdm2
(Fig. 2F, Lane 2) but were slightly elevated in cells expressing V4
(Lane 3) compared with those infected with the control vector (Lane
1). More obviously, p21Cip1 expression was significantly induced by
V4, whereas Wt Mdm2, but not V4, increased p19Arf levels. The latter
results are consistent with previous findings that active p53 feeds back
to repress Arf expression (61, 62). These data are consistent with the
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
idea that Mdm2 variants trigger a p53 response that slows cell growth,
and that growth retardation depends in part on p21Cip1.
The RING Domain Is Necessary and Sufficient for Growth
Inhibition by Mdm2 Variants. To define the domain(s) responsible
for growth arrest by the Mdm2 variants, we prepared additional
Mdm2 mutants (Fig. 3A). NH2-terminal truncation mutants lacked
197 (M1), 298 (M2), or 398 (M3) residues, leaving an intact RING
domain from amino acids 399 to 489 (M3). The truncation mutant
(M4) differed from M3 by codeletion of COOH-terminal residues
464 – 489. Each construct was tagged at its NH2 terminus with a
FLAG or HA epitope preceded by an initiator methionine codon,
cloned into the MSCV-IRES-GFP retroviral vector, and introduced
Fig. 3. The RING finger domain of Mdm2 inhibits cell growth. A, schematic structure
of Mdm2 deletion mutants (M1–M4) and M3 RING domain point mutants (M5 and M6).
p53-binding, p19Arf-binding, and RING finger (RD) domains are designated by unshaded
boxes. The NLS, NES, and sequences required for nucleolar localization (NrLS) are shown
as black boxes. Epitopes recognized by Mdm2 antibodies, 2A10 and SMP14, are indicated
as black bars above the top schematic. Arrows, predicted ATG translation start codons.
RING finger domain mutations generated by PCR are indicated for M5 and M6. B,
primary WT MEFs infected with retroviruses expressing full-length Mdm2 (WT, f),
mutants (M1, ‚; M2, E; M3, Œ; M4, ⽧; M5, ⫻; M6, 䡺), or an empty vector (F) were
grown and counted as described in Fig. 2. Bars, SE. C, protein expression was confirmed
in lysates made from WT MEFs after infection with retroviruses encoding Mdm2 mutants
(M1–M6, Lanes 1– 6). Immunoblotting (IB) was performed with antibody 2A10 to Mdm2
for Mdm2 mutants M1 and M2 or with an antibody to the FLAG tag for FLAG-M3 to
FLAG-M6.
into early-passage MEFs (Fig. 3B). Expression of each mutant construct was confirmed by immunoblotting with antibodies to Mdm2 or
the epitope tags, revealing variable levels of protein overexpression
(Fig. 3C). Similar to the natural Mdm2 variants recovered from B-cell
tumors, mutants M1, M2, and M3 each inhibited cell growth when
expressed in WT MEFs (Fig. 3B). However, M4, containing a disrupted RING finger domain, was completely devoid of inhibitory
activity.
We therefore used M3 as a backbone to generate two point mutations that should affect RING activities (Fig. 3A). In M5 and M6,
lysine 444 (446 in Hdm2) and cysteine 462 (464 in Hdm2) were
replaced by alanine and arginine, respectively; these residues are
required to maintain the functional integrity of the RING domain, and
its mutation cancels the E3 ubiquitin protein ligase activity of Mdm2,
both toward itself and toward p535 (15). When expressed in MEFs,
M5 inhibited cell growth, whereas M6 lacked inhibitory activity (Fig.
3B). Together, these results indicated that inhibition of cell proliferation by Mdm2 variants required an intact RING finger, although
lysine 444 was likely dispensable for this function.
Interaction of RING-containing, Truncated Mdm2 Isoforms
with Full-length Mdm2. Although growth inhibition by Mdm2 variants is strictly p53 dependent, all such isoforms lacked a functional
p53-binding domain, implying that Mdm2 variants interact with regulators of p53 rather than p53 itself. Given that p19Arf was not
required for growth inhibition, endogenous Mdm2 was the most
obvious candidate. Indeed, MEFs coinfected with the smallest RINGcontaining construct M3 together with full-length Mdm2 grew at
almost the same rate as MEFs infected with full-length Mdm2 alone
(Fig. 4A), indicating that M3 and Mdm2 functionally compete with
one another in this regard. The integrity of the Mdm2 RING domain
was essential for full-length Mdm2 to override the inhibitory effects
of M3, because Mdm2 mutants lacking the complete COOH terminus
(⌬464 – 489) or containing an alanine for cysteine mutation at codon
462 were unable to reverse M3-induced growth inhibition (Fig. 4A).
Because all inhibitory Mdm2 variants retained an intact RING
finger domain, we determined whether these variants could interact
with full-length Mdm2 through their respective RING domains. In
preliminary experiments, we found that tagged truncated Mdm2 isoforms isolated from B-cell tumors could coprecipitate with full-length
Mdm2 after their coexpression in insect Sf9 cells (data not shown). In
the simplest and most informative iteration of these experiments, we
infected Sf9 insect cells with baculoviruses expressing FLAG-M3 or
HA-tagged full-length Mdm2 (WT) and precipitated with either antiFLAG, with a control antibody to a Myc epitope (9E10), or with
SMP14, a monoclonal antibody recognizing an epitope in the mid
portion of Mdm2 that is absent in FLAG-M3 (Fig. 4C). Precipitated
proteins electrophoretically separated on denaturing gels were then
coimmunoblotted with monoclonal antibody 2A10 to detect Mdm2
and with anti-FLAG to detect FLAG-M3 (Fig. 4C). As expected,
anti-FLAG specifically precipitated FLAG-tagged M3, SMP14 reacted only with Mdm2, and 9E10 reacted with neither. By contrast, in
lysates of coinfected cells, SMP14 precipitated M3 (Fig. 4C, Lane 2),
and anti-FLAG coprecipitated Mdm2 (Fig. 4C, Lane 3). A COOHterminal deletion (FLAG-tagged M4) abolished the interaction of the
RING with full-length Mdm2 (Fig. 4C, Lanes 7–9). However, coexpression of full-length Mdm2 deleted in its RING domain (⌬464 –
489) together with FLAG-M3 still allowed complexes to form (Fig.
4C, Lanes 4 – 6). Despite the ability of these two proteins to interact,
the Mdm2 COOH-terminal truncation mutant was unable to override
the inhibitory effects of M3 (Fig. 4A). Finally, when we coexpressed
FLAG-tagged M3 with HA-tagged M3, the two RING domains were
observed to strongly bind to one another (Fig. 4D). These results
indicated that the RING domains of truncated Mdm2 isoforms were
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
Fig. 4. Mdm2 rescues RING finger domain-mediated growth inhibition. A, primary
WT MEFs were infected with retroviruses expressing full-length Mdm2 (WT, f) or
FLAG-tagged M3 (Œ), or were coinfected with both (⫻), with M3 plus Mdm2 (⌬464 –
489; F), or with M3 plus Mdm2 (C462A; ‚). Growth rates were determined by counting
cell numbers from triplicate cultures every day for 8 days. B, NIH3T3 cells containing a
zinc-inducible Arf protein under the control of the metallothionein promoter
(pMTCB6HA-ARF) were infected with a retrovirus encoding FLAG-tagged M3 (Lane 2).
Uninfected cells were used as controls (WT, Lane 1). Zinc (100 ␮M) was added to the
cultures for an additional 24 h to induce p19Arf, after which cell lysates were prepared.
Proteins precipitated with anti-FLAG (M2, FLAG) were separated on denaturing gels and
blotted with antibody to Mdm2 (2A10) and anti-FLAG. IP, immunoprecipitation; IB,
immunoblot. C, insect Sf9 cells were coinfected with baculoviruses expressing FLAGtagged M3 and HA-tagged full-length Mdm2 (HA-WT), FLAG-M3 and HA-tagged Mdm2
with a truncation of the RING domain (HA-⌬464 – 489), or HA-tagged full-length Mdm2
and FLAG-tagged M4 (FLAG-M4). Cell lysates were precipitated with anti-Myc (9E10)
used as a negative control (Lanes 1, 4, and 7), antibodies to Mdm2 (SMP14, Lanes 2 and
5; 2A10, Lane 9), or with anti-FLAG (M2, Lanes 3, 6, and 8). Electrophoretically
separated proteins were immunoblotted with anti-Mdm2 (2A10) and anti-FLAG (M2). IP,
immunoprecipitation; IB, immunoblot. D, insect Sf9 cells were coinfected with baculoviruses expressing HA-tagged M3 (HA-M3) and FLAG-tagged M3 (FLAG-M3, Lanes
1–3) or infected with individual baculoviruses (Lanes 4 –7) and lysed 48 h after infection.
Cell lysates were precipitated with anti-Myc (9E10, Lane 1), anti-HA (HA, Lanes 2, 4, and
6), and anti-FLAG (Lanes 3, 5, and 7), and immunoblotted with anti-FLAG (Lanes 1–5)
or anti-HA (Lanes 6 and 7). IP, immunoprecipitation; IB, immunoblot. E, insect Sf9 cells
were coinfected with baculoviruses expressing HA-tagged Mdm2 mutants, HA ⌬1–50,
469 – 489 (Lanes 1–3), or HA ⌬305– 489 (Lanes 4 – 6), together with FLAG-M3. Cells
were lysed 48 h after infection. Lysates were precipitated with anti-Myc (9E10, Lanes 1
and 4), anti-HA (Lanes 2 and 5), and anti-FLAG (Lanes 3 and 6), and separated proteins
were immunoblotted with anti-FLAG and anti-HA. IB, immunoblot.
required for their interactions with full-length Mdm2. However, the
intact RING domain (M3) can also recognize a second binding site
retained in the COOH terminally truncated Mdm2 ⌬464 – 489.
To identify the second interaction site outside of the Mdm2 RING
domain, two additional Mdm2 deletion mutants were coexpressed
with FLAG-tagged M3 in Sf9 cells (Fig. 4E). One of these contained
deletions in both the p53-binding and RING domains (⌬1–50, 464 –
489), and the other lacked residues 305– 489, leaving the p53-binding
and acidic domains intact. Coprecipitation experiments revealed that
both mutants interacted with FLAG-M3 (Fig. 4E), suggesting that, in
addition to RING-RING interactions, FLAG-M3 can also bind to the
acidic domain of Mdm2.
To confirm that similar interactions could occur in mammalian
cells, we introduced M3 alone into the NIH-3T3 cell line that conditionally expressed zinc-inducible p19Arf. Induction of p19Arf strongly
increased p53 levels and led to Mdm2 expression. Precipitation with
the anti-FLAG antibody demonstrated that endogenous Mdm2 protein
coprecipitated with M3 under these conditions (Fig. 4B, Lane 2).
Similar results were obtained using HA-tagged M3 (data not shown).
Mdm2 Variants Do Not Affect Ubiquitination of p53 Mediated
by Mdm2. If inhibition of cell proliferation by the overexpressed
Mdm2 RING domain depends upon direct binding to endogenous
Mdm2, a potential consequence might be disruption of Mdm2 E3
ubiquitin protein ligase activity, leading, in turn, to p53-dependent
growth retardation. We therefore tested whether Mdm2 variants could
inhibit Mdm2-directed p53 ubiquitination in reconstituted in vitro
enzyme reactions containing E1 and E2 (UbcH5) enzymes plus recombinant full-length Mdm2. Multiple ubiquitinated forms of p53
were resolved on denaturing gels and detected with an antibody to p53
(Fig. 5A, Lane 1). This assay does not distinguish between forms of
p53 mono-ubiquitinated on multiple lysine acceptor sites from forms
containing tandem ubiquitin chains at single sites (polyubiquitination). However, only mono-ubiquitinated forms have been documented in such assays (20). Expression of Mdm2, V4, M3 and M4
proteins in Sf9 cells was confirmed by immunoblotting (Fig. 5B).
Addition of full-length Mdm2 to the reaction (10 ␮g of Sf9 lysate as
shown in Fig. 5B) was essential for ubiquitination of p53 (Fig. 5A,
Lane 1), which was completely inhibited by addition of 1 or 10 ␮g of
a peptide representing the NH2-terminal 37 amino acids (N37) of
p19Arf (Fig. 5A, Lanes 2 and 3). Mutant Mdm2 proteins failed to
induce ubiquitination of p53 (Fig. 5A, Lanes 6, 9, and 11) and did not
inhibit p53 ubiquitination mediated by full-length Mdm2 (Fig. 5A,
Lanes 4, 5, 7, 8, and 10). It should be noted that lysates containing
equal quantities of total protein (10 ␮g) expressed far more V4 than
full-length Mdm2 (Fig. 5B). Moreover, the mutant Mdm2 proteins
were unable to reverse inhibition of Mdm2-dependent ubiquitination
by p19Arf N37 (Fig. 5, C and D). Therefore, induction of p53 in cells
overexpressing the growth-inhibitory Mdm2 variants is not likely to
be attributable to their ability to directly inhibit p53 ubiquitination by
full-length Mdm2.
DISCUSSION
Despite the frequent disruption of the ARF-Mdm2-p53 pathway in
both human and mouse cancers, the contribution, if any, of Mdm2
variants to tumorigenesis has remained puzzling. Among six alternatively spliced Mdm2 variants that we isolated from E␮-Myc-induced
B-cell lymphomas, five failed to bind p53. Variant V6 was the only
isoform that contained an intact p53-binding domain, but because of
a frame shifting deletion, it did not encode a detectable protein
product. Each variant retained the central p19Arf-binding domain and
an intact COOH-terminal RING finger domain. Counterintuitively,
the enforced expression of these Mdm2 variants in primary MEFs and
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
Fig. 5. In vitro ubiquitination of p53 is unaffected by truncated Mdm2 isoforms. A, in
vitro ubiquitination of p53 was performed with recombinant proteins expressed in Sf9
cells, including full-length Mdm2, the V4 variant, the RING domain (M3), or a COOHterminally truncated RING domain mutant (M4). N37, representing the first 37 NH2terminal amino acids of p19Arf, was prepared in bacteria using a chemically synthesized
minigene template (58). Ladders of ubiquitinated forms of p53 were visualized with
antibody to p53. Sf9 lysates containing Mdm2 (10 ␮g of total protein) or lysates
expressing M3, M4, or V4 (1 or 10 ␮g of total protein as indicated at the top of the panel)
were used. B, expression of proteins in Sf9 cells infected with full-length Mdm2 and
variant V4 was confirmed by immunoblotting (IB) with antibody 2A10 to Mdm2, whereas
protein expression from Sf9 cells infected with FLAG-tagged M3 and FLAG-tagged M4
was confirmed by immunoblotting with anti-FLAG (M2). Uninfected cells were used as
controls. C, in vitro p53 ubiquitination assay with full-length Mdm2 alone (Lanes 3–5) or
together with variant Mdm2 V4 (Lanes 8 –10) with increasing concentration of N37p19Arf (Lanes 4, 5 and 9, 10). D, p53 ubiquitination assay with full-length Mdm2 together
with the RING domain (M3, Lanes 3–5) or a RING domain mutant (M4, Lanes 7–9) with
increasing quantity of N37-p19Arf (Lanes 4, 5 and 7–9).
bone marrow-derived pre-B cells and in immortalized NIH-3T3 fibroblasts inhibited rather than enhanced cell proliferation. Isoforms
that predominantly localized to the cell nucleus (e.g., V2 and V4) as
well as those that remained cytoplasmic (e.g., V5) were active when
overexpressed. Introduction of Mdm2 variants into cells appreciably
stabilized p53 and induced both endogenous Mdm2 and p21Cip1.
Growth inhibition was strictly p53 dependent and was only partially
alleviated in cells lacking either p21Cip1 or p19Arf.
Because the ability of NH2-terminally truncated Mdm2 isoforms to
inhibit cell proliferation required p53 but could not be mediated
through a direct interaction with p53 itself, we reasoned that their
interaction with full-length Mdm2 might be required. In agreement
with this idea, Mdm2 variants were observed to associate directly with
full-length Mdm2. Growth inhibition was completely abrogated by a
single point mutation at cysteine 462 that prevents Mdm2 from
ubiquitinating both p53 and itself (16, 37) as well as by a partial
COOH-terminal deletion (amino acids 464 – 489), both of which disrupt the structure of the RING finger domain. Therefore, the integrity
of the RING domain was required for a functional interaction. Moreover, the isolated RING domain, when expressed alone, was found to
be growth inhibitory. In contrast, elimination of lysine 444 was
without effect, implying that such modification is not required for
growth inhibition. Mutation of the cryptic nucleolar localization sequence (amino acids 464 – 471) within the RING finger domain of
Mdm2, which is required for p19Arf-mediated sequestration in the
nucleolus, did not affect growth inhibition, suggesting that nucleolar
translocation of Mdm2 must not be necessary either (data not shown;
Refs. 18, 57, and 63).
Deletion mapping studies indicated that the isolated RING domain
of Mdm2 was capable of binding the full-length Mdm2 protein.
Binding was mediated by direct RING-RING interactions as well as
by an association of the isolated RING domain with the acidic domain
of Mdm2. Other investigators reported that full-length Hdm2 was
growth inhibitory when expressed in NIH-3T3 cells (60), whereas we
obtained the opposite result, i.e., overexpression of Mdm2 accelerated
proliferation. In their studies, the inhibitory domain of Hdm2 was
mapped to residues 155 to 324, which includes the central Mdm2
acidic domain but not the COOH-terminal RING. Again, our results
do not agree, and the basis for these discrepancies remains unresolved.
Because RING fingers appear to have no intrinsic E3 protein ligase
activity of their own, the simplest interpretation is that an interaction
between the full-length and variant Mdm2 proteins might impair the
E3 ligase activity of Mdm2. However, this now seems unlikely,
because neither the expression of variant Mdm2 V4 nor expression of
the RING domain alone inhibited Mdm2-mediated p53 ubiquitination
in an in vitro assay. Another possibility is that overexpression of
Mdm2 RING domains in cells can sequester the E2 Ubc enzymes that
are required for Mdm2 E3 ligase activity. We do not favor this for
several reasons: (a) E2 enzymes interact with many E3s and are not
generally thought to be rate-limiting in vivo; and (b) the crystal
structure of the Cbl E3 ligase indicates that E2 binding is coordinated
both by residues within the RING domain and through additional
contacts elsewhere in the protein (64). In addition, overexpression of
UbcH5 or UbcH7 together with Mdm2 variants failed to rescue their
growth inhibitory effects (data not shown). Therefore, the mechanism
by which truncated Mdm2 isoforms inhibit cell growth remains undefined.
Despite the ability of truncated Mdm2 isoforms to associate directly
with Mdm2, an important caveat is a lack of formal proof that Mdm2
is, in fact, their critical target. There is no simple way to test whether
Mdm2 is required, because cells from Mdm2-null mice cannot be
propagated unless they also lack p53 (6, 7). It may well prove that
Mdm2-related Mdm4 (MdmX) or other proteins that interact with
Mdm2 are responsible for the observed effects. In agreement with data
of others (65), we confirmed that the RING finger domain of Mdm2
could interact with Mdm4 and vice versa (data not shown). The exact
role of Mdm4 in inhibiting p53 function remains unclear, but unlike
Mdm2, Mdm4 is not a p53-inducible gene, does not seem to be
expressed at particularly high levels during p53 stress responses, and
although it inhibits p53-dependent transcription, Mdm4 is not thought
to catalyze p53 degradation (34, 39). Disruption of Mdm4 in the
mouse germ-line leads to embryonic lethality accompanied by growth
arrest, but not apoptosis, and these effects are rescued on a p53-null
background (36). Intriguingly, disruption of the Mdm4 gene resulted
in production of a truncated product that likely encodes the RING
domain. This formally leaves open the possibility that embryonic
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Mdm2 ISOFORMS INHIBIT PROLIFERATION
lethality results from a gain of function (overexpression of the Mdm4
RING) versus Mdm4 loss.
Whatever the exact mechanisms, enforced overexpression of the
truncated Mdm2 variants led to p53 activation, which could be reversed by simultaneous overexpression of full-length Mdm2. Similarly, a recent study showed that Mdm2 rescues cell growth arrest
mediated by another Mdm2 binding protein, MTBP (66). MEFs
transduced by Mdm2 variants alone arrested irreversibly after several
days, remained viable for as long as 4 weeks in culture, and assumed
an enlarged and flat morphology reminiscent of senescent fibroblasts.
Interestingly, although p21Cip1 induction in response to Mdm2 variants was relatively robust, the induced levels of endogenous Mdm2
were significantly lower than those usually observed in cells undergoing a p53-dependent stress response. It may be that the ability of
Mdm2 variants to interact directly with full-length Mdm2 somehow
affects the ability of p53 to activate the Mdm2 feedback loop that
normally cancels the p53 response.
Several studies showed that alternatively spliced and mutant Mdm2
variants are found in many types of human tumors, including invasive
breast cancer (47), late-stage and high-grade ovarian and bladder
carcinomas (42), and liposarcomas (48). Although the role of these
Mdm2 variants in the onset or late stages of tumors remains elusive,
their occurrence and persistence in both human and mouse tumors
suggest that they somehow contribute to tumorigenicity. Our results
reveal that when overexpressed in primary MEFs or pre-B cells, these
Mdm2 variants paradoxically inhibit rather than promote cell growth.
We have considered several possibilities to rationalize these results:
(a) it may prove that, similar to activated Ras (67), the enforced
expression of these Mdm2 variants triggers growth arrest in primary
cells, but in collaboration with Myc, promotes proliferation; and (b)
alternatively, expression of truncated Mdm2 variants might allow
certain cells to escape E␮-Myc-induced apoptosis during early stages
of lymphomagenesis, after which subsequent genetic changes then
allow the expansion of this resistant population. However, we found
that enforced expression of Mdm2 variants in early-passage MEFs did
not inhibit Myc-ER-induced apoptosis in response to tamoxifen (data
not shown). Conceivably, the variants might even be induced as part
of a surveillance mechanism to prevent cell proliferation in response
to oncogenic signals, whereas subsequent selection for Mdm2 overexpression, Arf loss, or p53 mutations would bypass their effects,
leaving them as inert vestigial markers during later stages in tumor
development. Whatever the explanation, RING finger domains can act
as potent growth inhibitors.
ACKNOWLEDGMENTS
We thank Dr. John Cleveland for many helpful discussions and critical
review of the manuscript, Jason Weber for initial help designing the oligonucleotides for PCR amplification of the Mdm2 variants from B-cell tumors,
Frederique Zindy for preparing MEFs, and David Randle for preparing mouse
bone marrow-derived pre-B cells. We are indebted to Rose Mathew and
Camulous Hornsby for excellent technical assistance.
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