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g e n o m ic s
43, 3 4 - 4 2 (1997)
a r t i c l e NO. GE9 74775
Isolation and Identification of the Human Homolog
of a New p53-Binding Protein, Mdmx
Avi Shvarts,1 Merlijn Bazuine, Patrick Dekker, Yolande F. M. Ramos, Wilma T. Steegenga,
Gerard Merckx,* Reinier C. A. van Ham, Willemien van der Houven van Oordt,
Alex J. van der Eb, and A. G. Jochemsen2
Laboratory o f Molecular Carcinogenesis, Sylvius Laboratory, Leiden University, P.O. Box 9503, 2300 RA Leiden , The Netherlands;
and * Department o f Human Genetics, University Hospital Nijmegen, The Netherlands
Received November 4, 1996; accepted April 17, 1997
We recently reported the identification of a mouse
cDNA encoding a new p53-associating protein that
w e called Mdmx because of its structural sim ilarity
to Mdm2, a well-known p53“binding protein. Here we
report the isolation of a cDNA encoding the human
hom olog of Mdmx. The ORF of the cDNA encodes a
protein of 490 amino acids, 90% sim ilar to mouse
Mdmx. The homology betw een Mdmx and Mdm2 is
m ost prom inent in the p53-binding domain and the
p u tative m etal-binding domains. The Mdmx protein,
w hich, based on SDS-PAGE, has a MW of 80 kDa, can
bind p53 in vitro. The human MDMX gene is tran­
scribed in all tissues tested, w ith high levels in th y­
m us. By fluorescence in situ hybridisation analysis
w e m apped the mouse mdmx gene to chromosome 1
(region F -G ) and the human MDMX gene to chromosom e lq32. ©1997 Academic Press
INTRODUCTION
The loss of wild-type p53 in human malignancies re­
sults in aberrant cell-cycle progression, escape from
apoptosis, and enhanced angiogenesis, which all con­
tribute to tumor growth. These biological processes are
most likely controlled by p53 through its transcriptionregulating function, which, in human tumors, is usu­
ally altered due to gene mutations (Hollstein et al
1991, 1994), but can also take place via cytoplasmic
retention (Moll et al., 1992, 1995) and by association
with other proteins. Tumor antigens of several DNA
tumor viruses have been shown to inactivate the function(s) of p53, by a tight association (SV40-LT, adenovi­
rus type 5 large E1B protein, HBV X-antigen; Lane
and Crawford, 1979; Linzer and Levine, 1979; Samow
etal., 1982; Zantemaei a l, 1985; Feitelsonei a l, 1993),
1 P resent address: Division of Molecular Carcinogenesis, N ether­
lands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
2 To whom correspondence should be addressed. Telephone; (31) 71
5276136. Fax: (31) 715276284. E-mail: A.G.Jochemsen@biochemistry.
MedFac.LeidenUniv.NL.
0888-7543/97 $25.00
Copyright © 1997 by Academic Press
All rights of reproduction in any form reserved.
by enhanced degradation of the p53 protein (HPV-E6;
Scheffner et a l , 1990; Werness et a l , 1990), or by as
yet unknown ways, possibly involving altered oligomer­
ization of p53 (adenovirus type 12 large E1B, adenovi­
rus type 5 E l A; Steegenga et a l, 1995, 1996). Impor­
tantly, HPV and HBV have been implicated in human
cervical carcinoma and liver carcinoma (Srivastava et
a l, 1992; Ueda et al., 1995), respectively. In addition,
some cellular proteins have been found to bind to p53
and influence its properties as a transcription factor.
First, the Wilms’ tumor protein WT1, a transcription
factor itself, is able to modulate p53-regulated tran­
scription (Maheswaran et al, 1993). Recently, the E2F1
and DPI proteins were also shown to interact directly
with p53 and inhibit its transcription-stimulation func­
tion (O’Connor et al, 1995; Martin et a l, 1995). Last,
p53 was found to complex with the Mdm2 protein
(Barak and Oren, 1992; Momand et a l, 1992). The asso­
ciation of Mdm2 with p53 completely abrogates all
transcription-regulating properties of p53 (Momand et
al., 1992; Chen et a l, 1995). Mdm2 most likely func­
tions by concealing the transactivation domain of p53
(Oliner et al, 1993). Most interestingly, overexpression
of Mdm2 has been found in a variety of tumors, in
general correlating with the absence of mutations in
the p53 gene (Oliner et a l , 1992; Reifenberger et a l,
1993; Habuchi et al, 1994; Lianes et a l, 1994; Corvi
et a l, 1995). Overexpression of Mdm2 is apparently
sufficient for inhibition of the p53 tumor-suppression
function. Thus, inactivation of p53 in human tumors
can be achieved by mutation, cytoplasmic retention, or
overexpression of Mdm2, or by several viral proteins.
We recently reported the identification of a new p53associating protein. The translation product of this
gene, named mdmx, shows significant homology with
the Mdm2 protein, especially in the p53-binding do­
main and in the putative functional domains, located
in the C-terminal part of the protein. We reported that
the murine Mdmx protein binds to p53 in vivo and, like
Mdm2, inhibits the activation of transcription by p53
(Shvarts et a l, 1996). Unlike mdm2, which is a p5334
35
IDENTIFICATION OF HUMAN Mdmx
responsive gene, mdmx transcription appears not to be
regulated in a p53-dependent manner, at least not after
UV irradiation. This might suggest that mdmx is not
a modifier of p53 after UV irradiation. We now report
the isolation of a human cDNA of 2216 bp that encodes
the human Mdmx protein of 490 aa. The mouse and
human Mdmx proteins have 90% identity at the protein
level. We show that the human Mdmx protein binds to
p53 in vitro. By FISH analysis we mapped the mouse
mdmx gene to chromosome 1 region F -G and its hu­
man homolog to chromosome lq32. We report the iden­
tification of a new member of the mdm family that
might be involved in human carcinogenesis.
MATERIALS AND METHODS
Northern analysis. A hum an m ulti tissue N orthern blot (Clontech) was hybridized with a2P-labeled probes according to th e m anu­
facturer’s m anual.
Isolation and sequencing of h u m a n M D M X cDNA. A cDNA li­
brary from the colonic epithelial cell line T84 in a UNI-Zap-XR vector
(Stratagene) was screened with mouse m dm x cDNA probe according
to the protocol supplied by the m anufacturer. pBSK plasm ids con­
taining the hum an M D M X cDNA were obtained w ith R408 helper
phage (Stratagene).
Double-stranded DNA was sequenced by a T7 polymerase sequenc­
ing kit and AutoRead Sequencing k it (Pharmacia), The sequence
reactions were subsequently analyzed m anually on polyacrylam ide
gel and by the ALF (automatic laser fluorescence) DNA sequencing
machine (Pharm acia). All the sequence prim ers were obtained from
Isogene Bio-Science B.V.
Isolation o f genom ic m d m x DNA clones. To obtain genomic mouse
m dm x sequences, we screened a genomic mouse library derived from
129/OLA mice in EMBL3A phage using the stan d ard procedure
(Sambrook et a l, 1989). Subclones were generated by digestion w ith
S a il and by cloning the fragm ents into £>a£I-digested pIC20R vector,
To obtain h u m a n M D M X genomic sequences, library NO. 700 (P I
human), ligated into pAdlOSacBII (Francis et al., 1994), was
screened w ith a fragm ent of approximately 560 bp obtained by RTPCR on mRNA isolated from the hum an tum or cell line G401 (Weism ann et al., 1987) with a prim er set corresponding to n t 4 6 0 -4 7 8 /
1003-1022. The presence of MDMXT-specific sequences in th e geno­
mic clones was established by Southern blot analysis w ith th e use
of specific cDNA probes (data not shown).
In vitro associations between M dm x and p53 . To obtain in vitro
translated Mdmx proteins, the full-length coding regions of mouse
and hum an Mdmx were cloned into pcDNA3 vector (Invitrogen). H u ­
man p53 protein was translated in uiti'o from the modified pET 15b/
p53 vector (Shvarts et ah, 1996) and th e hum an cJu n protein from
a pBAT vector (Annweiler et al., 1991) containing th e h u m an cJun
coding region (pBAT-cJun, a gift from Dr. P. Angel, K arlsruhe, Ger­
many).
In vitro transcription/translation was performed w ith th e use of
the coupled transcription/translation system obtained from Prom ega
(Madison, WI) in th e presence of [3BS]methionine. In vitro tran slated
Mdmx proteins w ere incubated in th e presence or absence of bacterially produced p53 proteins (Shvarts et al.} 1996) for 30 m in on ice.
Subsequent im m unoprécipitations were performed as described pre­
viously (Steegenga et al.} 1995) with a m ixture of th e monoclonal
antibodies PAbl22/PAb421 directed against p53, th e polyclonal rab ­
bit antiserum pAblOO directed against a peptide of mouse Mdmx,
or the polyclonal rabbit antiserum pAb55 raised against full-length
hum an Mdmx.
To obtain G S T -h u m a n Mdmx fusion protein, the to tal coding re­
gion of hum an Mdmx was cloned into vector pRP259, a modified
pGEX expression vector (Smith and Johnson, 1988), A pproxim ately
1 fig of G ST -M dm x, bound to beads, was tumbled for 4 h a t 4°C
with labeled in vitro tran slated p53. Subsequently the beads were
washed three tim es with binding buffer. Im m unoprecipitated and
G ST-M dm x-bound proteins were separated on a 9% SDS-polyacrylam ide gel, prepared for fluorography w ith 22.5% PPO (2,5- diphenyloxazole) in dimethylsulfoxide, dried, and exposed to Kodak XAR-5
film a t -80°C,
Fluorescence in situ hybridization (FISH). F IS H was performed
on mouse cells regenerated from a muscle biopt and on hum an lym­
phocytes. M etaphase spreads were prepared via th e stan d ard proce­
dures. A total of 400 and 150 ng labeled m dm x probe for mouse and
h u m an chromosomes, respectively, and 5Ox mouse Cot I and 5Ox
h u m an Cot I DNA (Gibco, Life Technology) was dissolved in 12 //I of
a hybridization solution (50% v/v deionized form amide, 10 % dextran
sulfate, 2x SSC, 1% v/v Tween 20, pH 7.0). P rior to hybridization,
the probe was denatured at 80°C for 10 min, chilled on ice, and
incubated at 37°C for 30 m in allowing preannealing. After denaturation of the slides, probe incubations were carried out u n d er an 20
x 20 m m coverslip in a moist cham ber for 45 h. Immunocy to chemical
detection of the hybridizing probe was achieved with FITC-conjugated sheep-anti-digoxigenin (Boehringer).
For evaluation of the chromosomal spreads a Zeiss epifluorescence
microscope equipped with the appropriate filter for visualization of DAPI
and FITC fluorescence was used. Digital images were acquired with a
high-performance cooled CCD camera (Photometries, Tucson) and fur­
ther processed on a computer with the help of the BDS-Image FISH
software package (Biological Detection Systems Inc, Rockville, MD).
RESULTS
Molecular Cloning of mdmx cDNA
To identify the human M DM X cDNA we screened
a cDNA library from the colonic tumorigenic cell line
Construction of human MDMX cDNA
AUG
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clone 1
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UAA
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Human mdmx; homologous to human mdm2
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Human mdmx Intron sequence?
F IG . 1- Schem atic representation of hum an M D M X cDNA clones
obtained from a Lam bdaZap cDNA library from colon carcinoma cell
line T84. The Xmn I site in the middle of the cDNA has been used
to obtain the h u m an M D M X cDNA containing th e complete open
reading frame.
36
SHVARTS ET AL.
A
1
61
121
3
181
23
241
43
301
63
361
83
421
103
481
123
541
143
601
163
661
183
721
203
781
223
841
243
901
263
961
283
1021
303
1081
323
1141
343
CGGCACGAGCTAGGATCTGTGACTGCCACCCCTCCCCCCACCCGGGCTCGGCGGGGGAGC
GACTCATGGAGCTGCCGTAAGTTTTACCAACAGACTGCAGTTTCTTCACTACCAAAATGA
M T
60
120
CATCATTTTCCACCTCTGCTCAGTGTTCAACATCTGACAGTGCTTGCAGGATCTCTCCTG
S
F
S T S A Q C S T S D S A C R I S P G
180
22
GACAAAT CAAT CAGG TACGA CCAAAAC TG CCGCT TT TGAAGATTTTG CATG CAGCAGGTG
Q X N Q V R
P K L P L L K I L H A A G A
CGCAAGGTGAAATGTTCACTGTTAAAGAGGTCATGCACTATTTAGGTCAGTACATAATGG
Q G E M F T V K E V M H Y L G Q Y
I M V
TGAAGCAACTTTATGATCAGCAGGAGCAGCATATGGTATATTGTGGTGGAGATCTTTTGG
K Q
L Y D Q Q E Q H M V Y C G G D L L G
240
42
GAGAACTACTGGGACGTCAGAGCTTCTCCGTAAAGAACCCAAGCCCTCTCTATGATATGC
E L
L G R Q S F S V K N P S P L Y D M L
2
300
62
360
82
420
102
TAAGAAAGAATCTTGTCACTTTAGCCACTGCTACTACAGATGCTGCTCAGACTCTCGCTC
R K N
L V T
L A T A T T D A A Q T L A L
TCGCACAGGATCACAGTATGGATATTCCAAGTCAAGACCAACTGAAGCAAAGTGCAGAGG
A Q
D H S M D I P S Q D Q L K Q S A E E
480
122
AAAGTTCCACTTCCAGAAAAAGAACTACAGAAGACGATATCCCCACACTGCCTACCTCAG
S
S
T S R K R T T E D D I P T L P T S E
600
162
AG CATAAATGCATACAT TCTAGAGAAGATGAAGACTTAATTGAAAATT TAGCC CAAGATG
H K C
I
H S R E D E D L
I
E N L A Q D E
AAACATCTAGGCTGGACCTTGGATTTGAGGAGTGGGATGTAGCTGGCCTGCCTTGGTGGT
T S R L D L G F E E W D V A G L P W W F
TTTTAGGAAACTTGAGAAGCAACTATACACCTAGAAGTAATGGCTCAACTGATTTACAGA
L G N L R S N Y T P R S N G S T D L Q T
CAAATCAGGATGTGGGTACTGCCATTGTTTCAGATACTACAGATGACTTGTGGTTTTTGA
N Q D V G T A I V S D T T D D L W F L N
660
182
540
142
720
2 02
780
222
840
242
ATGAGTCAGTATCAGAGCAGTTAGGTGTTGGAATAAAAGTTGAAGCTGCTGATACTGAAC
E
S
V S E
Q L G V G I
K V E A A D T E Q
900
262
AAA CAAG TGAAGAAG TAGGGAAAGT AAGTGACAAAAAGGTGATTGAAGTGGGAAAAAATG
T S
E
E V G K V S D K K V I E V G K N D
960
282
ATGACCTGGAGGACTCTAAGTCCTTAAGTGATGATACCGATGTAGAGGTTACCTCTGAGG
D L E
D S K S L S D D T D V E V T S E D
1020
302
1080
322
1140
342
ATGAGTGGCAGTGTACTGAATGCAAGAAATTTAACTCTCCAAGCAAGAGGTACTGTTTTC
E W Q C T E C K K F N S P S K R Y C F R
GTTGTTGGGCCTTGAGGAAGGATTGGTATTCAGATTGTTCAAAGTTAACCCATTCTCTCT
C W A L R K D W Y S D C S K L T H S L S
CCACGTCTGATATCACTGCCATACCTGAAAAGGAAAATGAAGGAAATGATGTCCCTGATT
T S
D I
T A I
P E K E N E G N D V P D C
1200
362
1201
363
GTCGAAGAACCATTTCGGCTCCTGTCGTTAGACCTAAAGATGCGTATATAAAGAAAGAAA
R R T
I
S A P V V R P K D A Y I
K K E N
1260
382
1261
383
1321
403
1381
423
1441
443
1501
463
1561
483
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
ACTCCAAACTTTTTGATCCCTGCAACTCAGTGGAATTCTTGGATTTGGCTCACAGTTCTG
S
K L
F D
P C N S V E F - L D L A H S S E
1320
402
AAAG C CAAGAGAC CA TCT CAAG CATGGGAGAACAGT TAGATAAC CT TT CTGAACAGAGAA
S Q E T I S S M G E Q L D N L S E Q R T
1380
422
CAGATACAGAAAACATGGAGGATTGCCAGAATCTCTTGAAGCCATGTAGCTTATGTGAGA
D T E N M E D C Q N L L K P C S L C E K
AAAGACCACGAGACGGGAACATTATTCATGGAAGGACGGGCCATCTTGTCACTTGTTTTC
R P R
D G N
X I
H G R T G H L V T C F H
1440
442
1500
462
156 0
482
1620
490
1680
1740
1800
1860
1920
1980
2040
2100
2160
2216
ACTGTGCCAGAAGACTAAAGAAGGCTGGGGCTTCATGCCCTATTTGCAAGAAAGAGATTC
C A R R L K K A G A S C P
I C K K E I Q
AGCTGGTTATTAAGGTTTTTATAGCATAATGGTAGTACGAACATAAAAATGCATTTATTC
L V I K V F I A
AGTTCACTTACCACATTATTTGAAAATCAATCCTTTATTTAATTTTATTTCCAACCTGTC
AGAGAATGTTCTTAGGCATCAAAATCCAAGGTAGCTGTAAGAAAAATACTGGAGCTAACA
ATG AAGAACAGAAGTAAT CTGATTAGT CAAATTATTAAGTG CCATGGATTACTTTATGCA
GCAGTCAGGTACATAGTTAGGTGAACCCAAAAGAAAAACTCTTGAAAACAAGAGATTTCT
TCCATGCACATTTACAATATTGAGGTATAATTAACATGATAAAGTGTTTCCTTCTAACGA
GTTGTAGAAATCTGAGTAACCACCCAAAAAAGCAATAGAATGTTTGTGTCACCCCAAAAC
ACTCCCTTCTGCCCCTCTTCAGACAGTCCTTCAGCTATTTCATGGCTCTCACCCTAGTTT
TTTTTTTTTTTTGCACTTTTTTTTTTCCGGGGGTATAGGGGAGGTGTGGGGOGACAGGGT
CTGTCTTGTTCTGTCTCCCAGGCTGAAGTGCAGTGAGTCAAGATTGAGCCACTGCACTCC
AG CCTGGGTG ACAG CGCGAGACTC CATCTCAGAAAAAAAAAAAAAAAAAAAAAAA
P IG . 2. Nucleotide an d derived am ino acid sequence of h u m a n MDMX. (A) Nucleotide sequence of M D M X cDNA and predicted protein
sequence The putative AUG codon is un derlined and boldface; putative alternative translation s ta rt sites are shown in boldface. Conserved
cysteines in th e putative zinc-iinger a n d in the RING finger domains together with the core amino acids are given in boldface italics, and
a p u tativ e nuclear localization signal is underlined. (B) Sequence alignment of Mdm-like proteins.
T84 (Stratagene) with a mouse m dm x cDNA probe
(see Materials and Methods). Two independent phage
clones, which remained positive after three rounds
of purification, were isolated and purified to homoge­
neity. Sequence analysis showed inserts ofl260 and
2260 bp (clone 1 and clone 2, respectively, in Fig. 1).
The shortest construct shows a strong homology with
the 5' part of the previously identified mouse m d m x
cDNA (Shvarts et aL, 1996), including the putative
translation start site. Sequencing of the longest con­
struct revealed that in the 3' part of the insert a
stretch of 1143 bp shows a strong similarity with the
37
IDENTIFICATION OF HUMAN Mdmx
B
1
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
Hum an
M ouse
Hum an
M ouse
MDMX
MDMX
MDM2
MDM2
H um an
M ouse
H um an
M ouse
MDMX
MDMX
MDM 2
MDM2
M .. TSFSTSA
M . . TSHSTSA
MCNTNMSVPT
MCNTNMSVST
81
DLLGELLGRQ
DLLGDLLGCQ
D LL G D LFG V P
DLLGDVFGVP
161
TLPTSEHKCI
TLPTSRHKCR
RPSTSSRRRA
RLSTSSRRRS
241
SDTTDDLW FL
SD TTD D LW FL
E H S G D . . WLD
E H S G D . .C L D
321
TECKKFNSPS
TECKKFNSPS
T SC N E M N P P L
TSCNEMNPPL
401
LFDPCNSVEF
. FDPCNSVGF
IT Q A SQ SQ ES
AEQTPLSQES
481
FH C A R R L K K A
FH C A R R L K K S
FTCAKKLK KR
F T CAKKLKKR
QCSTSDSACR ISPG Q IN Q V R
QCSASDSACR I S S E Q I S Q V R
D G A V T T S Q IP A S . E Q E T L V R
E G A A ST SQ IP AS . EQETLVR
SFSVKNPSPL
SFSVKDPSPL
SFSVKEHRKI
SFSVKEHRKI
YDMLRKNLVT
YDMLRKNLVT
Y TM IY R N LW
YAM IY RN LVA
AAGAQGEMFT
AAGAQGEVFT
SVGAQKDTYT
SVGAQNDTYT
VKEVMHYLGQ
MKEVMHYLGQ
MKEVLFYLGQ
M K EIX FY IG Q
YIMVKQLYDQ
YIMVKQLYDQ
YIM TKRLYDE
Y IM T K R L Y D E
LATATTDAAQ TLALAQDHSM
SASNNTDAAQ TLALAQDHTM
V N Q Q E SS D S G T SV SE N R C H L
V S Q Q . . . DSG T S L S E S R R Q P
D I P S Q . . DQL
D F P S Q . . DRL
E G G SD Q K D L V
EGGSDLKDPL
KQSAEESSTS
K H G A T E Y SN P
QELQEE. . . .
CAP P E E . . . .
P K L P L L K IL H
PK L Q L L K IL H
PK PLLLK LLK
PK PLLLK LLK
H SR E D ED L IE N LA Q D E T 5R .
. . LDLG FE EWDVAGLPWW F L G N L R S N Y T P R S N G S T D L Q
D S R A D E D L I E HLS Q D E T S R .
. L D L D F E EWDVAGLPWW F L G N L R N N C I P K S N G S T D L Q
I S E T E E N S D E LSGERQRKRH K S D S I S L S F D E S L A L C V I R E I C C E . . . . . R S S S S E S T G T P
I S E T E E N T D E LPGERHRKRR R . . . . S L S F D P SL G L C E L R E M C S G G T S S S S S S S S E S T E T P
NESVSEQLGV
N E T V SE Q L G V
QDSVSDQFSV
QDSVSDQFSV
G IK V E A A D TE
G IK V E A A N S E
EFEVESLDSE
EFEVESLDSE
Q T S . . . . . EE
Q T S ................ E
D Y SL SE E G Q E
D Y SL SD E G H E
VGKVSDKKVI
VGKTSNKKTV
LSDEDDEVYQ
LSD ED D EV Y R
EVGKNDDLED
EVGKDDDLED
V T V Y Q A G E SD
VTVYQTGESD
SKSLSDDTDV
SRSLSDDTDV
TDSFEEDPEI
TDSFEGDPEI
KRY CFRCWAL
KRYCFRCWAL
PSHCNRCWAL
PSHCKRCWTL
RKDWYSDCSK
RKDWYSDCSK
RENWLPEDKG
RENWLPDDKG
LTHSLSTSDI
LTHSLSTSNI
K D K G E IS E K A
K D K V E IS E K A
T A IP E .K E N E
T A IP E K K D N E
K L E N ST Q A E E
KLENSAQAEE
GNDVPDCRRT
GXDVPDCRRT
GFDVPDCKKT
GLDVPDGKKL
ISA P W R P K D
ISA P W R P K D
I . . . VNDSRE
T . . . ENDAKE
LDLAHSSESQ
LDLAHSSESQ
EDYSQPSTSS
DDYSQPSTSS
ETISSM G EQ L
E IISSA R E Q T
SIIY SSQ E D V
SIV Y SSQ E SV
D N L S E Q R T D . . TENM EDC. .
D IFSE Q K A E . . T E S M E D F ..
R E FE R E E T Q D K E E S V E S S L P
K E L .K E E T Q H K D E S V E S S F S
QNLLKPCSLC
QNVLKPCSLC
L N A IE PC V IC
L N A IE PC V T C
EK R PR D G N II
E K R PR D G N II
Q G R P K N G C IV
Q G R P K N G C IV
80
QEQHMVYCGG
QEQHKVYCGG
K Q Q H IV Y C SN
K Q Q H IV Y C SN
160
R K R TT ED D IP
RK R TEEEDTH
. KPSSSHLVS
. K P SSSD L IS
240
T N Q D V G T A IV
T N Q D IG T A IV
SNPDLDAGVS
SHQDLDDGVS
320
EVTSEDEW Q C
E L T SE D E W Q C
S L A . . DYWKC
S L A ..D Y W K C
4 00
A Y IK K E .N S K
G Y L K E E . KPR
S C V E E N . DDK
PCAEEDSEEK
480
HGRTCJiLVTC
H G K T SH L T T C
HGKTCHLMAC
HGKTGHLMSC
510
G A SC PICK K E
GA SC PV CK K E
NKPCPVCRQP
NKPCPVCRQP
IQ L V IK V FIA
IQ L V IK V FIA
IQ M IV L T Y FP
IQ M IV L SY F N
F IG . 2 — C ontinued
3' part of the mouse m dm x cDNA, including the
translation stop codon. Clone 1 and clone 2 showed
a 700-bp identical sequence (Fig. 1). Comparing these
available sequences with the mouse m dm x cDNA and
the human M DM 2 cDNA sequence suggested that
these 700 bp are a real overlap, allowing us to gener­
ate a human M D M X cDNA with the use of an unique
X m n l recognition site present in the 700-bp overlap.
RT-PCR analysis on mRNAs from normal human
cells and a human tumor cell line with primers from
outside the overlapping region yielded a fragment of
a length expected from the combined cDNA (data not
shown). The human M D M X cDNA is at the 3' end,
approximately 500 bp longer than the thus far identi­
fied mouse m d m x cDNA because of a longer 3 '-un­
translated region. This 3' UTR is similar to that of
the human M DM 2 cDNA in that it contains an Alu
repeat as has been described for the MDM2 cDNA.
This human M D M X cDNA contains an open reading
frame of 490 amino acids with strong homology to
the mouse Mdmx protein sequence (Fig. 2A).
At the moment we do not know for sure the exact
nature of the 5' part of the cDNA insert of clone 2.
However, we propose this part to be intron sequences,
because comparison with the genomic organization of
the mouse m dm 2 gene indicates that the junction be­
tween the unknown 5' sequences and the Mdmx-homology region is exactly at a splice acceptor site (Jones et
al., 1996; Montes de Oca Luna, 1996). Another explana­
tion for the presence of these sequences is not excluded.
Sequence alignment of human M DM X with human
MDM2 revealed, analogous to mouse mdmx, , a signifi­
cant homology at both the DNA and the protein levels.
Figure 2A shows the cDNA sequence of human MDMX
and the putative open reading frame, encoding a pro­
tein of 490 aa. The sequence alignment of the Mdmlike proteins is shown in Fig. 2B. All proteins share the
p53-binding site, which is located in their N-terminus
(Chen et al., 1995; Brown et al., 1993; Haines et al.,
1994; Shvarts et al., 1996). The Mdm2 mRNAs contain
additional AUG codons at positions +50 and -1-62,
which have been shown to be real alternative transla­
tion start sites in both in vivo and in vitro translation
assays (Barak et al., 1994; Zauberman et al., 1995),
These AUGs are also present in the mdmx sequence at
approximately the same positions (+46, +53, and +61).
We have no direct evidence that they function as alter­
native starts in the MDMX mRNA, although in vitro
translation yields protein products of the expected size,
but also of a smaller size (see below). It suggests that
the MDMX mRNA might give rise to different protein
products, as has been described for Mdm2. At the Cterminus the putative metal-binding domains, residues
306 to 323 and residues 439 to 479 in the human Mdmx
protein, are highly conserved between the different
Mdm family members. The latter domain has recently
been reported to be structurally related to a zinc-bind­
ing domain called the RING finger (Boddy et al., 1994).
All Mdm proteins contain a putative nuclear localiza­
tion signal (Figs. 2A and 2B). However, the nuclear
SHVARTS ET AL.
38
ut
v4>
on
kb
?
9
P "t
r
,
A
A
« v
i
J—L ?
d
fi
u
J
*
«
« S £ 50
VI
» »S yI
1 J__L
g
_L
9.5 .
In Vitro Interaction between Mdmx and p53
7.5 4.4 2.4
1.35
9.5 .
é •
7.5 4.4 -
in pancreas and lung (Ladanyi et al., 1993). A 5.5-kb
MDM2 transcript was detected, but we find the MDM2
gene to be almost equally expressed in all tissues on
this blot with only a somewhat higher expression in the
thymus, but certainly not as prominent as the MDMX
transcript.
A&:
*
>>7 '
:
- ïïy k : .
'■ -%
r
2.4
1.35
9.5 _
7.5 4.4 2.4
¿\*
.Si?-
1.35
FIG« 3. Expression of MDMX mRNA in human tissues, (A)
N orthern blot containing poly(A)+-selected mRNA from human tis­
sues was probed with hum an MDMX cDNA. (B) The same blot was
probed with human MDM2 cDNA. (C) Human /?-actin cDNA was
used as control for the amount of mRNA loaded. Positions of molecular-weight markers (kb) are indicated.
localization signal of the Mdm2 protein is found at posi­
tions 178 -183 (RKRHK), in the middle part of the pro­
tein, whereas in the human Mdmx protein it is found
at positions 442-445 (KRPR) in the C-terminal part
Expression of MDMX mRNAs in Human Tissues
To determine the expression pattern of human
MDMX mRNA we performed Northern blot analysis on
a human multi tissue blot. Figure 3A shows that a
10-kb human MDMX transcript is synthesized in all
tissues, with the highest amount in the thymus and
a very low expression in colon. An additional shorter
transcript of approximately 2.2 kb is detectable in tes­
tis. We do not know the exact nature of the different
mRNAs, but the mRNA expression pattern of the
mouse and the human is rather conserved and will be
discussed in more detail later. Furthermore, expression
of human MDMX mRNA was found in all tissues and
cell lines tested by RT-PCR (data not shown). We com­
pared the expression of human MDMX with that of
MDM2 by reprobing the same blot with an MDM2
probe (Fig. 3B). The human MDM2 gene was pre­
viously shown to be expressed in a 5.5-kb transcript,
first identified by Oliner et a l (1992) predominantly in
skeletal muscle and liver, with somewhat lower levels
We have shown previously that mouse Mdmx can
bind p53 in vivo (Shvarts et a l , 1996). To investigate
the in vitro interaction between human and mouse
Mdmx and human p53, we performed two types of ex­
periments. First, we mixed in vitro translated, 3GS-labeled Mdmx with bacterially produced and purified p53
protein (see Materials and Methods). After this incuba­
tion immunoprécipitations with anti-p53 and antiMdmx antibodies were performed.
The results presented in Figs. 4A and 4B demon­
strate that in the presence of p53 protein significant
amounts of the in vitro translated human and mouse
Mdmx proteins are coimmunoprecipitated with a mix­
ture of the anti-p53 antibodies PAb 122/PAb 421. Fur­
thermore, it seems that only the upper band of mouse
Mdmx, which is probably generated from the first in­
frame AUG, interacts with p53, suggesting that the
protein lacking the N-terminal part does not bind p53.
Although difficult to see in Fig. 4, the same seems to
be true for the human Mdmx. To verify the in vitro
binding association between human Mdmx and p53, a
GST-Mdmx fusion protein was coupled to glutathione
beads and incubated with 35S-labeled, in vitro trans­
lated human p53 or cjun as a control. As can be seen in
Fig* 4C, the GST-Mdmx protein specifically interacts
with human p53 and not with cJun, while GST alone
binds neither protein.
We conclude from these results that both human
and mouse Mdmx proteins associate with p53 protein
in vitro and that the binding domain is probably lo­
cated in the extreme N-terminal part of the Mdmx
proteins.
Chromosomal Mapping of the mdmx Genes
Amplification of the human MDM2 gene has been
observed in a variety of tumors (Oliner et a l , 1992;
Reifenberger e t a l , 1993;Habuchie£ a l } 1994; Lianes
e t a l 3 1994; Corviet a l , 1995), Since the MDMX gene
might be present in amplicons described in human
tumors, we set out to localize the gene. To begin with,
the mouse mdmx gene was mapped with FISH to
chromosome 1, region F -G (Lyon and Searle, 1989;
Figs. 5A and 5B). This region is in synteny with a
region on the long arm of the human chromosome 1.
This possible location of the human MDMX gene was
verified by FISH analysis with the PI clone con­
taining human MDMX sequences. The human
MDMX gene was mapped to human chromosome
lq32 (Figs. 5C and 5D), This region was recently re­
ported to be amplified in a subset of liposarcomas
39
IDENTIFICATION OF HUMAN Mdmx
A
^proteins
MMDMX
IVMDMX+ p53
Ab's
a-MMDN/K
+
+
V
N.I. mouse
+
+
a-p53
+
.' ' '
t»
B
+
' ' ^,
'
*
"\j3ro teins
HMDMX + p53
HMDMX
Ab's
oc-HMDMX
N.I. mouse
+
+
+
+
C
10% Input
a-p53
GST
GST-HMDMX
+
cJun
p53
+ cJun
+ p53 + cJun
t •«
+ p53
\
•
%
1
i
FIG. 4. In vitro association between Mdmx and p53. (A, B) Im m unoprécipitations on a m ixture of in vitro tran slated , 3(5S-labeled Mdmx
proteins and bacterially produced p53 w ith a mouse Mdmx-specific polyclonal antibody pAblOO (aMMDMX), a polyclonal antibody recognizing
human Mdmx pAb55 (aHMDMX), or a m ixture of PAb 122/421 (crp53); nonim m une mouse antibody (N.I. mouse) was used in control
immunoprécipitations as indicated. (C) G ST-H M D M X fusion protein or only GST bound to glutathione beads w as mixed w ith eith er in
vitro translated ,r}BS-labeled p53 protein or cJun protein. After th is incubation th e beads were washed and bound proteins were separated
on SDS-PAGE.
(Szymanska et ah, 1996; Forus et al., 1995). The pos­
sible amplification of the M D M X gene in these tu ­
mors is currently under investigation.
D IS C U S S IO N
We report here the isolation and identification of the
human counterpart of the mouse mdmx gene. The hu­
man MDMX gene encodes a protein of 490 aa that is
90% homologous to mouse Mdmx (Shvarts et aL, 1996),
The amino acid sequence predicts a protein with a theo­
retical molecular weight of 54 kDa. However, on S D S PAGE human Mdmx was found to migrate as a protein
with a molecular weight of approximately 80 kDa. This
behavior corresponds to the observation that the Mdm2
protein also runs at a much higher molecular weight
on SDS-PAGE than expected from its amino acid se­
quence. We expect the Mdmx amino acid sequence to
be as presented here, since upstream from the putative
AUG, in-frame stop codons are found. The apparent
molecular weight of human Mdmx is somewhat larger
than that earlier found for mouse Mdmx. This might
be caused by different posttranslational modifications,
e.g., phosphorylation, which is known to occur on the
Mdm2 proteins.
A sequence comparison between human MDMX and
human MDM2 reveals conservation that is strongest
in the p53-binding domain located in the N-terminal
part of both proteins. Furthermore, putative functional
domains in the C-terminal part of the proteins are
highly conserved. These domains consist of a zinc finger
and a zinc-binding domain called the RING finger. The
latter is thought to be involved in protein-protein in­
teraction (Boddy et ah, 1994).
SHVARTS ET AL
40
D
Mouse chromosome 1
FIG . 5 . Chromosomal localization of th e m dm x genes by fluorescence in situ hybridization. (A, C) Mapping of the m dm x gene on mouse
and hum an metaphase chromosomes, respectively» Probe DNA, light spots; chromosomes are counter stained w ith DAPI. (B, D) Giemsalike banding to identify the chromosome and localize the probe on mouse chromosome 1 F - G and on hum an chromosome lq32. At least
20 informative chromosome spreads were used to determine the localization of the mdmx. genes.
By Northern blot analysis we detect a 10-kb tran­ primed mRNAs. Second, an entry in the NCBI EST
script of human MDMX in most tissues, with a rela­ database of 275 bp (L44283), derived from human thy­
tively high expression in thymus, Also mouse thymus mus RNA, is identical over a stretch of 113 bp with the
was found to express relatively high levels of the mu­ human MDMX cDNA sequence shown here (Fig, 6).
rine mdmx mRNAs (data not shown). The murine From studies on the genomic organization of the mouse
mdmx gene is expressed prominently into two long gene we know this to be exon 2. Both 5' and 3' of these
mRNAs of approx. 10 and 7.5 kb in all tested tissues exon sequences the sequence of the EST diverges from
and a mRNA of 2.2 kb, which is highly expressed in the cDNA sequence (Fig. 6), but now shows similarity
testis and to a very low level in other tissues (Shvarts with intron sequences of the mouse mdmx gene (not
et a/., 1996). An MDMX mRNA of approx. 2.2 kb is also shown). These results already suggested that unspliced
found in human testis. This mRNA is not detected in or not fully spliced mdmx mRNAs are expressed. In
the other tissues, but if the expression of this mRNA addition, we performed an RT-PCR assay on mouse
in the other tissues is as low relative to the longer thymus mRNA with one primer derived from exon 2
mRNA as in mouse tissues, it might be a detection and another from the following intron. A product of
problem. Thus, the mRNA expression patterns of the the expected length could be detected, again suggesting
human and mouse mdmx genes are rather similar, with that (partly) unspliced mdmx mRNAs are expressed.
The 5' end of cDNA most likely represents the ap­
the possible exception of the shorter mRNAs in tissues
other than testis. It is not firmly established whether proximate 5' end of the mRNA, as indicated by 5'the mRNAs detected all contain the coding region here RACE experiments (Shvarts et aL, 1996) and because
presented. However, we hypothesize that the longer in the mouse genome just upstream of this region pro­
hybridizing RNA band represents not (fully) processed motor activity was found (R.C.A. van Ham and A.G.
mRNA because of the following reasons. First, the Jochemsen, unpublished results). From these results
cDNA clone 2 we isolated most likely contains intron we hypothesize that the long mRNAs represent not
sequences, while the library was made from oligo(dT)- fully processed mRNA and that the cDNA sequence we
hu
EST
hu
ATGTGTCATATG TGTGTTTTACTTCTGCTGGTTGCCTTTGTGTGAATGCTAAATAGGGAATTTTCTGCATTAGAATAGATGTTATAAA TTTTTTTTTCTATTTAG
mdmx
EST
TTTTACCAACAGACTGCAGT
II!I II III I! IIIM I
EST
hu
1-GGCACGAGCTAGGATCTGTGACTGCCACCCCTCCCCCCACCCGGGCTCGGCGGGGGAGCGACTCATGGAGCTGCCGTAAG
mdmx
TACCAACAGACTGCAGT
mdmx
CT CACTACCAAAATGACATCATT
ÍII
CAC
m
u
ACCAAAATGACATCATT
llllll
CCACC CTGCTCAG GT CAACA
GACAGTGC
Mill
Mil Mil
CCACCTC GCTCAG
Mi l l
II
GT CAACA
GACAGTGC
GCAGGATC
I
iT
M
GCAGGAl
CC GGACAAA CAA CAG
U
M i l l II
GGACAAA CA
u
GTACGACCAAAACTGCCGCTTTTGAAGATTTT GCATGCAGCAGGTGCGCAAGGTGA
GTAAATCATTTTCGGTATTTCTAGTTTTTTGG TTTTTTTTTTTTTAAATTTTAAAA
FIG . 6 . Alignment of the 5 '-part of the hum an MDMX cDNA sequence with an EST from the NCBI database (Accession No. L44283).
IDENTIFICATION OF HUMAN Mdmx
now show is a representation of the smaller mRNA of
2.2 kb, although other possibilities cannot be excluded.
Thus, although we find the human MDMX 10-kb
mRNA to be highly expressed in thymus, it remains to
be seen whether this has consequences at the protein
level. It is not excluded that the unspliced mRNAs also
code for Mdmx-like proteins, since in clone 2 an inframe ATG codon is present just eight amino acids in
front of the Mdmx-homology region. It will be interest­
ing to find out whether this relatively high mRNA ex­
pression will turn out to be important in the differentia­
tion of T-cells or be reflected in a role of MDMX (over)expression in hematological malignancies, as has been
reported for MDM2 (Bueso-Ramos e t a l ., 1993; Maestro
et al., 1995; Zhou et aL} 1995). MDM2 amplification is
found in approximately 30% of human sarcomas
(Oliner et al., 1992). A recent analysis of liposarcomas
shows 50% amplification in 12ql4, most probably in­
volving MDM2 . In several liposarcomas (4/14) amplifi­
cation around lq32 has also been found, possibly indi­
cating involvement of M DM X (Szymanska et al., 1996).
Whether the M D M X gene is present in this amplicon
is currently under investigation.
ACKNOW LED GM ENTS
We th a n k Dr. R. V ersteeg for screening of th e P I library. We also
th a n k Dr. H.van D am for critical comments and suggestions, Drs.
C. T erleth and H. v an O rm ondt for critically reading th e m anuscript,
and Ron Schouten for photography.
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SHVARTS ET AL.
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