ACCELERATED PUBLICATION
Pex19p Dampens the p19ARF-p53-p21WAF1 Tumor Suppressor
Pathway*
Takashi
Sugihara
,
Sunil C.
Kaul§,
Jun-ya
Kato¶,
Roger R.
Reddel
,
Hitoshi
Nomura
, and
Renu
Wadhwa
**
From the
Chugai Research Institute for Molecular
Medicine, 153-2 Nagai, Niihari-Mura, Ibaraki 300-41, Japan, the
§ Institute of Molecular and Cell Biology, National
Institute of Advanced Industrial Science and Technology (AIST),
1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, the
¶ Graduate School of Biological Sciences, Nara Institute of
Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan,
and the
Children's Medical Research Institute, Westmead, New
South Wales 2145, Australia
Received for publication, January 11, 2001, and in revised form, March 1, 2001
 |
ABSTRACT |
We isolated a 33-kDa protein, Pex19p/HK33/HsPXF,
as a p19ARF-binding protein in a yeast two-hybrid screen. We
demonstrate here that Pex19p interacts with p19ARF in the cell
cytoplasm and excludes p19ARF from the nucleus, leading to a concurrent
inactivation of p53 function. Down-regulation of Pex19p by its
antisense expression resulted in increased levels of p19ARF, increased
p53 function, and a p53/p21WAF1-mediated senescence-like cell cycle
arrest. The data demonstrated a novel mechanism of down-regulation of the p19ARF-p53 pathway.
 |
INTRODUCTION |
The INK4a (MTS1, CDKN2) locus on
chromosome 9p21 is frequently altered in human cancers. It encodes two
unrelated tumor suppressor proteins: p16INK4a, an inhibitor of the
cyclin D-dependent kinases that acts upstream
of pRb, and p19ARF, an alternative reading frame protein that acts
upstream of p53 (1-3). Both of these proteins have roles in
replicative senescence and ras-induced premature
senescence of primary cells (4-7). Analysis of p19ARF knock-out mice
suggested that this protein functions as a tumor suppressor (3,
8, 9). Recently, it has been shown that p19ARF acts by obstructing
degradation and transcriptional silencing of p53 by
mdm2 (10, 11). It retains Mdm2 in the nucleolus, preventing its export to the cytoplasm, which is required for mdm2-mediated p53 degradation (12-16). Because p19ARF
shares no amino acid homology with known proteins and lacks any
decisive functional protein motifs, other cellular factors that might
regulate its activity and thereby its execution of growth arrest via
the p19ARF-p53 pathway remain poorly defined. Using a yeast interactive screen, we have identified the farnesylated protein Pex19p/HK33/HsPXF (essential for peroxisomal biogenesis) (17-19) as a p19ARF-binding protein. In the present study, we report that the two proteins interact
in the cell cytoplasm leading to exclusion of p19ARF from the nucleus
and inactivation of p53 function, which constitutes a novel mechanism
of down-regulation of the p19ARF-p53 pathway. Neutralization of the
Pex19p function by its antisense expression led to an accelerated
activation of p19ARF function and p53-p21WAF1-mediated cell cycle
arrest that resembled cellular senescence.
 |
MATERIALS AND METHODS |
Yeast Two-hybrid Screen--
cDNAs encoding full-length
p19ARF (p19ARF-F), amino-terminal 80 amino acids (p19ARF-N) and
carboxyl-terminal 89 amino acids (p19ARF-C) were cloned into the
BamHI, SalI site of the yeast expression vector
pODB8 (a kind gift from O. Louvet) (20). For library screening the
yeast reporter strain PJ69/2A
(Trp
/Leu
/His
) was
sequentially transformed with the plasmid pODB8/p19ARF and a human
testis cDNA library (CLONTECH) according to the
manufacturer's protocol. The cDNA-derived plasmids were recovered
from yeast and reintroduced into the yeast reporter strain Y187 to
confirm specificity of the interactions. To determine
-galactosidase activity in yeast, five colonies of simultaneously transformed Y187
yeast cells were grown overnight in Leu
/Trp
plates. Cell extracts were prepared using standard conditions, and
enzyme activity was determined using the GAL-Tropix kit according to
the manufacturer's protocol (Tropix Inc.). The clones were sequenced
using an ABI sequencer (PerkinElmer Life Sciences).
Cell Culture and Transfections--
Mouse embryonic fibroblasts
and monkey kidney cells were cultured in Dulbecco's modified Eagle's
minimal essential medium supplemented with 10% fetal bovine serum.
Transfections were performed using LipofectAMINETM (Life Technologies,
Inc.). Typically, 1 µg of plasmid DNA was used per well of a 24-well
dish, and 3 µg was used per 6-cm dish.
Plasmid Constructions--
Full-length Pex19p was cloned
from mouse testis by reverse transcription-polymerase chain
reaction using sense (5'-gaa ttc atg gcg gct gct gag gaa ggt-3')
and antisense (5'-gtc gac tca cat gat cag aca ctg ttc-3') primers with
EcoRI and SalI sites, respectively. Polymerase
chain reaction amplification product (94 °C for 30 s, 55 °C
for 30 s, and 72 °C for 3 min) was purified and sequentially
ligated to pGEM-T Easy (Promega), pEGFPC1
(CLONTECH), and pcDNA4/HisMax (Invitrogen)
vectors. Mouse p19ARF and its deletion mutants were cloned into the
indicated vectors by a similar strategy. The integrity of the plasmids
was confirmed by sequencing.
Mammalian Two-hybrid Analysis--
COS-7 cells were
seeded at 50-60% confluence in 24-well plates and transfected with 1 µg of DNA containing pG5 reporter plasmid, pM/mHK33, VP16/p19ARF, and
pM or VP16 control vectors as indicated in the relevant figure legends.
After 3 h of transfections, cells were refed with fresh medium and
then lysed in universal lysis buffer (Promega) after 48 h.
Luciferase activity was measured using the
Dual-LuciferaseTM reporter assay system (Promega). The
results presented are the means of at least three transfections.
In Vivo Coimmunoprecipitation--
Cell lysates (400 µg of
protein) in 400 µl of Nonidet P-40 lysis buffer were incubated
at 4 °C for 1-2 h with an antibody used for immunoprecipitation, as
indicated in figure legends. Immunocomplexes were separated by
incubation with protein A/G-Sepharose, Western blotting was performed
with the indicated antibodies by standard procedures, and detection was
done using ECL chemiluminescence.
Reporter Assays--
NIH 3T3, NIH-ARF,
NIH-ARF/pcDNA4-HisMAX- Pex19p (sense), and
NIH-ARF/pcDNA4-HisMAX-Pex19p (antisense) derivatives (selected in 1 mg/ml zeocine, Invitrogen) were transfected with the p53-responsive luciferase reporter plasmid, PG-13luc (kindly provided by Dr. Bert
Vogelstein). As a control, pRL-TK vector (Promega) was co-transfected in each assay to correct for variations in transfection efficiency. Cells were lysed and measured for luciferase activity as described above.
 |
RESULTS AND DISCUSSION |
To isolate p19ARF interacting proteins, a Gal4BD (Gal4 binding
domain)-p19ARF fusion protein was used as a bait to screen a library of
human cDNAs cloned into a Gal4AD (activation domain) yeast
two-hybrid plasmid. One of the five clones isolated was strongly
positive as determined by His prototrophy and induction of
-galactosidase expression. The nucleotide sequence of this clone
revealed its identity to the 33-kDa farnesylated protein Pex19p/HK33/HsPxF/mPxF, which is essential for peroxisomal biogenesis (17-19, 21). The amino- and carboxyl-terminal halves of p19ARF were
cloned into the Gal4BD vector. Pex19p, either full-length or carboxyl-
but not amino-terminal, strongly activated the
-galactosidase reporter, indicating that it is the carboxyl terminus of p19ARF that
interacts with Pex1p (Fig.
1A). Pex19p and p19ARF
interacted strongly in a mammalian two-hybrid assay (Fig.
1B). To further detect their interaction in mammalian cells,
we transfected COS-7 cells with expression plasmids encoding tagged
Pex19p and p19ARF and found that the proteins coimmunoprecipitated
(Fig. 1C).
GFP1-tesmin and MPD-myc were
used as respective unrelated negative controls. As seen in Fig.
1C (left panel), GFP-Pex19p coprecipitated with
p19ARF-myc (immunoprecipitated with anti-myc antibody) but not with
MPD-myc or with isotype-matched control antibody (Fig. 1C,
cf. lanes 1 and 4). GFP-tesmin (negative control)
did not precipitate with p19ARF-myc. Furthermore, immunoprecipitation of hemagglutinin (HA)-tagged p19ARF from NIH-ARF cells with anti-HA tag
antibody resulted in coprecipitation of a protein at ~35-kDa, along
with several others including Mdm2 (22). It is likely that the
35-kDa p19ARF coprecipitate is Pex19p.

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Fig. 1.
p19ARF interacts with Pex19p.
A, activation of yeast two-hybrid - galactosidase
reporter by Pex19p and either the full-length p19ARF or its
carboxyl-terminal half. Yeast cells were transformed with plasmids
encoding Pex19p and full-length (F), amino-terminal
(N), or carboxyl-terminal (C) p19ARF.
-Galactosidase ( -Gal) activity was measured
by liquid assay. B, activation of mammalian two-hybrid
luciferase reporter by transfection of cDNAs encoding mouse Pex19p
and p19ARF in COS-7 cells. C, in vivo
coimmunoprecipitation of Pex19p and p19ARF. Cells were transfected with
plasmids encoding a GFP-Pex19p fusion protein or GFP-tesmin (a negative
control) and myc epitope-tagged p19ARF
(p19ARF-myc) or MPD-myc (a negative control).
Immunoprecipitation was performed with a polyclonal anti-myc
antibody, and the myc immunocomplexes were analyzed for the
presence of GFP-Pex19p or GFP-tesmin by Western blotting with a
monoclonal anti-GFP antibody. GFP-Pex19p coprecipitated with p19ARF-myc
(lane 1) but not in the absence of p19ARF-myc (lane
3), and not with MPD-myc (lanes 4 and 5) or
control isotype-matched antibody (con.IgG). GFP-tesmin was
not precipitated with p19ARF-myc. Input panel shows the
signal from 10% of the lysate used for immunoprecipitation.
|
|
To determine whether Pex19p and p19ARF colocalize within intact cells,
we visualized the proteins by immunostaining COS-7 (not shown) and NIH
3T3 cells transfected with tagged p19ARF and/or Pex19p. p19ARF
localized mainly in the nucleolus, but there was some diffuse staining
in the cytoplasm (Fig. 2A,
a and b). Pex19p was seen only in the cytoplasm
(Fig. 2A, c). Most notably, cells expressing both
p19ARF-myc and GFP-Pex19p showed p19ARF in the cytoplasm in more than
90% of cells (Fig. 2A, d) where it colocalized with Pex19p (Fig. 2A, d-f) suggesting that the
overexpression of Pex19p causes nuclear exclusion of p19ARF (Fig.
2A). As p19ARF has been shown to bind to and inactivate
mdm2 in the nucleus (11, 16) resulting in activation of p53,
we predicted that exclusion of p19ARF from the nucleus would lead to
inactivation of p53 function via increased mdm2-mediated
degradation.

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Fig. 2.
Pex19p sequesters p19ARF in the
cytoplasm and causes inactivation of p53 function. A,
immunolocalization of p19ARF (a, b, and d) and
Pex19p (c and e). NIH 3T3 cells were transfected
with one (upper row) or two (lower row)
plasmids encoding the proteins indicated and were stained with
tag-specific antibodies. Pex19p was seen only in the cytoplasm
(panels c and e) with or without p19ARF. p19ARF
was localized in the nucleolus and cytoplasm in the absence
(a and b) and in the cytoplasm only in the
presence (d) of overexpressed Pex19p. B, NIH 3T3
cells transfected with increasing amounts of plasmid encoding V5-tagged
p19ARF showed up-regulation of p53-dependent luciferase
activity (an expected activity) (a). Cotransfection of
plasmid encoding Pex19p with p19ARF blocked the p19ARF-induced
up-regulation of p53-dependent reporter activity
(b). The induction of p19ARF in NIH-ARF cells by
ZnSO4 led to an increase in p53-dependent
reporter activity. Expression of Pex19p resulted in a decrease in p53
function, whereas its antisense expression led to a dramatic increase
(c). NIH 3T3 cells that lack expression of p19ARF did not
show any effect of expression of Pex19p and its antisense construct on
p53-dependent reporter activity (d).
|
|
NIH 3T3 cells lack endogenous p19ARF because of biallelic loss of the
INK4a locus. As expected, transfection of these cells with
p19ARF resulted in a dose-dependent increase in p53
activity (Fig. 2B, a). Cotransfections of Pex19p
with p19ARF blocked p53 activation (Fig. 2B, b).
We ruled out the possibility that p53 might be inactivated by direct
interaction with Pex19p by performing in vivo
coimmunoprecipitation of Pex19p and p53 wherein no Pex19p was seen to
precipitate with p53 (data not shown). We next used stably transfected
NIH 3T3 cells (NIH-ARF) that express exogenous HA-tagged p19ARF under
the control of the heavy metal-inducible metallothionein promoter (22).
The addition of 100 µM ZnSO4 to the culture
medium resulted in the expression of HA-p19ARF (detectable by Western
blotting) and a concurrent increase in p53-dependent
luciferase reporter activity (Fig. 2B, c). NIH-ARF cells
were stably transfected with expression plasmids encoding sense (S) and
antisense (AS) His-tagged Pex19p protein and were selected in medium
supplemented with zeocine (1 mg/ml). These derivatives were analyzed
for endogenous p53 activity when cultured in the presence of increasing
amounts of ZnSO4; 100 µM induced p19ARF
expression detectable by Western blotting. As predicted and consistent
with the results of transient transfections (Fig. 2B,
b), Pex19p transfectants
(NIH-ARF/Pex19p-S) showed down-regulation of p53
function and notably, the antisense derivative
(NIH-ARF/Pex19p-AS) showed dramatic up-regulation (Fig.
2B, c). NIH 3T3 cells that lacked p19ARF did not
show any effect of transfections of Pexp19-S and-AS constructs on
p53-dependent reporter activity (Fig. 2B, d). Taken together these results demonstrated that: (i) an
overexpression of Pex19p blocks p19ARF enhancement of p53-mediated
transcriptional activation; (ii) this occurs most likely because of
nuclear exclusion of p19ARF and abrogation of its interactions with
Mdm2, resulting in active degradation of p53; and (iii) such an effect
of Pex19p on the transcriptional activity of p53 is mediated by p19ARF.
We next analyzed the effect of Pex19p-p19ARF interactions on the
expression of p21WAF1, a gene that is transactivated by p53. Induction of p19ARF led to up-regulation of p21WAF1 expression in
NIH-ARF cells as has been described (22). Notably, Western blot
analysis of p19ARF and Pex19p with respective tag-specific antibodies revealed that the induction of p19ARF led to stabilization of Pex19p (Fig. 3A,
a). On the other hand, antisense derivatives of
NIH-ARF/Pex19p had more p19ARF than sense derivatives when cultured in
the presence of ZnSO4 for an equal time (Fig.
3A, a and b) suggesting that Pex19p
may cause destabilization/degradation of p19ARF by a mechanism that
remains to be defined. Accordingly, NIH-ARF/Pex19p antisense
derivatives showed a high level of p21WAF1 expression, whereas Pex19p
sense derivatives showed a lower level as compared with the control
NIH-ARF cells (Fig. 3A, a). The data suggested
that antisense Pex19p decreased the level of endogenous Pex19p,
resulting in activation of the p19ARF-p53-p21WAF1 pathway. This
interpretation was supported by the finding that transfection of the
antisense construct into NIH-ARF cells that stably express His-tagged
Pex19p resulted in decreased levels of His-Pex19p protein (Fig.
3A, c). We next studied the growth of NIH-ARF
cells and their Pex19p sense and antisense derivatives for Pex19p
expression when cultured with and without ZnSO4. As
expected, the induction of p19ARF in NIH-ARF cells led to growth
retardation (Fig. 3, B and C). Increased
expression of Pex19p decreased p19ARF-induced growth retardation (Fig.
3, B and C), seemingly because of inactivation of
the p53 function via nuclear exclusion of p19ARF by Pex19p, as
demonstrated above. NIH-ARF expressing antisense Pex19p showed severe
retardation of growth, exhaustion of their replicative potential, and a
senescence-like morphology (Fig. 3, B and C). On
the other hand, growth of NIH 3T3 cells that lack p19ARF expression was
not affected by transfections of Pex19p-S and -AS, constructs demonstrating that the effect was mediated by p19ARF-Pex19p
interactions (Fig. 3C and data not shown). This data implies
that p19ARF function is blocked, at least in part, by its interactions
with endogenous Pex19p, and abrogation of these interactions by
antisense expression of Pex19p led to activation of p19ARF-p53-p21WAF1
pathway and execution of a senescence-like growth arrest.

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Fig. 3.
Down-regulation of p19ARF-p53-p21WAF1 pathway
by Pex19p. Expression of antisense Pex19p led to activation of the
p19ARF-p53-p21WAF1 pathway and induction of a senescence-like
phenotype. A, NIH-ARF cells were stably transfected with
plasmids encoding sense (Pex19p-S) and antisense
(Pex19p-AS) His-tagged Pex19p (His-Pex19p). A
high level of expression of Pex19p was seen when cells were induced for
p19ARF expression (a). On the contrary, p19ARF was less in
NIH-ARF/Pex19p-S cells as compared with either control NIH-ARF or
NIH-ARF/Pex19p-AS cells (a and b). Induction of
p21WAF1 by p19ARF was less in NIH-ARF/Pex19p-S and more in
NIH-ARF/Pex19p-AS as compared with the NIH-ARF (a).
Antisense expression of Pex19p neutralized the effects of expressing
His-Pex19p in NIH-ARF/Pex19p-S cells (c). B,
growth characteristics of NIH-ARF cells stably transfected with Pex19p
or its antisense expression construct. Cells maintained in the presence
(+) or absence ( ) of 100 µM ZnSO4 were
fixed, stained with Giemsa and photographed just after the addition of
ZnSO4 (Day 0) or after 3 days of culture
(Day 3). Note the senescence-like morphology of
NIH-ARF/Pex19p-AS cells when cultured in the presence of
ZnSO4. C, equal numbers of cells of the types
indicated were plated and cultured with (+) or without ( )
ZnSO4. Cells were counted in six independent fields after 3 days of culture. Error bars represent standard deviations
from six experiments.
|
|
As p19ARF and its human homologue p14ARF are important mediators of
cellular senescence (5, 6, 9, 23, 24), understanding its precise
mechanism of action and how it is controlled is clearly important. p53
has a central role in many aspects of the cell's response to its
environment and control of proliferation (25), in part because of
transcriptional control of effectors such as p21WAF1; understanding the
factors that regulate its activity is also of critical importance. We
have described here a novel mechanism of down-regulation of the
p19ARF-p53-p21WAF1 pathway.
 |
FOOTNOTES |
**
To whom correspondence should be addressed. Tel.: 81-298-30-6211;
Fax: 81-298-30-6270; E-mail: renu@cimmed.com.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.C100011200
 |
ABBREVIATIONS |
The abbreviations used are:
GFP, green
fluorescent protein;
HA, hemagglutinin;
MPD, mevalonate
pyrophosphate decarbonylase;
ARF, alternative reading frame.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.