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J. Biol. Chem., Vol. 277, Issue 17, 15053-15060, April 26, 2002
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From the Harvard University School of Public Health, Department of
Cancer Cell Biology, Boston, Massachusetts 02115
Received for publication, January 9, 2002
The p53-family of proteins, including p53, p63,
and p73, shares a high degree of structural similarity and can carry
out some redundant functions. However, mechanisms that regulate the
localization and activity of these proteins have not been fully
clarified. In this study, a nuclear localization signal (NLS) was
identified in p73, which is required for p73 nuclear import and which
could promote the nuclear import of a heterologous, cytoplasmic
protein. Mutants lacking the NLS localized to the cytoplasm and
displayed diminished transcriptional activity. A nuclear export signal
(NES) was also recognized in p73s C terminus, the deletion of which caused p73 to display a more nuclear localization pattern. This NES was
sensitive to leptomycin B and could function as an independent export
signal when fused to a heterologous protein. Interestingly, p73 mutant
proteins lacking the NLS or the NES were more stable than wild-type
p73, suggesting that nuclear import and nuclear export are required for
efficient p73 degradation. Our results indicate that p73 localization
is controlled by both nuclear import and export and suggest that the
overall distribution of p73 is likely to result from the balance
between these two processes. Proper control of nuclear import and
export is likely to be an important regulatory determinant of p73.
The p53 family of proteins includes three members, p53, p63, and
p73. Each of these three proteins share a high degree of amino acid
sequence similarity and contain common functional domains, including an
N-terminal transactivation domain, a central DNA binding domain, and a
C-terminal oligomerization domain (1-3) (reviewed in Ref. 4). Given
these similarities, it is of interest to determine whether members of
the p53 family function in similar or distinct metabolic pathways. p53,
p63, and p73 can each activate transcription from reporter genes
harboring p53 responsive elements in transient expression assays (1, 3,
5-7). Further, each of the p53 family members can activate apoptosis
when overexpressed, though to varying extents (2, 5). These results
suggest that p53 family members may carry out some redundant functions.
Despite these similarities, the consequence of loss of p53, p63, and
p73 on development and cancer susceptibility are strikingly different.
p53 loss-of-function mutations are observed in over 50% of all human
cancers, and p53-deficient mice are highly susceptible to the
development of cancer (8-10). Further, Li-Fraumeni syndrome patients
are born with germ line mutations in p53 and display an increased
susceptibility to multiple cancers. These findings and others have
confirmed the role of p53 as a bona fide tumor suppressor.
In contrast, mutations in p63 and p73 are not commonly associated with
cancer, and p63- and p73-deficient mice display various developmental
deficiencies without an apparent increased cancer incidence.
p63-deficient mice have severe defects in limb and skin development
(11), while p73 deficiency leads to neurological, pheromonal, and
inflammatory defects (12). Germ line mutations in p63 have been
causally linked to electrodactyly-ectodermal dysplasia-clefting
and Hays-Wells syndrome in humans, syndromes characterized by
ectrodactyly, ectodermal dysplasia, and facial clefts (13, 14).
Thus, p63 and p73 appear to play distinct roles in development that are
not attributed to p53.
Mechanisms that regulate the levels and activity of p53 family members
have not been fully clarified. Given their ability to function as
transcription factors, one would predict the activity of the p53 family
may be tightly correlated with their nuclear localization. Indeed, p53
contains three nuclear localization signals
(NLSs)1 located in the C
terminus of the protein, and mutation or deletion of these NLSs leads
to the cytoplasmic sequestration of p53 and a consequent decrease in
p53 transcriptional activity (15-18). In addition to nuclear import,
the active export of p53 from the nucleus to the cytoplasm has also
emerged as an important determinant of activity. p53 contains two
nuclear export signals (NESs), one located in its C terminus and the
other located in its N terminus (19, 20). Disruption of the C-terminal
NES causes p53 to display a more pronounced nuclear localization (20).
Murine double minute (MDM) 2, the product of a p53-inducible gene, can
bind p53 and promote its ubiquitination and subsequent degradation by
the proteasome (21, 22). Recent studies indicate that the ability of
MDM2 to ubiquitinate p53 promotes the export of p53 from the nucleus to
the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes (23, 24). In this case, the addition of ubiquitin moieties
to p53 is thought to expose the C-terminal NES of p53 to the nuclear
export machinery. Mechanisms that regulate the subcellular localization
of other p53 family members have not been determined.
The purpose of the current study was to examine the nuclear import and
export of p73. Toward this end, a bipartite NLS was identified in the C
terminus of p73, which is required for p73 nuclear import and which can
promote nuclear import when fused to a heterologous, cytoplasmic
protein. Mutants lacking the NLS localized to the cytoplasm and
displayed less transcriptional activity than wild-type p73 in
transfected cells. An NES was also identified in the C terminus of p73,
the deletion of which caused p73 to display a more nuclear localization
pattern. This NES was sensitive to the nuclear export inhibitor
leptomycin B (LMB) and could function as an independent export signal
when fused to a heterologous protein. These results indicate that p73
localization is controlled by both nuclear import and nuclear export
and suggest that the overall distribution of p73 results from the
balance between these two processes. Interestingly, p73 mutant proteins lacking either the NLS or the NES were more stable compared with wild-type p73, suggesting that both nuclear import and nuclear export
are required for efficient p73 degradation.
Plasmid DNAs--
Hemagglutinin (HA)-tagged wild-type
p73 Myc-tagged Pyruvate Kinase (PK), an NLS Fusion
Protein--
Myc-tagged chicken muscle PK expression DNA was obtained
from Gideon Dreyfuss (Howard Hughes Medical Institute) (25). DNA fragments corresponding to amino acids 327-348, 327-344, 329-348, and 91-138 of wild-type p73 Two Yellow Fluorescent Protein (2YFP), an NES Fusion
Protein--
The p2YFP vector was obtained from Yanping Zhang
(University of Texas). DNA fragments corresponding to amino acids
337-355 of wild-type p53 or p53 NES Tissue Culture and Immunofluorescence--
U2OS, Saos-2, or
35-2 cells were grown in minimum essential medium supplemented with
10% fetal bovine serum (FBS), 100 µg/ml penicillin and streptomycin.
Transfections for U2OS and Saos-2 cells were done using the calcium
phosphate method in 35 mm2 dishes when the cells were
~80% confluent. Sixteen h after addition of the DNA precipitate,
cells were washed and refed with minimum essential medium plus 10%
fetal bovine serum. Cell extracts were prepared 8 h later. To
measure p73 half-life, cycloheximide (CHX) was added to a final
concentration of 25 µg/ml after washing and refeeding, and cell
lysates were prepared at various time points after CHX addition. For
immunofluorescence staining, cells were plated on glass coverslips and
were transfected using the calcium phosphate method for U2OS and Saos-2
cells or using FuGENE 6 (Roche Molecular Biochemicals) for 35-2 cells
according to the manufacturer's instruction. 16 h after
transfection, cells were either untreated or treated with 30 ng/ml of
LMB (Sigma) for 8 h. Cells were then fixed and stained as
described previously (26). Antibodies used for immunostaining of HA
p73 Immunoblots--
Cell lysates were prepared as described
elsewhere (26). Protein extracts were resolved by SDS-PAGE and
transferred to a PolyScreen polyvinylidene difluoride transfer membrane
(PerkinElmer Life Sciences). The membrane was probed with either
an anti-HA monoclonal antibody (HA.11 from Babco), an anti-MDM2
monoclonal antibody (SMP-14 from Santa Cruz), or an anti-p21 monoclonal
antibody (PharMingen).
Mechanisms that regulate the subcellular distribution of p73 have
not been determined. To address this question, epitope-tagged (HA-tagged) wild-type p73 and various N- and C-terminal deletion mutants of p73 were transiently expressed in U2OS cells. Localization of the HA-tagged p73 proteins was then determined by immunofluorescence staining using an anti-HA antibody. As shown in Fig.
1B, HA wild-type p73 Recent studies indicate that the primary NLS of p53 is bipartite in
structure and includes basic amino acid residues between positions
316-322, as well as basic residues at positions 305 and 306 (15, 16).
Alignment of the p53 and p73 sequences suggested that the p73 NLS may
also have a bipartite structure that includes the basic residues
between positions 345 and 348, as well as basic residues at positions
327 and 328 (Fig. 2A). To test
this possibility, p73 residues from 327-348 were fused to the
cytoplasmic protein PK, and localization of the PK·p73 fusion
proteins was determined. In these experiments, the PK·p73 fusion
proteins were Myc-tagged, allowing analysis of their localization by
immunofluorescence staining with antibodies against the Myc-epitope
(25). As shown in Fig. 2B, Myc-tagged PK localized almost
entirely to the cytoplasm of cells in which it was expressed. In
contrast, the Myc-tagged PK protein was relocalized to the nucleus when
fused to p73 residues 327-348. To test whether residues 345-348 were
required for this effect, these residues were deleted from the
Myc-PK·p73 fusion protein to generate the clone
Myc-PK·p73-(327-344). As shown in Fig. 2B, the
Myc-PK·p73-(327-344) fusion protein failed to enter the nucleus,
providing further proof that residues 345-348 are required for the
function of the p73 NLS. To test whether residues 327 and 328 also
contribute to p73 NLS function, these two residues were deleted from
the PK·p73 fusion protein to generate the clone PK·p73-(329-348).
As shown in Fig. 2B, this fusion protein also failed to
enter the nucleus. Taken together, these results confirm that residues
327-348 can function as an independent NLS when fused to the
cytoplasmic protein PK and that residues 327 and 328, as well as
residues 345-348, are required for this activity. The ability of
residues 91-138 to function as an NLS when fused to PK was also
tested. As shown in Fig. 2B, the Myc-tagged PK protein
remained cytoplasmic when fused to p73 residues 91-138. These results
indicate that p73 residues 91-138 do not contain any independent
nuclear import sequences. Given these results, we suspect that deletion
of p73 residues between positions 91-138 may inadvertently affect p73
protein structure in such a way that inhibits its proper nuclear
localization.
Nuclear Import and Export Signals in Control of the p53-related
Protein p73*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expression DNA was obtained from Frank McKeon (Harvard Medical
School). Wild-type MDM2 DNA was obtained from Steve Grossmann (Dana
Farber Cancer Institute). HA-tagged p73 
1-90 and HA p73
1-138 were generated by PCR using HA wild-type p73 as a
template. The 3' primer for PCR was the SP6 primer, and the 5' primers
were 5'-GGCGGATCCATGTACCCTTACGATGTACCGGATTACGCAGCCAGCGTGCCCACCCACTCG-3' for HA p73 1-90 and
5'-GGCGGATCCATGTACCCTTACGATGTACCGGATTACGCATCAGCCACCTGGACGTACTCC-3' for
HA p73 1-138. HA p73 1-348 and HA p73 1-344 were also made by PCR using HA wild-type p73 as a template. The 5' primer for
PCR was the T7 primer, and the 3' primers were
5'-GGCTCTAGATCACCGCCGCTTCTTCACACCGG-3' for HA p73 1-348 and
5'-GGCTCTAGATCACACACCGGCACCAAGGGC-3' for HA p73 1-344. DNAs
encoding HA p73 NNII (K345N, K346N, R347I, R348I) and HA p73
NES
(L375A, L377A) were generated using the QuikChange
mutagenesis kit (Stratagene). HA wild-type p73 was used as a
template, and the following oligonucleotides and their complementary
oligonucleotides were used for the mutagenesis: for HA p73 NNII,
5'-GGTGCCGGTGTGAATAATATTATTCATGGAGACGAGGAC-3'; for HA p73
NES, 5'-GCTGAAAGAGAGCGCTGAGGCGATGGAGTTGGTGC-3'. The double
mutant HA p73 NNII NES was similarly constructed using HA p73
NES as a template and the same oligonucleotides used for making HA
p73 NNII. To construct green fluorescent protein (GFP)-tagged
wild-type p73 and p73 NES, the HA wild-type p73 and HA
p73 NES DNAs were used as PCR templates with the following primers: 5'-GTACGCTAGCAGATCTACCATGGCCCAGTCCACCGCC-3' as the 5' primer and 5'-GGCGGATCCAAGTGGATCTCGGCCTCCG-3' as the 3' primer. The resulting PCR products were digested with BglII and
BamHI and cloned into the corresponding sites in the
pEGFP-N1 vector (CLONTECH laboratories).
were generated by PCR. The resulting PCR products were digested with KpnI and XbaI and
subsequently cloned into the corresponding sites in the C terminus of
the Myc-PK expression plasmid.
(L348A, L350A) (23), amino acids 364-382 of wild-type p73 or p73 NES, or amino acids 7-27 of wild-type p73 were made by PCR. The resulting PCR products were digested with NheI and AgeI and subcloned into
the corresponding sites in the p2YFP vector.
and Myc-PK fusion proteins were the anti-HA monoclonal antibody
HA.11 (Babco) and the anti-c-Myc monoclonal antibody Ab-1 (Calbiochem)
as the primary antibody, respectively, and fluorescein
isothiocyanate-conjugated anti-mouse antibody (Jackson Labs) as the
secondary antibody. Antibodies used for MDM2 staining were the
anti-MDM2 polyclonal antibody N-20 (Santa Cruz) as the primary antibody
and rhodamine red-conjugated anti-rabbit antibody (Jackson Labs) as the
secondary antibody.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
localized exclusively to the nucleus in ~75% of cells in which it
was expressed, while in the majority of other cases HA wild-type p73
localized mostly to the nucleus with only weak cytoplasmic staining. To
identify sequences that may affect p73 localization, we first examined
the p73 primary amino acid sequence for the presence of a potential
nuclear localization signal, characterized by a clustering of the basic
residues arginine or lysine (Fig. 1A). This analysis
revealed a potential NLS (KKRR) located between residues 345 and 348 (Fig. 1A). To test whether this sequence contributed to
p73 nuclear import, two deletion mutants were generated that lacked
the C-terminal amino acids 349-636 (HA p73 1-348) or 345-636 (HA
p73 1-344), and their localization patterns were assessed. As shown
in Fig. 1B, HA p73 1-348 localized almost exclusively to
the nucleus, similar to wild-type p73. In contrast, HA p73 1-344
localized equally to both the nucleus and the cytoplasm. These results
are consistent with the hypothesis that residues 345-348 contribute to
p73 nuclear import. To test this further, the KKRR sequence between
residues 345 and 348 were converted to asparagine and isoleucine to
create the clone HA p73 NNII, and localization of this clone was
examined. As shown in Fig. 1B, HA p73 NNII localized
exclusively to the cytoplasm of cells in which it was expressed,
confirming a role for the KKRR sequence motif in p73 nuclear import. We
also examined whether N-terminal sequences might affect p73
localization. Toward this end, deletion mutants of p73 were
generated that lacked the N-terminal 90 (HA p73 1-90) or 138 (HA
p73 1-138) amino acids, and the localization patterns of these
mutants were tested. As shown in Fig. 1B, HA p73 1-90
localized almost exclusively to the nucleus, similar to HA wild-type
p73. In contrast, HA p73 1-138 localized equally to both the
nucleus and the cytoplasm. These results suggested that deletion of one
or more sequence elements between residues 91 and 138 can inhibit
proper p73 nuclear localization. However, amino acid analysis of
residues 91-138 did not reveal any putative NLSs characterized by a
clustering of basic amino acids.
View larger version (24K):
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Fig. 1.
N-terminal and C-terminal mutations
affect p73 localization. A, schematic of the p73
protein. Indicated are the transactivation domain (TAD), the
DNA-binding domain (DBD), the oligomerization domain
(OD), and a stretch of basic amino acids (KKRR) located
between residues 345 and 348. B, U2OS cells were
transfected with DNAs encoding HA-tagged versions of either wild-type
p73 or the indicated p73 mutants. HA p73 NNII is a mutant in
which residues 345-348 (KKRR) were converted to NNII. Localization of
HA p73 was determined by immunofluorescence staining with an anti-HA
antibody. Shown are representative pictures illustrating the
localization of the various p73 proteins. The staining pattern for
p73 was scored for 100 cells in two separate experiments as
described previously (23). The graph shows the percentage of
cells with the indicated HA p73 staining patterns.
View larger version (35K):
[in a new window]
Fig. 2.
C-terminal p73 sequences function as
an independent NLS. A, amino acid sequence alignment of
p53 bipartite NLS with p73. Illustrated in bold are
basic-charged p53 amino acids that have been reported to comprise the
bipartite NLS and similarly positioned basic amino acids in p73.
B, p73 amino acid sequences can promote nuclear import
of the normally cytoplasmic PK protein. p73 residues 327-348,
327-344, 329-348, and 91-138 were fused in frame to Myc-tagged PK
protein. U2OS cells were transfected with DNAs encoding wild-type PK
only or PK fused to the indicated p73 sequences. Localization of PK was
then determined by immunofluorescence staining with an anti-Myc
antibody. Wild-type PK localizes exclusively to the cytoplasm. The
results indicate that p73 sequences between 327 and 348 can promote PK
nuclear import and that this nuclear import requires basic residues at
positions 327 and 328, as well as residues between 345 and 348.
p53 contains a leucine-rich NES located in its C terminus (20).
Mutations within this NES inhibit p53 nuclear export, and thus mutants
lacking the NES display a more pronounced nuclear localization (20, 23,
24). p73 contains a sequence similar to the p53 NES in its C terminus
(Fig. 3A). However, it has not yet been clarified whether p73 is subject to active nuclear export and
whether this putative NES in p73 plays a role in this process. To
address these questions, we utilized the nuclear export inhibitor LMB.
First, U2OS cells were transfected with DNAs encoding either HA-tagged
or GFP-tagged p73 and were subsequently untreated or exposed to LMB
to inhibit nuclear export. Localization of the transfected p73 proteins
was then determined by immunofluorescence staining. In these
experiments, HA-tagged p73 localized exclusively to the nucleus in
~60% of untreated cells in which it was expressed, and GFP-tagged
p73 localized to the nucleus in ~75% of transfected cells (Fig.
3B). In contrast, the percentage of cells in which the
HA-tagged p73 displayed an exclusively nuclear staining pattern increased to ~80% following LMB treatment, and the percentage of
cells in which GFP-tagged p73 localized to the nucleus only increased to ~95%. These results suggested that p73 is actively exported from the nucleus to the cytoplasm and that this occurs in a
LMB-sensitive manner. To investigate this further, the putative NES in
the C terminus of p73 was mutated to generate the clone HA p73
NES, and localization of the resulting mutant protein was monitored
in transfected cells. As shown in Fig. 3B, HA p73 NES
displayed a more pronounced nuclear staining pattern than did HA
wild-type p73 (97% nuclear only for HA p73 NES
versus 62% nuclear only for HA wild-type p73).
Similarly, GFP p73 NES also displayed a more nuclear staining
pattern than did GFP wild-type p73 (99% versus 75%).
These results suggested that p73 nuclear export requires an intact NES
in the p73 C terminus. To gain further evidence for the role of the
C-terminal NES in p73 nuclear export, we made use of the p73 mutant HA
p73 NNII (Fig. 1B), which localizes exclusively to the
cytoplasm but maintains an intact NES. As shown in Fig. 3C,
the HA p73 NNII mutant localized almost exclusively to the cytoplasm
when expressed in either U2OS or 35-2 (p53-null/MDM2-null) cells. In
contrast, LMB-treatment caused a marked shift of this mutant toward a
more nuclear localization pattern in both cell types. These results
suggest that the cytoplasmic localization of HA p73 NNII results
from both diminished nuclear import and continued nuclear export that
is mediated by the NES. To test this further, we mutated the NES in the
HA p73 NNII mutant, to generate the double-mutant HA p73 NNII
NES. As shown in Fig. 3C, mutation of the NES also caused
the HA p73 NNII mutant to shift toward a more nuclear localization
pattern. Taken together, the results in Fig. 3 indicate that p73
undergoes active nuclear export in transfected cells and that this
export depends on the p73 NES.
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We next wished to test whether the p73 NES can function as an
autonomous export signal when fused to another protein. Toward this
end, expression DNAs were generated in which wild-type or mutated NES
sequences from p53 or p73 were fused to tandem copies of the YFP(2YFP)
(19). Localization of the resulting fusion proteins was then examined
in transiently transfected U2OS cells. As shown in Fig.
4, 2YFP alone displayed a diffuse
localization pattern in both the nucleus and cytoplasm of individual
cells, with slightly more nuclear accumulation. When residues 337-355 of wild-type p53 (p53 wild-type NES) was fused to 2YFP, this fusion protein was exported from the nucleus and relocalized to the cytoplasm in greater than 50% of cells in which it was expressed,
consistent with previous studies (19). In contrast, a fusion protein of 2YFP with residues 337-355 of p53 in which leucines 348 and 350 were
converted to alanines (p53 NES) was not exported, confirming the
importance of these two leucines in the functionality of the p53
C-terminal NES (20). Similarly, fusion of 2YFP with residues 364-382
of wild-type p73 (p73 wild-type NES) resulted in striking nuclear
export of the fusion protein in greater than 80% of transfected cells
(Fig. 4). In contrast, conversion of leucines 375 and 377 in the p73
NES to alanines inhibited export of the 2YFP fusion protein, and LMB
treatment also inhibited export of the 2YFP·p73 NES fusion
protein. Taken together, these results demonstrate that the p73 NES can
function as an autonomous export signal when fused to 2YFP. Similar
results were obtained using 35-2 cells (p53 null/MDM2 null),
indicating that the autonomous activity of this p73 C-terminal NES
requires neither p53 nor MDM2 (data not shown). A recent study
demonstrated that p53 contains a second NES in its N terminus between
residues 11 and 27 (19). We fused the corresponding region of p73
(residues 7-27) to 2YFP to test whether this region of p73 can also
function as an autonomous NES. As shown in Fig. 4, the N-terminal
residues 7-27 in p73 failed to promote nuclear export when fused to
2YFP, indicating that these residues cannot function as an autonomous
NES.
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MDM2 can bind the N terminus of both p53 and p73 and has been shown to
promote p53 nuclear export. Given that p73 undergoes active nuclear
export, we wished to test the effect of MDM2 on the nuclear export of
p73. Accordingly, U2OS cells were transfected with DNAs encoding HA
wild-type p53 or HA wild-type p73 alone or co-transfected with DNAs
encoding MDM2. p53 and p73 localization was then examined by
immunofluorescence staining using an anti-HA antibody. As shown in Fig.
5A, HA wild-type p53 was
largely nuclear when expressed alone but was exported from the nucleus
and relocalized to the cytoplasm with co-expression of MDM2. This
effect was inhibited by treatment of transfected cells with LMB,
consistent with previous reports (23, 24). HA wild-type p73 also
localized to the nucleus when expressed alone. Interestingly, however,
HA p73 was relatively resistant to MDM2-mediated nuclear export,
remaining mostly nuclear in cells in which it was expressed with MDM2
(Fig. 5B). It should be noted that p73 and MDM2 could form a
complex in cells expressing both proteins as determined by
co-immunoprecipitation studies (data not shown), indicating that the
resistance of p73 to nuclear export by MDM2 did not result from an
inability of these two proteins to interact. In a high percentage of
cells, HA p73 appeared to co-localize in nuclear aggregates with
MDM2 (Fig. 5B), consistent with previous studies (27). The
significance of nuclear aggregate formation between p73 and MDM2 is
unknown. Nonetheless, these results indicate that p73 is a relatively
poor substrate for MDM2-dependent nuclear export when
compared with p53 and suggests therefore that the nuclear export of p53
and p73 may be regulated through different mechanisms.
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Finally, we wished to determine whether the stability and/or activity
of p73 was correlated with its subcellular localization. To examine p73
protein stability, U2OS cells were transfected with either HA wild-type
p73, HA p73 NES, or HA p73 NNII. Transfected cells were then
treated with CHX to inhibit de novo protein synthesis, and
the steady-state levels of p73 were monitored at different time
points after CHX addition. The rate at which p73 levels decrease under
these conditions is a measure of protein stability. As shown in Fig.
6A, the half-life
(t1/2) of HA wild-type p73 was less than 3 h, similar to previous studies which have reported the half-life of
p73 to be between 0.5-2 h (28, 29). In contrast, HA p73 NES
and HA p73 NNII were more stable than HA wild-type p73, with
half-lives in each case between 6 and 9 h. The fact that both
mutants are more stable than wild-type p73 suggests that nuclear import
and nuclear export are both required for efficient p73 degradation.
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p73 can bind to p53-responsive elements and activate transcription of
p53-responsive genes, including the genes encoding p21 and MDM2.
Accordingly, p21 and MDM2 protein levels were monitored in cells
expressing either HA wild-type p73, HA p73 NES, HA p73 NNII,
or HA p73 NNII NES to determine the relative ability of these
nuclear and cytoplasm-localized HA p73 proteins to activate gene
transcription. As shown in Fig. 6B, p21 and MDM2 protein levels were low in mock-transfected cells. Expression of HA wild-type p73 induced the expression of p21 and MDM2, indicating that p73 could activate transcription of the endogenous p21 and MDM2 genes in
these transfected cells. In contrast to wild-type p73, HA p73
NNII localized largely to the cytoplasm (Fig. 1B) and was markedly less able to induce p21 and MDM2 expression (Fig.
6B). These results are consistent with nuclear localization
being required for efficient p73 transactivation function.
Interestingly, HA p73 NES was also less active than HA wild-type
p73 at inducing p21 and MDM2 expression (Fig. 6B),
despite the fact that HA p73 NES had a more nuclear localization
pattern (Fig. 3B) and longer half-life (Fig. 6A)
than the wild-type p73 protein. The double mutant HA p73 NNII NES
was also less able to induce p21 and MDM2 expression than HA p73
NNII (Fig. 6B), although HA p73 NNII NES displayed a
more nuclear localization than HA p73 NNII (Fig. 3C).
These results suggest that mutations in the NES of p73 may have a
secondary effect on p73 transcriptional activity in addition to
inhibiting nuclear export.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Wild-type p53 is expressed at low levels in most normal cells and, at least in some cell types, is localized in the cytoplasm (20, 30). In response to DNA damage and various other stresses, p53 levels increase and the p53 protein accumulates in the nucleus. The stress-induced, nuclear accumulation of p53 is likely to result from both diminished nuclear export and continued nuclear import. Two NESs have been identified in p53, one located within the C-terminal oligomerization domain and the second located within the N-terminal MDM2-binding domain (19, 20). Various studies have suggested that each of these p53 NESs may be susceptible to inhibition following DNA damaging stress. For example, DNA damage-induced phosphorylations in the C terminus of p53 have been reported to increase the formation of p53 tetramers (31). This tetramerization of p53, in turn, is predicted to render the C-terminal NES inaccessible to the export machinery and thus unable to promote nuclear export (20). More recent studies have demonstrated that amino acid residues within the N-terminal NES of p53 become phosphorylated following DNA damaging stress and that this phosphorylation inhibits the NES function (19). Taken together, these findings suggest that the nuclear accumulation of p53 following DNA damage may result, at least in part, from an inhibition of nuclear export mediated by either the N-terminal or C-terminal NESs.
p73 is a member of the p53-family of proteins, and shares a high degree of amino acid sequence similarity with p53. Given the similarity between these two proteins, it is of interest to determine whether p73 and p53 are regulated through common or distinct pathways. Like p53, the p73 protein is stabilized and activated in response to certain DNA damaging agents. For example, p73 activation has been demonstrated following exposure to either cisplatin or ionizing radiation, and stabilization of the p73 protein has been reported in response to cisplatin treatment (28, 32). The stabilization and activation of p73 in response to these treatments is thought to require its phosphorylation by the tyrosine kinase c-Abl (28). In the current study, we identified a bipartite NLS, which is required for p73 nuclear localization and which can promote the nuclear localization of a normally cytoplasmic protein. In addition, we demonstrate that p73 undergoes active nuclear export and that this export is mediated, at least in part, by an NES located in the p73 C terminus. Presumably, p73 modifications that either promote nuclear import or inhibit nuclear export could also contribute to the activation of p73 in cisplatin or ionizing radiation-treated cells. In this regard, it will be interesting to determine whether the activation and stabilization of p73 in response to these agents is accompanied by its nuclear accumulation, as is the case for p53.
Gu et al. (27) have generated p53·p73 chimeric proteins to examine the functional differences between various p53 and p73 protein domains. In that study, it was found that a p53·p73 chimera in which the C-terminal p53 NES was replaced by the p73 NES was resistant to MDM2-mediated nuclear export. Based on these findings it was proposed that the p73 NES is non-functional. In the current study, we find that p73 displays a more nuclear localization pattern when its C-terminal NES is disrupted by mutation or when nuclear export is inhibited by LMB treatment. We further show that the p73 C-terminal NES can function as an autonomous nuclear export signal when fused to a heterologous protein. In these experiments the p73 C-terminal NES was a stronger NES when compared with the C-terminal NES of p53 (Fig. 4). These results clearly demonstrate that the NES of p73 is functional and that the p73 protein is subject to active nuclear export. However, the factors and conditions which regulate nuclear export of p53 and p73 are likely to be different. For example, p53 is efficiently exported from the nucleus to the cytoplasm when expressed with MDM2, whereas p73 forms nuclear aggregates with MDM2 and is largely resistant to MDM2-mediated nuclear export (Fig. 5). Why p73 is resistant to nuclear export by MDM2 is not clear. In this regard, a recent study demonstrated that p53 contains a second NES located in its N terminus between residues 11 and 27. Mutations in this NES inhibited p53 nuclear export, and this NES could function as an autonomous export signal when fused to a heterologous protein (19). In contrast, the corresponding N-terminal region of p73 (residues 7-27) lacked nuclear export function when fused to 2YFP (Fig. 4). Therefore, lack of an active NES in the N terminus of p73 may account, at least in part, for the resistance of p73 to MDM2-mediated nuclear export. A second possibility is that nuclear aggregate formation between p73 and MDM2 may inhibit p73 nuclear export. In any event, the fact that MDM2 is relatively inefficient at promoting p73 nuclear export raises the possibility that the export of p73 is normally mediated by a novel, as yet unidentified factor.
Given that p73 can function as a transcription factor, we considered
that nuclear import and export might affect p73 transactivation function. Not surprisingly, a nuclear import-deficient form of p73 was
less able to activate gene expression in transfected cells (Fig. 6).
However, a p73 protein deficient in nuclear export displayed a more
nuclear localization pattern but was less able to activate gene
expression. These results indicate that mutations within the NES of p73
affect the transactivation function of p73 in addition to blocking
nuclear export. p53 binds DNA as a tetramer, and mutations in the p53
NES also inhibit the transactivation function of p53 by blocking or
preventing the efficient formation of p53 tetramers (20). We suspect
that mutations in the p73 NES probably have a similar inhibitory effect
on p73 tetramerization and that this might account for the relatively
low transcriptional activity of the p73 NES mutant. We also
considered that nuclear import and export may play an important role in
regulating p73 protein stability. It has been suggested that p73 is
degraded through the ubiquitin proteasome pathway, though factors which
might promote p73 degradation through this pathway have not been
recognized (29). In the current study, p73 mutant proteins deficient in either nuclear import or nuclear export were more stable than wild-type
p73. These results suggest that both nuclear import and nuclear export
are required for efficient p73 protein degradation. This may be
analogous to p53 in which it has been proposed that p53 is
ubiquitinated in the nucleus and then exported to the cytoplasm for
degradation by cytoplasmic proteasomes (23, 24). p73 degradation may
occur through a similar mechanism in which p73 enters the nucleus where
it is ubiquitinated, followed by its export and degradation in the
cytoplasm. Such a model may explain the requirement for nuclear import
and nuclear export in p73 degradation.
Our results indicate that p73 localization is controlled by both
nuclear import and export signals and suggest that the overall distribution of p73 is likely to result from the balance between these
two processes. Proper control of nuclear import and export is likely to
be an important regulatory determinant of p73.
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FOOTNOTES |
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* This work was supported by NCI, National Institutes of Health Grant 1R01CA80918-01 and American Cancer Society Grant RSG-01-042-01 (to C. G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Harvard University
School of Public Health, Dept. of Cancer Cell Biology, 665 Huntington
Ave., Bldg. 1, 4th Floor, Boston, MA 02115. Tel.: 617-432-2532; Fax:
617-432-0107; E-mail: cmaki@hsph.harvard.edu.
Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M200248200
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ABBREVIATIONS |
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The abbreviations used are: NLS(s), nuclear localization signal(s); NES(s), nuclear export signal(s); MDM, murine double minute; LMB, leptomycin B; HA, hemagglutinin; GFP, green fluorescent protein; PK, pyruvate kinase; CHX, cycloheximide; YFP, yellow fluorescent protein.
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