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Molecular and Cellular Biology, May 2001, p. 3534-3546, Vol. 21, No. 10
Center for Cell Signaling, University of
Virginia, Charlottesville, Virginia 22908,1 and
Laboratory for Physiological Chemistry and Centre for
Biomedical Genetics, University Medical Center, 3584CG Utrecht, The
Netherlands2
Received 21 November 2000/Returned for modification 28 December
2000/Accepted 27 February 2001
AFX belongs to a subfamily of Forkhead transcription factors that
are phosphorylated by protein kinase B (PKB), also known as Akt.
Phosphorylation inhibits the transcriptional activity of AFX and
changes the steady-state localization of the protein from the nucleus
to the cytoplasm. Our goal was threefold: to identify the cellular
compartment in which PKB phosphorylates AFX, to determine whether the
nuclear localization of AFX plays a role in regulating its
transcriptional activity, and to elucidate the mechanism by which
phosphorylation alters the localization of AFX. We show that
phosphorylation of AFX by PKB occurs in the nucleus. In addition,
nuclear export mediated by the export receptor, Crm1, is required for
the inhibition of AFX transcriptional activity. Both phosphorylated and
unphosphorylated AFX, however, bind Crm1 and can be exported from the
nucleus. These results suggest that export is unregulated and that
phosphorylation by PKB is not required for the nuclear export of AFX.
We show that AFX enters the nucleus by an active, Ran-dependent
mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical
nuclear localization signal (NLS). S193, contained within this atypical
NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a
negative charge at S193 by mutating the residue to glutamate reduces
nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore,
attenuates the import of the transcription factor, which shifts the
localization of the protein from the nucleus to the cytoplasm and
results in the inhibition of AFX transcriptional activity.
Protein kinase B (PKB), also known
as Akt, promotes cell survival in many different cell types (24,
38, 40, 66). Following its initial cloning (17,
34), PKB was isolated as the transforming gene of v-Akt, hence
the name c-Akt and its classification as a proto-oncogene (6,
67). Activation of PKB requires the lipid phosphatidylinositol
3,4,5-triphosphate (PIP3) (15) and phosphorylation by an upstream kinase, PDK1 (2, 69, 72). PIP3 is produced by phosphatidylinositol 3-kinase in
response to signals from extracellular growth factors (for a review see reference 60). PKB has been shown to phosphorylate and
regulate the activity of transcription factors in response to survival factors. Genetic studies of Caenorhabditis elegans have
demonstrated that the PKB signal transduction pathway inhibits the
activity of the Forkhead transcription factor, daf-16, a
gene that regulates longevity (55). There are three human
orthologues of daf-16, AFX (13), FKHR
(27), and FKHRL1 (3), that were first
identified as chromosomal breakpoints in human tumors.
AFX is phosphorylated by PKB in response to insulin and serum at three
sites: T28, S193, and S258 (42). Phosphorylation of these
residues by PKB leads to both inhibition of the transcriptional activity of AFX and cytoplasmic retention and/or nuclear exclusion of
the protein. Withdrawal of serum or insulin results in AFX dephosphorylation, nuclear localization, and target gene activation. In
the absence of survival factors, Forkhead family members have been
shown to induce the transcription of proapoptotic genes, such as the
FasL gene (14) and the Bim gene
(22), thus triggering a cascade of events that lead to
apoptosis. In addition, overexpression of AFX blocks cell cycle
progression at G1 by a mechanism that is independent of
functional retinoblastoma protein but dependent on the cell cycle
inhibitor p27kip (49). Dysregulation of
Forkhead proteins may, therefore, comprise an important step in
oncogenic transformation by both inhibiting apoptosis and promoting
progression through the cell cycle.
Efficient regulated nuclear localization of transcription factors in
response to extracellular signals is essential for their function
(41). For example, in unstressed cells, p53 continuously shuttles into and out of the nucleus, and its subcellular distribution varies throughout the cell cycle. In response to certain stresses, however, p53 relocalizes to the nucleus to promote gene transcription. In some p53-related tumors, however, there is a defect in p53 localization (52, 65). In these cells, p53 is
constitutively cytoplasmic due to an increase in the export rate of the
protein. As a result, p53 cannot accumulate in the nucleus and carry
out its normal activities. This defect leads to unregulated cellular proliferation. Likewise, the transcriptional activity of Forkhead family members may be regulated not only by phosphorylation but also by
changes in their subcellular localization.
Many proteins are transported constitutively into and out of the
nucleus by members of the In contrast to constitutive transport, regulated transport occurs
only in response to specific cellular signals, such as
phosphorylation (35). For example, mitogen-activated
protein kinase-activated protein kinase 2 is a nuclear protein in
unstimulated cells. In response to specific extracellular signals, it
is phosphorylated, binds p38 stress-activated protein kinase, and is
only then exported from the nucleus (7). Regulated
transport mechanisms play critical roles in augmenting or attenuating
signaling information by controlling the cellular localization of
proteins. Many proteins that have regulated transport mechanisms are
shuttling proteins that move continuously between the cytoplasm and
nucleus. Thus, their steady-state localization is determined by the
relative rate constants for import and export. Shuttling coordinates
nuclear and cytoplasmic events by providing a rapid and reversible
method to regulate a nuclear and/or cytoplasmic activity. Nuclear
transport often works in tandem with other mechanisms to regulate
the function of proteins. Some transcription factors require
phosphorylation in addition to changes in nuclear localization to
completely regulate their activity. This redundancy increases the
strength of cellular switches and provides additional ways to integrate
environmental cues with cellular signals. Finally, proteins that alter
their cellular localization in response to extracellular signals need a
mechanism for turning that signal off when it is no longer required. Protein degradation, dephosphorylation, and nuclear transport are all
mechanisms used to terminate activity. Although the regulated transport
mechanisms for several yeast proteins have been identified, including
those for Pho4 (36, 37) and Mig1 (20, 21),
few regulated transport mechanisms have been described for mammalian proteins.
Our goal, therefore, was to elucidate how PKB phosphorylation and the
resulting cellular relocalization of AFX alter its transcriptional activity. In this study, we show that PKB phosphorylation of AFX in the
nucleus followed by Crm1-dependent nuclear export is required to
inhibit the transcriptional activity of the Forkhead family member.
Although phosphorylation by PKB does not significantly alter AFX export
from the nucleus, it appears that nuclear import of phosphorylated AFX
is regulated. Amino acids 180 to 221 of AFX are necessary and
sufficient for nuclear import but comprise a nonclassical nuclear
localization signal (NLS). Addition of a negative charge at S193 by
mutating the residue to a glutamate inhibits nuclear import. Therefore,
phosphorylation of AFX by PKB likely reduces the nuclear import rate of
AFX. This results in a shift in the steady-state localization of AFX
from the nucleus to the cytoplasm, thereby inhibiting its
transcriptional activity.
Plasmids and recombinant proteins.
pMT2-HA-AFX,
pMT2-HA-SASA, pMT2-HA-A3, pSG5-gagPKB, p1205-luc, and pCMV-lacZ have
been described previously (42, 49). pMT2-HA-AFX
( Cell culture.
Insulin receptor-overexpressing NIH 3T3 cells
(A14) were grown as described previously (15). All other
cells were passaged in Dulbecco's modified Eagle medium (supplemented
with 5% [vol/vol] fetal calf serum and penicillin-streptomycin).
Baby hamster kidney cells (BHK21), human embryonic kidney cells
(HEK293), and HeLa cells were cultured in a humidified, 37°C-5%
CO2 incubator. tsBN2 cells were grown at 33.5°C. Where
indicated, the tsBN2 cells were shifted to 39.5°C for 3 h after
the addition of 50 µM cycloheximide. The leptomycin B (LMB) used in
some cell-based assays was a gift from Barbara Wolff (Novartis).
Transfections.
Hemagglutinin (HA)-tagged AFX constructs were
transfected into A14, BHK21, HEK293, HeLa, and tsBN2 cells by the
calcium phosphate method. At 24 h posttransfection, the
transfection medium was replaced with fresh medium and the cells were
incubated at 37°C for an additional 24 h. Transfected HEK293
cells were harvested for immunoprecipitations at 48 h posttransfection.
Transfected A14 cells were serum starved overnight where indicated. For
immunofluorescence, A14 cells were either untreated, treated with 10 µM LY294002 (Calbiochem) for 10 min, or treated with 2 ng of LMB per
ml for 30 min prior to the addition of 1 µg of insulin per ml. The
cells were then incubated with insulin for an additional 30 min before
fixation. Transfected tsBN2, HeLa, and BHK21 cells were plated on
poly-L-lysine-coated coverslips. Transfected tsBN2 and HeLa
cells, where indicated, were serum starved for 1.5 h prior to fixation.
BHK21 cells were transfected with all pKGFP3 constructs using Effectene
transfection reagent (Qiagen) according to the manufacturer's
instructions. These cells were processed 20 h after transfection.
After the indicated treatments, the transfected cells were fixed and
permeabilized with 4% paraformaldehyde-2% sucrose in
phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4
[pH 7.4]) and ice-cold methanol as described previously (54,
58, 73). Fixed cells were then prepared for analysis by
fluorescence microscopy.
Fluorescence microscopy.
After fixation and
permeabilization, cells expressing GFP fusions were incubated with
4',6-diamidino-2-phenylindole (DAPI; 10 ng/ml) to stain the nuclei, and
then the coverslips were mounted on glass slides by using GelMount
(Biomeda). Images of the cells were captured using a 60×
water-immersion objective lens on a Nikon inverted microscope equipped
with a Hamamatsu charge-coupled device camera. All immunofluorescence
data were obtained and processed using Openlab (Improvision) and Adobe
Photoshop software. Images for each set of experiments were obtained
using the same camera settings. The relative nuclear and cytoplasmic
fluorescence levels of the GFP3 constructs were measured using
Openlab (Improvision). Nuclear fluorescence was calculated as a
percentage of the total cellular fluorescence
[N/(N+C)]. All fluorescence measurements were corrected for
background fluorescence levels. Each data point represents the mean
fluorescence obtained from 12 randomly chosen cells. Error is expressed
as the standard deviations of the means. All cells expressing HA-tagged
AFX constructs were blocked in 10% bovine serum albumin (BSA)-PBS at
room temperature incubated with monoclonal antibody 12CA5 (2 µg/ml)
and Texas red-conjugated donkey anti-mouse immunoglobulin G (1:1,500
dilution; Jackson ImmunoResearch Laboratories), both in 3% BSA;
stained with DAPI; and mounted and viewed as described above. Cells
expressing gag-PKB were detected using anti-gag and anti-PKB antisera
(15).
Immunodetection.
BHK21 cells expressing GFP3 constructs were
lysed with an equal volume of boiling Laemmli sample buffer. Equal
volumes of lysate (10 µl) were then analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting for GFP3 with polyclonal anti-GFP (1:1,000 dilution;
Molecular Probes) and horseradish peroxidase (HRP)-conjugated
anti-rabbit secondary antibody (1:20,000 dilution; Jackson
Laboratories). Proteins were revealed by chemiluminescence (Kirkegaard
& Perry Laboratories). Subcellular fractionation of insulin-treated (1 µg/ml) A14 cells was carried out according to published protocols
(47, 76). Anti-phosphoT32 FKHR-L1, anti-c-cbl, and
anti-RNA pol II were gifts from Anne Brunet (Harvard University), Kris
Reedquist (University Medical Center, Utrecht, The Netherlands), and
Marc Timmers (University Medical Center), respectively.
Anti-phosphoS193 AFX and anti-phosphoS473 PKB were obtained from New
England Biolabs.
Luciferase assays.
A14 cells were cotransfected with the
1205-luc reporter, pMT2-HA-AFX, and pCMV-lacZ. Cells were also
transfected where indicated with pSG5-gagPKB. Cells were preincubated
with LMB where noted for 30 min prior to the addition of insulin. Cells
were then treated with insulin for 16 h in the absence or presence
of LMB. Transcriptional activity was measured 48 h after
transfection. Luciferase and Crm1 binding assays.
HEK293 cells were either mock
transfected or transfected with pMT2-HA-AFX or pMT2-HA-A3. Where
indicated, cells were treated with LY294002 (10 µM) for 2 h
prior to lysis. All cells were then washed twice with ice-cold PBS,
placed on ice, and lysed by the addition of 400 µl of lysis buffer
(25 mM HEPES [pH 7.4], 300 mM NaCl, 1.5 mM MgCl2, 20 mM
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.10.3534-3546.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of Nuclear Import by Protein Kinase B
(Akt) Regulates the Subcellular Distribution and Activity of the
Forkhead Transcription Factor AFX
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-importin family of nuclear transport receptors (28, 48). These receptors recognize specific
localization signals within their cargoes, and their association with
these signal sequences is controlled by the small GTPase Ran. The
regulators of Ran are distributed asymmetrically within the cell: the
RanGTPase-activating protein (RanGAP) is cytoplasmic
(10), whereas the Ran exchange factor (RanGEF) is
nuclear (11). Therefore, most of the Ran present in the
nucleus is predicted to be GTP bound, while cytoplasmic Ran is
predicted to be GDP bound. The directionality of transport depends on
this RanGTP gradient. Unloading of cargo from an import receptor in the
nucleus is triggered by the binding of RanGTP to the receptor
(29, 31, 61). Conversely, an export receptor can only load
its cargo in the presence of RanGTP. Release of export cargo into the
cytoplasm occurs by the hydrolysis of RanGTP to RanGDP, stimulated by
RanGAP and other required cofactors (39).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
198-216) was constructed using PCR-based mutagenesis. To construct
the vectors encoding the fusions of green fluorescent protein (GFP) to
the C termini of various AFX sequences, pKGFP3 was used. To create
pKGFP3, GFP-GFP was PCR amplified and inserted as a
BglII-BglII fragment into pKGFP. For
pKGFP3-AFX(180-197), pKGFP3-AFX(198-221), and
pKGFP3-AFX(180-221), the indicated residues of AFX were PCR amplified
and ligated into pKGFP3 as XbaI-XbaI fragments.
Site-directed mutagenesis of residues within pKGFP3-AFX(180-221) to
produce pKGFP3-AFX(180-221)I-V, pKGFP3-AFX(180-221)S193A, and pKGFP3-AFX(180-221)S193E was carried out using the
Quikchange mutagenesis kit (Stratagene) according to the
manufacturer's recommendations. All mutations were verified
by sequencing. pQE60-Crm1 was a gift from Iain Mattaj (EMBL,
Heidelberg, Germany) (5). Crm-His6 was
purified using Talon beads (Clontech) as described previously (39). The addition of 14 mM -mercaptoethanol was
required to retain the stability of the protein. pQE32-RanQ69L was a
gift from Dirk Görlich (University of Heidelberg).
His6-RanQ69L was prepared as described earlier
(31).
-galactosidase measurements were
performed as described (42).
-glycerophosphate, 1 mM sodium orthovanadate, 0.1% Triton X-100, 1 mM okadaic acid, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg
of leupeptin per ml, and 20 µg of aprotinin per ml). Lysates were
cleared by centrifugation (5 min at 14,000 × g,
4°C). The lysate was used for immunoprecipitation with 12CA5 at 4°C
for 1 h. Protein A-Sepharose beads were added to the samples for
1 h at 4°C. Immunoprecipitates were washed three times with buffer A (PBS, 1% NP-40, 20 mM -glycerophosphate, 2 mM sodium orthovanadate, 1 mM okadaic acid) and once with buffer B (50 mM morpholine propanesulfonic acid [pH 7.5], 500 mM lithium chloride, 20 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM okadaic acid).
To release endogenous 14-3-3 protein bound to HA-AFX,
immunoprecipitates were washed three times with PBS containing 1 M
MgCl2 where indicated. Controls were washed three times
with PBS without added MgCl2.
-mercaptoethanol, 0.05% ovalbumin, 1 mM sodium
orthovanadate, 20 mM -glycerophosphate, 1 mM okadaic acid, and 1 mM
PMSF). Crm1-His6 was added to each assay to yield a final
concentration of 500 nM. His6-RanQ69L was added as
indicated to a final concentration of 3 µM. Samples were incubated
for 2 h at 4°C and then were washed three times with binding
buffer. Beads were resuspended in Laemmli sample buffer and the
proteins were separated by SDS-PAGE and immunoblotted with
HRP-conjugated 12CA5 (1:5,000 dilution), monoclonal
anti-His6 (1:2,000 dilution; BabCo), or polyclonal
anti-14-3-3 (1:100 dilution; Santa Cruz Biotechnology), and
with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody
(1:20,000 dilution; Jackson Laboratories). Proteins were revealed by
chemiluminescence (Kirkegaard & Perry Laboratories).
Heterokaryon fusion assays. BHK21 cells were transfected with pMT2-HA-A3. An acceptor cell line, GSN2, was a gift from Bryce Paschal (University of Virginia) (12). This stably transfected HeLa cell line expresses the nondiffusible nuclear protein GFP-streptavidin-SV40NLS. BHK21 and GSN2 cells were coplated onto poly-L-lysine-coated coverslips overnight. Cells were treated with 50 µM cycloheximide for 30 min. The plasma membranes were then fused for 2 min with 50% polyethylene glycol (molecular weight, 8,000) prewarmed to 37°C. Cells were washed four times with medium and were incubated at 37°C for an additional 1 h in the presence of cycloheximide. Cells were then fixed and processed for immunocytochemistry as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PKB-dependent phosphorylation of AFX triggers relocalization of AFX
from the nucleus to the cytoplasm.
The subcellular distribution of
Forkhead family members is altered on addition of insulin or serum to
cells (9, 14, 71). This relocalization is dependent on the
phosphorylation of the Forkhead protein by PKB. As a basis for
subsequent experiments, we confirmed that the addition of insulin
results in a change in the steady-state distribution of HA-AFX in serum
starved cells from the nucleus to the cytoplasm within 30 min (Fig.
1A). Mutation of PKB phosphorylation
sites S193 and S258 to alanine (HA-SASA) inhibited this relocalization,
as did treatment with the phosphatidylinositol 3-kinase inhibitor
LY294002 prior to the addition of insulin (Fig. 1A). Additionally, the
coexpression of constitutively active PKB (gagPKB) resulted in the
redistribution of HA-AFX but not HA-SASA to the cytoplasm (Fig. 1B). A
significant fraction of gagPKB, however, is localized to the nucleus
(Fig. 1B). This result is consistent with previous studies that have
shown gagPKB localization at the plasma membrane (40%), in the
cytoplasm (30%), and within the nucleus (30%) (1, 16).
These results confirm that phosphorylation of AFX by PKB results in a
rapid redistribution of AFX from the nucleus to the cytoplasm.
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Nuclear export subsequent to phosphorylation by PKB in the nucleus
is required for the inhibition of AFX transcriptional activity.
We
wanted to test the hypothesis that PKB-dependent redistribution of AFX
to the cytoplasm is required to regulate the transcriptional activity
of the protein. The best-characterized nuclear export pathway uses a
leucine-rich nuclear export signal (NES), which is bound by the export
receptor Crm1 in the presence of RanGTP in the nucleus (25, 26,
43, 56, 68). The trimeric complex is then exported to the
cytoplasm and is disassembled. Crm1-dependent export can be inhibited
by LMB (74), which specifically inactivates Crm1 by
covalent modification of a key cysteine residue in the NES-binding
region of the protein (44). We tested, therefore, whether
LMB could inhibit the cytoplasmic relocalization of HA-AFX in response
to insulin (Fig. 2A)
and serum (data not shown). As shown
in Fig. 2A, LMB completely blocked the nuclear export of AFX. Thus,
nuclear export of AFX in response to insulin or serum proceeds via a
Crm1-dependent pathway.
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Regulation of localization of AFX by PKB does not occur at the level of export. There are several distinct mechanisms by which PKB-dependent phosphorylation of AFX could trigger cytoplasmic accumulation of the protein. First, PKB phosphorylation could facilitate nuclear export by promoting the binding of Crm1 to AFX. In a second model, AFX may be exported from the nucleus constitutively but be reimported rapidly when it is unphosphorylated, so that its steady-state distribution is predominantly nuclear. PKB phosphorylation would then inhibit or decrease the rate of import, leading to a redistribution to the cytoplasm. Third, both nuclear import and export of AFX could be constitutive, and phosphorylation would permit cytoplasmic retention by increasing the affinity of AFX for a cytoplasmic anchor protein. Finally, phosphorylation by PKB could release AFX from a nuclear anchor.
To distinguish between these possibilities, we performed Crm1 binding assays. AFX cannot be expressed in appreciable amounts as a recombinant protein in Escherichia coli. Therefore, HA-AFX and HA-A3 (HA-AFX with the following mutations: T28A, S193A, and S258A) were immunoprecipitated from mammalian cell lysates and incubated with recombinant Crm1 in the absence or presence of RanQ69L, a mutant of Ran locked in its GTP bound conformation. RanQ69L increases the affinity of Crm1 for its export cargoes. Interestingly, both PKB-phosphorylated (HA-AFX) and -unphosphorylated (HA-AFX plus LY294002 and HA-A3) AFX bound to Crm1 in the presence of RanQ69L (Fig. 3A). These data demonstrate that binding of Crm1 to AFX is independent of phosphorylation by PKB.
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, Crm1, and RanQ69L (data not shown).
The observation that Crm1 binding to AFX is independent of PKB
phosphorylation is supported by the results of heterokaryon fusion
assays. HA-A3 has a steady-state nuclear localization but nonetheless
may actively shuttle into and out of the nucleus. Nucleocytoplasmic
shuttling can be observed by the use of cell fusion assays in which the
donor and acceptor nuclei of the fused cells can be distinguished.
Redistribution of a tagged protein from one nucleus to another can only
occur if the protein exits the donor nucleus and is reimported into the
acceptor nucleus. To perform heterokaryon fusion assays, we used BHK21
cells expressing HA-A3 as the donor and a reporter cell line, GSN2,
that expresses GFP-streptavidin-NLS as the acceptor. The
GFP-streptavidin-NLS fusion protein, which is constitutively nuclear
and does not shuttle, acts as a marker for the acceptor nuclei. HA-A3
shuttling would lead to its equilibration into the nucleus of a fused
GSN2 cell. On the other hand, if HA-A3 does not shuttle, no HA-A3 would
be detected in the nuclei of the GSN2 cells. Figure 3C shows an AFX donor nucleus surrounded by three GSN2 acceptor nuclei that also stained positive for HA-A3. Therefore, even though unphosphorylated AFX
is predominantly nuclear in its steady-state distribution, it is
constitutively shuttling into and out of the cytoplasm. AFX
phosphorylation by PKB, however, results in a shift in the steady-state
distribution of AFX to the cytoplasm. Taken together these results
suggest that the regulation of AFX localization in response to PKB
phosphorylation occurs not at the level of nuclear export or nuclear
retention but instead at the level of nuclear import or through
cytoplasmic retention of phosphorylated AFX.
AFX import into the nucleus proceeds via an active, Ran-dependent mechanism. On account of the results described above, it was important to identify the mechanism by which AFX is imported into the nucleus. In principle, AFX could accumulate in the nucleus either by an active mechanism or by diffusion followed by nuclear retention. Most active nuclear transport pathways studied to date require energy and an intact Ran gradient across the nuclear envelope. To determine whether the nuclear accumulation of AFX observed in serum-starved cells proceeds via an active, Ran-dependent mechanism, we used tsBN2 cells. These cells have a temperature-sensitive RanGEF (RCC1) allele (70). When grown at the permissive temperature (33.5°C), tsBN2 cells have an intact Ran gradient. However, incubation at the nonpermissive temperature (39.5°C) causes collapse of the Ran gradient and prevents Ran-dependent nuclear transport. As we have shown previously, incubation for 3 h at 39.5°C in the presence of cycloheximide (to prevent new RanGEF synthesis) completely inhibits the import of other substrates known to utilize a classical import pathway, such as the glucocorticoid receptor (62).
To test the Ran dependence of AFX import, we transfected tsBN2 cells with a plasmid encoding HA-AFX. In the presence of cycloheximide, cells were grown at either 33.5°C or were shifted to 39.5°C for 3 h. The cells were then serum starved. Immunofluorescence revealed that at the permissive temperature HA-AFX entered the nucleus on serum starvation (Fig. 4A, 33.5°C). In cells incubated at 39.5°C, however, HA-AFX did not accumulate in the nucleus (Fig. 4A, 39.5°C). We conclude, therefore, that import of AFX into the nucleus proceeds via an active, Ran-dependent mechanism.
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Basic residues on both sides of S193 are necessary and sufficient
for nuclear import of AFX.
To determine whether PKB
phosphorylation regulates the nuclear import of AFX, we needed to
identify the NLS within the AFX sequence. Clusters of basic residues
are often predictive that a region of a protein may act as an NLS. AFX
contains two clusters of basic residues flanking the PKB
phosphorylation site S193. Therefore, deletion of these clusters would
test whether they are required for AFX nuclear import. Deletion of
residues 198 to 216 from HA-AFX that are C terminal to S193 results in
a protein that is unable to enter the nucleus under the same conditions of serum starvation that result in HA-AFX accumulation in the nucleus
(Fig. 4B). Thus, this region is necessary for nuclear import of AFX. To
test whether this region is sufficient to mediate import, we expressed
residues 198 to 221 as an amino-terminal fusion to GFP-GFP-GFP
[AFX(198-221)-GFP3] in mammalian cells. The triple GFP construct
(~80 kDa) was used to prevent passive diffusion through the nuclear
pore complex (NPC) which has a diffusion limit of ~60 kDa. (These
fusion proteins could not be expressed in E. coli, because
the NLS is cleaved stoichiometrically during the induction of
expression under all conditions tested). As shown in Fig.
5A, AFX(198-221)-GFP3 did not accumulate
in the nucleus. The basic region upstream of S193 was also not able to
act as an NLS in isolation [Fig. 5; AFX(180-197)-GFP3]. However, a
construct containing both basic regions accumulated efficiently in the
nucleus [AFX(180-221)-GFP3]. Expression of these constructs as
carboxy-terminal fusions to GFP-GFP-GFP resulted in the same
localization (data not shown). We conclude, therefore, that residues
180 to 221 of AFX constitute an NLS and that basic residues on both
sides of S193 are required for the nuclear import of AFX.
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1, importin 3, or importin to this region of the protein
could not be detected (data not shown). Therefore, we produced a series of mutations in AFX(180-221)-GFP3 to identify residues required for
import (Fig. 6). Quantification of
relative nuclear and cytoplasmic fluorescence levels demonstrated that
mutation of arginines 188 to 190 to alanine had the most deleterious
effect on import (Fig. 6, mutant II), followed by the mutation of
lysines 209 and 210 (Fig. 6, mutant V). This result reinforces the
requirement for basic residues both upstream and downstream of S193 for
nuclear import and confirms that the sequence is an atypical NLS.
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Phosphorylation of S193 reduces the rate of nuclear import.
Phosphorylation of S193 adds a negative charge to a positively charged
region of AFX. This may affect the binding of AFX to its nuclear import
receptor and reduce its rate of import. To test this hypothesis, we
created two AFX(180-221)-GFP3 mutants: S193A, which cannot be
phosphorylated, and S193E, which may mimic phosphorylation at this
site. Nuclear import of S193A was comparable to that of wild-type
AFX(180-221)-GFP3 (Fig. 7A and C).
This demonstrates that mutation of
S193 results in a stable fusion protein that is able to enter the
nucleus. The S193E mutation, however, substantially reduced nuclear
accumulation (Fig. 7A and C). This result suggests that PKB
phosphorylation of S193 reduces the rate of nuclear import of AFX. It
is important to note that these GFP3 constructs do not contain an NES.
Therefore, over a 20-h transfection period, even with a greatly reduced
rate of import some nuclear accumulation would be expected, since the
construct cannot be reexported. Therefore, a phosphorylation-dependent
reduction in the import rate of AFX, combined with constitutive nuclear
export, would shift the steady-state distribution of the protein from
the nucleus to the cytoplasm. This mechanism is consistent with the
known redistribution of AFX to the cytoplasm on addition of insulin or
serum.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nuclear transport of the Forkhead transcription factor, AFX, plays
a critical role in the regulation of its transcriptional activity.
Phosphorylation of AFX by PKB results in a rapid change in the
steady-state distribution of the protein from the nucleus to the
cytoplasm. However, the mechanism by which this relocalization occurs
has not been determined, although it has been proposed by others that
export may be regulated by PKB and that 14-3-3 may function as a
chaperone for export, as has also been suggested for the regulation of
cdc25 export (19). Based on the data presented in this
paper, we propose a different model, in which nuclear import, not
export, of AFX is the principal target of regulation by PKB (Fig.
8).
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Unphosphorylated AFX is predominantly nuclear at steady state. However, heterokaryon fusion assays (Fig. 3C) demonstrated that the protein is, in fact, continually shuttling between the nucleus and the cytoplasm. The rate constant for unphosphorylated AFX import, therefore, must exceed the rate constant for its export (Fig. 8A). Importantly, the nuclear export of AFX appears to be unaltered by PKB phosphorylation. Nuclear export of both phosphorylated and unphosphorylated AFXs is likely mediated by the export receptor, Crm1. Both wild-type HA-AFX and the triple mutant, HA-A3, which cannot be phosphorylated by PKB, bind Crm1 in the presence of RanGTP (Fig. 3A). In support of the identification of Crm1 as the export receptor, we show that AFX export from the nucleus in response to insulin is blocked by the addition of LMB (Fig. 2A), a potent and specific inhibitor of Crm1 function.
The addition of insulin to cells results in the activation of PKB and its translocation into the nucleus, where it phosphorylates Forkhead family members (Fig. 8B). Treatment of cells with LMB traps AFX and FKHRL1 in the nucleus. However, both are phosphorylated in response to insulin to the same extent and at the same residues as in cells not treated with LMB (Fig. 2B). This result, therefore, strongly indicates that PKB enters the nucleus when activated by insulin to phosphorylate Forkhead transcription factors and other target proteins. Phosphorylation alone, however, is not sufficient to inhibit the transcriptional activity of AFX. We have shown, using transcriptional activation assays, that blockade of AFX nuclear export by the addition of LMB results in a loss of insulin-induced transcriptional control (Fig. 2B). Therefore, both phosphorylation by PKB and nuclear export mediated by Crm1 are essential for complete inhibition of AFX transcriptional activity.
We have found that AFX enters the nucleus by an active, Ran-dependent mechanism (Fig. 4A) and that import requires a basic region in AFX encompassing PKB phosphorylation site S193 (Fig. 5). Importantly, the addition of a negative charge at S193 substantially attenuates nuclear import (Fig. 7), most likely by reducing the affinity of AFX for its nuclear import receptor. We propose, therefore, that phosphorylation by PKB at S193 reduces the rate of AFX import. Since the steady-state localization of a protein is determined by its relative rate constants for import and export, the localization of AFX would shift from the nucleus to the cytoplasm (Fig. 8B). This alteration in the transport rate constants in response to phosphorylation by PKB, in conjunction with proposed cytoplasmic retention by binding 14-3-3 proteins (14), would result in both the efficient nuclear exclusion of AFX and the inhibition of its transcriptional activity. In conclusion, we propose that this nuclear exclusion mechanism is required to regulate the activity of AFX.
These data are consistent with the results of transcriptional activation assays reported previously for FKHR (30). The PKB phosphorylation site S256 in FKHR is analogous to S193 in AFX. Mutation of S256 to an alanine in the context of full-length FKHR abolished the ability of insulin and PKB to inhibit FKHR activity. In contrast, mutation of S256 to an aspartate resulted in a substantial inhibition of its transcriptional activity. These effects were not observed when the other two PKB sites were mutated independently. Although the localization of the S256D mutant was not assayed, our data suggest that the inhibition of FKHR activity may be due, in part, to a deficit in nuclear import.
"Classical" nuclear localization sequences are characterized by
short amino acid stretches that are enriched in basic amino acids. The
NLS of the large T antigen of simian virus 40 was identified by
deletion analysis that resulted in mislocalization of the protein to
the cytoplasm (45). It was later defined as a
seven-amino-acid sequence (PKKKRKV) sufficient to confer
nuclear localization when conjugated to a carrier protein
(46). Some proteins contain similar sequences that are
referred to as monopartite NLSs. Other proteins, such as nucleoplasmin,
contain bipartite NLSs that consist of two patches of basic residues
separated by a 10-amino-acid spacer (23). Generally,
proteins carrying classical or bipartite NLSs bind a cytoplasmic
receptor, importin (28, 48). Importin associates
with importin , a protein that docks import complexes at the NPC and
translocates import cargo into the nucleus. There are, however, many
exceptions to this type of import, and there is a large family of
importins and exportins that can recognize distinct NLSs and mediate
transport of different subsets of cargo. For example, ribosomal
proteins (33) and histones (32) have been
shown to bind directly to several different importin family members
and dock at the NPC independently of importin . In addition, other
proteins, such as hnRNP K (51) and -catenin
(75), can translocate through the NPC in the absence of
any soluble factors.
We have delineated residues 180 to 221 of AFX as a novel type of nuclear import signal. This region contains a small portion of the DNA binding domain. Therefore, like other Forkhead family members, the DNA binding domain of AFX contributes to DNA binding and nuclear localization (59). This region of AFX contains 12 lysine and arginine residues. Although several groups of these basic residues could act as monopartite or bipartite NLSs, the information provided by mutational analysis suggests that AFX contains a nonclassical NLS. Mutation of arginines 188 to 190 in the NLS of AFX has the greatest inhibitory effect on nuclear import, followed by the mutation of lysines 209 to 211. These groups of basic residues are separated by 18 amino acids, and both are contained within similar sequence repeats (KAPRRR and KAPKKK). These repeats may be important for import receptor binding.
The identity of the nuclear import receptor of AFX remains to be
established. Since we do not observe binding of AFX to several classical import receptors, including importins 1, 3, and , it
may bind to a novel member of the importin family (which comprises >20
members in mammalian cells). Alternatively, AFX may enter the nucleus
in other ways, for instance, by piggybacking on another protein that
contains a classical NLS. The role of PKB-mediated phosphorylation and
14-3-3 binding in altering AFX import can be more thoroughly studied
once the receptor is identified.
Binding of Forkhead family members to 14-3-3 proteins has been proposed
to play a role in the retention of phosphorylated Forkheads in
the cytoplasm (14). Phosphorylated FKHRL1
(14) and AFX (Fig. 3A) both bind 14-3-3 proteins.
However, we have never observed any specificity of AFX binding to a
particular 14-3-3 isoform. In fact, we have observed binding to
14-3-3, 14-3-3
, and 14-3-3 (data not shown). Recently, it has
been proposed that 14-3-3 protein binding may impart no specific
information about subcellular targeting (53). Instead, a
14-3-3 protein may obscure the NLS or NES of the protein to which it
binds. In this way, 14-3-3 proteins would affect the subcellular
localization of their target proteins by interfering with the binding
of transport receptors. 14-3-3 binding to Forkhead family members may
sterically inhibit import receptor binding and, thereby, act to prevent import.
The proposal has also been made that 14-3-3 possesses an NES that is recognized by Crm1 and participates directly in the export of binding partners through a Crm1 interaction (63). In our hands, however, 14-3-3 does not bind Crm1 directly, nor is its association with AFX necessary for the interaction of AFX with Crm1. Therefore, in this case at least, 14-3-3 isoforms appear to play no part in regulating AFX nuclear export.
What is the AFX NES? Leucine-rich regions found in several Forkhead family members conform to the consensus sequence for a Crm1-dependent NES (9). This region in AFX corresponds to residues 300 to 308 (LELLDGLNL). Deletion of residues 298 to 308 results in a protein that is unable to relocalize from the nucleus to the cytoplasm on stimulation with insulin (unpublished data). This region, therefore, likely represents the Crm1-dependent NES.
Distinct chromosomal translocations in pediatric alveolar rhabdomyosarcoma and acute lymphoblastic leukemia (ALL) involve two human Forkhead genes. Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations that arise from fusion of PAX3 or PAX7 to the FKHR gene (27). The PAX3-FKHR fusion protein is a more potent transcriptional activator than PAX3 alone (8) and transforms primary cells (64). Several chromosomal translocations in ALL occur at the 11q23 locus, and all convey a poor prognosis that is usually associated with a high rate of treatment failure and relapse. These breakpoints affect the MLL gene (also called HRX, ALL, or Htrx1) that is disrupted midway through the coding region (18, 77). A well-documented translocation arises from the fusion of MLL to the AFX gene on chromosome X (13).
Importantly, the fusions of AFX and FKHR to their breakpoint partners occur at identical amino acid positions within the Forkhead proteins. The resulting fusion proteins contain the N-terminal DNA binding region of the fusion partner and the C-terminal transactivation domain of the Forkhead protein. The MLL-AFX fusion protein expresses residues 148 to 501 of AFX (57). This preserves the NLS of AFX identified in this study (residues 180 to 221) but deletes the T28 phosphorylation site. We have shown that deletion of residues N terminal to S193 causes a loss of transcriptional regulation by insulin (42). In addition, potential loss of S193 phosphorylation in the context of the fusion protein would allow constitutive import into the nucleus. Therefore, both the deregulation of Forkhead activity and a constitutive nuclear localization likely contribute to the oncogenic properties of these fusion proteins.
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ACKNOWLEDGMENTS |
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We thank all members of our laboratories for their continued advice throughout the experimental and preparatory phases of the manuscript. In particular, we thank Mark Lindsay, Michael Nemergut, Kendra Plafker, Scott Plafker, Alicia Smith, Katie Welch, Nancy de Ruiter, and Hans Bos. We also thank Iain Mattaj and Dirk Görlich for their generosity in providing expression plasmids used in this study (Crm1, RanQ69L), Barbara Wolff for her gift of LMB, Anne Brunet for the anti-phospho T32 FKHRL1 antibody, Kris Reedquist for the c-cbl antibody, Marc Timmers for the RNA pol II antibody, and Bryce Paschal for the GSN2 cell line.
This work was supported by a grant awarded to I.G.M. from the National Institutes of Health, DHHS (GM-50526). A.M.B. is supported by a Postdoctoral National Service Award from the National Institutes of Health, DHHS (GM-20017). G.K. and B.B. are supported by grants from NWO and the Dutch Cancer Foundation (KWF).
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Virginia, Center for Cell Signaling, P.O. Box 800577, Charlottesville, VA 22908. Phone: (804) 982-0083. Fax: (804) 924-1236. E-mail: amb7c{at}Virginia.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, May 2001, p. 3534-3546, Vol. 21, No. 10
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.10.3534-3546.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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