From the Department of Biochemistry, Flanders
Interuniversity Institute for Biotechnology, Faculty of Medicine and
Health Sciences, Ghent University, Rommelaere Institute, Albert
Baertsoenkaai 3, B-9000 Ghent, Belgium and Flanders Interuniversity
Institute for Biotechnology, the
Institut für
Biochemie I, Medizinische Fakultät, Universität zu
Köln, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany, and
the ** Laboratory of Experimental Cancerology, Department of
Radiotherapy and Nuclear Medicine, Ghent University Hospital, De
Pintelaan 185, B-9000 Ghent, Belgium
Received for publication, September 27, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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Despite thorough structure-function analyses, it
remains unclear how CapG, a ubiquitous F-actin barbed end capping
protein that controls actin microfilament turnover in cells, is able to reside in the nucleus and cytoplasm, whereas structurally related actin-binding proteins are predominantly cytoplasmic. Here we report
the molecular basis for the different subcellular localization of CapG,
severin, and fragminP. Green fluorescent protein-tagged fragminP
and severin accumulate in the nucleus upon treatment of transfected
cells with the CRM1 inhibitor leptomycin B. We identified a nuclear
export sequence in severin and fragminP, which is absent in CapG.
Deletion of amino acids Met1-Leu27
resulted in nuclear accumulation of severin and fragminP. Tagging this
sequence to CapG triggered nuclear export, whereas mutation of single
leucine residues (Leu17, Leu21, and
Leu27) in the export sequence inhibited nuclear export.
Based on these findings, a nuclear export signal was identified in
myopodin, a muscle-specific actin-binding protein, and the Bloom
syndrome protein, a RecQ-like helicase. Deletion of the myopodin
nuclear export sequence blocked invasion into collagen type I of C2C12 cells transiently overexpressing myopodin. Our findings explain regulated subcellular targeting of distinct classes of actin-binding proteins.
In eukaryotic cells, the nucleus and cytoplasm are separated by
the nuclear envelope. Macromolecular traffic occurs via nuclear pore
complexes, huge proteinaceous structures that span the double membrane
that allow transport in essentially two modes: energy-independent passive diffusion and energy-dependent facilitated
translocation (for reviews see Refs. 1 and 2). Passive diffusion is
efficient for small molecules but becomes slow and inefficient as the
size approaches a limit of 20-40 kDa. In contrast, facilitated
translocation allows passage of macromolecules as large as several
megadaltons and proceeds via specific nuclear transport receptors able
to associate with components of the nuclear pore complex as well as
cargo molecules that are translocated across the pore. Cargo molecules
are recognized via import or export targeting signals, referred to as
nuclear localization signals or nuclear export signals
(NES),1 respectively
(1-3).
There are several types of nuclear localization signals, which mediate
nuclear import by direct binding to one or more importin receptors of
the An increasing number of actin-binding proteins has been reported to
shuttle between nucleus and cytoplasm. Already in 1993, Onoda et
al. (10) showed that CapG (Mbh1 or gCap39), a ubiquitous 39-kDa
barbed end F-actin-binding protein particularly abundant in macrophages
(11), is a nuclear and cytoplasmic protein. CapG does not contain a
canonical nuclear localization signal, but it has been suggested that
phosphorylation of CapG may be involved in controlling the subcellular
localization of the protein (12). More recent data obtained from CapG
null mice showed that CapG plays a role in phagocytosis and receptor
membrane ruffling (13). Cofilin is a major actin depolymerizing
protein, and its nuclear translocation is regulated by phosphorylation
in some cells (14-17).
There is evidence in favor of a role for actin in nuclear processes,
ranging from chromatin remodelling (18) to nuclear export (19). The
presence of actin-binding proteins in the nucleus suggests that actin
may not merely be present in the nucleus but that its polymerization is
controlled as well. Alternatively, nuclear actin-binding proteins may
display functions distinct from controlling actin polymerization. For
instance, supervillin, an F-actin bundling protein, contains nuclear
localization signals (20) and associates with the androgen receptor,
modulating its transcriptional activity (21). Zyxin, a component of
focal adhesions, is involved in controlling mitosis through association
with h-warts/LATS1, a serine/threonine kinase and constituent of the
mitotic apparatus (22).
CapG is structurally related to Dictyostelium discoideum
severin and Physarum polycephalum fragminP (FrgP). These
proteins are characterized by repeats of 125-150-amino acid segments.
In CapG, severin, and FrgP, the subdomains are organized in triplicate, whereas gelsolin contains six repeats. CapG displays 54% similarity with severin (23) and FrgP (24). In contrast to CapG, however, they
have not been reported to shuttle between nucleus and cytoplasm (25,
26). In addition to binding the fast growing end of actin filaments,
severin and fragminP display F-actin severing activity in the presence
of calcium, enabling these proteins to modulate cell morphology and
cell motility processes.
We show here that severin and FrgP, but not CapG, contain Rev-like NESs
at their N termini that control nucleo-cytoplasmic trafficking of these
proteins. Mutation of single leucine residues in the export sequence
abrogated export activity. As such, these findings may provide a
molecular basis for the observed nuclear and cytoplasmic localization
of CapG as compared with the predominantly cytoplasmic localization of
severin and FrgP. Myopodin, a muscle-specific actin bundling protein,
was recently shown to shuttle between nucleus and cytoplasm in a
developmentally regulated manner (27). Delineation of a nuclear export
sequence in severin and fragminP enabled us to identify a nuclear
export sequence in myopodin, as well as in the Bloom syndrome protein.
Reagents--
Texas Red-X-phalloidin and Alexa 488-conjugated
goat anti-mouse IgG conjugate were obtained from Molecular Probes
(Eugene, OR). Restriction enzymes were from New England Biolabs, Inc.
(Beverly, MA). Platinum Taq HIFI polymerase, pcDNA3.1
V5-His-TOPO, pCR® T7 CT-TOPO, and anti-V5 antibody were purchased from
Invitrogen. Pfu Turbo polymerase and Quikchange
site-directed mutagenesis kit were from Stratagene (La Jolla, CA).
pEGFP-N1 was purchased from Clontech (BD
Biosciences, Palo Alto, CA). Leptomycin B and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma. DEAE-cellulose (DE-52) was purchased from Whatman (Maidstone, UK).
Molecular mass markers for SDS-PAGE were from Bio-Rad.
Phenylmethylsulfonyl fluoride was from Serva (Heidelberg, Germany).
Other protease inhibitors were from Amersham Biosciences.
Cell Culture and Transfection--
MDCK-AZ (28), C2C12, and
HEK293T cells were maintained at 37 °C in a humidified 10%
CO2 incubator and grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg of
streptomycin (Invitrogen). HEK293T cells seeded on rat tail
collagen-coated cover slips were transfected with cDNA constructs
using calcium phosphate. MDCK-AZ and C2C12 cells were transfected with
LipofectAMINE (Invitrogen) according to the manufacturer's
instructions. The cells were grown for 24-72 h before processing for
microscopy. For quantitation of experimental data, 200 cells were
counted and scored for GFP localization (cytoplasmic only, or
cytoplasmic and nuclear). KS100 software (Zeiss) was used to calculate
the ratio of nuclear fluorescence versus cytoplasmic
fluorescence in transfected cells. CapG-EGFP as well as fragminP NES
mutants showed a nuclear/cytoplasmic ratio of 1.40 (arbitrary units), whereas FrgP(M1-L27)-CapG-EGFP and other constructs with a similar subcellular distribution were characterized by a nuclear/cytoplasmic ratio of 0.71 (arbitrary units). The experiments were performed in
triplicate. The mean values and standard deviations were calculated.
cDNA Cloning--
Total RNA was isolated from C2C12
myoblasts (generously provided by Dr. J. C. Adams, Lerner Research
Institute, Cleveland, OH) with the RNeasy kit (Qiagen), and mRNA
was reverse transcribed into first strand cDNA. For PCR,
50-µl reactions were set up containing 2.5 units of Taq
polymerase, 2 µl of template cDNA, and 0.8 µM primer. 35 cycles of PCR were performed using the following primers to
obtain full-length myopodin:
5'-cgaattcgccaccatgattccttgcagtcatcaattcag-3' and
5'-cggtggatcccgctcttccacaactgacggtttccac-3'. For myopodin lacking the
NES, the primer 5'-cgaattcgccaccatgtctgaaaagcaagtgaaagaagc-3' was used.
cDNAs were cloned into pcDNA3.1/V5-His (Invitrogen). All of the
constructs were verified by sequencing.
cDNA clones encoding FrgP, CapG, or severin were cloned into
pEGFP-N1. FrgP was subcloned into CapG-pEGFP-N1 as a
KpnI/AgeI fragment. CapG-FrgP chimeras were
created by a unique procedure using partially complementary primers in
a PCR with CapG-FrgP-pEGFP-N1 as template. In the example of swapping
the CapG S3 subdomain for FrgP S3, the forward primer contains 21 nucleotides at the end of the S2 domain of CapG and 24 nucleotides at
the beginning of the S3 domain of FrgP. The reverse primers contains 45 nucleotides at the end of the S2 domain of CapG, including the
complementary nucleotides of the forward primer. After the addition of
Pfu Turbo polymerase and temperature cycling (18 cycles),
the nonmutated, parental cDNA was digested with DpnI.
Following transformation, XL1-Blue supercompetent cells repair the DNA
as a consequence of the 21 complementary nucleotides. Using
CapG-pEGFP-N1 as a template, the coding sequence for the FrgP NES was
fused N-terminally to CapG in a similar manner. The forward primer
encodes amino acids 18-27 of the NES sequence, a Kozak sequence, an
extra ATG codon, and 24 nucleotides of the N terminus of CapG, starting with the second codon, omitting the start codon of CapG, to prevent incorrect initiation of translation. The reverse primer contains the
complementary nucleotides of the NES sequence and 24 nucleotides starting from the HindIII-cloning site in the pEGFP-N1
vector. Additional sequences were fused to CapG in separate PCRs.
N-terminal tagging of the myopodin or Bloom NES to CapG-EGFP was
performed according to the same strategy. Mutations in the
NES-FrgP-CapG construct were generated using the Quikchange
site-directed mutagenesis kit (Stratagene).
Recombinant Protein Expression and Purification--
The
full-length CapG sequence was cloned into the pLT10T3 expression vector
(29) and transformed into Escherichia coli MC1061 that
already contained the pSCM26 plasmid (30) and harbors the T7 polymerase
gene (see also Ref. 24). The cells were grown in 500 ml of LB medium
containing 100 µg/ml ampicillin. The cultures were grown to an
A600 of 0.6 at 37 °C. Protein expression was induced by the addition of 0.5 mM isopropyl
thio- Fluorescence Microscopy and Immunocytochemistry--
The cells
were viewed directly for EGFP fluorescence after fixation with 3%
para-formaldehyde in phosphate-buffered saline (Invitrogen)
for 20 min at room temperature and staining with DAPI. For myopodin
staining, the cells were fixed, permeabilized in 0.1% Triton X-100,
washed in phosphate-buffered saline, incubated with anti-V5 antibody
for 1 h at 37 °C, and following several washes in
phosphate-buffered saline incubated with Alexa 488-conjugated goat
anti-mouse antibody for 30 min at room temperature. Microscopic images
were captured with a Zeiss Axioplan epifluorescence microscope (×40
objective) equipped with an Axiocam cooled CCD camera and processed
using KS100 software (Zeiss).
Capping of Actin Filaments--
The experiments were performed
essentially as described earlier (31, 32). Briefly, preassembled
unlabeled actin filaments (final concentration, 1 µM)
were used as "nuclei." They were subsequently mixed with 3 µM G-actin (25% pyrene-labeled) in the absence or presence of untagged recombinant CapG or Myc-V5-His6-tagged
CapG, in a total volume of 300 µl. The increase in fluorescence
( Nuclear Export Inhibition--
MDCK-AZ cells transfected with
fragminP-EGFP, CapG-fragminP-EGFP or NES fragminP-CapG-EGFP were
treated with 10 ng/ml leptomycin B (Sigma) and fixed 360 min after the
addition of the drug.
Collagen Invasion Assay--
Invasion into collagen Type I was
performed as described previously (33). Six-well plates were filled
with 1.25 ml of neutralized type 1 collagen (0.09%; w/v) (Upstate
Biotechnology, Inc.) and incubated for at least 1 h at 37 °C to
allow gelification. The cells were harvested using Moscona buffer and
Trypsin/EDTA and seeded on top of the collagen gel. The cultures were
incubated at 37 °C for 24 h. The depth of cell migration inside
the gel was measured using a phase contrast microscope controlled by a computer program. Invasive and superficial cells were counted in 12 fields of 0.157 mm2. The invasion index is the percentage
of cells invading the gel over the total number of cells.
Miscellaneous--
The protein concentrations were determined by
the method of Bradford (34) using bovine serum albumin as a standard.
CapG-EGFP, Severin-EGFP, and fragminP-EGFP Localize Differentially
in Transfected Cells--
The molecular mass of native CapG (39 kDa) is compatible with passive transport of the protein to the nucleus
(2). Despite their similar size, severin and FrgP are predominantly
cytoplasmic, and these proteins are not observed in the nucleus under
normal growth conditions. We cloned CapG upstream of EGFP, resulting in
a fusion protein with a predicted molecular mass of 67 kDa. Transient
expression of CapG-EGFP in HEK293T cells showed a distribution identical to wild type CapG (10); fluorescence was detected in the
cytoplasm as well as in the nucleus (Fig.
1A). By contrast, FrgP-EGFP
and severin-EGFP were present in the cytoplasm under these conditions.
In a limited number of cells, very weak nuclear staining could be
observed (Fig. 1, B-D). Western blots on crude extracts of
transfected HEK293T cells showed a band corresponding with the expected
molecular mass of the fusion proteins (not shown). Because passive
diffusion of bovine serum albumin, a protein with similar mass (68 kDa), is extremely slow (2), we considered it unlikely that CapG-EGFP
(67 kDa) translocates to the nucleus by passive diffusion.
Leptomycin B Induces Nuclear Accumulation of fragminP--
We
further examined nuclear trafficking of these proteins by generating a
CapG-FrgP-EGFP fusion polypeptide. We expected that CapG-FrgP-EGFP
would display a nuclear and cytoplasmic localization similar to those
of CapG-EGFP. Surprisingly, however, the CapG-FrgP-EGFP fusion protein
was present in the cytoplasm following transfection in HEK293T or MDCK
cells (Fig. 2, A and
B). This may be due to inhibition of interaction between
CapG and a putative nuclear import factor by FrgP in the large fusion
protein. Alternatively, FrgP may contain a nuclear export sequence that
prevents nuclear accumulation of the CapG-FrgP-EGFP fusion protein. The
second possibility was further investigated using leptomycin B (LMB), a
drug that interferes with the nuclear export factor CRM1 (35). When
MDCK cells transiently expressing CapG-FrgP-EGFP were incubated with
LMB, we observed nuclear enrichment of the fusion protein (Fig.
2C). Similarly, FrgP-EGFP (Fig. 2, D and
E) accumulated in the nucleus upon incubation of transfected
MDCK cells with LMB. These findings suggest that FrgP contains a
nuclear export sequence. If CapG should lack such a regulatory
sequence, it could explain why this actin-binding protein is
constitutively present in the nucleus and cytoplasm under normal growth
conditions. This was further investigated by transfection studies using
CapG-FrgP chimerical proteins (domain swapping).
The N-terminal Region of Severin and fragminP Constitutes a Nuclear
Export Sequence That Is Absent in CapG--
To identify the putative
regulatory sequences in severin, FrgP, and CapG controlling their
subcellular localization, we generated CapG-FrgP chimerical proteins
whereby CapG subdomains were exchanged for structurally similar FrgP
subdomains (see "Experimental Procedures"). Because FrgP is cloned
downstream of CapG, hybrid proteins always start with the CapG
methionine residue (Table I). We
predicted that when a nuclear export sequence in FrgP is deleted, the
hybrid protein would be nuclear. Surprisingly, all of the chimeras
showed a distribution identical to CapG-EGFP, displaying cytoplasmic as
well as nuclear distribution. Even the chimera that contained 95% of
the FrgP sequence and barely 5% of the CapG sequence (CapG M1-L27/FrgP
Q52-D371) showed nuclear localization (Fig.
3A).
Aligning the N-terminal regions of CapG, severin, and FrgP revealed
several conserved hydrophobic residues in severin and FrgP, a hallmark
of nuclear export sequences (36). This stretch of amino acids is absent
in CapG (Fig. 3B). This sequence resembles the Rev-type
export signal (37, 38), but the spacing between the hydrophobic amino
acids is different from the Rev NES signal.
We tested the functionality of this region by deleting FrgP amino acids
Met1-Leu27 in CapG-FrgP-EGFP, FrgP-EGFP, and
the corresponding 27 amino acids in severin-EGFP and analyzed the
distribution of these truncated fusion proteins following transfection
in HEK293T cells. Interestingly, fluorescence was detected in the
nucleus as well as in the cytoplasm (Fig. 3, C-E), similar
to CapG-EGFP. This result shows that the N-terminal region of severin
and FrgP constitutes a functional nuclear export sequence.
Three Leucine Residues in the Severin/FrgP NES Are
Indispensable for Directing Nuclear Export--
To ascertain whether
the FrgP/severin nuclear export sequence is able to export an otherwise
nuclear protein, we cloned the FrgP export sequence upstream of
CapG-EGFP. Appendage of amino acids
Gly18-Leu27 of FrgP to the N terminus of
CapG-EGFP followed by transfection in HEK293T cells did not affect the
localization of the fusion protein (not shown), suggesting that this
region alone is not sufficient for nuclear export. This finding also
demonstrates that modification of the CapG N terminus per se
does not interfere with nuclear trafficking. Furthermore, a CapG fusion
protein containing an N-terminal Myc tag and a C-terminal V5 epitope in
addition to a His6 tag was found to cap the barbed ends of
actin filaments as efficiently as the untagged recombinant protein
(Fig. 4). Therefore, N-terminal
attachment of amino acids does not interfere with the F-actin capping
activity of the actin-binding protein. However, appendage of FrgP amino
acids Leu17-Leu27, containing three leucine
residues, promoted export of CapG-EGFP in 22% of the cells (Fig.
5, A and D). FrgP
M1-L27-CapG-EGFP, containing the full stretch of the 27 N-terminal
amino acids of FrgP, promoted cytoplasmic localization of the fusion
protein in nearly 100% of the cells (Fig. 5, B and
D), demonstrating that the FrgP N-terminal region represents
a functional nuclear export sequence able to export CapG from the
nucleus. Transfection of this construct into MDCK cells also resulted
in a cytoplasmic localization (Fig. 5C) in the absence of
LMB. However, the addition of LMB to the cells resulted in nuclear
enrichment of FrgP NES-tagged CapG-EGFP (Fig. 5C').
The FrgP nuclear export sequence contains two isoleucines and three
leucine residues (Fig. 3B). In severin, Ile14 is
substituted by a Val residue. To examine the contribution of these
hydrophobic residues in directing nuclear export, we generated single
and double point mutants in the CapG-EGFP cDNA construct that
harbors the FrgP nuclear export sequence (FrgP M1-L27-CapG-EGFP).
Single mutants in which either Ile9, Ile14, or
Val25 were substituted by an alanine residue did not
significantly prevent nuclear export of FrgP M1-L27-CapG-EGFP (I9A,
96.5% cytoplasmic; I14A, 96% cytoplasmic; and V25A, 99.5%
cytoplasmic). Expression of the I9A/I14A double mutant yielded similar
results (Fig. 5, E and I); 96.5% of the cells
showed cytoplasmic localization of the fusion protein. These results
suggest that both isoleucine residues as well as Val25 do
not play a significant role in export activity of the fragminP NES.
Significantly, mutation of Leu17, Leu21, or
Leu27 resulted in a subcellular distribution virtually
identical to that of wild type CapG, tagged to EGFP (Fig. 5,
F-H). These results demonstrate that the
Leu17/Leu21/Leu27 triad is a
critical determinant in the FrgP/severin nuclear export sequence. Fig.
5I shows that the distribution of these NES mutants reverted
to the typical nuclear-cytoplasmic localization pattern of CapG-EGFP in
100% of the cells observed.
Identification of a Nuclear Export Sequence in Myopodin and Bloom
Syndrome Protein--
Recently, Weins et al. (27) reported
that mouse myopodin, a muscle-specific actin-bundling protein and
synaptopodin homolog, accumulates in the nucleus under stress
conditions. Furthermore, myopodin shows
differentiation-dependent nuclear-cytoplasmic
redistribution. Nuclear export of myopodin is sensitive to LMB, despite
the absence of a classical NES (27). Sequence alignment between
myopodin and FrgP/severin showed conservation of the three leucine
residues, which, incidentally, are also located at the N terminus of
myopodin (Fig. 6A). To examine
the role of this myopodin region as a potential nuclear export
sequence, we tagged this sequence onto CapG-EGFP following the same
strategy as for FrgP and analyzed its distribution in transfected
HEK293T cells. Surprisingly, fusion of myopodin amino acids
Leu14-Leu24 to CapG-EGFP yielded no change in
the subcellular localization (Fig. 6B), in contrast to FrgP
L17-L27-CapG-EGFP (see above). However, the myopodin
Pro3-Val13 segment on its own exported
CapG-EGFP in 10% of the cells (Fig. 6C). Finally, myopodin
P3-L24-CapG-EGFP gave rise to cytoplasmic localization of the fusion
protein in 16% of the cells (Fig. 6, D and F).
This finding shows that the N-terminal region of myopodin displays
weaker nuclear export activity as compared with the severin/FrgP NES.
Furthermore, despite the similarity between FrgP and myopodin N-terminal regions at the primary structure level, both export sequences are probably different because the myopodin region containing the leucine-rich stretch displays no export activity, in contrast to
the corresponding region in fragminP. Finally, our data do not exclude
the presence of other regions in myopodin involved in controlling the
subcellular distribution of this actin bundling protein.
Henderson and Eleftheriou (37) previously predicted a putative Rev-like
NES in the Bloom syndrome protein
(Lys1146-Asp1163). However, this region was
unable to export EGFP from the nucleus, suggesting that it is not a
functional NES. Based on our data we examined the Bloom sequence for a
potential NES. An 11-amino acid region in the center of the Bloom DEAD
box domain (Ile749-Ile759) contains one
leucine and two isoleucine residues, spaced at identical intervals as
in the FrgP NES (Fig. 6A). Although the corresponding
myopodin segment (Leu14-Leu24) showed no
significant export activity (Fig. 6B), the Bloom amino acid
sequence Ile749-Ile759 was able to export
CapG-EGFP from the nucleus with an efficiency comparable with that of
myopodin Pro3-Leu24; in 20% of the cells,
CapG-EGFP was exported from the nucleus (Fig. 6, E-F).
Secondary structure predictions did not reveal particular features that
are common to the export signals of Bloom, FrgP, or myopodin, apart
from the hydrophobic residues. The presence of this NES in an
ATP-dependent helicase is not fully clear at present but
opens up new avenues for future research. Of note, the Bloom NES and
the surrounding residues are conserved in a number of other helicases.
The NES of Myopodin Modulates Collagen Invasiveness--
The
partial effect of the myopodin NES on nuclear exclusion of CapG-EGFP,
as compared with the efficiency of the FrgP/severin NES, is not
unexpected. Indeed, as reported earlier (37-38), different NES
sequences do not export EGFP from the nucleus with the same efficiency.
These differences in relative NES strength are physiologically relevant
in that some shuttling proteins require more rapid export from the
nucleus than others. Other regions in the protein may also affect the
subcellular localization, as illustrated in the case of myopodin. When
amino acids Met1-Leu24 of myopodin were
deleted, no significant enrichment in the nucleus was observed as
compared with the wild type protein following overexpression in
differentiating C2C12 muscle cells (Fig.
7A). In both cases,
co-localization with F-actin was observed as reported previously by
Weins et al. (27), most likely because of the actin-binding
region in myopodin (amino acids 410-563). Therefore, we performed
collagen type I invasion experiments as an independent method to
explore a potential role for the myopodin NES. Whereas HEK293T or C2C12
cells did not invade a collagen matrix (Fig. 7B, lanes
1, 2, 5, and 6), cells expressing
full-length myopodin invaded into collagen type I (Fig. 7B,
lanes 3 and 7). Significantly, the myopodin
deletion mutant lacking Met1-Leu24 was unable
to induce collagen invasion (Fig. 7B, lanes 4 and 8). This result argues in favor of a regulatory role of the
myopodin export sequence in mediating collagen invasion, although its
exact mode of action in this mechanism is not clear at present.
Possibly, deletion of the NES perturbs the natural ability of myopodin
to shuttle between nucleus and cytoplasm in a subtle manner, causing the mutant to reside longer in one cellular compartment. This hypothesis requires analysis of putative myopodin interaction partners
that are involved in promoting invasion by the actin-binding protein.
In summary, we have shown that the occurrence of a nuclear export
sequence can account for the differential localization in cells of
several representatives of a subfamily of gelsolin-related actin-binding proteins. The observations that CapG lacks such a NES and
that it does not translocate to the nucleus in a passive manner may
suggest that it plays an active role in that cell compartment. Possibly, CapG is involved in transmitting changes in actin
treadmilling to the nucleus, because changes in actin dynamics are
known to affect gene transcription mediated by serum response factor
(39). However, as shown previously for zyxin (22) and supervillin (21),
actin may not necessarily be the target of nuclear CapG. Further
studies are aimed at elucidating this question.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
or
type (reviewed in Ref. 3). In contrast, the pathways and
details of signal-mediated nuclear export are less well defined. The
best characterized nuclear export signal is the small, hydrophobic,
leucine-rich NES, identified initially in the human immunodeficiency
virus type 1 Rev protein (4) and the heat-stable inhibitor of
cAMP-dependent protein kinase (PKI) (5). Structurally and
functionally related export sequences have since been detected in many
cellular and viral proteins (6, 7), including actin (8). Direct
interaction with the export factor CRM1 (exportin 1) is essential for
the export of proteins containing a leucine-rich NES (9).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactoside for 4 h at 30 °C. The cells
were collected by centrifugation at 5,000 rpm for 15 min and lysed in
lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM
NaCl) by lysozyme (0.1 mg/ml, 40 min at room temperature) and
sonication. Soluble CapG was further purified by ion-exchange chromatography on a DEAE and monoQ column. Full-length CapG cDNA was also amplified by PCR, whereby a sequence encoding a Myc tag was
introduced N-terminally. This construct was cloned into the pCR T7
CT-TOPO vector, resulting in a fusion protein with a C-terminal V5 and
His6 tag. Expression of this fusion protein was achieved as
described above, and purification was accomplished by ion-exchange chromatography on a DEAE column.
ex, 365 nm;
em, 388 nm) was measured
over time.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Subcellular localization of CapG-EGFP,
FrgP-EGFP, and severin-EGFP. HEK293T cells were transfected with
CapG-EGFP (A), FrgP-EGFP (B), or severin-EGFP
(C). CapG shows a nuclear and cytoplasmic localization, in
contrast to FrgP and severin (predominantly cytoplasmic). The right
panels (A'-C') show corresponding DAPI images.
Bar, 40 µm. D, histogram showing the proportion
of transfected cells displaying cytoplasmic (C) or
cytoplasmic + nuclear (CN) localization of the CapG-, FrgP-,
and severin-EGFP fusion proteins. The data are the means ± S.E.
of three independent experiments.
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Fig. 2.
Nuclear enrichment of CapG-FrgP-EGFP and
FrgP-EGFP upon leptomycin B treatment. Cytoplasmic localization of
CapG-FrgP-EGFP following expression in HEK293T (A) or MDCK
cells (B). Treatment of MDCK cells with 10 ng/ml LMB
promotes nuclear enrichment of CapG-FrgP-EGFP (C).
D and E, localization of FrgP-EGFP before
(D) or after (E) the addition of LMB.
A'-E', DAPI staining. Bar, 40 µm.
CapG-FrgP-EGFP hybrid proteins
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Fig. 3.
Deletion of severin or FrgP
Met1-Leu27 results in nuclear
accumulation. A, the CapG(M1-L27)-FrgP(Q52-D371)-EGFP
chimera shows nucleo-cytoplasmic distribution following expression in
HEK293T cells. B, amino acid sequence alignment between the
N-terminal regions of FrgP, severin, and CapG, including the consensus
of hydrophobic residues. Conserved amino acids in the predicted NES are
shaded; hydrophobic residues in the NES are
black. C-E, deletion of FrgP amino acids 1-27
in CapG-FrgP-EGFP (C) or FrgP-EGFP (D) or
deletion of the corresponding region in severin-EGFP (E)
allows nuclear enrichment of the fusion proteins. Right panels
(A' and C'-E') show corresponding DAPI staining.
Bar, 40 µm.
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Fig. 4.
F-actin capping activity of recombinant
Myc-V5-His6-tagged CapG. Top panel,
SDS-PAGE analysis of purified untagged recombinant CapG (lane
1) and Myc-CapG-V5-His6 (lane 2). The
molecular mass markers are indicated. The gel was stained with
Coomassie Brilliant Blue. Bottom panel, F-actin capping
activity of untagged recombinant CapG and Myc-CapG-V5-His6.
The experiments were performed using pyrene-labeled actin as described
under "Experimental Procedures." The Control curve
represents actin polymerization in the absence of CapG.
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Fig. 5.
Role of leucine residues Leu17,
Leu21, and Leu27 in the ability of the FrgP NES
to export CapG to the cytoplasm. A, localization of
FrgP (L17-L27)-CapG-EGFP in HEK293T cells and of FrgP(M1-L27)-CapG-EGFP
following expression in HEK293T (B) or MDCK cells
(C). C', LMB promotes nuclear accumulation of
FrgP(M1-L27)-CapG-EGFP. D, histogram showing quantitation of
data presented in A and B. E-H,
HEK293T cells expressing FrgP(M1-L27)-CapG-EGFP point mutants: I9A/I14A
(E), L17A (F), L21A (G), and L27A
(H). Amino acid numbering is according to Fig.
3B. Bar, 40 µm. I, histogram showing
the proportion of cells with a cytoplasmic (C) or
cytoplasmic and nuclear (CN) distribution of FrgP M1-L27
CapG-EGFP (CONTROL) and point mutations that were created in
the NES. The values are representative of three independent experiments
(means ± S.E.).
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Fig. 6.
Identification of a nuclear export signal in
myopodin and Bloom. A, sequence alignment of FrgP,
severin, CapG, myopodin, and Bloom syndrome protein. B-D,
HEK293T cells expressing myopodin(L14-L24)-CapG-EGFP (B),
HEK293T cells expressing myopodin(P3-V13)-CapG-EGFP (C), or
myopodin(P3-L24)-CapG-EGFP (D) show nuclear exclusion of the
fusion protein in a subset of the cell population, in contrast to
myopodin(L14-L24)-CapG-EGFP. E, HEK293T cells transfected
with Bloom(I749-I759)-CapG-EGFP. The right panels
(B'-E') show DAPI staining. Bar, 40 µm.
F, quantification of the results presented in
B-E. The data are the means ± S.E. of three
independent experiments.
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Fig. 7.
Role of the myopodin NES in controlling
collagen invasion by myopodin. A, C2C12 cells
transfected with full-length myopodin
(Met1-Glu758). A', C2C12 cells
transfected with myopodin Ser25-Glu758
(lacking the NES). The cells were stained with monoclonal anti-V5
antibody. Bar, 40 µm. B, collagen invasion
induced by myopodin. Expression of full-length (FL) myopodin
induces invasion of HEK293T (lane 3) and C2C12 cells
(lane 7). This effect is abrogated in cells expressing a
myopodin Ser25-Glu758 truncation mutant
(lanes 4 and 8). EV, empty vector;
Myop., myopodin. The data are the means ± S.E. of
three independent experiments.
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FOOTNOTES |
---|
* This work was supported by Interuniversity Attraction Poles Grant IUAP/P5, by the Concerted Actions Program of Ghent University, by the Fund for Scientific Research-Flanders, by Fortis Bank Verzekeringen, and by Köln Fortune.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.
§ Recipient of a fellowship from the Instituut voor Wetenschap en Technologie.
¶ Postdoctoral Fellow of the Fund for Scientific Research-Flanders (Belgium).
To whom correspondence should be addressed: Dept. of
Biochemistry, Faculty of Medicine and Health Sciences, Flanders
Interuniversity Institute for Biotechnology, Ghent University,
Rommelaere Institute, Albert Baertsoenkaai 3, B-9000 Gent, Belgium.
Tel.: 32-9-33-13340; Fax: 32-9-33-13597; E-mail:
jan.gettemans@rug.ac.be.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M209946200
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ABBREVIATIONS |
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The abbreviations used are: NES, nuclear export sequence; EGFP, enhanced green fluorescent protein; FrgP, plasmodial fragmin; LMB, leptomycin B; DAPI, 4,6-diamidino-2-phenylindole; MDCK, Madin-Darby canine kidney.
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