From the Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, Pennsylvania 18840
Received for publication, November 5, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The armadillo protein SmgGDS promotes guanine
nucleotide exchange by small GTPases containing a C-terminal polybasic
region (PBR), such as Rac1 and RhoA. Because the PBR resembles a
nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has
significant NLS activity when it is fused to green fluorescent protein
(GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR
has very poor NLS activity when it is fused to GFP or in the context of
full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is
enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR.
Conversely, SmgGDS nuclear accumulation is diminished by interactions
with RhoA. An SmgGDS nuclear export signal sequence that we identified
promotes SmgGDS nuclear export. These results suggest that
SmgGDS· Rac1 complexes accumulate in the nucleus because the
Rac1 PBR has NLS activity and because Rac1 supplies the appropriate
GTP-dependent signal. In contrast, SmgGDS·RhoA complexes
accumulate in the cytoplasm because the RhoA PBR does not have NLS
activity. This model may be applicable to other armadillo proteins in
addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA
also provide stimulatory and inhibitory signals, respectively, for the
nuclear accumulation of p120 catenin. These results indicate that small
GTPases with a PBR can regulate the nuclear transport of armadillo proteins.
Armadillo (ARM)1 family
proteins that contain multiple copies of the ~42-amino acid (aa) ARM
motif include SmgGDS, p120 catenin (p120ctn), Several ARM proteins interact with the Rho family of small GTPases
(8-15) or with guanine nucleotide exchange factors (GEFs) for these
GTPases (16, 17). SmgGDS promotes guanine nucleotide exchange by small
GTPases containing a C-terminal polybasic region (PBR), which is a
series of adjacent lysines or arginines (11-15, 18). We noticed a
striking sequence similarity between the PBR of small GTPases that
interact with SmgGDS and the NLS sequence of proteins that associate
with the ARM protein karyopherin We tested this hypothesis by examining the nuclear
accumulation of transiently transfected SmgGDS in cells co-transfected with mutant or wild-type Rac1 or RhoA. We show that the nuclear accumulation of SmgGDS is enhanced by interactions with Rac1 but diminished by interactions with RhoA. The PBR of Rac1, but not RhoA,
was found to have NLS activity. These findings support a model in which
the ability of the Rac1 PBR to act as an NLS promotes the nuclear
accumulation of SmgGDS·Rac1 complexes. Conversely, the inability of
the RhoA PBR to act as an NLS promotes the cytoplasmic accumulation of
SmgGDS·RhoA complexes. This model may be applicable to other ARM
proteins in addition to SmgGDS, because we demonstrate that the nuclear
accumulation of endogenous p120ctn is similarly enhanced by Rac1 but
not by RhoA. These findings identify a new function of Rac1 and RhoA
and define a novel mechanism for the nuclear accumulation of SmgGDS and
potentially other ARM proteins.
cDNA Constructs--
The majority of the cDNA constructs
coding for hemagglutinin (HA)- or myc-tagged proteins that were
generated for this study have been deposited in the Guthrie cDNA
Resource Center (available at www.cDNA.org), which provides these
and other cDNAs to the research community as a non-profit service.
Three mammalian expression vectors were used to subclone cDNAs
coding for wild-type or mutant SmgGDS, Rac1, or RhoA in this study. The
myc-pcDNA3.1 vector was made by inserting cDNA coding for two
copies of the myc epitope (EQKLISEEDL), which is recognized by the 9E10
monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), into
the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The HA-pcDNA3.1
vector, which contains cDNA coding for three copies of the HA
epitope (YPYDVPDYA) that is recognized by HA antibody (Covance,
Berkeley, CA), was a generous gift of the Guthrie cDNA Resource
Center. The pEGFP-C1 vector was purchased from
Clontech (Palo Alto, CA). The pEFBOSp120-3AB and
pEFBOSp120-3A plasmids, coding for the 3AB and 3A isoforms of p120ctn,
respectively (6), were generously provided by Dr. Frans van Roy
(University of Ghent) and were used without modification. The
pSR
As depicted below in Fig. 1a, we generated cDNAs coding
for wild-type SmgGDS (Sg) or mutant SmgGDS containing alanine
substitutions in the N-terminal NES sequence (SgNA2 or SgNA4), the
C-terminal NES sequence (SgCA2 or SgCA4), or in both the N-terminal and
C-terminal NES sequences (SgNA4CA4). These cDNAs were inserted into
the HA-pcDNA3.1 vector to generate constructs coding for proteins
with an N terminus 3× HA tag. Alternatively, the cDNAs were
inserted into the myc-pcDNA3.1 vector to generate constructs coding
for proteins with an N terminus 2× myc tag.
As depicted below in Fig. 2a, the pEGFP-C1 vector was used
to generate constructs coding for green fluorescent protein (GFP) fused
to the N terminus of the following aa sequences: PPPVKKRKRK to generate
the GFP-PBR(Rac1) construct, PPPVKKRKRKCLLL to generate the
GFP-PBR(Rac1)CAAX construct, QARRGKKKSG to generate the GFP-PBR(RhoA) construct, and QARRGKKKSGCLVL to generate the GFP-PBR(RhoA)CAAX construct. To facilitate expression, a codon for methionine was inserted at the C terminus of the GFP coding sequence in the pEGFP-C1 vector, immediately preceding the cDNA insert.
We obtained from the Guthrie cDNA Resource Center the
HA-pcDNA3.1 vector containing cDNA inserts coding for wild-type
Rac1 (Rac1), constitutively active Rac1 containing a valine
substitution at aa 12 (CA-Rac1), or dominant negative Rac1 containing
an asparagine substitution at aa 17 (DN-Rac1). We replaced the six
basic aa comprising the Rac1 PBR in these cDNAs with glutamine to
yield the Rac1(PBRQ), CA-Rac1(PBRQ), and DN-Rac1(PBRQ) cDNAs, as
depicted below in Fig. 3a. We also obtained the
HA-pcDNA3.1 vector containing cDNA inserts coding for wild-type
RhoA (RhoA), constitutively active RhoA containing a valine
substitution at aa 14 (CA-RhoA), and dominant negative RhoA containing
an asparagine substitution at aa 19 (DN-RhoA) from the Guthrie cDNA
Resource Center. The five basic aa comprising the RhoA PBR in these
constructs were replaced with glutamine to yield the RhoA(PBRQ),
CA-RhoA(PBRQ), and DN-RhoA(PBRQ) cDNAs, as depicted below in Fig.
3d. The cDNA inserts were placed into the pEGFP-C1
vector to generate constructs coding for proteins with an N terminus
GFP tag. Alternatively, the cDNAs were inserted in the
HA-pcDNA3.1 vector to generate constructs coding for proteins with
an N terminus 3× HA tag.
Transfection of Cells with cDNA Constructs--
The CHO-m3
cell line, which was used for all of the experiments in this study, is
a Chinese hamster ovary (CHO) cell line stably transfected with the
M3 muscarinic acetylcholine receptor. This extensively
characterized cell line (15, 20-22) was used because we are
investigating the effects of muscarinic acetylcholine receptor
activation on the observed Rac1- and RhoA-dependent
responses, which will be the subject of a future report. In the absence
of agonist for the muscarinic acetylcholine receptor, which is the case
in this study, the CHO-m3 cells do not exhibit any detectable differences from parental CHO cells (20, 21). The cells were transfected by suspension in Ham's F-12 medium (6 × 106 cells/200 µl of medium) containing 8 µg of the
indicated cDNA, and electroporation by a single electric pulse (200 V, 50 ms) using a BTX Electro Square Porator (Genetronics, Inc., San
Diego, CA). The electroporated cells were transferred to tissue culture plates and incubated for 16-24 h (37 °C, 5% CO2) in
complete CHO medium consisting of Ham's F-12 medium, heat-inactivated
fetal bovine serum (5%), glutamine (0.3 µg/ml), penicillin (20 units/ml), and streptomycin sulfate (20 µg/ml). The cells were
then used in the indicated assays or plated onto glass coverslips
in complete CHO medium (2 × 104 cells/ml medium) and
incubated for an additional 16-24 h (37 °C, 5% CO2)
before being examined for the intracellular localization of the
indicated proteins.
Intracellular Localization of Proteins--
Cells expressing
GFP-tagged proteins were examined by fluorescence microscopy while the
cells were still alive. To co-localize GFP-tagged proteins with
HA-tagged proteins or with endogenous p120ctn, the cells were fixed
with 3% formaldehyde in phosphate-buffered saline (PBS) (15 min,
4 °C) and incubated with 50 mM ammonium chloride in PBS to quench formaldehyde fluorescence (10 min, 25 °C).
The fixed cells were permeabilized with acetone (10 s, 4 °C) or with
0.2% Triton X-100 (TX-100) in PBS (10 min, 25 °C), as described
previously (15, 22). After incubating with PBS containing 1% bovine
serum albumin (30 min, 25 °C), the cells were incubated with mouse
antibody to HA (Covance) or to p120ctn (BD Transduction Laboratories,
San Diego, CA) (1 h, 25 °C), followed by incubation with
TRITC-anti-mouse IgG (1 h, 25 °C). The cells were mounted in PBS
containing 90% glycerol and 0.1% p-phenylenediamine and
examined using a Nikon Optiphot fluorescence microscope, as previously
described (22). Digital images of the cells were collected using a
Kodak DC 290 zoom digital camera and Adobe Photoshop software.
In each assay, investigators ranked the nuclear localization of
proteins in at least 20 different cells transfected with the same
cDNA, the identity of which was unknown to the investigators. The
relative amount of a protein that was detectable in the nucleus of each
cell was ranked using the following scale: 1 = undetectable or
very low nuclear level and high cytoplasmic level; 2 = moderate nuclear level and high cytoplasmic level; 3 = similar nuclear and
cytoplasmic levels; 4 = high nuclear level and moderate
cytoplasmic level; 5 = high nuclear level and undetectable or very
low cytoplasmic level.
We found that TX-100 solubilizes some cytosolic proteins, causing the
ratio of nuclear protein to cytosolic protein in TX-100-permeabilized cells to sometimes be greater than the value of these ratios in non-permeabilized and acetone-permeabilized cells. This effect of
TX-100 becomes apparent when the subcellular distributions of GFP and
GFP-tagged proteins in TX-100-permeabilized cells (see Figs. 6-8) are
compared with those in living cells (see Figs. 2b, 3b, and 3e). This effect also becomes apparent
when the subcellular distributions of HA-Sg and HA-SgNA4 in
TX-100-permeabilized cells (see Figs. 6 and 7) are compared with those
in acetone-permeabilized cells (see Fig. 1, b and
c). Despite this limitation, TX-100 permeabilization was
used because it was found to be significantly better then acetone
permeabilization in preserving the subcellular distributions of the
expressed wild-type or mutant Rac1 and RhoA proteins.
Immunoprecipitation of 35S-Labeled
Proteins--
Twenty-four hours after electroporation, the transfected
cells were suspended in labeling medium consisting of methionine- and
cysteine-free Dulbecco's modified Eagle's medium,
[35S]methionine (10 µCi/ml), and 2% heat-inactivated
fetal calf serum. The cells were cultured for an additional 16 h
(37 °C, 5% CO2) in the presence or absence of compactin
(Sigma, St. Louis, MO) and then suspended in ice-cold lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2.5 mM
EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µM
leupeptin, pH 7.4) containing phosphatase inhibitors (15). The cells
were incubated in the lysis buffer for 20 min on ice with periodic
vigorous vortexing of the cell suspension, followed by centrifugation
(13,000 × g, 10 min, 4 °C). Nuclear proteins are
solubilized by this method, as indicated by our ability to detect
nuclear proteins such as RCC1 in Western blots of the lysate
supernatants (data not shown). The supernatants from the centrifuged
lysates were immunoprecipitated using the 9E10 myc antibody (Santa Cruz
Biotechnology), the HA antibody (Covance), or the p120ctn antibody (BD
Transduction Laboratories), as previously described (15). The
immunoprecipitates were subjected to SDS-PAGE followed by
autoradiography as previously described (15).
Binding of [35S]GTP Statistical Analyses--
The means of the measured values of
each treatment group were compared by using Student's t
test. Means were considered to be significantly different from one
another if p was <0.05 in the Student's t
test. In all comparisons, original measured values, rather than
percentages, were used in the analyses.
SmgGDS Has a Nuclear Export Signal Sequence--
Because SmgGDS
interacts with nuclear proteins (23, 24) and cytoplasmic proteins
(11-15, 18), we looked for sequences in SmgGDS that might promote
nucleocytoplasmic shuttling. Although classic NLS sequences are not
apparent in SmgGDS, putative NES sequences, consisting of
LX(2-3)LXXLXL in
which the leucines are sometimes replaced by isoleucines (reviewed in
Ref. 6), are present near the N terminus at aa 4-13
(LSDTLKKLKI) and near the C terminus at aa 465-473
(LALIAALEL), as
indicated by the underlined aa. We generated cDNA constructs coding
for HA-tagged wild-type SmgGDS (HA-Sg) or HA-tagged mutant SmgGDS
proteins containing alanine substitutions in the putative
N-terminal or C-terminal NES sequences (Fig.
1a). Transient expression of
these cDNA constructs in CHO-m3 cells indicates that the nuclear
accumulation of SmgGDS is significantly increased by disrupting the
N-terminal NES sequence (aa 4-13) but not by disrupting the C-terminal
NES sequence (aa 465-473) (Fig. 1, b and c).
Inactivation of the nuclear export protein exportin 1 by leptomycin B
(25) dramatically increases the nuclear accumulation of both wild-type
SmgGDS and mutant SmgGDS missing the C-terminal NES sequence but only
modestly increases the nuclear accumulation of SmgGDS missing the
N-terminal NES sequence (Fig. 1, b and c). These
results indicate that SmgGDS shuttles between the nucleus and
cytoplasm. The nuclear export of SmgGDS is dependent on the N-terminal
NES sequence and functional exportin 1.
The PBR of Rac1, but Not RhoA, Functions as an NLS--
We
hypothesize that SmgGDS enters the nucleus when it associates with
small GTPases containing a PBR that functions as an NLS. The PBR is
present in the 10 amino acids immediately preceding the terminal
CAAX region in Rac1 (PPPVKKRKRK) and RhoA
(QARRGKKKSG), as indicated by the underlined
basic aa. cDNA constructs coding for these 10 aa fused to GFP were
generated (Fig. 2a) and
expressed in CHO-m3 cells (Fig. 2b). GFP accumulates in both
the nucleus and cytoplasm of the transfected cells (Fig.
2b), probably reflecting the unfacilitated diffusion of GFP
(~27 kDa) through nuclear pores. The nuclear localization of the GFP
chimeric protein is increased significantly by the PBR of Rac1 but only
modestly by the PBR of RhoA (Fig. 2b). cDNA constructs
coding for GFP fused to the last 14 amino acids of each GTPase were
also generated, to yield GFP chimeras containing the PBR plus the
CAAX sequence of each GTPase (Fig. 2a).
Expression of these cDNA constructs in CHO-m3 cells indicates that
the isolated PBR of Rac1, but not RhoA, acts as a potent NLS even in
the presence of the CAAX sequence (Fig. 2b).
To determine whether the PBR acts as an NLS in the context of
full-length Rac1 or RhoA, we generated cDNA constructs coding for
GFP-tagged wild-type, constitutively active, or dominant negative Rac1
or RhoA proteins containing either a normal or mutant PBR (Fig.
3, a and d).
Transient transfection of these constructs in CHO-m3 cells indicates
that Rac1 nuclear accumulation is enhanced by activation of Rac1, as
indicated by the enhanced nuclear accumulation of GFP-CA-Rac1 (Fig. 3,
b and c), and diminished by disruption of the
PBR, as indicated by the reduced nuclear accumulation of the GFP-tagged
Rac1(PBRQ) proteins (Fig. 3, b and c). Thus, Rac1 nuclear accumulation is promoted both by its conversion to the GTP-bound state and by an intact PBR, consistent with the PBR acting as
an NLS. The unique intracellular distributions of the wild-type and
mutant Rac1 proteins are not changed by replacing the GFP tag with a
much smaller 3× HA tag (~3 kDa) (data not shown). This finding
indicates that the PBR enhances Rac1 nuclear accumulation even when
Rac1 is presumably small enough to diffuse through nuclear pores. As
expected, the GFP-tagged RhoA proteins exhibit much less nuclear
accumulation than do the GFP-tagged Rac1 proteins (Fig. 3, e
and f), consistent with the inability of the RhoA PBR to act
as an NLS. Disruption of the RhoA PBR does not diminish, but instead
can actually enhance, the nuclear accumulation of GFP-tagged RhoA
proteins (Fig. 3f), providing further evidence that the RhoA
PBR does not act as an NLS.
It is possible that Rac1(PBRQ) proteins do not accumulate in the
nucleus because they are sequestered in the cytoplasm due to abnormal
interactions with cytoplasmic proteins. To examine this possibility,
HA-tagged wild-type and mutant Rac1 proteins were immunoprecipitated
from 35S-labeled cells and examined for
co-precipitating proteins (Fig. 4a). RhoGDI preferentially
co-precipitates with HA-Rac1, whereas IQGAP1· calmodulin complexes
preferentially co-precipitate with HA-CA-Rac1 (Fig. 4a,
lanes 1 and 3), consistent with our previous findings (22). These proteins do not co-precipitate with HA-Rac1(PBRQ) or HA-CA-Rac1(PBRQ) (Fig. 4a, lanes 2 and
4), indicating that disruption of the PBR diminishes the
interaction of Rac1 with cytoplasmic proteins. The reduced interaction
of Rac1(PBRQ) proteins with cytosolic proteins may account for our
observations that mutation of the PBR diminishes the ability of
CA-Rac1 to localize at membrane ruffles (Fig. 3b) and
releases DN-Rac1 from a restricted juxtanuclear localization (Fig.
3b). The reduced interaction of Rac1(PBRQ) proteins with
cytoplasmic proteins also lessens the possibility that Rac1(PBRQ)
proteins remain in the cytoplasm because they are sequestered by
cytoplasmic proteins. Instead, these findings are consistent with the
model that Rac1(PBRQ) proteins remain in the cytoplasm because the NLS
has been lost by disrupting the PBR.
The prenylation state of the Rac1(PBRQ) proteins was determined by
examining their migration rates in SDS-PAGE gels. Non-prenylated small
GTPases migrate slower than prenylated small GTPases in SDS-PAGE gels
(11, 26). We found that Rac1 and Rac1(PBRQ) proteins exhibit similar
rates of migration on SDS-PAGE gels (Fig. 4b). Rac1 and
Rac1(PBRQ) proteins isolated from cells treated with the prenylation
inhibitor compactin (27) migrate slower than proteins isolated from
untreated cells (Fig. 4b). These results indicate that the
Rac1(PBRQ) proteins are prenylated. Thus, the unique intracellular
distributions of the Rac1(PBRQ) proteins are probably not due to
changes in prenylation of these proteins.
Mutation of the PBR Alters Rac1 Protein Interactions and Guanine
Nucleotide Exchange--
To further define the functions of the Rac1
PBR, we compared the abilities of the HA-tagged Rac1 and Rac1(PBRQ)
proteins to bind [35S]GTP
The results of the [35S]GTP
The HA-tagged Rac1(PBRQ) proteins do not detectably
co-precipitate with myc-Sg (Fig. 5a, lanes 3,
5, and 7), indicating that mutation of the Rac1
PBR diminishes the stability of the Rac1·SmgGDS complex. Diminished
stability of the Rac1(PBRQ)·SmgGDS complex is consistent with
an enhanced rate of guanine nucleotide exchange by Rac1(PBRQ) compared
with Rac1, as indicated by the [35S]GTP
Myc-SgNA4 does not detectably co-precipitate any of the HA-tagged Rac1
or Rac1(PBRQ) proteins (Fig. 5a, lanes 10-15),
indicating that mutation of the SmgGDS N terminus NES diminishes the
stability of the Rac1·SmgGDS complex. This instability of
Rac1·SgNA4 complexes may contribute to the enhanced rate of guanine
nucleotide exchange exhibited by HA-Rac1 in the presence of myc-SgNA4,
compared with myc-Sg (Fig. 4d).
Previous studies indicate that SmgGDS interacts more effectively with
RhoA than with Rac1 (12-15, 22). Consistent with these studies, we
found that both myc-Sg and myc-SgNA4 co-precipitate HA-tagged RhoA
proteins (Fig. 5b) much more effectively than they co-precipitate HA-tagged Rac1 proteins (Fig. 5a), indicating
that SmgGDS associates more strongly with RhoA than with Rac1. These findings are consistent with our previous reports that endogenous SmgGDS co-precipitates more readily with HA-DN-RhoA than with HA-DN-Rac1 in stably transfected CHO-m3 cells (15, 22). Mutation of the
RhoA PBR or the SmgGDS N terminus NES diminishes the stability of
RhoA·SmgGDS complexes (Fig. 5b), similar to the effects of these mutations on Rac1·SmgGDS complexes (Fig. 5a).
Rac1 and RhoA Regulate SmgGDS Localization--
Our hypothesis
predicts that SmgGDS will accumulate in the nucleus when it interacts
with Rac1, but not with RhoA, because the PBR of Rac1, but not RhoA,
has NLS activity. To test this prediction, we examined the nuclear
localization of HA-Sg and HA-SgNA4 in cells
co-expressing GFP-tagged Rac1 or RhoA
proteins with a normal or mutant PBR (Figs. 6 and
7). The nuclear accumulation of HA-Sg is
significantly enhanced by GFP-CA-Rac1 (Fig. 6, a
(panel 4) and b), suggesting that Rac1-GTP
supplies a signal that promotes SmgGDS nuclear accumulation. Mutation
of the PBR diminishes the ability of CA-Rac1 to stimulate SmgGDS
nuclear accumulation (Fig. 6, a (panel 5) and
b), consistent with the Rac1 PBR acting as an NLS for
Rac1·SmgGDS complexes. Expression of dominant negative GFP-DN-Rac1(PBRQ), which supplies neither a GTP-dependent
signal nor a PBR-dependent signal, diminishes HA-Sg nuclear
accumulation (Fig. 6, a (panel 7) and
b). The inhibitory effects of GFP-DN-Rac1 and
GFP-DN-Rac1(PBRQ) on SmgGDS nuclear accumulation are most noticeable in
the presence of diminished SmgGDS nuclear export, as exhibited by
HA-SgNA4 (Fig. 6, a (panels 13 and 14)
and c).
Interestingly, the nuclear accumulation of GFP-Rac1 is greater in cells
transfected with HA-SgNA4 (Fig. 6a, panel 9) than in cells transfected with HA-Sg (Fig. 6a, panel
2). This result may occur because conversion of Rac1 to the
GTP-bound state, which promotes the nuclear accumulation of Rac1 (Fig.
3c), is stimulated more by co-transfected SgNA4 than by Sg
(Fig. 4d).
As predicted, the GFP-tagged RhoA, CA-RhoA, and DN-RhoA proteins do not
enhance the nuclear localization of HA-Sg and HA-SgNA4 but actually
diminish it (Fig. 7). Interestingly, in some cases the inhibitory
effects of the RhoA proteins on HA-Sg and HA-SgNA4 nuclear accumulation
are lessened when the RhoA PBR is mutated (Fig. 7). This result may
occur because mutation of the PBR lessens the ability of RhoA to
interact with SmgGDS (Fig. 5b).
Rac1 and RhoA Regulate p120ctn Localization--
The abilities of
Rac1 and RhoA to regulate the nuclear localization of other ARM
proteins were tested by examining the distribution of endogenous
p120ctn in CHO-m3 cells transiently transfected with the wild-type or
mutant GTPases (Fig. 8). The nuclear
accumulation of p120ctn is increased significantly by GFP-CA-Rac1 (Fig.
8, a (panel 2) and b) but only
modestly by GFP-CA-Rac1(PBRQ) (Fig. 8, a (panel
3) and b). This finding indicates that both
GTP-dependent and PBR-dependent signals from
Rac1 enhance the nuclear accumulation of p120ctn. In contrast, the
nuclear accumulation of p120ctn is diminished by GFP-CA-RhoA (Fig. 8,
a (panel 4) and b). This inhibitory effect of GFP-CA-RhoA on p120ctn nuclear accumulation is most noticeable when the cells are treated with leptomycin B (Fig. 8,
a (panel 9) and c), which was
previously reported to inhibit p120ctn nuclear export (6).
To investigate the physical association of p120ctn with Rac1, we
transiently co-expressed either the 3A or 3AB isoform of p120ctn (Ref.
6) with HA-tagged Rac1, CA-Rac1, or DN-Rac1 in CHO-m3 cells. Although
we could immunoprecipitate the endogenous p120ctn and the expressed 3A
and 3AB isoforms of p120ctn from the 35S-labeled
cells using the p120ctn antibody, we could not detect any
co-precipitation of the HA-tagged Rac1, CA-Rac1, or DN-Rac1 proteins
with the immunoprecipitated p120ctn proteins (data not shown).
Similarly, although we could immunoprecipitate the HA-tagged Rac1,
CA-Rac1, or DN-Rac1 proteins using the HA antibody, we could not detect
any co-precipitation of endogenous p120ctn or the expressed 3A or 3AB
isoforms of p120ctn with the immunoprecipitated HA-tagged proteins
(data not shown).
Our findings support a model in which SmgGDS nuclear
accumulation is regulated by opposing Rac1- and
RhoA-dependent pathways (Fig.
9). According to our model, the
association of SmgGDS with Rac1-GDP converts it to Rac1-GTP, which
generates a GTP-dependent signal that promotes the nuclear
accumulation of the Rac1·SmgGDS complex (Fig. 9A). This
GTP-dependent signal may involve the activation of Rac1
effectors. Our model depicts Rac1-GTP entering the nucleus with SmgGDS
(Fig. 9A). This proposed entry of Rac1-GTP into the nucleus
is consistent with the enhanced nuclear accumulation of GFP-CA-Rac1 in
CHO-m3 cells (Fig. 3, b and c) and the nuclear accumulation of Rac1 in other cell types (28-30).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, plakoglobin,
APC, karyopherin
(also known as importin
), and several other
proteins (reviewed in Refs. 1-3). Nucleocytoplasmic shuttling by many
ARM proteins allows them to regulate events in different cellular
compartments, including gene transcription and cell adhesion (reviewed
in Refs. 2-7). ARM proteins enter the nucleus by different mechanisms
(reviewed in Refs. 2-7). Karyopherin
enters the nucleus when it
associates with proteins containing a nuclear localization signal (NLS)
sequence consisting of a series of adjacent lysines or arginines
(reviewed in Ref. 3). The NLS sequence is believed to anchor within the long surface groove formed by the multiple ARM repeats of karyopherin
, promoting the nuclear import of both the NLS-containing protein and karyopherin
(3, 5). APC possesses two NLS sequences and may
enter the nucleus by associating with karyopherin
or related
proteins (4, 7). The mechanisms by which other ARM proteins enter the
nucleus are less clear, because some of these proteins neither possess
classic NLS sequences, nor have they been reported to associate with
NLS-containing proteins.
. It is possible that the 11 ARM
repeats of SmgGDS form a surface groove that binds the PBR of small
GTPases, just as the 10 ARM repeats of karyopherin
form a groove
that binds the NLS sequences of different proteins (3). Based on this
possibility, we hypothesized that the PBR of small GTPases acts as an
NLS, promoting the association of these GTPases with SmgGDS and the
nuclear accumulation of the SmgGDS·GTPase complex.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-smg GDS plasmid (19), which was generously provided by Dr.
Yoshimi Takai, Osaka University Medical School, was used as the
original source of the full-length SmgGDS coding sequence (SmgGDS
isoform 2, NCBI Protein Data base accession number AAA21876). Site-directed mutagenesis of the cDNA constructs was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA).
S by HA-tagged Wild-type and
Mutant Rac1 Proteins--
These assays were performed exactly as
described by Strassheim et al. (15). Twenty-four hours after
electroporation, the cells were permeabilized by a freeze/thaw cycle,
incubated for 10 min (30 °C) with buffer containing 150 nM [35S]GTP
S (1 mCi/mmol), solubilized
with detergent, and centrifuged (13,000 × g, 10 min,
4 °C). The resulting supernatants were immunoprecipitated with HA
antibody, and the amounts of [35S]GTP
S bound to the
immunoprecipitated HA-tagged Rac1 proteins were determined by liquid
scintillation counting.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SmgGDS has a functional NES at the N
terminus. a, the cDNAs that were used to test the
function of the putative NES sequences in SmgGDS are depicted. The
relevant leucines and isoleucines in the putative NES sequences near
the N terminus (aa 4-13) and C terminus (aa 465-473) of wild-type
SmgGDS (Sg) are shown in the top diagram. The
alanines that replace these relevant leucines and isoleucines in the
different SmgGDS mutants (SgNA2, SgNA4,
SgCA2, SgCA4, and SgNA4CA4) are shown
in the lower diagrams. b, CHO-m3 cells transiently
expressing the indicated HA-tagged wild-type or mutant SmgGDS proteins
were incubated in the absence (top panels) or presence
(bottom panels) of 10 nM leptomycin B for 90 min. The fixed and acetone-permeabilized cells were immunofluorescently
stained with HA antibody. The intracellular distribution of HA-SgCA4
(left panels) was found to be indistinguishable from that of
HA-SgCA2 (not shown). The intracellular distribution of HA-SgNA4
(center panels) was found to be indistinguishable from those
of HA-SgNA2 and HA-SgNA4CA4 (not shown). Results shown are
representative of four independent experiments. The bar
represents 10 µm. c, the nuclear localization of the
indicated HA-tagged proteins in fixed and acetone-permeabilized cells
was scored by investigators who did not know the identities of the
HA-tagged proteins expressed by the cells, nor the drug treatment of
the cells. Results shown are the means ± 1 S.E. from 80 cells
scored in four independent experiments. Brackets above the
columns indicate a statistical comparison between the two
bracketed samples. Symbols within a column
indicate a statistical comparison between the sample and the control
sample of cells transfected with HA-Sg and treated without drug
(first column). (*, p < 0.001;
NS, not significant.)
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Fig. 2.
The isolated PBR of Rac1, but not RhoA, has
NLS activity. a, the cDNAs that were used to test
the NLS activity of the isolated PBR of Rac1 or RhoA are depicted. The
PBR(Rac1) and PBR(RhoA) cDNAs code for the 10 aa immediately
preceding the CAAX region of Rac1 or RhoA, respectively. The
PBR(Rac1)CAAX and PBR(RhoA)CAAX cDNAs code for the last 14 aa of
Rac1 or RhoA, respectively. b, the intracellular
distributions of transiently expressed GFP or the indicated GFP chimera
proteins in living CHO-m3 cells were determined by fluorescence
microscopy. The results shown are representative of four independent
experiments. The bar represents 10 µm.
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Fig. 3.
The PBR enhances the nuclear accumulation of
Rac1, but not RhoA. a, the cDNAs that were used to
test the function of the PBR in Rac1 are depicted. The top
diagram shows the lysines and arginines in the PBR of Rac1 (aa
183-188), and the glycine and threonine (aa 12 and 17) that regulate
Rac1 activity. The lower diagrams show the valine
substitution at aa 12 in the CA-Rac1 mutants, the asparagine
substitution at aa 17 in the DN-Rac1 mutants, and the glutamine
substitutions at aa 183-188 in the Rac1(PBRQ) mutants. b,
the distributions of the transiently transfected GFP-tagged wild-type
or mutant Rac1 proteins in living CHO-m3 cells were determined by
fluorescence microscopy. The results shown are representative of four
independent experiments. The bar represents 10 µm.
c, the nuclear localization of the GFP-tagged proteins in
fixed and TX-100-permeabilized CHO-m3 cells was scored by investigators
who did not know the identities of the GFP-tagged proteins expressed by
the cells. Results shown are the means ± 1 S.E. from 60 cells
scored in three independent experiments. Brackets above the
columns indicate a statistical comparison between the two
bracketed samples. Symbols within a column
indicate a statistical comparison between the sample and the control
sample of cells transfected with GFP-Rac1 (first column).
(**, p < 0.001; NS, not significant.)
d, the cDNAs that were used to test the function of the
PBR in RhoA are depicted. The top diagram shows the lysines
and arginines in the PBR of RhoA (aa 182-187), and the glycine and
threonine (aa 14 and 19), which regulate RhoA activity. The lower
diagrams show the valine substitution at aa 14 in the CA-RhoA
mutants, the asparagine substitution at aa 19 in the DN-RhoA mutants,
and the glutamine substitutions at aa 182-187 in the RhoA(PBRQ)
mutants. e, the intracellular distributions of the
transiently expressed GFP-tagged wild-type or mutant RhoA proteins in
living CHO-m3 cells were determined by fluorescence microscopy. The
results shown are representative of four independent experiments. The
bar represents 10 µm. f, the nuclear
localization of the indicated GFP-tagged proteins in fixed and
TX-100-permeabilized CHO-m3 cells was scored by investigators who did
not know the identities of the GFP-tagged proteins expressed by the
cells. The results shown are the means ± 1 S.E. from 60 cells
scored in three independent experiments. Brackets above the
columns indicate a statistical comparison between the two
bracketed samples. Symbols within a column
indicate a statistical comparison between the sample and the control
sample of cells transfected with GFP-RhoA (first column). (**,
p < 0.001; *, p < 0.005;
NS, not significant.)
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Fig. 4.
Mutation of the PBR alters Rac1 protein
interactions and guanine nucleotide exchange but not prenylation.
a, to assess Rac1 protein interactions, the indicated
proteins were immunoprecipitated with HA antibody from lysates of equal
numbers of 35S-labeled CHO-m3 cells transiently expressing
the HA-tagged proteins (lanes 1-6) or transiently
transfected with the HA-pcDNA3.1 vector (control, lane
7). A representative autoradiograph from five independent
experiments is shown. Proteins that were identified by Western blotting
or peptide sequencing previously (22) are indicated at the
left. b, to assess Rac1 prenylation, the
indicated HA-tagged proteins were immunoprecipitated from transiently
transfected CHO-m3 cells that were incubated for 16 h with
[35S]methionine in the absence (odd-numbered
lanes) or presence (even-numbered lanes) of 5 µM compactin. A representative autoradiograph from three
independent experiments is shown. c, the guanine nucleotide
exchange activities of the wild-type and mutant Rac1 proteins were
assessed by measuring [35S]GTP S binding by the
HA-tagged proteins transiently expressed in CHO-m3 cells. The control
value in each experiment was the amount of [35S]GTP
S
bound to HA-Rac1 (first column). Results shown are the
means ± 1 S.E. from three independent experiments conducted with
triplicate samples in each experiment. Brackets above the
columns indicate a statistical comparison between the two
bracketed samples. Symbols within a column
indicate a statistical comparison between the sample and the control
sample of cells transfected with HA-Rac1 (first column).
(**, p < 0.005; *, p < 0.05;
NS, not significant.) d, the effects of
co-transfected SmgGDS on Rac1 guanine nucleotide exchange was assessed
by measuring [35S]GTP
S binding by the HA-tagged
proteins in CHO-m3 cells that were co-expressing the indicated
myc-tagged proteins. In each experiment, the control value was the
amount of [35S]GTP
S bound to HA-Rac1 in cells
co-transfected with the myc-pcDNA3.1 vector (first
column). Results shown are the means ± 1 S.E. from three
independent experiments conducted with triplicate samples in each
experiment. Brackets above the columns indicate a
statistical comparison between the two bracketed samples.
(*, p < 0.05; NS, not significant.)
S (Fig. 4, c
and d). Constitutively active HA-CA-Rac1 binds less
[35S]GTP
S than does HA-Rac1 (Fig. 4c),
consistent with constitutively active CA-Rac1 having a slower rate of
GTP hydrolysis, which would result in a slower rate of
[35S]GTP
S binding (22). Dominant negative HA-DN-Rac1
also binds less [35S]GTP
S than does HA-Rac1 (Fig.
4c), consistent with the mutation of aa 17 in DN-Rac1
diminishing the ability of DN-Rac1 to bind GTP (22). Interestingly,
HA-Rac1(PBRQ) binds more [35S]GTP
S than does HA-Rac1
(Fig. 4, c and d), indicating that disruption of
the PBR does not diminish [35S]GTP
S binding but can
actually enhance it. Consistent with reports that SmgGDS promotes
guanine nucleotide exchange by Rac1 (12-14), we found that expression
of either myc-Sg or myc-SgNA4 enhances [35S]GTP
S
binding by HA-Rac1 and HA-Rac1(PBRQ) in transiently transfected CHO-m3
cells (Fig. 4d).
S binding assays indicate
that Rac1 and Rac1(PBRQ) proteins are able to interact with either
SmgGDS or mutant SmgGDS containing a disrupted N-terminal NES. To
investigate the stability of these protein interactions, we
co-precipitated the HA-tagged Rac1 proteins with either myc-Sg or
myc-SgNA4 transiently expressed in 35S-labeled CHO-m3 cells
(Fig. 5a). We found that
myc-Sg weakly co-precipitates HA-Rac1 and HA-CA-Rac1 but strongly
co-precipitates HA-DN-Rac1 (Fig. 5a, lanes 2,
4, and 6). These results agree with the generally
accepted model that GTPases form relatively unstable complexes with
SmgGDS, because the GTPases only transiently associate with SmgGDS
during guanine nucleotide exchange (15, 22). Conversely, dominant
negative GTPases form relatively stable complexes with SmgGDS, because
dominant negative GTPases do not readily bind GTP and thus do not
readily dissociate from SmgGDS during guanine nucleotide exchange (15,
22).
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Fig. 5.
The ability of SmgGDS to form strong
complexes with RhoA and weak complexes with Rac1 is affected by
mutating the PBR of the GTPases or the N terminus NES of SmgGDS.
a and b, the interactions of SmgGDS with Rac1
(a) and RhoA (b) were examined by
immunoprecipitating myc-Sg (lanes 1-7) or myc-SgNA4
(lanes 9-15) from lysates of equal numbers of
35S-labeled CHO-m3 cells that were co-expressing the
indicated HA-tagged proteins. As a control, lysates of cells
co-transfected with the myc-pcDNA3.1 and HA- pcDNA3.1 vectors
were also immunoprecipitated with myc antibody (lane 8). A
representative autoradiograph from six independent experiments is
shown. The names of the identified proteins in the
immunoprecipitates are indicated at the left. The
identity of the protein that migrates with a relative molecular mass of
24 kDa has not yet been established.
S binding
assays (Fig. 4, c and d). If Rac1(PBRQ)·SmgGDS complexes are less stable than Rac1·SmgGDS complexes, then
Rac1(PBRQ)·SmgGDS complexes may turnover more rapidly than
Rac1·SmgGDS complexes, resulting in a faster rate of
[35S]GTP
S binding by Rac1(PBRQ) proteins than by Rac1 proteins.
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Fig. 6.
The nuclear accumulation of SmgGDS is
enhanced by CA-Rac1 and diminished by DN-Rac1. a,
CHO-m3 cells were co-transfected with cDNAs coding for HA-Sg
(panels 1-7) or HA-SgNA4 (panels 8-14) and the
indicated GFP-tagged wild-type or mutant Rac1 proteins. Control cells
were co-transfected with the pEGFP-C1 vector (panels 1 and
8). The HA-tagged proteins (red) and GFP-tagged
proteins (green) were detected by the fluorescence of
TRITC-labeled antibodies and GFP, respectively, in the fixed and
TX-100-permeabilized cells. In each panel, two images of the
same field of cells show the distribution of the HA-tagged proteins
(left) and the GFP-tagged proteins (right).
Results shown are representative of four independent experiments. The
bar represents 10 µm. b and c, the
nuclear localization of HA-Sg (b) or HA-SgNA4 (c)
in fixed and TX-100-permeabilized CHO-m3 cells that were co-expressing
the GFP-tagged proteins was scored by investigators who did not know
the identities of the HA-tagged or GFP-tagged proteins expressed by the
cells. Results shown are the means ± 1 S.E. from 80 cells scored
in four independent experiments. Brackets above the columns
indicate a statistical comparison between the two bracketed
samples. Symbols within a column indicate a statistical
comparison between the sample and the control sample of cells
transfected with GFP (first column). (**, p < 0.001; *, p < 0.05; NS, not
significant.)
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Fig. 7.
The nuclear accumulation of SmgGDS is
diminished by RhoA. a, CHO-m3 cells were co-transfected
with cDNAs coding for HA-Sg (panels 1-7) or HA-SgNA4
(panels 8-14) and the indicated GFP-tagged wild-type or
mutant RhoA proteins. Control cells were co-transfected with the
pEGFP-C1 vector (panels 1 and 8). The HA-tagged
proteins (red) and GFP-tagged proteins (green)
were detected by the fluorescence of TRITC-labeled antibodies and GFP,
respectively, in the fixed and TX-100-permeabilized cells. In each
panel, two images of the same field of cells show the
distribution of the HA-tagged proteins (left) and the
GFP-tagged proteins (right). Results shown are
representative of three independent experiments. The bar
represents 10 µm. b and c, the nuclear
localization of HA-Sg (b) or HA-SgNA4 (c) in
CHO-m3 cells that were co-expressing the GFP-tagged proteins was scored
by investigators who did not know the identities of the HA-tagged or
GFP-tagged proteins expressed by the cells. Results shown are the
means ± 1 S.E. from 60 cells scored in three independent
experiments. Brackets above the columns indicate a
statistical comparison between the two bracketed samples.
Symbols within a column indicate a statistical comparison
between the sample and the control sample of cells transfected with GFP
(first column). (**, p < 0.001; *, p < 0.05; NS, not significant.)
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Fig. 8.
The nuclear accumulation of p120ctn is
enhanced by CA-Rac1 and diminished by CA-RhoA. a,
CHO-m3 cells transiently expressing the indicated GFP-tagged proteins
were incubated in the absence (panels 1-5) or presence
(panels 6-10) of 10 nM leptomycin B for 90 min.
Endogenous p120ctn (red) and the GFP-tagged proteins
(green) were detected by the fluorescence of TRITC-labeled
antibodies and GFP, respectively, in the fixed and TX-100-permeabilized
cells. In each panel, two images of the same field of cells
show the distribution of p120ctn (left) and the GFP-tagged
proteins (right). Results shown are representative of four
independent experiments. The bar represents 10 µm.
b and c, the nuclear localization of p120ctn in
fixed and TX-100-permeabilized CHO-m3 cells that were previously
incubated in the absence (b) or presence (c) of
10 nM leptomycin B for 90 min was scored by investigators
who did not know the identities of the GFP-tagged proteins expressed by
the cells, nor the drug treatment of the cells. Results shown are the
means ± 1 S.E. from 80 cells scored in four independent
experiments. Brackets above the columns indicate a
statistical comparison between the two bracketed samples.
Symbols within a column indicate a statistical comparison
between the sample and the control sample of cells transfected with GFP
(first column). (**, p < 0.001; *, p < 0.05; NS, not significant.)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model of the regulation of SmgGDS nuclear
accumulation by Rac1 and RhoA. A spatial diagram of the cell is
depicted, with the cytoplasm (top) separated from the
nucleus (bottom) by the nuclear membrane (gray
rectangles). a, when Rac1-GDP associates with SmgGDS,
it converts to Rac1-GTP. The nuclear accumulation of SmgGDS is promoted
by GTP-dependent signals from Rac1-GTP (represented by the
single asterisk) and by the PBR of Rac1 (shaded
area on Rac1), which provides an NLS (represented by
the double asterisks) for Rac1·SmgGDS complexes. Rac1-GTP
accumulates with SmgGDS in the nucleus, contributing to the greater
nuclear accumulation of Rac1-GTP than Rac1-GDP. b,
Rac1(PBRQ) is able to associate with SmgGDS and convert to the
GTP-bound form. However, Rac1(PBRQ)-GTP is less effective than Rac1-GTP
in promoting SmgGDS nuclear accumulation, because Rac1(PBRQ) associates
more transiently with SmgGDS and because Rac1(PBRQ) is unable to
provide an NLS for SmgGDS. The diminished nuclear accumulation of
Rac1(PBRQ)·SmgGDS complexes contributes to the reduced nuclear
accumulation of Rac1(PBRQ) compared with Rac1. c, DN-Rac1
associates with SmgGDS but cannot convert to the GTP-bound form,
resulting in the formation of stable DN-Rac1·SmgGDS complexes. The
formation of these complexes reduces the nuclear accumulation of
SmgGDS, because DN-Rac1 cannot provide a GTP-dependent
signal and because DN-Rac1 competitively inhibits SmgGDS from
interacting with endogenous Rac1. The retention of DN-Rac1·SmgGDS
complexes in the cytoplasm may contribute to the reduced nuclear
accumulation of DN-Rac1. d, based on our results, SmgGDS
associates more strongly with RhoA than with Rac1. The formation of
RhoA·SmgGDS complexes diminishes the nuclear accumulation of SmgGDS,
because the PBR of RhoA does not provide an NLS and because RhoA
competitively inhibits SmgGDS from interacting with endogenous Rac1.
The conversion of RhoA-GDP to RhoA-GTP may release SmgGDS, allowing
SmgGDS to interact with endogenous Rac1.
Our model proposes that Rac1·SmgGDS complexes dissociate in the nucleus (Fig. 9A). This proposal is consistent with our inability to co-precipitate any of the HA-tagged Rac1 or Rac1(PBRQ) proteins with myc-SgNA4 (Fig. 5a, lanes 5-10), which is retained in the nucleus (Fig. 1, b and c). According to our model, the retention of SgNA4 in the nucleus contributes to its reduced association with Rac1 and Rac1(PBRQ) proteins.
We propose that Rac1(PBRQ) is less effective than Rac1 in promoting SmgGDS nuclear accumulation because Rac1(PBRQ) has a more transient interaction with SmgGDS and because Rac1(PBRQ) does not provide an NLS for SmgGDS (Fig. 9B). These attributes of Rac1(PBRQ) may also contribute to the diminished nuclear accumulation of Rac1(PBRQ) compared with Rac1 (Fig. 3, b and c).
According to our model, DN-Rac1 associates with SmgGDS but cannot convert to the GTP-bound form, resulting in the prolonged association of DN-Rac1 with SmgGDS (Fig. 9C). This proposal is consistent with our ability to co-precipitate stable complexes of DN-Rac1·SmgGDS (Fig. 5a). The association of DN-Rac1 with SmgGDS may reduce the nuclear accumulation of SmgGDS because DN-Rac1 cannot provide a GTP-dependent signal and because DN-Rac1 competitively inhibits the association of endogenous Rac1 with SmgGDS (Fig. 9C). The stable association of DN-Rac1 with SmgGDS in the cytoplasm may also contribute to the reduced nuclear accumulation of DN-Rac1 (Fig. 3, b and c).
Our model also provides an explanation for the inhibitory effects of RhoA on SmgGDS nuclear accumulation (Fig. 9D). Our results indicate that SmgGDS associates more strongly with RhoA than with Rac1 (Fig. 5). We propose that RhoA diminishes the nuclear accumulation of SmgGDS because the PBR of RhoA does not provide an NLS and because RhoA competitively inhibits the association of endogenous Rac1 with SmgGDS (Fig. 9D). The conversion of RhoA-GDP to RhoA-GTP may release SmgGDS, allowing SmgGDS to interact with Rac1 (Fig. 9D). Our finding that SmgGDS associates more strongly with RhoA than with RhoA(PBRQ) (Fig. 5b) may account for our observations that, in some cases, the nuclear accumulation of SmgGDS is inhibited more by RhoA proteins than by RhoA(PBRQ) proteins (Fig. 7).
Our findings indicate that Rac1 supplies both an NLS- and a GTP-dependent signal to promote the nuclear accumulation of Rac1·SmgGDS complexes. The importance of these signals in relation to each other has not yet been established. Interestingly, there is greater nuclear accumulation of SmgGDS in the presence of CA-Rac1(PBRQ) than in the presence of Rac1(PBRQ) or DN-Rac1(PBRQ) (Fig. 6). This finding indicates that the enhanced GTP-dependent signal from CA-Rac1(PBRQ) can promote the nuclear accumulation of SmgGDS even when CA-Rac1(PBRQ) cannot supply an NLS. One possible explanation for this finding is that CA-Rac1(PBRQ) activates an effector that subsequently interacts with complexes of SmgGDS associated with endogenous Rac1. According to this proposal, endogenous Rac1 supplies the NLS for the SmgGDS·endogenous Rac1 complex, whereas CA-Rac1(PBRQ) activates an effector to enhance the nuclear accumulation of the SmgGDS·endogenous Rac1 complex. In this way, CA-Rac1(PBRQ) may enhance the nuclear accumulation of SmgGDS·endogenous Rac1 complexes, even though CA-Rac1(PBRQ) lacks an NLS and may not accompany these complexes into the nucleus.
An important aspect of our model is that the Rac1 PBR functions as an
NLS for complexes of Rac1·SmgGDS, promoting the nuclear entry of
Rac1·SmgGDS complexes. This proposed mode of SmgGDS nuclear entry is
very similar to the enhanced nuclear entry of karyopherin when it
associates with NLS-containing proteins such as open reading frame 57 (5). The 11 ARM repeats of SmgGDS may create a platform that has
structural and functional similarities to the NLS-binding platform
created by the 10 ARM repeats of karyopherin
. Karyopherin
is
believed to bind two NLS-containing proteins simultaneously at NLS
binding sites indicated by the aa sequence WXXXN (reviewed
in Ref. 3). Karyopherin
has three WXXXN sequences at the
major NLS binding site, and two WXXXN sequences at the minor
NLS binding site (reviewed in Ref. 3). We observed that SmgGDS has one
WXXXN sequence (WIPSN), which is located at aa 275-279 in
SmgGDS isoform 2 and at aa 324-328 in SmgGDS isoform 1 (NCBI Protein
Data base accession number P52306). The presence of the
WXXXN sequence in SmgGDS is consistent with the Rac1 PBR acting as an NLS that promotes the formation and nuclear accumulation of SmgGDS·Rac1 complexes. The fact that SmgGDS has less
WXXXN sequences than karyopherin
is consistent with
SmgGDS interacting with a unique subset of NLS-containing proteins
(only small GTPases with PBRs), compared with karyopherin
, which
interacts with a large variety of NLS-containing proteins.
Although there are appealing similarities between the nuclear entry of
SmgGDS associated with Rac1 and the nuclear entry of karyopherin associated with an NLS-containing protein, there are also some
interesting differences. Karyopherin
must interact with importin
at the nuclear pore to enter the nucleus. This interaction depends
on the importin
-binding domain of karyopherin
(reviewed in Ref.
3). We have not detected this domain in SmgGDS, suggesting that SmgGDS
does not directly interact with importin
when entering the nucleus.
It was recently suggested that the interaction of importin with the
small GTPase Ran provides a model for the interaction of SmgGDS with
small GTPases (31). This suggestion, which is based on the binding of
Ran-GTP to the 10 ARM repeats of importin
(reviewed in Ref. 31),
has parallels to our model. However, the absence of a PBR in Ran
diminishes the similarities between the Ran/importin
interaction
and the Rac1/SmgGDS interaction. Despite this limitation, Ran/importin
interactions may provide additional insights into the structural
features of small GTPases and SmgGDS, which allow these proteins to interact.
The Rac1 PBR shares striking structural and functional similarities with a classic monopartite NLS sequence. However, the Rac1 PBR also has other functions, including regulating the ability of Rac1 to physically associate with RhoGDI, IQGAP1·calmodulin complexes (Fig. 5a) and the Rac1 effector PAK1 (32). It is possible that the diminished interactions of Rac1(PBRQ) with these proteins contribute to the diminished ability of Rac1(PBRQ) to accumulate in the nucleus, and the diminished ability of Rac1(PBRQ) to promote the nuclear accumulation of SmgGDS. Nevertheless, the ability of the Rac1 PBR to act as an NLS for SmgGDS and the inability of the RhoA PBR to act as an NLS provide a plausible explanation for our findings.
Our findings indicate that, like SmgGDS, the nuclear accumulation of
p120ctn is enhanced by GTP-dependent and
PBR-dependent signals supplied by Rac1 but not by RhoA.
Thus, our model of the Rac1- and RhoA-dependent regulation
of SmgGDS nuclear accumulation (Fig. 9) generally applies to the Rac1-
and RhoA-dependent regulation of 120ctn nuclear
accumulation. If Rac1 regulates p120ctn nuclear localization in the
same manner that it regulates SmgGDS nuclear localization, Rac1 should
physically interact with p120ctn. However, we have been unable to
precipitate complexes of Rac1 associated with either endogenous p120ctn
or with the transiently expressed 3A or 3AB isoforms of p120ctn.
Complexes of Rac1·p120ctn may form in the cells, but they may be too
unstable to isolate by immunoprecipitation. Complexes of Rac1·p120ctn
may be as unstable as complexes of Rac1·SmgGDS, which are also
difficult to immunoprecipitate (Fig. 5a). Even though
Rac1· SmgGDS and Rac1(PBRQ)·SmgGDS complexes are difficult to
immunoprecipitate (Fig. 5a), both Rac1 and Rac1(PBRQ)
interact with SmgGDS in the cells, as indicated by the enhanced binding of [35S]GTPS by Rac1 and Rac1(PBRQ) in the presence of
SmgGDS (Fig. 4d). Interestingly, it was previously reported
that p120ctn interacts with RhoA in vitro (9), consistent
with our model that p120ctn interacts with RhoA, as well as with Rac1,
in vivo. However, we cannot currently assess whether these
potential interactions involve the direct association of p120ctn with
RhoA and Rac1 or involve the participation of additional intermediary proteins.
We found that DN-Rac1 inhibits the nuclear accumulation of SmgGDS (Fig. 6) but does not inhibit the nuclear accumulation of p120ctn (Fig. 8). This difference may occur because of the different abilities of SmgGDS and p120ctn to serve as a GEF for Rac1. According to our model, the formation of stable DN-Rac1·SmgGDS complexes during attempted guanine nucleotide exchange by DN-Rac1 inhibits the ability of SmgGDS to interact with endogenous Rac1 and enter the nucleus (Fig. 9C). In contrast, DN-Rac1 may not form a stable complex with p120ctn, because p120ctn reportedly does not act as a GEF for Rac1 (9). The inability of DN-Rac1 to stably bind p120ctn would make DN-Rac1 a much less effective inhibitor of p120ctn nuclear accumulation, compared with the ability of DN-Rac1 to inhibit SmgGDS nuclear accumulation.
Our model depicts the conversion of Rac1-GDP to Rac1-GTP occurring in the cytoplasm, before complexes of Rac1·SmgGDS or Rac1·p120ctn arrive at the nuclear pore (Fig. 9A). However, the nuclear localization of several GEFs for Rac1 (33-35) and for other Rho family members (36, 37) suggests the interesting possibility that Rac1-GDP converts to Rac1-GTP when the Rac1·p120ctn complex arrives at the nuclear pore, where the Rac1·p120ctn complex might have access to nuclear GEFs. This possibility is consistent with p120ctn associating with Vav (16), which is a Rac1 GEF that is present in the nucleus (33, 34).
Nucleocytoplasmic shuttling allows ARM proteins to participate in many
cellular activities, including the regulation of small GTPases, gene
transcription, cell adhesion, and neoplastic transformation (reviewed
in Refs. 2, 4, 6, and 10). Our demonstration that Rac1 and RhoA
regulate the nucleocytoplasmic shuttling of SmgGDS and p120ctn raises
the intriguing possibility that these GTPases affect the nuclear
localization of other ARM proteins. Other small GTPases with a PBR,
which have been reported to interact with SmgGDS, include Cdc42 (13,
14), K-Ras4B (11, 14), Rap1A (18), Rap1B (11, 14), and RalA (18). The
PBRs of these GTPases may have functions similar to those of Rac1 and RhoA. This possibility is supported by a previous report that the
ability of Rap1 to interact with SmgGDS requires a Rap1 C-terminal region containing the PBR (38). The roles of these small GTPases in
regulating the nuclear accumulation of SmgGDS and other armadillo proteins need to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Takai for the gift of the
pSR-smg GDS plasmid and F. van Roy for the gift of the
pEFBOSp120-3AB and pEFBOSp120-3A plasmids.
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FOOTNOTES |
---|
* This work was supported by grants (to C. L. W.) from the NHLBI/National Institutes of Health (Grant RO1 HL63921) and the Pennsylvania/Delaware Affiliate of the American Heart Association (Grant 9951305U).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: Molecular Pharmacology
Laboratory, One Guthrie Square, Sayre, PA 18840. Tel.: 570-882-4650;
Fax: 570-882-4643; E-mail: williams_carol@guthrie.org.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211286200
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ABBREVIATIONS |
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The abbreviations used are:
ARM, armadillo;
aa, amino acid(s);
CHO, Chinese hamster ovary;
GEF, guanine nucleotide
exchange factor;
GFP, green fluorescent protein;
EGFP, enhanced green
fluorescent protein;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
HA, hemagglutinin;
NES, nuclear
export signal;
NLS, nuclear localization signal;
p120ctn, p120 catenin;
PBR, polybasic region;
TX-100, Triton X-100;
CAAX, aliphatic
amino acid;
DN, dominant negative;
PBS, phosphate-buffered saline;
TRITC, tetramethylrhodamine isothiocyanate.
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