 |
INTRODUCTION |
The insulin receptor substrate
(IRS)1 proteins are a family
of docking proteins, which include IRS-1-4, Gab-1, and
p62dok (1). IRS-1 was the first to be identified as a
docking protein for both the insulin and the IGF-I receptors. It
transmits a signal from both receptors by interacting with a number of
partners, including phosphatidylinositol 3-kinase, SHP2, Grb2,
Crk, and others (2, 3). Tyrosine kinase activity of the receptors and
phosphorylation of IRS-1 are essential steps in signal transduction. Of
the downstream signals generated by IRS-1, the best studied is the
phosphatidylinositol 3-kinase signaling pathway, which plays a major
role in a number of biological responses to growth factors (1, 4).
IRS-1 interacts directly with both the insulin and the IGF-I receptors,
and the domains required for their interaction have also been
identified (5). Because of its direct interaction with the receptors,
its size, and its downstream signaling, it has been generally assumed
that IRS-1 is an exclusively cytosolic (or plasma membrane) protein (6,
7). However, IRS-1 is known to interact with the SV40 T antigen (8, 9)
and nucleolin (10). T antigen and nucleolin are predominantly nuclear
proteins, although minor fractions of either protein can be found in
the cytosol (11-13). It has been therefore tacitly assumed that IRS-1, anchored to the receptor, was interacting with the minor cytosolic fractions of T antigen and nucleolin. There is evidence, however, that
signal-transduction molecules can translocate to the nucleus. They
include mitogen-activated protein kinase (14), p70S6K/TOR
(15, 16), the STAT proteins (17, 18), Akt (19), 
-catenin (20), the
epidermal growth factor receptor (21), phosphatases (22), IRS-3 (23),
and a cleaved ErbB-4 receptor (24). Indeed, Jans and Hassan (25) have
summarized in a review the evidence that growth factors and their
receptors can accumulate in the nuclei of cells. IGF-I, IGF-IR, and
IRS-1 are not mentioned in that review, but insulin is (26).
The first observation that IRS-1 can be translocated to the nuclei
should be credited to Lassak et al. (27), who used
medulloblastoma cells expressing the human JCV virus. The nuclear
translocation of IRS-1 in association with T antigen (this time, the
SV40 T antigen) was confirmed by Prisco et al. (28). In this
communication, we have extended the observations of Lassak et
al. (27) and Prisco et al. (28) to other cell lines and
have attempted to obtain information on the biological significance of
the nuclear translocation. Our results confirm that IRS-1 is
translocated to the nuclei and especially to the nucleoli of cells in
culture. Oncogenes like SV40 T antigen and v-src, as well as the
activated IGF-IR can induce nuclear/nucleolar translocation of IRS-1.
In addition, our data indicate that nuclear IRS-1 (but not cytosolic IRS-1) interacts with the upstream binding factor (UBF), a protein that
regulates RNA polymerase I activity and, therefore, ribosomal RNA
(rRNA) synthesis (29, 30). In agreement with this interaction, cells
with nuclear/nucleolar IRS-1 have a markedly increased rRNA synthesis
at levels superior to those found in cells with cytosolic localization
of IRS-1. Our findings, coupled with previous findings that IRS-1
increases cell size (see "Discussion"), suggest that nuclear IRS-1
may be involved in sustained cell growth, especially through the
production of rRNA.
| |
EXPERIMENTAL PROCEDURES |
Cell Lines--
The cell lines used in these experiments are
mostly mouse embryo fibroblasts (MEF) generated by a 3T3 protocol. The
parental cell line was derived from mouse embryos with a targeted
disruption of the IGF-IR genes (31). Designated as R
cells (32), this MEF cell line has been used extensively as a cell line unresponsive to
IGF-I stimulation. R+ cells were obtained from R cells by stable
transfection with a plasmid expressing the wild-type human IGF-IR. R+
cells express high levels of IGF-I receptors and respond to IGF-I with
growth (33). R/T cells were derived from R cells by transfection
with a plasmid expressing the simian virus 40 (SV40) T antigen (32).
R/v-src cells are R cells expressing the v-src oncogene (34).
R/IRS/FLAG cells and R+/IRS/FLAG cells were generated, respectively,
from R and R+ cells by transduction with a retroviral vector
expressing mouse IRS-1 (28) fused in frame with a FLAG epitope at its
3' end. Viral transduction was performed as previously described (35).
Selection was carried out with 1 µg/ml puromycin (Invitrogen). All
cell lines are mixed populations. Two cell lines, which do not express
IRS-1, 32D cells (36), and LNCaP cells (37) were used as negative
controls for anti-IRS1 staining. For experiments on rRNA synthesis, 32D IGF-IR, 32D/IRS-1, and 32D IGF-IR/IRS1 cells were used. These cells are
described in detail in Zhou-Li et al. (9) and Valentinis et al. (38). The cells used for the experiments shown in
Fig. 9 are LNCaP cells stably expressing a wild-type mouse IRS-1
(37).
Plasmid--
pIRS/FLAG was generated from pGR159 MSCV.pac
retroviral vector (28) by fusing in-frame the wild-type mouse IRS-1
sequence with the FLAG sequence (Eastman Kodak Co.) at the 3' end. The IRS-1 sequence fused in frame with the FLAG epitope at the 3' end was
produced by PCR. The detailed methodology for the construction of the
retroviral vector has been already described (35).
Immunofluorescence/Confocal Microscopy--
Cells plated on
glass coverslips were washed with PBS and fixed with 3.0%
paraformaldehyde in PBS for 20 min at room temperature, followed by
permeabilization with 0.2% Triton X-100 in PBS for 2 min at room
temperature. Coverslips were washed with PBS, and nonspecific binding
of IgG was blocked with 10% normal donkey serum (sc-2044, Santa Cruz
Biotechnology) in PBS for 20 min at room temperature. The cells were
then stained with the antibodies described below, as indicated. After
incubation with primary antibodies, coverslips were washed with PBS
three times. The cells were subsequently stained with fluorescein
isothiocyanate- and rhodamine-conjugated secondary antibodies
(sc-2078 and sc-2095, Santa Cruz) for 1 h at room temperature.
Finally, coverslips were washed with PBS three times and mounted on
glass slides with Vectashield mounting medium (H-1000, Vector
Laboratories Inc.). Fluorescent images were collected on a Zeiss
Axiovert 100 confocal microscope using a Zeiss 40× objective. In one
experiment, propidium iodide was used to stain the nuclei. In this
case, the cells were digested with RNase A (1 mg/ml) for 30 min before
staining with propidium iodide (2.5 µg/ml, Molecular Probes P-3566)
for 5 min.
Immunohistochemistry--
32D and 32D-derived cells were washed
three times with Hanks' buffer and seeded at a density of 5 × 104/2 ml of RPMI 1640 medium supplemented with 10% fetal
bovine serum plus or minus IGF-I (50 ng/ml). Cells were
harvested after 16 h, and cytospins were prepared. After fixing in
3.7% formaldehyde solution in PBS and permeabilization with 0.2%
Triton X-100 in PBS, the immunostaining was carried out using the
Histomouse SP kit (Zymed Laboratories Inc. 95-9541)
following the manufacturer's protocol. The magnifications for the
figures presented are 1000×.
Subcellular Fractionation--
For cell fractionation, cell
monolayers were trypsinized and collected in serum-containing medium.
Cells were washed with ice-cold PBS and taken up in buffer A (10 mM N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid
(HEPES), PH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride). After swelling on ice
for 10 min, plasma membranes were disrupted by adding 0.1% Nonidet
P-40 and mixing for 10 s. Cell breakage was examined under the
microscope. The nuclei were pelleted by centrifugation at 6000 rpm for
45 s at 4 °C, and a cytoplasmic fraction (supernatant) was
recovered. The pellet was then washed in 1 ml of ice-cold sucrose
buffer (0.32 M sucrose, 3 mM CaCl2,
2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl PH 8.0, 1 mM dithiothreitol, and
0.5 mM phenylmethylsulfonyl fluoride) and centrifuged at
3000 rpm for 5 min at 4 °C. The washing of nuclei was repeated three
times. The pellet was subsequently resuspended in buffer C (20 mM HEPES PH 7.9, 1 mM EDTA, 1 mM
EGTA, 400 mM NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). After rocking for 20 min
at 4 °C, the samples were centrifuged at 12,000 rpm for 15 min at
4 °C to recover a nuclear fraction (supernatant).
Western Blots--
Aliquots of either cytoplasmic or nuclear
extracts (50 µg/sample) were resolved by SDS-4-15% polyacrilamide
gels and transferred to nitrocellulose membranes. They were then probed
with the antibodies indicated in the figures and developed with the ECL
system (Amersham Biosciences). Equal amounts of cell lysates
(500 µg/sample, unless otherwise stated) determined by Bio-Rad
protein assay, were immunoprecipitated with an anti-IRS-1 antibody
(UBI) and 25 µl of protein A+G beads (Amersham Biosciences). After
washing, precipitates were directly subjected to Western blot analysis.
Antibodies--
The following antibodies were used: rabbit
polyclonal anti-IRS-1 antibody (06-248, Upstate Biotechnology), mouse
monoclonal anti-nucleolin antibody (MS-3, sc-8031, Santa Cruz
Biotechnologies), rabbit polyclonal anti-Id1 antibody (C-20, sc-488,
Santa Cruz Biotechnologies), mouse monoclonal anti-SV40 T antigen
antibody (Pab101, sc-147, Santa Cruz Biotechnologies), mouse monoclonal anti-FLAG antibody conjugated with FITC (16-177, Upstate
Biotechnology), mouse monoclonal antibody conjugated to PCNA (PC10,
sc-56, Santa Cruz Biotechnologies), and mouse monoclonal anti-Grb2
antibody (Transduction Laboratories). To confirm the specificity of
IRS-1 detection in the nuclei, we also used two other antibodies:
rabbit polyclonal IgG anti-IRS-1, preCT (06-52b, Upstate
Biotechnology), and rabbit polyclonal anti-IRS-1 (C-20, sc-559,
Santa Cruz Biotechnologies). The latter was provided with its blocking
peptide (C20p, sc-559P). For UBF, we used a mouse monoclonal UBF
antibody from Santa Cruz Biotechnologies (sc-13125).
Metabolic Labeling of rRNA--
32D IGF-IR, 32D/IRS-1, and 32D
IGF-IR/IRS-1 cells (38) were seeded at a density of 5 × 104 cells/ml in RPMI 1640 medium supplemented with
heat-inactivated 10% fetal bovine serum and 50 ng/ml of IGF-I (Life
Technologies) for the indicated times. In one experiment, 32D
IGF-IR/IRS1 cells were pre-treated with rapamycin (Sigma, 10 ng/ml) for
48 h. The cells were then labeled for 4 h with
[32P]orthophosphate at a final concentration of 250 µCi/ml (ICN Biochemicals) in phosphate-free RPMI 1640 medium (Life
Technologies). After labeling, the cells were washed and incubated in
fresh medium for 2 h. Total RNA was isolated using RNeasy MiniKit
(Qiagen) and separated by electrophoresis on 1% agarose formaldehyde
gels. After drying, the 32P-labeled rRNA was visualized by
autoradiography. The bands were also counted in a liquid scintillation counter.
| |
RESULTS |
Co-localization of IRS-1 and SV40 T Antigen in the Nuclei of R
Derived Cells--
In previous papers, we showed that IRS-1 could be
co-immunoprecipitated with the SV40 T antigen, either in R cells (8) or in 32D cells (9). We have investigated the localization of IRS-1 by
confocal microscopy, using the appropriate antibodies described under
"Experimental Procedures." Fig.
1A shows a confocal microscopy
picture of R/T cells, growing in fetal bovine serum. The cells
were stained for IRS-1 (rhodamine, left) or SV40 T antigen (FITC, center), and the right panel gives the
merged picture. T antigen is, as expected, almost exclusively localized
in the nucleus, but so is IRS-1. The merged picture gives a clear
co-localization (there are tiny specks of both proteins in the
surrounding cytoplasm, but both T antigen and IRS-1 are mostly
nuclear). The specificity of the immunohistochemistry for IRS-1 was
monitored in several ways (see also below in the section on R+ cells).
First, we determined the localization of IRS-1 in the parental R
cells by confocal microscopy. R cells have no IGF-I receptors, but
high levels of IRS-1, at least in comparison to other 3T3 cells. Fig.
1B shows R cells stained with rhodamine for IRS-1 and with
FITC for nucleolin. R cells had been stimulated with IGF-I for 8 h, but the same results were obtained with R in SFM or stimulated
with serum (data not shown). In R cells, IRS-1 (as detected by this
antibody) is essentially limited to the cytoplasm. The nuclei appear as dark centers, dotted by the nucleoli stained with the
anti-nucleolin antibody. The same antibody was completely negative when
used on two cell lines that do not express IRS-1, prostatic human
cancer cells LNCaP, and murine hemopoietic 32D cells (data not
shown).
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1.
Co-localization of IRS-1 and the SV40 T
antigen in the nuclei of R cells. Confocal microscopy of R/T
cells (32) growing in fetal bovine serum. The cells were stained with
an antibody to IRS-1 (rhodamine) and with an antibody to SV40 T antigen
(FITC). The pictures are merged in the last
subpanel. B, IRS-1 in R cells. Confocal
microscopy of R cells. Individual and merged pictures after staining
with antibodies to IRS-1 (rhodamine) and nucleolin (FITC). Quiescent
R cells had been stimulated for 8 h with IGF-I (50 ng/ml).
C, R+ cells in serum-free medium. D, R+ cells
8 h after stimulation with IGF-I. In both instances, the cells
were stained as R cells in panel B.
|
|
To further confirm the results obtained by confocal microscopy, we
carried out a subcellular fractionation of R and R/T cells (Fig.
2). After subcellular fractionation (see
"Experimental Procedures"), IRS-1 can be detected in the nuclear
fraction of R/T but not of R cells (lanes 3 and
4). In both cell lines, there is still IRS-1 in the cytosol.
The fractions are reasonably pure, as Grb2 is detectable in the cytosol
but not in the nuclear fraction of both cell lines. On the contrary,
the T antigen can only be detected in the nuclear fraction of R/T
cells (Fig. 2C). In Fig. 2D, we have taken the
cytosolic and nuclear fractions of both cell lines and
immunoprecipitated them with an antibody to IRS-1. T antigen was
detected only in the nuclear fraction of R/T cells.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Subcellular fractionation of R/T and R
cells. R/T and R cells were grown in fetal bovine serum.
Cytosol and nuclear fractions were prepared as described under
"Experimental Procedures." A, Western blot of the two
fractions (nuclear and cytosol) stained with an antibody to IRS-1.
IRS-1 is detectable in the cytosol of both cell lines, but in the
nucleus only in R/T cells. B, the same blot stained with
an antibody to Grb2. Only the cytosol fraction is positive for Grb2 and
in both cell lines. C, the same blot stained with an
antibody to SV40 T antigen. R/T cells show the presence of T antigen,
and only in the nuclear fraction, as expected. D, from the
same lysates, we immunoprecipitated IRS-1 with the appropriate
antibody. The Western blot of the immunoprecipitate shows that T
antigen is detectable only in the nuclear fraction of R/T
cells.
|
|
By confocal microscopy, it would seem that IRS-1 is, if not
exclusively, largely localized to the nucleus of R/T cells, while, in
Western blots, the majority of IRS-1 is cytosolic, even in R/T cells.
We will discuss this discrepancy in a later section.
IRS-1 Localizes in the Nuclei and Nucleoli of R+ Cells Stimulated
with IGF-I--
Either by confocal microscopy (Fig. 1) or by
subcellular fractionation (Fig. 2), IRS-1 is detectable in R cells
only in the cytosol. This is true regardless of the growth conditions
(SFM, IGF-I, or fetal bovine serum). We then asked whether IRS-1 would be detectable in the nuclei of R+ cells (33), which are R cells expressing a wild-type human IGF-IR. R+ cells were stained with antibodies to IRS-1 (rhodamine) or nucleolin (FITC). Fig. 1C
is a confocal microscope image of R+ cells kept in SFM for 48 h
(quiescent cells). Fig. 1D, shows the same cells 8 h
after stimulation with IGF-I (50 ng/ml). In quiescent cells, IRS-1 is
mostly, but not exclusively, localized in the cytoplasm. By comparing
Fig. 1, C and D, three things are noticeable. In
stimulated cells, there is a marked increase in the amount of IRS-1
translocated to the nucleus, the nucleoli are larger, and some of the
IRS-1 co-localizes with nucleolin in the nucleoli. These observations
were highly reproducible.
Subcellular Fractionation of R+ Cells--
We followed the same
methodology used for R and R/T cells. Fig. 3 shows that a
substantial fraction of IRS-1 is found in the nuclei of R+ cells
stimulated with IGF-I. The nuclear fraction is apparently pure, because
Grb2 is not detectable. There is much PCNA in the cytosol
fraction, which can only be due to leakage. The presence of PCNA in the
cytosol does not invalidate the conclusion that IRS-1 is present in the
nucleus. On the contrary, it suggests that its nuclear localization may
be underestimated by subcellular fractionation. There is a small amount
of IRS-1 in the nuclei of quiescent R+ cells in SFM (the original shows
a detectable albeit very weak band), which is in agreement with the
findings obtained by confocal microscopy. The nuclear presence of
minute amounts of IRS-1 in the nuclei of quiescent R+ cells is
explained in the "Discussion." Also in the "Discussion" will be
given the other controls used to validate the specificity of the
antibodies used to detect IRS-1.
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 3.
Subcellular fractionation of R+ cells.
Cytosol and nuclear fractions were isolated as in Fig. 1 from
unstimulated R+ cells and R+ cells stimulated with IGF-I for 8 h.
The Western blot was subsequently stained for IRS-1 (upper
panel), Grb2 (middle panel), and PCNA
(lower panel). The antibodies used are described under
"Experimental Procedures."
|
|
Localization of IRS-1 in the Nuclei of R v-src Cells--
IRS-1
has also been shown to interact with v-src (34, 39), and we predicted
that IRS-1 would also be translocated to the nuclei in Rv-src
cells. R v-src cells were originally described by Valentinis et
al. (34). In monolayer cultures, they grow in serum-free medium
and form foci in 10% serum. They also form colonies in soft agar,
v-src being one of two among several oncogenes tested that can
transform R cells (reviewed in Ref. 40). Fig. 4 shows a confocal microscopy picture of
R v-src cells in SFM for 48 h. There is staining for IRS-1 in
the nuclei of these cells, although the cytoplasm also stains
(rhodamine). In these cells, nucleolin stains the nucleoli, but also
gives a diffuse staining of the nucleoplasm (FITC). The merged picture
confirms the localization of IRS-1 in the nuclei, and the nucleoli, of
R v-src cells.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
IRS-1 localizes in the nucleus of R/v-src
cells. R/v-src cells (34) are shown in SFM. Confocal microscopy
of cells stained for IRS-1 (rhodamine), nucleolin (FITC), and the
merged picture (C).
|
|
Other Controls for IRS-1--
The nuclear translocation of IRS-1
has been confirmed by using a FLAG antibody to detect an IRS-1
expressing a FLAG epitope. For this purpose, we generated cell lines
from both R and R+ cells transfected with a plasmid in which the
mouse IRS-1 (41) had been tagged with a FLAG epitope (28). In R+
IRS-1/FLAG cells stimulated with IGF-I, the FLAG antibody gave
FLAG-positive nuclei. R cells were also transfected with the
IRS-1/FLAG plasmid. In these cells in SFM, the FLAG-stainable material
was in the cytoplasm and IGF-I did not cause nuclear translocation
(data not shown). The results were the same as in 32D cells transfected
with this construct (28). Other controls include the following. 1) We used three different commercially available antibodies to IRS-1. All of
them gave the same results. 2) One of these antibodies came with the
peptide used for immunization. Competition with this peptide completely
abrogated the staining for IRS-1, both in immunohistochemistry and in
confocal mucroscopy. 3) None of the antibodies used gave a reaction
with cells not expressing IRS-1, such as LNCaP cells (37) and parental
32D cells (36, 38). This was true by confocal microscopy and by Western
blots. The experimental data are not shown, but are available on request.
Time Course of IRS-1 Translocation in R+ Cells--
We have looked
at IRS-1 nuclear translocation in R+ cells at various times after
stimulation with IGF-I (50 ng/ml). The results are shown in Fig.
5A, where, for convenience,
only the merged pictures are given. In all instances, IRS-1 is
predominantly nuclear and especially nucleolar. The nucleoli become
more prominent after IGF-I stimulation, and the increase in nucleolar
size is accompanied by a co-localization of nucleolin and IRS-1 (yellow
staining of nucleoli). The increase in nucleolar size as visualized by
the anti-nucleolin antibody is dramatic, as confirmed in Fig.
5B. Compare for instance the size of the nucleoli in IGF-I
stimulated R+ cells versus the same cells in serum free
medium, or the R cells stimulated with IGF-I.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of IRS-1 translocation to the
nucleus in R+ cells. A, after 48 h in SFM, R+ cells
were stimulated with IGF-I for the indicated times. Compare with the R+
in SFM of Fig. 1. B, size of the nucleoli, as visualized by
an anti-nucleolin antibody. The cells used and the conditions are
indicated above the panels. R+ cells are slightly stimulated even in
serum-free medium (explanation in the text).
|
|
IRS-1 Co-precipitates UBF from Nuclear Extract--
A legitimate
question at this point is whether nuclear IRS-1 shows different
biological effects than cytosolic IRS-1. The confocal microscopy
pictures show strong evidence that IRS-1 localizes not just to the
nuclei, but also to the nucleoli. We have also mentioned above that the
nucleoli markedly increase in size in R+ cells stimulated with IGF-I,
and that IRS-1 often co-localizes with nucleolin in the nucleoli. The
interaction of IRS-1 with the nucleoli prompted us to ask whether
nuclear/nucleolar IRS-1 plays a role in rRNA synthesis and/or
processing. We addressed this question by two different approaches: 1)
interaction (by immuno-coprecipitation) of IRS-1 with nucleolar
proteins and 2) effect of subcellular localization of IRS-1 on rRNA synthesis.
We first asked whether IRS-1 would interact with UBF, a key regulator
of the rDNA promoter, and therefore of rRNA synthesis (29, 30). It
localizes exclusively to the nucleolus and stimulates RNA polymerase I
activity (29). To test the interaction of IRS-1 with UBF, whole-cell
lysates and cytosolic or nuclear fractions of R and R/T cells were
examined. The cells were growing in 10% serum. In Western blots of
whole-cell lysates, UBF was detectable in both R and R/T cells
(Fig. 6). When cytosolic and nuclear fractions of these MEF were immunoprecipitated with an antibody to
IRS-1, UBF was immunoprecipitated in the nuclei but not in the cytosol
of both cell lines (R cells are growing in serum). In R/T cells, an
antibody to IRS-1 co-precipitates both UBF1 and UBF2 (last
lane of Fig. 6), but UBF1 is by far the most abundant. This is an
important finding, as UBF1 is the active form (29). The reverse is also
true, i.e. an antibody to UBF co-precipitates IRS-1,
although this time IRS-1 is detectable only in the nuclei of R/T
cells (lane 2 of lower row). This is probably due
to the lower amount of IRS-1 (and UBF) in the growing R cells. The
purity of the fractions was monitored as usual (data not shown). In
this experiment, we used cells in which IRS-1 was localized to the nuclei. To verify the result, we repeated the experiment on 32D-derived cells. As already mentioned, 32D IGF-IRS1 cells grow exponentially in
IGF-I (42). 32D IGF-IR cells expressing mutant IRS-1 proteins have been
described in a previous paper (28). We have chosen the 32D IGF-IR cells
expressing the 
PTB IRS-1, an IRS-1 protein with a deletion of the
PTB domain (37). In these cells, IRS-1 remains cytoplasmic (28) and the
cells do not survive in IGF-I (40). Fig. 6B shows that UBF
is co-precipitated by an antibody to IRS-1 in 32D IGF-IR IRS-1 cells
(nuclear IRS-1) but not in the cells expressing the PTB
mutant of IRS-1 (cytoplasmic). The lane marked whole
lysates shows that UBF is present in the cells with the mutant
IRS-1, whereas the parental 32D cells are used as the usual control,
because they do not express IRS-1.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
IRS-1 immunoprecipitates UBF from the nuclear
fractions of cells. MEF were used for the experiments in
panel A, either R or R/T cells. UBF seems to
be slightly but consistently higher in lysates of R/T cells.
Cytoplasmic and nuclear fractions were immunoprecipitated with an
anti-IRS-1 antibody. UBF is present only in the nuclear fractions (the
experiments were done in 10% serum, where R cells also proliferate).
The lower row of panel A shows a reverse
experiment on the nuclear fraction, using an antibody to UBF to
immunoprecipitate IRS-1 (left lane R cells, second
lane R/T cells). IRS-1 is detectable in R/T cells (see text).
B, 32D-derived cells were used. 32D IGF-IR/IRS1 has a
wild-type IRS-1, whereas 32D IGF-IRS-1 PTB express an IRS-1 with a
deletion of the PTB domain (see text). UBF is co-precipitated by an
antibody to IRS-1 only in the cells expressing the wild-type IRS-1
(nuclear). The PTB IRS-1 does not translocate to the nucleus and
does not co-precipitate UBF. Parental 32D cells do not express
IRS-1.
|
|
The interaction of IRS-1 with UBF is important for two reasons. It
confirms the nucleolar localization of IRS-1 and places IRS-1 in close
contact with a major regulator of rRNA synthesis. A corollary of this
observation is that nuclear IRS-1 ought to increase rRNA synthesis.
This corollary is supported by three different experiments, described below.
Subcellular Localization of IRS-1 and rRNA Synthesis--
R
cells and R derived cells express substantial amounts of IRS-1. In
the circumstances, we thought it preferable to examine the effect of
IRS-1 on rRNA synthesis in 32D-derived cells. Parental 32D cells do not
express IRS-1 (36, 38). We have previously described two 32D-derived
cell lines, one expressing only IRS-1 (9) and the other expressing
IRS-1 and an increased level of IGF-IR (38). The former cell line,
because of the lower levels of IGF-IR, is not IL-3-independent. After
IL-3 withdrawal and IGF-I supplementation, 32D/IRS-1 cells survive
somewhat longer than parental cells, but eventually die (9). 32D
IGF-IR/IRS-1 cells, instead, are IL-3-independent and even form tumors
in mice (42). Fig. 7, A-C
shows an immunohistochemistry of these two cell lines plus the parental
32D cells. All cells were stained with an antibody to IRS-1 and with
hematoxylin to stain the nuclei. Parental 32D cells, as expected (36,
38), do not stain at all for IRS-1. 32D/IRS-1 cells stain strongly for
IRS-1, but the localization is essentially cytoplasmic. In 32D
IGF-IR/IRS-1 cells, most of the cells have both nuclear and cytoplasmic
IRS-1, with the nuclear localization being predominant (note the
different color of the nuclei, when the cell has little or no IRS-1).
These two cell lines were labeled with 32P at 16 h
after shifting from IL-3 to IGF-I. The incorporation of 32P
into rRNA was determined as detailed under "Experimental
Procedures." Fig. 7D shows rRNA synthesis in 32D/IRS-1 and
32D/IGF-IR/IRS-1 cells. Both cell lines were stimulated with IGF-I for
16 h. At this time (16 h after shifting to IGF-I) both cell lines
survive in the absence of IL-3, but rRNA synthesis is 10 times higher in the cells with nuclear IRS-1 than in the cells with cytosolic IRS-1
(confirmed by counting the radioactivity in the bands).
View larger version (98K):
[in this window]
[in a new window]
|
Fig. 7.
Subcellular localization of IRS-1 and rRNA
synthesis. Upper, immunohistochemistry of 32D-derived cells,
stained with an antibody to IRS-1. All cells were stimulated for
16 h with IGF-I (50 ng/ml). A, 32D cells, negative for
IRS-1. B, 32D/IRS-1 cells. IRS-1 is cytosolic, the nuclei
are stained pale blue. C, 32D IGF-IR/IRS-1 cells.
In most cells, IRS-1 is predominantly nuclear, although some IRS-1 is
clearly visible in the cytosol. D, rRNA synthesis in the
32D/IRS-1 and 32D IGF-IR/IRS-1 cells under the same conditions as in
the upper panel. The incorporation of
32P into RNA was carried out as described in
"Experimental Procedures." Lane 1, 32D/IRS-1 cells;
Lane 2, 32D IGF-IR/IRS-1 cells.
|
|
To confirm that nuclear localization of IRS-1 increases rRNA synthesis,
we carried out another experiment. Rapamycin specifically inhibits mTOR
(43) and, therefore blocks (albeit not always completely) IRS-1
signaling at the level of p70S6K activation (3). In 32D
IGF-IR/IRS-1 cells, rapamycin inhibits transformation and
causes differentiation (42). In 32D IGF-IR/IRS-1 cells treated with
rapamycin, IRS-1 is exclusively localized to the cytosol (Fig.
8B). In this experiment, the
nuclei were stained with propidium iodide (red) and IRS-1
with FITC (green). There is no overlap between the two
stains in the merged picture. Fig. 8A shows that rapamycin
markedly inhibits rRNA synthesis in these cells, thus confirming the
importance of nuclear IRS-1 in increasing rRNA synthesis.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of rapamycin on rRNA synthesis and
cellular localization of IRS-1. A, rRNA synthesis in
32D-derived cells treated with rapamycin. 32D IGF-IR/IRS-1 cells were
used and are described in the text. The determination of rRNA synthesis
was carried out as in Fig. 6. Lane 1, cells
treated with rapamycin; Lane 2, untreated cells.
B, confocal microscopy picture of 32D IGF-IR/IRS-1 cells
treated with rapamycin. Nuclei stained red with propidium
iodide, IRS-1 stained green. The cells are differentiating
into granulocytes, and IRS-1 is found only in the cytosol. There is no
overlapping between the two stains in the merged picture.
|
|
Effect of IRS-1 on rRNA Synthesis--
We then compared two cell
lines that are identical, except that one expresses IRS-1 (32D
IGF-IR/IRS1 cells) and the other does not (32D IGF-IR cells). Both cell
lines grow exponentially in the first 48 h after shifting from
IL-3 to IGF-I, although 32D IGF-IR/IRS-1 cells continue to grow (and
form tumors in animals), whereas 32D IGF-IR cells eventually
differentiate along the granulocytic lineage (38, 42). The previous
experiment showed that IRS-1 is present in the nuclei of 32D
IGF-IR/IRS-1 cells (Fig. 7C). Fig.
9 shows that rRNA synthesis is lower in
32D IGF-IR cells than in 32D IGF-IR/IRS1 cells at both times. When the
bands were counted, rRNA synthesis was increased in 32D IGF-IR/IRS1
cells from 2- to 3-fold relative to 32D IGF-IR cells. The labeling of nascent rRNA was essentially abolished when the cells where treated with 0.05 µg/ml actinomycin, which specifically inhibits rRNA synthesis (Fig. 11A).
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 9.
IRS-1 increases rRNA synthesis. The
synthesis of rRNA was determined by the incorporation of
32P into RNA for 4 h. The cells chosen were 32D IGF-IR
cells (lanes 1), which do not express IRS-1, and
32D IGF-IR/IRS1 cells (lanes 2), which express
IRS-1. The cells were stimulated with IGF-I (50 ng/ml), for either
24 h (upper panel) or 48 h
(lower panel). RNA amounts were monitored with
rRNA (top row of each panel).
|
|
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 10.
Immunoprecipitation of nucleolin by an
anti-IRS-1 antibody. Lysates were prepared from LNCaP/IRS-1 cells
(see text) after fractionation into a nuclear and a cytosolic fraction.
The first two lanes are Western blots of the fractions (C
for cytosol, N for nuclear). The amounts of nucleolin are
roughly similar in the two fractions. All other lanes are blots of
immunoprecipitates, using an antibody to IRS-1 to co-precipitate
nucleolin. Nucleolin is immunoprecipitated only in the nuclear
fraction. Different growth conditions (fetal bovine serum, serum-free
medium, or IGF-I) gave the same results. The lower
panel shows that IRS-1 was immunoprecipitated in both
fractions, although in two conditions, IRS-1 is higher in the nuclear
than in the cytosolic fraction. For the lysates, we used 40 µg of
protein (5 µg for IRS-1), and for the immunoprecipitates, 400 µg of
protein.
|
|
Co-precipitation of Nucleolin and IRS-1 Is Limited to the
Nuclei--
We have already mentioned that IRS-1 and nucleolin
interact with each other (10). We have asked whether subcellular
localization of IRS-1 plays a role in their interaction, as it seems to
do with the SV40 T antigen and UBF. For this purpose, we used
LNCaP/IRS-1 cells (37). These are human prostate-cancer cells stably
transfected with a plasmid expressing IRS-1 (the parental cells do not
express IRS-1). We selected these cells because they express unusually high amounts of nucleolin (roughly10-15 times the levels in MEF), thus
making it easier to detect.
After subcellular fractionation, both the cytosol and the nuclear
fractions were monitored by Western blot for the presence of nucleolin.
Fig. 10 shows that nucleolin is
abundant in these cells and is present in roughly equal amounts in the
cytosolic and nuclear fractions (the purity of the fractions was
monitored as in Fig. 1). When the two fractions were immunoprecipitated with an antibody to IRS-1, nucleolin was found only in the
immunoprecipitate from the nuclei, although IRS-1 was found in both
fractions. This experiment was repeated several times, both with this
cell line and another LNCaP cell line in which, besides IRS-1, IGF-IR
expression was increased (37). In all instances, and regardless of
growth conditions, nucleolin was immunoprecipiated by an anti-IRS-1
antibody only in the nuclear fraction. It should be noted that LNCaP
cells (parental or derived) were grow in serum-free medium, although IGF-I partially increases their growth (37).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of Actinomycin D on rRNA synthesis
and UBF in 32D IGF-IR IRS-1 cells. The concentration of
actinomycin D was 0.05 µg/ml. Ribosomal RNA synthesis was determined
as in the previous figures in cells growing exponentially or in cells
treated for 24 h with actinomycin D (panel A). The
bands from autoradiography were counted in a liquid scintillation
counter. In panel B, aliquots of the same cells
treated in similar manner were stained for UBF before or after
treatment with actinomycin D. UBF is detectable in the exponentially
growing cells, but not in the cells treated with actinomycin D.
|
|
Effect of Actinomycin D on rRNA synthesis and UBF in 32D IGF-IR
IRS-1 Cells--
Actinomycin D, at very low concentrations, inhibits
almost exclusively rRNA synthesis (48), a finding confirmed in 32D
IGF-IR IRS-1 cells (Fig. 11A). The effect is dramatic, as
incorporation of 32P decreases to a level less than 1% of
untreated cells. Fig. 11B shows that at the same time, UBF
is no longer detectable in the treated cells, whereas it is clearly
visible in the untreated cells. Thus, IRS-1 has no effect on rRNA
synthesis in cells, where UBF has been decreased by treatment with
Actinomycin D.
| |
DISCUSSION |
Our data confirm and extend the results of Lassak et
al. (27) and Prisco et al. (28) that IRS-1 can
translocate to the nuclei of cells. New findings in this communication
include: 1) IRS-1 translocates not only to the nuclei but especially to
the nucleoli of R+ cells stimulated with IGF-I; 2) nuclear/nucleolar translocation is confirmed in different cell lines, like R+ and R/v-src cells; 3) more important, we find that nuclear IRS-1 immunoprecipitates at least two nucleolar proteins, UBF and nucleolin. The finding with UBF is especially significant, since this protein resides exclusively in the nucleolus, and an interaction with IRS-1 can
only occur in the nucleolus, or, at the most, the nucleus; 4)
nuclear/nucleolar translocation of IRS-1 correlates with an increase in
rRNA synthesis. The two most important questions raised by our
observations are the evidence for nuclear translocation and its
biological significance.
Most important for us is to determine the biological significance of
IRS-1 nuclear translocation. In other words, what does nuclear IRS-1 do
different from cytoplasmic IRS-1? A clue to a biological function of
nuclear IRS-1 is its preferential localization to the nucleoli and the
accompanying dramatic increase in nucleolar size. The increase in
nucleolar size does not occur in R cells stimulated by IGF-I. The
nucleolus is the site of ribosomal RNA synthesis, which, in turn,
depends on the activity of RNA polymerase I (29). The activity of the
rDNA promoter and its polymerase is regulated by a few proteins, among
which is prominent is the role of UBF (29, 30). It is therefore
intriguing that IRS-1 in the nucleus co-precipitates UBF; the
appropriate controls indicate that this interaction does occur.
Interestingly, IRS-1 immunoprecipitates largely the active form of UBF
(29), UBF1. IRS-1 also immunoprecipitates nucleolin, and only in
nuclear extract. However, the interaction with UBF1 is the most
significant. It confirms the nucleolar localization of IRS-1 (there is
no UBF in the cytosol), which is further supported by the finding that
a mutant IRS-1 that does not translocate to the nucleus (28) also fails
to co-precipitate UBF. This result suggests a role of nuclear IRS-1 in
rRNA synthesis. In agreement with this suggestion, we show a
correlation between nuclear/nucleolar localization of IRS-1 and
increased rRNA synthesis compared with cells with cytosolic IRS-1 or
without IRS-1. Thus, there is a substantial difference in rRNA
synthesis between 32D IRS-1 (cytoplasmic IRS-1) and 32D IGF-IR/IRS-1
cells (nuclear IRS-1). Second, in rapamycin-treated 32D IGF-IR/IRS-1
cells, the cytosolic localization of IRS-1 is accompanied by a marked
reduction in rRNA synthesis with respect to untreated cells. Rapamycin
inhibits p70S6K and causes the differentiation of 32D
IGF-IR/IRS-1 cells (42). TOR proteins, which are inhibited by
rapamycin, are known to stimulate rRNA synthesis and rRNA processing
(reviewed in Ref. 43). Our findings are in agreement with the fact that
rRNA synthesis decreases in differentiated cells (44) and that the
nucleolus actually disappears in terminally differentiated cells. The
chick erythrocyte nucleus is the best example of nucleolar involution
in differentiated cells (45). Finally, rRNA synthesis is much lower in
32D IGF-IR cells that do not express IRS-1 than in 32D IGF-IR/IRS-1
cells, where IRS-1 is in the nucleus. Interference with UBF inhibits the effect of IRS-1 on rRNA synthesis (Fig. 11). We therefore suggest that nuclear IRS-1 plays an important role in increasing rRNA synthesis
and that it does so by interacting with the regulator of RNA polymerase
I activity, UBF1.
Increased rRNA synthesis results in increased cell size. Cell size is
usually determined by the amount of protein/cell (46, 47), which in
turn requires an increase in the amount of rRNA (47, 48). An increased
synthesis of rRNA implies a larger number of ribosomes, hence more
protein synthesis and an enlargement of cells (48). Indeed, in murine
hemopoietic 32D cells, we have shown that cells expressing IRS-1 are
larger than 32D cells not expressing IRS-1, even when both cell types
are growing exponentially (42). Interestingly, the size of 32D
IGF-IR/IRS1 cells decreases when the cells are treated with rapamycin.
The importance of IRS-1 and its downstream signaling on cell size
in vivo are also supported by several reports in the
literature. Mice with deleted IRS-1 (49) or p70S6K (50)
genes are smaller than their wild-type littermates. But the importance
of IRS-1 and p70S6K in cell-size regulation was rigorously
demonstrated by the observations that homologues of either IRS-1 (51)
or the S6 kinase (52) regulate cell size in Drosophila.
Clearly, IRS-1 cannot be an absolute requirement for rRNA synthesis and
cell size; otherwise 32D cells would not grow in IL-3. Indeed, deletion
of IRS-1 or chico in Drosophila or mice results in
animals that are smaller, but viable. Our studies offer for the first
time a molecular explanation. Even in cells without IRS-1, there is
rRNA synthesis, but a nuclear IRS-1 increases it considerably,
presumably by interacting with UBF1. In other words, in animals with
compromised IRS-1 signaling, body size is reduced, indicating that
IRS-1 does indeed contribute an additional (and not redundant) stimulus
to growth in size. On the basis of our results, we suggest that
nuclear/nucleolar IRS-1 is the important contributor to cell growth.
Nuclear translocation of IRS-1 is now rigorously demonstrated. The
evidence from this and previous papers (27, 28) can be summarized as
follows. 1) The IRS-1 antibodies show nuclear localization in R cells
expressing T antigen or v-src that are known to interact with IRS-1.
Parental R cells are negative for nuclear IRS-1. 2) Nuclear
localization is increased in R+ cells after stimulation with IGF-I.
IRS-1 is not detectable in the nuclei of R+ cells stimulated with
epidermal growth factor (data not shown) or in R cells, regardless of
growth conditions. 3) The anti-IRS-1 antibodies failed to give a
positive stain with two cell lines (LNCaP and parental 32D cells) in
which IRS-1 is not expressed (36, 37). 4) Translocation into the nuclei
is also supported by the use of an IRS-1 with a FLAG tag (Ref. 28, and this paper). The FLAG antibody fails to stain cells that do not express
the IRS-1/FLAG construct. In R cells expressing this construct, FLAG
is found essentially in the cytoplasmic fraction. Only in R+ cells
stimulated with IGF-I is FLAG-tagged material detectable in the nuclei.
5) Controls with different antibodies and competing peptides confirm
that the protein detected in Western blots, in confocal microscopy and
in immunohistochemistry is bona fide IRS-1. R+
cells show some IRS-1 staining even in SFM (see Fig. 1). R+ cells,
however, are known to secrete small amounts of IGF-I, and their IRS-1
is tyrosyl-phosphorylated (albeit weakly) even when the cells are in
SFM (34).
There is a discrepancy between subcellular fractionation and confocal
microscopy in terms of the fraction of IRS-1 translocated to the
nuclei. For instance, in R/T cells, it would seem, from confocal
microscopy, that all or most of IRS-1 is in the nucleus. There are
specks of material stained with the IRS-1 antibody outside the nuclei,
but much too little in comparison to the amount revealed by subcellular
fractionation. Our explanation is leakage from the nuclei during
fractionation. It is true that the T antigen is recovered only in the
nuclear fraction, but PCNA is not. PCNA is a nuclear protein, and yet
it is recovered in equal amounts in both fractions. Perhaps some
proteins leak out of the nuclei more than others, and IRS-1 could be
one of them. A second possibility is that IRS-1 could be present in the
nucleus in the form of cleavage products, as in the case of ErbB-4
(24). However, in our subcellular fractionation studies, the size of
IRS-1 was full length (see Figs. 1 and 2), and no other bands were
detected, even though some smaller bands could be detected in the
cytosol. Although we cannot exclude the presence of small amounts of
nuclear IRS-1 even in R cells, the general evidence is that under
certain circumstances, IRS-1 can translocate to the nuclei. We suggest
that, in this case, confocal microscopy may be more reliable than
subcellular fractionation.
It has recently been reported that IRS-3 (23) translocates to the
nucleus. In this paper, the authors reported that IRS-1 did not
translocate to the nucleus. The discrepancy between the data of Kabuta
et al. (23) and those of Lassak et al. (27), Prisco et al. (28), and ours is probably due to the
different cells used.
Finally, the co-localization of IRS-1 and nucleolin is in agreement
with the reported interaction of IRS-1 with nucleolin (10). Nucleolin
plays a role in rRNA processing (53). We have shown in this paper that
IRS-1 co-precipitates nucleolin in the nuclei, but not in the cytosol,
despite the fact that in LNCaP cells, nucleolin is abundant in both
fractions. The biological significance of the IRS-1/nucleolin
interaction is less obvious than the UBF/IRS-1 interaction. However, we
find it interesting that their close interaction may be limited to the
nuclear environment.
In conclusion, our data give rigorous evidence that IRS-1 can be
translocated to the nuclei of mouse embryo fibroblasts by two oncogenes
and by stimulation of the wild-type IGF-IR with IGF-I. From these
experiments, it is reasonable to hypothesize a role of nuclear IRS-1 in
rRNA synthesis as indicated by its localization in the nucleoli, its
interaction with UBF1 in the nuclear fractions, and its correlation to
increased rRNA synthesis. Finally, it is unlikely that nuclear
translocation of IRS-1 is only an artifact of tissue cultures, as
nuclear IRS-1 has been reported in tissue sections from human breast
cancers (54) and human medulloblastomas (27).
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Thomas Jefferson
University, 233 S. 10th St., 624 BLSB, Philadelphia, PA 19107. Tel.:
215-503-4507; Fax: 215-923-0249; E-mail: R Baserga@jci.tju.edu.
