(Received for publication, April 14, 1995; and in revised form, September 15, 1995)
From the
Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.
Acidic fibroblast growth factor (aFGF), ()which
promotes cell differentiation and proliferation (Burgess and Maciag,
1989; Basilico and Moscatelli, 1992), binds to transmembrane receptors
containing a cytoplasmic tyrosine kinase domain that is activated upon
binding (Johnson and Williams, 1993; Ruta et al., 1989; Mason,
1994). It has remained controversial if aFGF transmits signal only
through the receptor or if the growth factor is also translocated into
the cells and to the nucleus to stimulate DNA synthesis (Imamura et
al., 1990; Cao et al., 1993). We recently demonstrated
that aFGF fused to diphtheria toxin A fragment could be translocated
(``microinjected'') by the diphtheria toxin pathway to the
cytosol and to the nucleus and stimulate DNA synthesis in cells lacking
functional aFGF receptors (Wied
ocha et al., 1994). Clearly, therefore, intracellular growth factor
can convey a biological signal.
Morphological and biochemical
evidence for nuclear targeting of externally added aFGF and certain
other growth factors has been provided by several groups (Imamura et al., 1990; Baldin et al., 1990; Kimura, 1994;
Wiedocha et al., 1994; Moroianu
and Riordan, 1994; Imamura et al., 1994). A major problem in
such studies is to demonstrate that the protein has really crossed
cellular membranes to gain access to the cytosol or to the nucleoplasm.
Alternatively, it could be present in intracellular vesicles or
cisternal compartments that may have a juxtanuclear location (Prudovsky et al., 1994), or it could be trapped in cytoskeletal material
adhering to the nucleus upon disrupting or dissolving the cells.
To test for translocation in a more rigorous manner, we have developed a novel principle to study whether or not a protein is exposed to the cytosol. We added to aFGF a C-terminal CAAX (C = Cys, A = an aliphatic amino acid, and X = any amino acid) motif, which, upon exposure to the cytosol, will be modified by attachment of a prenyl group followed by proteolytic removal of the last three amino acids and carboxyl methylation of the appearing C-terminal cysteine (Clarke, 1992; Cox and Der, 1992). Prenylating enzymes have been observed only in the cytosol (Reiss et al., 1990; Schaber et al., 1990; Casey et al., 1991) and possibly in the nucleus (Lutz et al., 1992; Sinensky et al., 1994), and there is no indication that prenylation occurs at the cell surface or inside compartments of the endocytic pathway. In the present study, we have found that externally added CAAX-tagged aFGF is efficiently farnesylated by cells, indicating that translocation of the growth factor to the cytosol and to the nucleus does indeed take place.
pTrc-dtA-cax in E. coli DH5 was induced as
above, and the bacteria were harvested, washed once with PBS (10 mM sodium phosphate, pH 7.4, 0.14 M NaCl), resuspended in
PBS, and sonicated. After centrifugation, the clear supernatant was
chromatographed on a Sephacryl S-100HR column equilibrated with 20
mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM dithiothreitol. Fractions containing dtA-CAAX were
collected, dialyzed against 20 mM phosphate buffer, pH 6.0, 1
mM dithiothreitol, and chromatographed on a Q-cartridge
(Bio-Rad) with a linear NaCl gradient (0-1 M) in the
same buffer.
To study partitioning of proteins from cells, the cells were lysed directly in the Triton/PBS mixture in the presence of 1 mM phenylmethylsulfonyl fluoride, and, after removal of the nuclei by centrifugation, the procedure for phase separation described above was followed.
Figure 1:
Ability of aFGF-CAAX to be
farnesylated in vitro. aFGF-CAAX (lanes
2-5) and aFGF (lanes 6 and 7) were
incubated in a reticulocyte lysate in the presence of
[H]farnesyl pyrophosphate in the absence and
presence of microsomes and 50 µM B581. The samples were
then immunoprecipitated with immobilized anti-aFGF antibodies and
analyzed by SDS-PAGE and fluorography. In lane 1 is shown
non-farnesylated [
S]methionine-labeled
aFGF-CAAX for comparison.
To test the biological
activity of aFGF-CAAX, we measured its ability to bind to and
stimulate DNA synthesis in NIH3T3 cells, which express functional FGF
receptors (Imamura et al., 1990). Scatchard analysis of
binding data obtained in the presence of 10 units/ml heparin showed
that NIH3T3 cells contain 74,000 binding sites for aFGF with a K of 150 pM (data not shown). As shown in Fig. 2A, aFGF and aFGF-CAAX were equally
efficient in competing out the binding of
I-aFGF to the
cells. Furthermore, aFGF-CAAX, like aFGF, stimulated DNA
synthesis in serum-starved NIH3T3 cells in a dose-dependent manner,
both in the absence and presence of heparin (Fig. 2B).
Although aFGF-CAAX was less active than aFGF at low
concentrations, it appears that the addition of a C-terminal CAAX motif does not seriously impair the biological functions of the
growth factor.
Figure 2:
Ability of aFGF-CAAX to bind to
and stimulate DNA synthesis in NIH3T3 cells. A, to measure
binding, 5 ng/ml I-aFGF and increasing concentrations of
unlabeled aFGF or aFGF-CAAX in the presence of 10 units/ml
heparin were incubated for 2 h at 4 °C with cells growing as
monolayers in 24-well microtiter plates at a density of 5
10
cells/well. The cells were washed, and the bound
radioactivity was measured. B, to measure
[
H]thymidine incorporation, cells were
preincubated for 48 h in serum-free medium at 37 °C; then,
increasing amounts of aFGF or aFGF-CAAX were added with and
without 10 units/ml heparin, and the incubation was continued for 24 h
at 37 °C. During the last 6 h, the cells were incubated with 1
µCi/ml [
H]thymidine, and the incorporated
radioactivity was measured.
Figure 3:
Farnesylation of aFGF-CAAX in
NIH3T3 cells. A, serum-starved cells were incubated with 10
units/ml heparin and 100 ng/ml aFGF or aFGF-CAAX overnight in
the presence of lovastatin and labeled mevalonic acid. The cells were
lysed, and the postnuclear supernatant was divided into three parts.
One part was treated with anti-aFGF adsorbed to protein A-Sepharose,
and one part was treated with heparin-Sepharose, which was subsequently
washed with 0.7 M NaCl. The third part was precipitated with
5% trichloroacetic acid. The precipitated material was analyzed by
SDS-PAGE and fluorography. The position of small GTP-binding proteins (Small G) and of farnesylated aFGF-CAAX (asterisk) are indicated. B, increasing amounts
of aFGF or aFGF-CAAX were added to 2 10
serum-starved cells in the absence and presence of 50 µM B581. The cells were incubated overnight and lysed. Growth factor
in the cytoplasmic fraction and in material extracted with 0.5 M NaCl from the sonicated nuclei was precipitated with
heparin-Sepharose (upper panel) or with anti-aFGF-protein
A-Sepharose (lower panel). In all cases, the material was
analyzed by SDS-PAGE and fluorography.
To compare the labeling of aFGF-CAAX with that of endogenous prenylated proteins, we analyzed the total trichloroacetic acid-precipitable material in the cytoplasmic fraction. The results (lane 6) showed a labeled band migrating as expected for prenylated aFGF-CAAX (position indicated with an asterisk). There was no band at the same position in cells treated with aFGF (lane 7). The labeling extent of externally added aFGF-CAAX was comparable to that of prenylated endogenous proteins in the cells, such as the small G proteins (lane 6). There was no labeling when aFGF-CAAX was added to lysed cells on ice, excluding the possibility that the labeling seen in Fig. 3A could have occurred after lysis of the cells (data not shown).
We and others (Zhan et
al., 1992; Wiedocha et al.,
1994) have earlier found that when NIH3T3 cells are incubated with
labeled aFGF, part of the growth factor is recovered in the nuclear
fraction. In the experiments in Fig. 3B, we therefore
analyzed both the cytoplasmic and the nuclear fractions for material
adsorbing to heparin-Sepharose and to immobilized anti-aFGF. When cells
were incubated with aFGF in the presence of labeled mevalonate, as
expected no labeled growth factor was precipitated with
heparin-Sepharose (upper panel) or with immobilized anti-aFGF (lower panel) either from the cytoplasmic (lanes 2 and 3) or from the nuclear fractions (lanes 8 and 9). When aFGF-CAAX was used instead, labeled
growth factor was precipitated from both fractions, both with
immobilized heparin and immobilized anti-aFGF (lanes 4, 5 and 10, 11). The relative amount of growth
factor in cytoplasm and nucleus varied somewhat between experiments. In
the presence of the farnesylation inhibitor B581, no labeling was
observed (lanes 6 and 12). The results indicate that
externally added aFGF-CAAX is indeed farnesylated by the cells
and that part of the modified protein migrates to the nucleus.
Figure 4:
Requirement of specific FGF receptors for
farnesylation of aFGF-CAAX. A, serum-starved NIH3T3
cells were incubated overnight in serum-free medium containing
lovastatin and labeled mevalonic acid. Then, aFGF-CAAX (10
ng/ml) was added in the absence and presence of 1 µg/ml aFGF, and
the incubation was continued for 24 h more. The cells were then lysed,
and the cytoplasmic fraction and material extracted with 0.5 M NaCl from the sonicated nuclei was mixed and submitted to
immunoprecipitation with anti-aFGF-protein A-Sepharose. B,
NIH3T3 (lanes 1-9) and U2OS Dr1 cells (lanes
10-13) were incubated overnight with lovastatin and labeled
mevalonic acid as above. Then, 10 units/ml heparin and increasing
amounts of aFGF-CAAX, dtA-CAAX, or dtA were added.
When indicated, the cells were exposed to the process of scrape loading
and, without change in medium, incubated further overnight. Finally,
the cells were lysed and treated as above and immunoprecipitated with
anti-aFGF (lanes 1-4 and 10-14) or
anti-diphtheria toxin adsorbed to protein A-Sepharose (lanes
5-9). The immunoprecipitated material was analyzed by
SDS-PAGE and fluorography. Lane 14, aFGF-CAAX labeled in vitro with [H]farnesyl pyrophosphate
in the presence of microsomes.
The possibility existed that the
labeling obtained was derived from a subpopulation of cells that,
although gently treated, could have been damaged but not killed, and
that the growth factor could have entered the cells by a mechanism
similar to that in scrape loading (Frankel and Pabo, 1988; Leevers and
Marshall, 1992; Morris et al., 1993). To test this, we first
measured the ability of NIH3T3 cells to modify a protein that does not
bind to the cells, viz. diphtheria toxin A fragment with a
C-terminal CAAX motif (dtA-CAAX). This protein is
efficiently farnesylated in vitro (data not shown). The cells
were incubated with increasing concentrations of dtA-CAAX and
subjected to the process of scrape loading. A faint 20-kDa band was
observed when 100 µg/ml of the construct had been present in the
medium (Fig. 4B, lane 8). This is 10 times the concentration required to detect labeled aFGF-CAAX in these cells (lane 2).
U2OS Dr1 cells lack specific
aFGF receptors, but the growth factor binds extensively and with high
affinity to surface heparans on the cells (Sakaguchi et al.,
1991; Wiedocha et al., 1994). In
these cells farnesylation of the growth factor was observed only after
exposure to 100 µg/ml aFGF-CAAX and scrape loading (Fig. 4B, lanes 10-13). These data
indicate that the farnesylation of aFGF-CAAX in lanes
2-4 was not due to ``leakage'' of the growth
factor into wounded cells, and they support the finding above that
specific FGF receptors are required for translocation.
When the U2OS Dr1 cells were transfected with FGF receptor 4 (FGFR4), they bound aFGF specifically in the sense that excess unlabeled aFGF in the presence of heparin prevented the binding (Fig. 5A). The untransfected cells bound very little aFGF in the presence of heparin. When aFGF-CAAX was added to these cells in the presence of labeled mevalonic acid, a strong band corresponding to the growth factor appeared (Fig. 5B, lane 2), which was absent in the untransfected cells (lane 1). In the presence of excess unlabeled growth factor, only a weak band appeared (lane 3). Clearly, farnesylation of externally added aFGF-CAAX was dependent upon binding to the specific receptor in U2OS Dr1 R4 cells.
Figure 5:
Farnesylation of aFGF-CAAX in
U2OS Dr1 cells transfected with FGFR4. A, to untransfected and
FGFR4-transfected U2OS Dr1 cells growing in 24-well microtiter plates,
which had been coated with gelatine, were added increasing amounts of I-aFGF (specific activity, 15,000 counts/min/ng) in the
presence of 10 units/ml heparin. After 4 h at 4 °C, the cells were
washed and dissolved in 0.5 M NaOH, and the cell-associated
radioactivity was measured. B, serum-starved untransfected and
transfected U2OS Dr1 cells were incubated overnight in serum-free
medium containing lovastatin and labeled mevalonic acid. Then,
aFGF-CAAX (100 ng/ml) was added in the absence and presence of
1 µg/ml unlabeled aFGF, as indicated, and the incubation was
continued for further 24 h. The cells were then lysed, and the
cytoplasmic fraction and material extracted with 0.5 M NaCl
from the sonicated nuclei was mixed and submitted to
immunoprecipitation with anti-aFGF-protein
A-Sepharose.
Figure 6:
Ability of aFGF-CAAX farnesylated in vitro and in vivo to partition into Triton X-114. A, aFGF-CAAX was synthesized in a rabbit reticulocyte
lysate in the presence of [S]methionine and then
incubated with farnesyl pyrophosphate under conditions as in Fig. 1, in the absence and presence of 50 µM B581.
2 µl were subjected to Triton X-114 partitioning. The water phase (W) and the detergent phase (D) were treated with
anti-aFGF adsorbed to protein A-Sepharose, and the immunoprecipitated
material was analyzed by SDS-PAGE and fluorography. B, NIH3T3
cells were incubated overnight with 10 units/ml heparin and 5 ng/ml
[
S]methionine-labeled aFGF (lanes 1 and 2) or aFGF-CAAX in the absence (lanes 3 and 4) and presence of B581 (lanes 5 and 6). The
cells were lysed, and the cytoplasmic fraction and material extracted
with 0.5 M NaCl from the sonicated nuclei were mixed and
submitted to Triton X-114 partitioning. Then, the water phase and the
detergent phase were treated with heparin-Sepharose, and the adsorbed
material was analyzed by SDS-PAGE and
fluorography.
When
NIH3T3 cells were incubated with
[S]methionine-labeled aFGF and then treated with
Triton X-114, all the labeled protein partitioned into the water phase (Fig. 6B, lanes 1 and, 2). On the
other hand, when aFGF-CAAX was used, part of the protein
partitioned into the detergent phase (lanes 3 and 4).
This was not the case when the cells had been incubated with labeled
aFGF-CAAX in the presence of the farnesylation inhibitor B581 (lanes 5 and 6). Densitometric analysis indicated
that approximately one-third of the cell-associated aFGF-CAAX was present in the detergent phase and therefore farnesylated.
Experiments with I-aFGF-CAAX indicated that
200,000 molecules were associated with each cell after overnight
incubation under conditions as in Fig. 6B. Since not
all aFGF-CAAX molecules that enter the cytosol may be
farnesylated (see ``Discussion''), this indicates that at
least 70,000 molecules of aFGF-CAAX per cell entered the
cytosol.
Figure 7: Time course of labeling of aFGF-CAAX after addition to cells. Serum-starved U205R4 cells were incubated with 100 ng/ml aFGF-CAAX and 10 units/ml heparin for the indicated periods of time in the presence of lovastatin and labeled mevalonic acid. The cells were lysed, and the postnuclear supernatant and material extracted from the nuclei with 0.5 M NaCl was treated with heparin-Sepharose, which was subsequently washed with 0.7 M NaCl. The adsorbed material was analyzed by SDS-PAGE and fluorography.
The main conclusion from the present work is that externally
added aFGF-CAAX (and therefore also aFGF) is able to cross
cellular membranes to gain access to the cytosol and/or the
nucleoplasm. FGF receptors appear to be required for this, as
aFGF-CAAX was not farnesylated when added to U2OS Dr1, a human
osteosarcoma cell line, which does not express measurable amounts of
functional FGF receptors and which does not respond to addition of aFGF
with increased growth or with increased thymidine incorporation
(Wiedocha et al., 1994). While
these cells bind aFGF extensively in the absence of heparin, no binding
was detected in the presence of heparin
(Wied
ocha et al., 1994),
indicating that binding occurs only to surface heparans. The data
therefore indicate that binding to specific FGF receptors is required
for translocation to the cytosol.
Scrape loading has been used to
introduce exogenous proteins into the cytosol, such as the Tat-encoded
protein from human immunodeficiency virus (Frankel and Pabo, 1988),
human papillomavirus E7 protein (Morris et al., 1993), and
p21 (Leevers and Marshall, 1992). In these cases,
extracellular protein concentrations in the range of 0.1-1 mg/ml
were required to obtain measurable biological effects. This is similar
to the concentration of aFGF-CAAX here found to be required to
detect farnesylation in cells lacking FGF receptors, as well as of
dtA-CAAX, which does not bind to cells. We therefore consider
it unlikely that the farnesylation detectable in NIH3T3 cells at 10
ng/ml of extracellular aFGF-CAAX is due to growth factor that
enters through occasional and transient membrane defects in the cells.
The Triton X-114 partitioning experiments indicated that about
one-third of the total cell-associated aFGF-CAAX is
farnesylated, which may amount to 70,000 molecules per cell. The
rest may either still be bound to cell surface receptors or it could be
located in endocytic vesicles and other membrane-bounded compartments.
Also, some of the CAAX-tagged growth factor present in the
cytosol or in the nucleus may for some reason not be farnesylated. If
farnesylation of aFGF-CAAX can only occur in the cytosol,
transport of the unmodified growth factor to the nucleus could prevent
farnesylation from reaching completion. Our recent data demonstrate
that farnesylation of CAAX-modified diphtheria toxin A
fragment is completed only 1 h after translocation to the cytosol
(Falnes et al., 1995).
Although farnesylation can only be
taken as evidence for transport to the cytosol, once present in the
cytosol the growth factor can migrate into the nucleus (Imamura et
al., 1992; Cao and Petterson, 1993). Also, the farnesylated growth
factor is partly found in the nucleus. In the experiment shown in Fig. 3B, the major part is in the cytoplasmic fraction,
whereas in other experiments more was found in the nuclear fraction.
The reason for this variation is not clear. The ability of aFGF to
locate to the nucleus has been found to depend upon the presence of a
positively charged sequence near the N terminus, and removal of this
sequence rendered the growth factor unable to stimulate DNA synthesis
but not to bind to and activate the specific FGF receptors (Imamura et al., 1990; Wiedocha et
al., 1994). Unmodified aFGF could migrate more efficiently into
the nucleus than the farnesylated growth factor, which may adhere to
cellular membranes by its hydrophobic tail. It should be noted,
however, that a secondary signal, such as a polybasic motif or
palmitoylation, in addition to prenylation is usually required to
confer membrane association of the protein (Hancock et al.,
1991a).
The use of a CAAX tag to study translocation of a protein to the cytosol should be useful for many purposes. We have recently shown that dtA-CAAX reconstituted with diphtheria toxin B fragment becomes farnesylated upon translocation to the cytosol by the toxin pathway and that the appearing modification can be used to monitor toxin entry under various conditions (Falnes et al., 1995). We are currently studying if CAAX-tagged growth factors other than aFGF become farnesylated when added to cells.
aFGF is synthesized as a cytoplasmic protein without a signal sequence, and it is not clear how it is transported out of the cells (Muesch et al., 1990; Jackson et al., 1995). The present finding that it also enters into the cytosol when added to the medium suggests that there must be transport mechanisms in both directions. The nature of these mechanisms is not known, but it is tempting to speculate that proteins involved are related to proteins of the ABC transporter family, such as the multidrug resistance protein (Endicott and Ling, 1989; Sharma et al., 1992), the peptide-transporting Tap proteins in the endoplasmic reticulum (Roelse et al., 1994), and to the product of the STE6 gene, transporting a-factor in Saccharomyces cerevisiae (McGrath and Varshavsky, 1989).
The location of the transport in the cells is also not known. It could occur at the level of the cell surface membrane or across membranes of intracellular vesicles. It has recently been demonstrated that binding of aFGF induces translocation of the receptor from the cell surface to a juxtanuclear location (Prudovsky et al., 1994), which could be the site where membrane translocation of the growth factor takes place. Certain protein toxins, such as diphtheria toxin, ricin, Shigella toxin, and others (Olsnes et al., 1988; Olsnes and Sandvig, 1988), are able to cross membranes of intracellular vesicles to gain access to the cytosol. In some cases, such as diphtheria toxin, the receptor-binding B fragment of the toxin facilitates translocation of the A fragment into the cytosol. However, this may not be the case with all toxins, and in the case of ricin and Shigella toxin there is no evidence that the receptor binding subunits have a function beyond binding to the relevant receptors. As we have earlier speculated (Olsnes et al., 1974), the toxins may take advantage of transport mechanisms developed by the cells for other purposes, such as transport of certain growth factors.