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Physiol Rev
83: 163–182, 2003; 10.1152/physrev.00021.2002.
Transport of Exogenous Growth Factors and Cytokines
to the Cytosol and to the Nucleus
SJUR OLSNES, OLAV KLINGENBERG, AND ANTONI WIE˛DŁOCHA
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway
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I. Introduction
II. Translocation Mechanisms Used by Protein Toxins
A. Toxins that carry their own translocation apparatus
B. Toxins employing cellular mechanisms for entry
III. Problems in Demonstrating Translocation of Proteins From the Exterior to the Cytosol
and to the Nucleus
IV. New Approaches to Assess the Presence in the Cytosol and in the Nucleus of Externally
Added Proteins
V. External Proteins That Have Been Reported to Enter the Cytosol and Nucleus
A. aFGF
B. bFGF
C. HIV-Tat
D. Interferon-␥
E. Other external proteins that may enter the cytosol and nucleus
VI. Transport of Transmembrane Receptors to the Nucleus
VII. Concluding Remarks
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Olsnes, Sjur, Olav Klingenberg, and Antoni Wie˛dłocha. Transport of Exogenous Growth Factors and Cytokines
to the Cytosol and to the Nucleus. Physiol Rev 83: 163–182, 2003; 10.1152/physrev.00021.2002.—In recent years a
number of growth factors, cytokines, protein hormones, and other proteins have been found in the nucleus after
having been added externally to cells. This review evaluates the evidence that translocation takes place and
discusses possible mechanisms. As a demonstration of the principle that extracellular proteins can penetrate cellular
membranes and reach the cytosol, a brief overview of the penetration mechanism of protein toxins with intracellular
sites of action is given. Then problems and pitfalls in attempts to demonstrate the presence of proteins in the cytosol
and in the nucleus as opposed to intracellular vesicular compartments are discussed, and some new approaches to
study this are described. A detailed overview of the evidence for translocation of fibroblast growth factor, HIV-Tat,
interferon-␥, and other proteins where there is evidence for intracellular action is given, and translocation mechanisms are discussed. It is concluded that although there are many pitfalls, the bulk of the experiments indicate that
certain proteins are indeed able to enter the cytosol and nucleus. Possible roles of the internalized proteins are
discussed.
I. INTRODUCTION
Protein hormones, growth factors, and cytokines
bind to receptors at the cell surface and act by transmitting a signal to the interior of the cell. The receptors are
most often transmembrane proteins, and the signaling
often implies the activation of an enzymatic activity carried by the intracellular domain of the receptor protein as
such, or by a protein that is associated with the receptor
or that becomes associated with it upon binding of the
ligand to the extracellular part of the receptor. These
processes have been worked out in great detail and are
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not reviewed here. It is generally assumed that the protein
hormones and growth factors stay at the cell exterior or
that they are taken up by endocytosis and may continue to
signal for a while until they are eventually degraded (192).
In both cases it is assumed that the hormones, cytokines,
and growth factors are separated from the cytosol by a
membrane. Steroid hormones, thyroid hormones, retinoic
acid, and other lipophilic compounds are able to penetrate cellular membranes and act by binding to proteins in
the cytosol or in the nucleus, resulting in gene regulation.
The two classes of hormones and growth factors are
considered as fundamentally different in this respect.
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
II. TRANSLOCATION MECHANISMS USED
BY PROTEIN TOXINS
Before discussing translocation of growth factors
and cytokines, we briefly review how certain protein toxPhysiol Rev • VOL
ins from poisonous plants and from pathogenic bacteria
penetrate into the cytosol. It is possible that related mechanisms can be employed in the translocation of physiological proteins. Some of these toxins employ cellular
mechanisms for translocation, whereas others carry their
own translocation apparatus. In neither case is the translocation a simple process.
A. Toxins That Carry Their Own
Translocation Apparatus
Diphtheria toxin is the pathogenicity factor in diphtheria (36, 155). The active form of the toxin consists of
two disulfide-linked polypeptides with different functions.
The one polypeptide, the A-fragment, is the toxic component. It is an enzyme that enters the cytosol and inactivates elongation factor 2 required for protein synthesis.
This is achieved by adding an ADP-ribose residue onto a
unique amino acid, diphthamide, found in the elongation
factor. As a result, protein synthesis is blocked and the
cell eventually dies.
The A-fragment is by itself essentially nontoxic. Only
when given to cells in concentrations more than 106 times
higher than those required for whole toxin is some toxic
effect observed.
The B-fragment consists of two functionally different
domains. The R-domain, which is located at the COOH
terminus, binds the toxin to cell surface receptors that are
uncleaved precursors of heparin-binding epidermal
growth factor (EGF)-like growth factor (148). The remaining part of the B-fragment, the T-domain, is almost entirely ␣-helical (29) and inserts into membranes upon
triggering by low pH (183). The toxin meets low pH in
endosomes after the surface-bound toxin is taken up by
endocytosis.
The translocation process is initiated by the insertion
of two of the ␣-helices (8 and 9) of the T-domain into the
membrane (139, 190). At the tip of the loop separating
these helices there are two acidic amino acids that must
be protonated before the helices can insert into the membrane. This protonation normally occurs in the acidic
endosomes, but it can also occur at the cell surface if the
cells are exposed to medium with low pH (182). The
insertion of the helices into the membrane induces a
process that eventually pulls the A-fragment across the
membrane. A rather extensive unfolding of the A-fragment is required for the translocation to occur.
The toxin is also able to translocate passenger proteins. Thus acidic fibroblast growth factor (aFGF) (233)
and dihydrofolate reductase (93) fused to the NH2 terminus of the toxin A-fragment were efficiently translocated
into the cytosol by the toxin pathway. Even the passenger
protein must unfold for translocation to occur. The inability of many proteins to unfold at the low pH of the
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Over the years it was reported repeatedly that also
protein hormones, cytokines, and growth factors were
found intracellularly, usually in the nucleus. Many of these
claims were based on data obtained with immunofluorescence or cell fractionation, approaches that can easily give
misleading results, as discussed below. However, a number
of studies where more sophisticated techniques were employed have provided evidence that at least some of these
proteins are indeed able to penetrate into cells. Some evidence for intracellular action of the growth factors has also
appeared. Although it is still not generally accepted that
protein hormones and growth factors may act intracellularly, sufficient data have accumulated to justify a critical
review of the existing literature.
Transport of proteins across cellular membranes has
proven to be a complicated process in all cases where the
mechanism has been worked out in detail. Transport of
export proteins into the lumen of the endoplasmic reticulum
(85, 133, 171) and of mitochondrial proteins into mitochondria (98, 214) requires complicated translocation machines
involving a number of protein components. In both cases the
proteins must unfold to some extent before translocation
can take place. Folded proteins may be able to translocate
into chloroplasts (76) and peroxisomes (33), but also here
complicated machineries are involved. The idea that proteins should be able to translocate through the plasma membrane or across the membranes of intracellular membranebound organelles therefore seems unlikely unless a
translocation apparatus is available.
A number of short basic peptides have been reported to
induce translocation of proteins across membranes. These
peptides, which originate from antennapedia (41, 165–167),
from HIV-Tat transactivating protein (147), and from other
sources were chemically linked or fused by recombinant
technology to various proteins with known intracellular activities. When added to cells, they induced biological effects
consistent with their known function. The procedure has
been tested out in a large number of settings with positive
results. So far quantitative assessments of the amount of
translocated protein has not been reported, and the mechanism of entry remains largely unknown.
Recently, it has become clear that misfolded proteins
in the endoplasmic reticulum are not exported from the
cell, but retro-translocated into the cytosol where they are
degraded by proteasomes (234, 236). Several components
used for export of proteins into the endoplasmic reticulum, such as the sec61 complex, are involved in this
reverse translocation.
TRANSLOCATION OF GROWTH FACTORS
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sists of removing an adenosine residue from the 28S RNA
of the large ribosomal subunit (48 –50). Although this does
not result in disruption of the RNA backbone, elongation
factors are unable to bind properly to the modified ribosomes and protein synthesis stops.
The A-chain of cholera toxin is an ADP-ribosylating
enzyme that modifies Gs proteins and thereby increases
the level of cAMP in the cells (61).
In addition to the toxins here mentioned, there are a
large number of protein toxins that enter the cytosol (7).
In some cases this occurs in similar ways as here described, but in most cases the mechanism has not yet
been elucidated. The translocation appears, however, to
be a complicated process in all cases.
Because a large number of bacteria and plants have
been able to form protein toxins that are able to translocate an enzymatically active protein efficiently to the
cytosol using different approaches, this cannot be a too
difficult task. Therefore, it would a priori not be unlikely
that physiological molecules involved in transmembrane
signaling would use similar strategies. Furthermore, because in all cases the toxins are complicated molecules, it
is unlikely that simple protein molecules will be translocated unless they employ some preexisting cellular translocation mechanism.
B. Toxins Employing Cellular Mechanisms
for Entry
In many studies on the translocation of external proteins from the exterior to the nucleus, either cell fractionation or immunofluorescence has been used. Both methods involve serious problems of interpretation, and
appropriate controls are required.
In the cell fractionation approach it is not always
easy to control for the possibility that the association of a
protein with the nucleus could occur after the cells were
disrupted or dissolved. In many cases the proteins in
question contain positively charged stretches that have
affinity for nuclei.
Another problem with cell fractionation as a method
to assess the localization in the cell is that in some cases
the receptor is anchored to cytosceletal components that
are not solubilized upon mechanical disruption of the
cells or by treating them with nonionic detergents (31).
Upon fractionation they may therefore appear in the same
fraction as the nuclei.
A similar difficulty may arise in immunofluorescence
experiments. Here the cells are often first fixed with
paraformaldehyde and then permeabilized with detergents to make the intracellular proteins accessible to
antibodies. A problem in these experiments is that the
A number of toxins are not equipped with their own
translocation apparatus, and still they are able to translocate an enzymatically active subunit to the cytosol. To
this group belong the plant toxins abrin and ricin and the
bacterial toxins cholera toxin and Shiga toxin. Also these
toxins bind to receptors at the cell surface by their Bmoiety, which consists of a single polypeptide chain in the
case of abrin and ricin (153) and of a pentamer of identical polypeptides in the case of cholera toxin (60) and
Shiga toxin (151, 154). After binding, the toxins are endocytosed and transported retrograde to the Golgi apparatus (215) and from there to the endoplasmic reticulum
(40, 170, 181). Here the A-chain is liberated from the
B-moiety and translocated to the cytosol apparently by
the mechanism used by misfolded proteins (endoplasmic
reticulum-associated degradation; ERAD) (75, 102, 224,
226, 234). Once in the cytosol, the toxin A-chains are
somehow able to avoid degradation by the proteasomes
and can therefore carry out their enzymatic effects.
In the case of abrin, ricin, and Shiga toxin, this conPhysiol Rev • VOL
III. PROBLEMS IN DEMONSTRATING
TRANSLOCATION OF PROTEINS FROM
THE EXTERIOR TO THE CYTOSOL
AND TO THE NUCLEUS
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endosomes is probably the reason why only few proteins
are able to be translocated by the toxin.
Another toxin that carries its own translocation apparatus, anthrax toxin, has a completely different structure and mechanism of action. This toxin is synthesized
by Bacillus anthracis and consists of three protein entities (107). The protein called the protective antigen (because antibodies against it protect against the toxin) is
proteolytically cleaved to yield a polypeptide that aggregates to form a heptameric ring structure that binds to cell
surface receptors (21) and forms binding sites for the two
other components termed edema factor and lethal factor
(107). These two proteins compete for the same binding
site on the heptameric ring structure. After the binding
has taken place, the complex is endocytosed and, like in
the case of diphtheria toxin, the low pH in the endosomes
induces translocation of the toxic entities, the edema
factor and the lethal factor, into the cysosol (225). The
edema factor is an adenylate cyclase that is activated by
calmodulin (108), whereas the lethal factor is a metalloprotease that cleaves the mitogen-activated protein kinase kinase (MEK) (44, 217). Both actions interfere with
the ability of the macrophages to kill the anthrax bacilli,
which are therefore able to grow uninhibited in the blood
with production of large amounts of toxin with often fatal
results.
Also, anthrax toxin is able to translocate passenger
proteins to the cytosol, and also in this case, extensive
unfolding of the proteins is a requirement for translocation (225).
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
possible amino acids. The CAAX box found in K-Ras, viz.
Cys-Val-Ile-Met (231), is an example. When a protein carrying this signal is added to cells in the presence of
radioactive mevalonate, a precursor of the farnesyl group,
the protein will only be labeled if it reaches the cytosol or
the nucleoplasm in the living cell (Fig. 1).
PKC is also found only inside cells. There are many
isoforms of this enzyme, but they are all found in the
cytosol and in the nucleus (74, 136). A Golgi-associated
form (PKC␮) is found at the cytoplasmic side of the
membrane as it interacts with cytoplasmic proteins (164).
Therefore, if an externally added protein can be demonstrated to be phosphorylated by PKC, it follows that the
protein must have reached the cytosol or the nucleoplasm
in vivo (96, 97).
There are certainly many other intracellular enzymes
that could be used for the same purpose. The requirement
is that a similar enzymatic activity is not present at the
cell surface or in intracellular vesicular or tubular structures that the protein could reach after endocytosis. It is
also advantageous that the enzyme recognizes a simple
amino acid sequence that can be engineered into proteins
as required.
Targeting of cytosolic proteins to the peroxisomes
occurs from the cytosol and requires that the protein
carries a peroxisome targeting signal that may be the
tripeptide Ser-Lys-Leu (196). By engineering a peroxisome
targeting signal onto an external protein that is translocated into the cytosol, it is likely that it will end up in the
peroxisomes where it can be visualized if the protein is
fluorescently labeled. Colocalization with a peroxisomal
marker protein, such as catalase, indicates its presence in
the peroxisomes (193).
A fourth approach to study translocation of externally added proteins to the cytosol is to permeabilize
IV. NEW APPROACHES TO ASSESS THE
PRESENCE IN THE CYTOSOL AND IN
THE NUCLEUS OF EXTERNALLY
ADDED PROTEINS
To test in a more rigorous way if externally added
proteins are able to penetrate to the cytosol and nucleus,
it is useful to study if enzymes that are only found in the
cytosol and in the nucleus are able to modify the proteins
in vivo. Two such enzymes, farnesyl transferase (231) and
protein kinase C (PKC) (96, 97), have been employed.
Farnesyl transferase consists of two polypeptides that are
both found only in the cytosol (28, 185) and in the nucleus
(121, 191). In accordance with this, both subunits are
synthesized without signal sequences. The enzyme recognizes a COOH-terminal signal, a CAAX box, consisting of
four amino acids. Those are cysteine followed by two
aliphatic amino acids and ending with one out of several
Physiol Rev • VOL
FIG. 1. Farnesylation of CaaX-modified acidic fibroblast growth
factor (aFGF). aFGF was modified to contain the four amino acids
Cys-Ile-Val-Met in its COOH-terminal end. Upon binding to specific
fibroblast growth factor (FGF) receptors, the growth factor is translocated to the cytosol and nucleus. Farnesyl transferase adds a farnesyl
group to the cysteine residue and a protease then removes the three
COOH-terminal amino acids. In the presence of radioactive mevalonic
acid, a precursor of the farnesyl group, the translocated growth factor is
labeled.
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fixation does not immobilize all proteins in the cell (135).
In the case of small proteins, less than half of the molecules may be immobilized. Because the fixation may damage intracellular diffusion barriers, such as the membranes of intracellular compartments, proteins that were
located inside membrane-bonded vesicular structures in
the living cell may diffuse into the cytosol and to the
nucleus during the fixation period. Further diffusion
could occur during the subsequent incubation with antibodies. As discussed in section V, some protein hormones
and growth factors are, after endocytosis, transported to
juxtanuclear vesicular compartments and may therefore
be in an advantageous position for diffusion into the
nucleus once the permeability barriers are broken down.
To test if an externally added protein is translocated
to the cytosol is even more difficult than to measure
transport to the nucleus. Cell fractionation experiments
are here particularly difficult as both mechanical disruption and detergent lysis may liberate material from intracellular vesicular compartments such that it will appear in
the cytosol fraction.
A particular problem exists in the possibility that the
growth factor or a splicing variant of it may be expressed
in the cytosol as a response to activation of the receptors
for the same growth factor. This appears to be the case
with basic fibroblast growth factor (bFGF) where the
growth factor is expressed as a result of fibroblast growth
factor (FGF) receptor stimulation (158). The appearance
in the cytosol of the growth factor or of immunologically
cross-reacting material after treatment of the cells with
the same growth factor can easily be interpreted as translocation to the cytosol of the externally added protein.
Clearly, additional tests are required to demonstrate
that an externally added protein is indeed transported to
the cytosol and to the nucleus in vivo.
TRANSLOCATION OF GROWTH FACTORS
V. EXTERNAL PROTEINS THAT HAVE BEEN
REPORTED TO ENTER THE CYTOSOL
AND NUCLEUS
A. aFGF
aFGF is synthesized without a signal peptide, and it is
not known in detail how it is released from the cells. A
series of papers from the laboratory of Maciag and coworkers (80) has provided information about the mechanism. In the first place, inhibitors of transport through the
Golgi apparatus, such as brefeldin A, do not inhibit the
release, indicating that a Golgi-dependent route is not
involved. Furthermore, the release is induced by heat
shock such as incubating the cells at 42°C (80).
The released growth factor is not biologically active
as such, but it can be activated by treatment with high
concentrations of ammonium chloride. It is released as a
disulfide-linked dimer. When the cysteine residues in the
growth factor were mutated, the growth factor was not
released even at increased temperature (81). Further
studies revealed that the critical residue was Cys-30 (210).
Together with the growth factor is released the cytoplasmic domain of synaptotagmin-1, a protein involved in
membrane fusion (211). When ␤-galactosidase was fused
to the growth factor and expressed in the cells, the whole
fusion protein was exported (106). In cells transfected
with a deletion mutant of synaptotagmin, neither the
growth factor as such nor the fusion protein was exported. Also a member of the S100 family of calcium
binding proteins, S100A13, was found to be released together with the growth factor (27). Overexpression of
S100A13 allowed export even of the cysteine-free growth
factor that is normally not exported (105).
Interleukin-1 (IL-1) is also synthesized without a signal sequence. When IL-1␣ was expressed in cells, it inhibited the export of aFGF, suggesting that they, at least
Physiol Rev • VOL
partly, use the same export mechanism (212). Synaptotagmin is not required for the export of IL-1␣, because the
export was not inhibited by the deletion mutant that
prevented export of aFGF.
aFGF is synthesized as a molecule with an extended
free NH2-terminal end. The first 20 amino acids are
cleaved off in part of the molecules, but this does not
appear to affect the biological activity. For this reason the
shorter form is used in most experiments. The first indication that aFGF may have a function in the nucleus was
obtained by Imamura et al. (77). They observed that a
polybasic region close to the NH2 terminus of the protein
was required for mitogenic activity of the growth factor,
but not for the ability of the growth factor to stimulate
tyrosine phosphorylation and induce transcription of cfos. When the polybasic region was replaced by the nuclear localization sequence from yeast histone 2B, mitogenic activity was restored.
Subsequent studies from the same group demonstrated that point mutations in the same region did not
abolish the mitogenic effect of the growth factor and that
the deletion mutant was less stable than the unmodified
growth factor (59). Therefore, the NH2-terminal nuclear
localization sequence as such was not required for mitogenic activity.
Imamura et al. (79) found that the growth factor
lacking a nuclear localization sequence in the NH2-terminal region was unable to enter the nuclear fraction,
whereas aFGF containing this sequence was present in
the nuclear fraction obtained by cell fractionation. This
was also the case when it was replaced by the nuclear
localization sequence from yeast histone. However, they
did not provide direct evidence that growth factor purifying together with the nucleus was really inside the
nucleus in the living cell.
Later the same laboratory (78) provided immunofluorescence evidence that the growth factor really accumulated in the nuclei of BALB/3T3 cells and human umbilical vein endothelial cells, but only during the G1 phase
of the cell cycle. Surprisingly, Komi et al. (101) found that
a 26-amino acid peptide from the NH2 terminus of the
growth factor, including the nuclear localization sequence, was able to induce mitogenesis in cells when
added in concentrations 1,000 times higher than that required for the growth factor. The mechanism behind this
stimulation and if it is related to the mechanism of action
of the growth factor is not known.
Sano et al. (184) found that aFGF accumulated in the
nuclei in part of the cells in inflammatory arthritic joints.
Later, Cao et al. (26) found that when aFGF was overexpressed in cells it was to a large extent found in the
nuclei. On the other hand, they were not able to demonstrate transport of externally added growth factor to the
nuclei as measured by immunofluorescence.
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selectively the plasma membrane with streptolysin O.
This bacterial toxin acts by forming pores in cellular
membranes that are large enough to allow soluble proteins in the cytosol to diffuse out of the cell (17). It is
possible to manipulate the system in such a way that
pores are only formed in the plasma membrane and not in
membranes of intracellular vesicles (170). The toxin acts
by binding to cholesterol in the membrane, and this binding occurs even at 0°C. However, at the low temperature,
the toxin is unable to form pores in the membrane. The
cells are therefore extensively washed to remove unbound toxin and then exposed briefly to medium at 37°C
to allow pores to be formed. If the protein leaks out from
the cells under these conditions, it is likely that it is
present in the cytosol (96).
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
Physiol Rev • VOL
cytosol or the nucleus. It was found that the growth factor
was farnesylated under these conditions. Immunoprecipitation demonstrated that the labeled protein was indeed
aFGF. Binding to the specific FGF receptors was required
for translocation to occur. The much more abundant binding to surface heparans did not result in translocation as
measured by this procedure.
In an other approach, advantage was taken of the
observation that aFGF is readily phosphorylated by PKC
(95, 97). Mutation of the main site phosphorylated in vitro
resulted in inability of the growth factor to be phosphorylated in vivo when given to cells. This indicates that
there is a single PKC site that is phosphorylated in vivo.
Because PKC is an intracellular enzyme found only in the
cytosol and in the nucleus, phosphorylation of externally
added growth factor can be taken as evidence that the
growth factor was indeed translocated to the cytosol or
nucleus.
The translocation was inhibited by genistein (146)
and by wortmannin and LY294002 (94), indicating that
tyrosine kinase and phosphatidylinositol 3-kinase activity
are required for translocation. Mutation analysis of the
FGF receptor 4 demonstrated that the kinase domain of
the receptor is not necessary for translocation of the
growth factor to occur. On the other hand, a short sequence in the COOH terminus of the receptor was found
to be indispensable for translocation (96).
The mutant aFGF K132E referred to above (25),
which binds to and activates FGF receptors in an apparently normal way, was found to be able to enter the
cytosol and the nucleus (97). Furthermore, when it was
introduced into cells as a fusion protein with diphtheria
toxin (Fig. 2), it entered the nucleus like the wild type, but
it did not induce DNA synthesis. This indicates that the
mutant growth factor was unable to interact properly with
an intracellular target.
Experiments were therefore conducted to search for
proteins binding to the wild-type growth factor, but not to
the K132E mutant. Three proteins fulfilling this criterion
were indeed found. FIBP is a novel nuclear protein with
unknown function (99, 100). Casein kinase 2 (CK2) is
found in the cytosol and in the nucleus and consists of
two subunits, both of which bind to aFGF and to bFGF.
The strongest binding was seen with the enzymatically
active ␣-subunit (193). Furthermore, by comparing a number of aFGF mutants with varying mitogenic activity (95),
good correlation was found between mitogenicity and
binding to CK2␣ (193). P34 is a protein associated with
the cytoplasmic side of the endoplasmic reticulum (152),
which also binds selectively the mitogenic growth factor
(194).
Translocation of aFGF to the cytosol is inhibited by
bafilomycin A1 (128), which inhibits the vesicular-type
proton pumps. This indicates that the translocation
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Prudovsky et al. (168) found that FGF receptor
(FGFR)-1 moved from the cell periphery to a juxtanuclear
location upon treatment of the cells with aFGF. They also
found that only the receptor ␣1-isoform containing three
Ig-like loops, but not the receptor ␤1-isoform (containing
only two Ig-like loops), migrated to the juxtanuclear location (169). Furthermore, the FGFR-1␣ type of receptor
was much more efficient than the ␤-type in transferring
aFGF to the nuclear fraction as obtained after biochemical fractionation of the cells.
Burgess et al. (25) found that aFGF where residue
132 had been mutated to Glu (K132E) was very inefficient
at inducing mitosis, although it bound to the specific FGF
receptors, apparently in the same way as the wild-type
growth factor. Furthermore, it induced tyrosine kinase
activity of the receptor and transcription of immediate
early genes like the wild-type growth factor. One difference was that the mutant growth factor bound less firmly
to heparin than the wild type. Transfection of NIH 3T3
cells with the wild-type growth factor induced a transformed phenotype, but this was not the case with the
mutant (24). When cells were incubated with wild-type
growth factor, it was proteolytically processed to a somewhat shorter protein, but this was not the case with the
mutant (66). This processing was proposed to be required
for mitogenicity.
Wie˛ dłocha et al. (233) developed a system to introduce aFGF into cells lacking receptors for the growth
factor. aFGF was fused to the A-fragment of diphtheria
toxin, and the toxin was reconstituted with B-fragment.
This construct was able to inject the fusion protein into
cells that contain toxin receptors. With the use of cells
that were mutated to be resistant to the intracellular
action of the toxin, it could be demonstrated that the
fusion protein was translocated into the cytosol and that
it subsequently moved to the nucleus. When this construct was given to cells lacking FGF receptors, DNA
synthesis was induced (230). Despite this, there was no
proliferation of the cells.
In further experiments it was demonstrated that for
cell proliferation to occur it was also necessary that the
cells expressed FGF receptors and that these were activated by the growth factor (232). It was concluded that
both signals from the activated receptors and from the
intracellular growth factor are necessary to induce cell
proliferation.
To test if the growth factor as such, i.e., without the
assistance of diphtheria toxin, is able to translocate to the
cytosol and to the nucleus, two approaches were used. In
one approach a farnesylation signal, a CAAX box, was
added onto the COOH terminus of the growth factor
(231). Because the farnesyl transferase is located intracellularly, farnesylation of the externally added growth
factor indicates that the growth factor has reached the
TRANSLOCATION OF GROWTH FACTORS
169
occurs from an acidic vesicular compartment. However, not the acidic pH, but rather the transmembrane
potential of the vesicles, is required for the translocation to occur (128). Citores et al. (31) found that in cells
transfected with FGFR-4, aFGF is taken up by endocytosis that only partly is associated with coated pits. It is
subsequently transported to a juxtanuclear location
that was found to be identical to the recycling endosome compartment (Fig. 3). The receptor continued to
signal at this location as determined by its high phosphotyrosine content.
Further studies by Citores et al. (30) demonstrated
that the kinase activity of the receptor was required for
endocytic uptake by the coated pit pathway, but not for
uptake by an alternative pathway. The kinase activity in
full-length receptor was required for efficient transport to
the recycling endosomes. However, when the whole kinase domain was deleted, the growth factor was still
transported to the recycling endosomes, indicating that
there is a signal in the kinase domain that prevents the
receptor from entering the recycling endosomes and that
this signal is overruled by the tyrosine kinase activity of
the receptor.
For translocation of proteins across membranes, it is
usually required that the protein unfolds to a certain
extent. This is also the case when aFGF is translocated
into cells as a fusion protein with diphtheria toxin (230,
233). On the other hand, when aFGF as such was translocated into cells, extensive unfolding is not required
(227).
FIG. 3. Fluorescently labeled aFGF binds to the surface of cells transfected with FGF receptor 4. After incubation
at 37°C, it is taken up by endocytosis and finally accumulates in a juxtanuclear compartment identified as the recycling
endosome compartment.
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FIG. 2. aFGF but not aFGF-K132E stimulates DNA
synthesis in cells lacking FGF receptors when it is translocated into the cells with diphtheria toxin as a vector.
aFGF and aFGF-K132E were fused to the NH2 terminus of
diphtheria toxin A-fragment (DT-A) and reconstituted with
diphtheria toxin B-fragment (DT-B) to obtain a translocation competent molecule. The constructs were given to
cells lacking receptors for FGF but containing receptors
for the toxin. (The cells had previously been mutated to
become resistant to the intracellular action of diphtheria
toxin.)
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
B. bFGF
Physiol Rev • VOL
C. HIV-Tat
Tat is a protein encoded by the human immunodeficiency virus that binds to the 5⬘-long terminal repeats and
induces strongly the synthesis of viral RNA and protein.
Green and Loewenstein (63) and Frankel and Pabo (58)
found independently that HIV-Tat was able to stimulate
HIV-LTR-driven RNA synthesis in intact cells, particularly
when chloroquine was present. Although the authors did
not demonstrate directly that Tat was translocated into
the cytosol and nucleus, this appears as very likely. The
concentration of Tat required in the medium to obtain
maximal transactivating effect was in the micromolar
range. Scrape loading of the cells reduced the required
concentration by a factor of 20 –30. Mann and Frankel
(129) found that Tat is taken up by endocytosis and that
cells contain ⬎107 binding sites/cell. Binding, uptake, as
well as the transactivation was prevented in the presence
of heparin. They were unable to identify a specific receptor.
The question whether or not Tat is able to transport other proteins into cells has been approached by
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Very little work has been done on the endocytic
uptake and intracellular transport of bFGF. Gleizes et al.
(62) studied the uptake in BHK cells. The experiments
were carried out in the absence of heparin, and therefore,
the authors were unable to distinguish between growth
factor bound to specific receptors and growth factor
bound to surface heparans, which was probably the most
abundant binding (175). The authors found that the
growth factor was taken up from caveolae and that it was
seen in early endosomes, multivesicular bodies, and lysosomes. Marchese et al. (130) studied uptake of the related
keratinocyte growth factor under conditions where the
binding was carried out in the presence of 0.3 M NaCl to
inhibit binding to surface heparans. Keratinocyte growth
factor binds to a variant of FGFR-2. They found that the
growth factor was taken up from coated pits and transported to a juxtanuclear location.
Like aFGF, bFGF is synthesized without a signal
sequence and is transported out of the cells by a pathway
not involving the endoplasmic reticulum/Golgi route. The
27-kDa heat shock protein facilitates the export (163).
Probenicid (68) and ouabain (38, 57) inhibit the export,
suggesting that multidrug resistance associated protein
and the Na⫹-K⫹-ATPase are involved in the process.
bFGF is synthesized as several different molecular
entities due to several CUG initiation sites upstream of
the canonical AUG. The extended variants of the growth
factor contain in the NH2-terminal extensions several GR
repeats that act as nuclear targeting sequences (43).
Therefore, the extended forms of the growth factor are
mainly found in the nucleus and are not exported out of
the cells. The nuclear localization is mainly found in
sparse, rapidly dividing cells, whereas in dense or senescent cells the growth factor is mainly cytosolic (238). The
18-kDa, AUG-initiated, form is found mainly in the cytosol, although some protein may also be present in the
nucleus. In contrast to this, the homeoprotein engrailed
contains an extension that is of importance both for the
intracellular localization and for transport out of the cell
(127).
When a 24-kDa extended form of the growth factor
was added externally to cells, it appeared to have the
same action as the 18-kDa form (67). This could be due to
intracellular processing of the growth factor (141). It
appears that intracellular bFGF has a different role than
externally added bFGF. Thus, in neural crest-derived
Schwann cells, antibodies to bFGF inhibited pigmentation
caused by externally added bFGF, but it had no effect on
12-O-tetradecanoylphorbol 13-acetate-induced melanogenesis, which is dependent on intracellular bFGF (188).
Several authors have reported transport of bFGF to
the nucleus after it had been added to cells. Bouche et al.
(20) reported that the growth factor accumulated in the
nucleoli during the G1 stage and that this correlated with
the stimulation of ribosomal gene transcription in bovine
endothelial cells. Externally added bFGF was found to be
present in the nuclear fraction upon cell fragmentation
(13). Furthermore, bFGF was reported to stimulate transcription in cell-free systems (20, 149).
When bFGF is expressed in cells, the high-molecularmass forms are transported to the nucleus, whereas the
18-kDa form, which lacks a nuclear localization signal, is
not (22). The high-molecular-mass forms contain several
GR repeats (162) that are methylated and may be responsible for the nuclear localization. Transport of the endogenous growth factor to the nucleus appears to be necessary for stimulation of growth in serum-deprived cells (9,
88). The distribution of the growth factor between nucleus and cytosol was found to vary during early development (174).
By radiation fragmentation analysis it was found that
bFGF is present in cells in a larger complex (156). It was
later found that bFGF interacts with the ␤-subunit of CK2
and stimulates the kinase (19). The authors did not find
any binding to the enzymatically active ␣-subunit. Furthermore, they did not find any binding of aFGF to CK2.
Mutation of a single amino acid in bFGF (S117A) abolished the ability of the growth factor to bind to CK2 and
also abolished the mitogenic activity of the growth factor.
The mutation did not abolish the ability of the growth
factor to bind to and activate FGFR (11).
Altogether, there is good evidence that bFGF has an
intracellular mode of action in addition to its action on the
extracellular part of FGFR.
TRANSLOCATION OF GROWTH FACTORS
Physiol Rev • VOL
that the entry of endocytosed peptide into the nucleus
occurred after fixation and permeabilization are highly
desirable.
Tyagi et al. (213) reported that a fusion protein of
glutathione-S-transferase and Tat entered cells and induced transactivation. However, it is difficult to evaluate
these data because there was a thrombin cleavage site
between the two proteins and the experiment was carried
out in 10% serum. It is therefore possible that the fusion
protein was cleaved and that only the Tat moiety entered
the nuclei.
Tat produced in HIV-infected cells or in cells transfected with the Tat gene is released from the cells and is
able to stimulate growth in AIDS-KS cells at concentrations ⬍1 ng/ml (51). On the other hand, transport to the
nucleus and transactivation required a concentration of
100 ng/ml or more.
1. Tat-binding sites
The fact that Tat has angiogenic properties (4) raised
the suspicion that it might bind to the receptor for an
angiogenic factor. It was in fact found that Tat binds with
high affinity (in the picomolar range) to one of the two
receptors for vascular endothelial growth factor,
VEGFR-2 or Flk-1/KDR (6, 140, 142). Binding induced the
tyrosine kinase activity of the receptor. A basic peptide
(amino acids 46 – 60) derived from Tat was also able to
bind and activate the receptor, whereas another peptide
(amino acids 65– 80) comprising the RGD sequence was
not.
At micromolar concentrations, Tat binds to certain
integrins (14, 219). The basic domain of Tat was more
important than the RGD domain for this binding.
A 90-kDa protein was also found to bind Tat, but this
protein has so far not been identified (222). Also this
protein bound to the basic peptide, but not to the RGDcontaining peptide.
It has also been reported that Tat binds to the lowdensity lipoprotein receptor-related protein in neuronal
cells (115).
Finally, Tat binds to heparin and cell surface heparan
sulfate (5, 213). This is a high-capacity binding on most
cells, and the affinity is much lower than the binding to
the VEGF receptor. Still it is much stronger than that of
cytochrome c, another basic protein.
Altogether, it is clear that Tat undergoes many interactions with cell surface molecules and induces rapidly
activation of intracellular regulatory proteins such as
phosphatidylinositol 3-kinase (137). Therefore, it is difficult to draw conclusions with respect to entry of the
protein into the cytosol and nucleus on the basis of functional data.
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several authors. Fawell et al. (55) linked Tat chemically
to ␤-galactosidase and reported that when added to
cells the whole cell was stained blue upon development
with chromogenic substance. However, the resolution
in these experiments was far too low to decide whether
the conjugate was in the cytosol and nucleus rather
than contained in intracellular vesicles. The same holds
true for experiments where Schwarze et al. (187) fused
TAT-translocating peptide to ␤-galactosidase by recombinant technology and tested uptake in cells. The fact
that the cells took up the conjugate, but not ␤-galactosidase as such, reflects the ability of Tat to bind to
surface heparans and other cell surface molecules. No
evidence for penetration of the fusion protein into the
cytosol or nucleus was presented.
Bonifaci et al. (18) fused dihydrofolate reductase to
Tat and studied the ability of the construct to transactivate the HIV-LTR promoter of a plasmid transfected into
the cells. They reported that the fusion protein transactivated LTR-CAT equally well as Tat alone. They did not
exclude the, perhaps unlikely, possibility that some Tat
could have been cleaved off from the fusion protein and
that it entered the cytosol and nucleus without the passenger protein. Furthermore, they reported that the construct was present in the nuclear fraction when added to
cells alone, but in the cytoplasmic fraction (comprising
endosomes as well as cytosol) when methotrexate was
present as well. Methotrexate binds to dihydrofolate reductase and prevents its unfolding (46). Unfolding is required for translocation of proteins across the membrane
of the endoplasmic reticulum and of the mitochondria,
and it is required for translocation of toxins across the
membrane of endosomes. The data suggest that also the
translocation of the fusion protein of Tat and dihydrofolate reductase requires unfolding for translocation from
the exterior to the nucleus. It should, however, be kept in
mind that the presence of a protein in the nuclear fraction
of disrupted cells does not necessarily mean that the
protein is translocated into the nucleus in vivo.
Several authors have attempted to use a presumed
transducing amino acid stretch from Tat to transport
passenger proteins into cells. Nagahara et al. (147) reported that an 11-amino acid sequence of Tat translocated
p27kipI into cells. This protein is a signaling protein inhibiting Cdk and arresting cells in G1. They found that the
construct had this ability when added to cells, particularly
if the protein had been treated with urea immediately
before. They did not provide direct evidence that the
protein had really been translocated into the cells, but
controls with fusion protein with inactive p27kipI suggested that this was the case.
A basic domain of Tat has been reported to enter the
nucleus when added externally to cells (218). The entry
was found to occur even at 4°C. Experiments to exclude
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
D. Interferon-␥
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Interferon-␥ is a potent cytokine involved in regulation of many biological processes such as host defense,
inflammation, and autoimmunity. The mature protein consists of 143 amino acids after cleavage of the signal sequence (54). Two interferon-␥ molecules noncovalently
homodimerize in an antiparallel manner (45). The interferon-␥ receptor consists of a ligand-binding ␣-chain (3)
and a ␤-chain required for signal transduction (72, 131,
198). The intracellular part of the interferon-␥ receptor is
associated with the tyrosine kinases janus kinase (JAK) 1
and JAK2 (145, 221), which upon activation of the receptor phosphorylate and activate signal transducers and
activators of transcription (STAT) 1. Phosphorylated
STAT1 forms homodimers (189), which are capable of
binding to a specific DNA sequence, interferon-␥ activated
sequence (GAS), thereby regulating transcription (39).
However, also the interferon-␥ molecule itself has been
reported to enter the cytosol and nucleus to play a direct
role in signaling.
Using electron microscopy with an immunogold
technique, MacDonald et al. (122) reported rapid (within
1–2 min) translocation to the nucleus of prebound, extracellular murine interferon-␥ in mouse L929 cells. Immune
electron microscopy was also used to visualize interferon-␥ in the nucleus in Hep2 cells and in mouse liver
(104, 179). Similar results were obtained by cell autoradiography and by cell fractionation after incubation with
125
I-interferon-␥ (10).
Extracellularly added interferon-␥ shows strict species specificity between human and murine cells (56, 172).
This fact has been exploited in experiments suggesting
that interferon-␥ can trigger responses from a location in
the cytosol in addition to its ability to bind to the extracellular part of interferon-␥ receptors. Fidler et al. (56)
used liposome encapsulation to deliver interferon-␥ intracellularly. They studied interferon-␥-stimulated tumoricidal properties of purified human blood monocytes and
mouse peritoneal exudate macrophages. In a first set of
experiments to confirm the species specificity, they found
that extracellular human interferon-␥ (together with muramyl dipeptide) stimulated the tumoricidal activity of
human monocytes, but not of mouse macrophages, and
vice versa, extracellular mouse interferon-␥ stimulated
the mouse cells, but not the human ones. In contrast,
when liposome-encapsulated interferon-␥ was used, the
effects were no longer species specific. Furthermore, pronase treatment of the cells to remove cell surface interferon-␥ receptors rendered the cells irresponsive to unencapsulated interferon-␥, while liposome-encapsulated
interferon-␥ still stimulated tumoricidal activity.
Smith et al. (197) found that human interferon-␥ stimulated expression of a major histocompatibility complex
(MHC) class II molecule (Ia) when microinjected into
murine macrophages, but not when added extracellularly.
Sanceau et al. (180) took another approach by expressing in mouse L cell lines wild-type or a truncated
version of human interferon-␥ lacking the signal sequence
for secretion. Mouse cells expressing intracellular, nonsecreted human interferon-␥ were protected against viral
infection (varicella-zoster and Mengo virus), while transfectants secreting human interferon-␥ were susceptible to
infection. Protection by intracellular human interferon-␥
was slightly less efficient than protection by extracellular
murine interferon-␥. Also, expression of MHC class II
antigens was induced by intracellular, but not by secreted,
human interferon-␥ in these mouse cells.
Similar results were obtained by Lewis et al. (109),
who first expressed murine interferon-␥ lacking its signal
sequence in human A-549 cells, thereby obtaining cells
partially protected against viral infection. Next, these authors changed to using a similar approach in a murine cell
line, designated Ltk-aprt-cells, which is resistant to the
antiviral effects of both interferon-␥ and interferon-␤, but
becomes protected against viral infection by the simultaneous stimulation by these factors. Expression of nonsecreted human interferon-␥ was shown to cooperate with
extracellularly added murine interferon-␤. In control experiments they showed that this effect was not due to
leakage to the medium of human interferon-␥, since extracellular human interferon-␥ had no effect on the cells.
Also, the observed effect was not due to expression of
murine interferon-␥. The advantage of the rather complicated cell-ligand system used in those experiments is that
it rules out the possibility that secretion of endogenous
type I interferons (␣ or ␤) rendered the cells resistant.
Together, this work suggests that, in addition to the species-specific binding to and activation of cell surface receptors, interferon-␥ may also exert species nonspecific
effects in the cytoplasm.
This notion was supported by the finding of a nonspecies specific binding site for interferon-␥ on the cytoplasmic part of interferon-␥ receptor ␣-subunit (64, 208,
209). A sequence in the NH2 terminus of interferon-␥
bound to the extracellular part of the receptor, while a
COOH-terminal stretch of interferon-␥ was able to bind to
the intracellular part of the receptor. The intracellular
binding site for interferon-␥ on the receptor was reported
to be required both for internalization of the ligand and
for biological responsiveness (53). Furthermore, Szente et
al.(209) reported that a peptide derived from the COOHterminal part of interferon-␥ could induce viral resistance
and MHC class II upregulation in a murine macrophage
cell line in a rather species nonspecific manner.
The COOH termini of both murine and human interferon-␥ contain a basic amino acid stretch similar to the
nuclear localization signal (NLS) of simian virus 40 Tantigen. This sequence was shown to be a functional NLS
TRANSLOCATION OF GROWTH FACTORS
tated, the growth factor lost its mitogenic effect, although
it retained its ability to bind to and activate EGF receptors. This suggests that the growth factor has an intracellular site of action in addition to its activation of the
receptor. Further evidence for this is that Schwannomaderived growth factor was able to stimulate DNA synthesis in cells where the kinase domain of the EGF receptor
was inactivated by mutation, whereas EGF, which does
not contain a NLS, lacked this ability.
3. Heregulin
Heregulin is another growth factor of the EGF family.
This protein also contains a NLS. Li et al. (112) found that
the growth factor was accumulated in the nuclei 30 min
after being added to the cells. This was demonstrated by
immunofluorescence and by electron microscopy with
125
I-labeled growth factor. It would have been desirable to
demonstrate the nuclear labeling by additional methods
to exclude the possibility of artifacts.
4. Parathyroid hormone-related protein
E. Other External Proteins That May Enter the
Cytosol and Nucleus
1. Vascular endothelial growth factor
Li and Keller (111) demonstrated that when a monolayer of bovine adrenal cortex cells was wounded by a
scratch, the cells close to the wound started to take up
vascular endothelial growth factor (VEGF) in their nuclei.
Cells located more distantly from the wound took up the
growth factor by endocytosis, but in this case without
nuclear localization. The nuclear uptake lasted for a few
hours and resulted in induction of a number of proteins
that were not expressed in the more distantly located
cells. Binding of the growth factor to the specific VEGF
receptors was required for nuclear accumulation. Several
control experiments were conducted to exclude the possibility that the nuclear localization was due to injury of
the cell membrane in the cells close to the scratch, which
could conceivably have allowed entry of the growth factor through membrane wounds with subsequent accumulation in the nucleus. Interestingly, as the cultures grew
older, they lost the ability to respond to injury with transport of VEGF to the nuclei.
2. Schwannoma-derived growth factor
Kimura (90) studied the interaction of oligonucleotides with Schwannoma-derived growth factor, a member
of the EGF growth factor family (and the rat equivalent of
amphiregulin). They found that it interacts with AT-rich
sequences. Differently from EGF, the Schwannoma-derived growth factor contains a NLS. When this was muPhysiol Rev • VOL
Parathyroid hormone-related protein (PTHrP) has
been found in the nucleus by several authors. This protein
has several splice variants in the range of 139 –173 amino
acids. The NH2 terminus is identical to that of parathyroid
hormone, and therefore, it binds to the parathyroid hormone receptor, a G protein-linked heptahelical transmembrane protein. PTHrP is responsible for the common endocrine paraneoplastic syndrome humoral hypercalcemia
(23). When the protein was overexpressed in cells, it was
detected both at the cell surface, in the cytosol, and in the
nucleus (2, 132). The protein contains a nuclear targeting
sequence, and when this was mutated, both surface binding of the protein and accumulation in the nucleus were
eliminated. The authors found that when a small peptide
containing the nuclear targeting sequence was added to
cells, it was accumulated in the nucleus both in cells
containing PTHrP receptors and in cells lacking them.
The import of PTHrP into the nuclei is apparently due
to a direct interaction with importin-␤, whereas importin-␣ does not seem to be involved (103). PTHrP was
found to bind tightly to RNA (1).
Prepro-PTHrP expressed in cells was found to be
degraded by a process involving ubiquitinylation and degradation by proteasomes (134). This indicates that the
protein can be translocated retrograde from the endoplasmic reticulum to the cytosol. If some of it is not degraded,
it could accumulate in the nucleus. There is, however, no
evidence that externally added PTHrP is translocated by
this route as has been demonstrated in the case of ricin
(226). Altogether, there is so far insufficient data supporting the view that externally added PTHrP is translocated
to the nucleus in vivo.
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in a standard nuclear import assay in digitonin-permeabilized cells (207). Also, this sequence appears to be crucial
for the biologic activity of the molecule (8, 120, 195, 229).
Subramaniam et al. (206) studied nuclear translocation of STAT1 in response to interferon-␥. Preincubation
of interferon-␥ with polyclonal antibodies against its
COOH-terminal, NLS-containing part blocked STAT1 nuclear translocation. The same effect was obtained by
deleting the NLS of interferon-␥. Microinjection of the
antibodies into mouse L929 cells also inhibited STAT1
nuclear translocation in response to interferon-␥, but not
in response to interferon-␣, suggesting a direct role of
cytoplasmic interferon-␥ in the nuclear translocation of
STAT1. In support of this hypothesis, interferon-␥ and
STAT1 were coimmunoprecipitated with NPI-1 (importin-␣ homolog) from cell lysates after the cells had been
treated with interferon-␥ at 37°C. Less interferon-␥ was
coimmunoprecipitated from cells treated at 4°C or treated
with interferon-␥ with deleted NLS.
Altogether, there is considerable evidence for a role
of interferon-␥ in the cytosol and nucleus.
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OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
5. Prolactin
6. Growth hormone
Growth hormone and its receptor have also been
found to be associated with the nucleus (116, 117). The
evidence was based on cell fractionation experiments and
immunofluorescence. In addition, some experiments with
125
I-labeled growth hormone and electron microscopy
were carried out. Neither of these methods is sufficiently
sensitive to exclude the possiblity that the receptor and
the growth hormone were in juxtanuclear membranebound compartments rather than inside the nucleus. In
any event, the authors observed growth hormone-stimulated transport of the growth hormone receptor to the
nuclear region, consistent with, but not proving, that the
ligand induced translocation of the receptor to the nucleus.
7. IL-1␣
Grenfell et al. (65) found that IL-1␣ bound strongly to
isolated nuclei. Cell fractionation studies demonstrated
that IL-1␣ added to cells was endocytosed and accumulated in the nuclear fraction. Curtis et al. (37) and Weitzmann et al. (223) reported that IL-1␣ enters the nucleus
together with its receptor. In both cases it was not excluded that the lymphokine was bound to its receptor
located in a juxtanuclear area in such a way that the
Physiol Rev • VOL
8. Hepatoma growth factor
Hepatoma growth factor belongs to a new family of
growth factors characterized by a so-called HATH box
(91). It is synthesized without a leader sequence, but it has
a bipartite NLS. Externally added growth factor was reported to be internalized and enter the nuclei. The evidence for this was immunofluorescence studies (91), and
it will be interesting to see this corroborated by additional
evidence.
9. Cytokines and growth factors
Several cytokines and growth factors have been
found to act in an autocrine way. In typical experiments
antibodies against the factor added to the medium did not
inhibit the stimulatory effect, whereas the expression of
antisense nucleotides did inhibit it. This was found for
IL-6 (118, 177). However, this does not necessarily mean
that the cytokine acts in the cytosol or nucleus. An alternative explanation is that the cytokine binds to and signals from receptors present in intracellular vesicular compartments. On the other hand, the recent finding by Yu et
al. (237) that endocytosed IL-2 bound to its receptor is
degraded by proteasomes indicates that the interleukin is
translocated, presumably from the endoplasmic reticulum
to the cytosol. If some of the retrograde translocated
protein is not degraded, it could exert an intracellular
action either in the cytosol or in the nucleus.
In the case of platelet-derived growth factor, an alternative B-chain-like protein has been described that
lacks the signal sequence and is therefore expressed in
the cytosol (42) and possibly in the nucleus.
Wetmore et al. (228) found that brain-derived growth
factor is present in the nuclei of cells producing the
growth factor. Possibly, this is due to translation initiation
at an upstream CUG initiation site, thus initiating without
an NH2-terminal signal sequence.
Macrophage inhibitory factor was found to interact
with an intracellular protein Jab1, which is a coactivator
of AP-1 (92). Although direct evidence for translocation of
this 12-kDa protein from the exterior to the cytosol is
lacking, the data suggest that the cytokine has an intracellular action.
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Prolactin was reported to be present in the nucleus of
T lymphocytes that had been treated with IL-2 (35). The
data were obtained with immunofluorescence of fixed
cells. Antiprolactin prevented growth of IL-2-stimulated
cells, but when prolactin containing a NLS and no signal
sequence was expressed in the cells, they were growing in
the presence of IL-2 (34). Expression of prolactin only
lacking the signal sequence did not stimulate growth. This
suggests that prolactin has an intracellular, probably nuclear, action in addition to its interaction with the cell
surface receptors. Later work from the same group (15)
reported that the translocation of prolactin to the nucleus
was inibited by rapamycin, wortmannin, and LY294002,
indicating that phosphatidylinositol 3-kinase is involved.
Perrot-Applanat et al. (160) found no evidence for
transport of prolactin or its receptor to the nucleus, although both were extensively endocytosed and accumulated in a juxtanuclear position.
Rycyzyn et al. (178) reported that cyclophilin B binds
to prolactin and is endocytosed together with the prolactin-prolactin receptor complex. The complex is then
transported retrograde in the cell to the endoplasmic
reticulum where prolactin and cyclophilin are translocated to the cytosol and subsequently transported to the
nucleus. Removal of a nuclear targeting signal from cyclophilin abolished its ability to stimulate DNA synthesis.
interleukin was still inside a vesicular structure. Upon
fractionation of the cells, the complex could be present in
the nuclear pellet. Maier et al. (126) found that the precursor of IL-1␣ expressed in cells accumulated in the
nucleus and that this was necessary to inhibit growth in
some cells, whereas it acted as an oncoprotein in others
(205). The ability to accumulate in the nuclei was found to
reside in the NH2-terminal part of the precursor that is not
present in the exported protein. Also, the NH2-terminal
prodomain of IL-16 was found to localize the lymphokine
to the nucleus (239).
TRANSLOCATION OF GROWTH FACTORS
175
10. VP22
14. Ciliary neurotrophic factor
A protein from herpesvirus, VP22, was reported to
have the ability to exit the cell where it is synthesized and
enter adjacent cells where it was found in the nucleus in
immunofluorescence studies (47). The protein was also
reported to carry passenger proteins, such as p53, into
cells and deliver them to the nucleus (16, 161). However,
the protein was unable to carry diphtheria toxin A-fragment into the cytosol (52), and recent studies (119) have
raised questions whether the protein is redistributed after
the (incompletely) fixed cells were rehydrated. Further
experiments are required to settle this question.
Ciliary neurotrophic factor lacks a signal sequence. It
was found to be present in the nuclei of cells producing it
(12), despite the fact that it lacks a typical NLS. Perhaps
its small size allows it to enter the nucleus where it could
be bound to an acceptor molecule. Its role in the nucleus
is not known.
It has long been discussed whether or not insulin has
a cytosolic mechanism of action. Immunofluorescence
and other indirect methods have indicated the presence of
the hormone in the cytosol, and an intracellular binding
protein has been identified (70). Microinjection of insulin
stimulated RNA and protein synthesis in frog oocytes
(138). However, more definitive evidence is required before it can be concluded that insulin is able to penetrate
into cells.
Also, insulin-like growth factor has been reported to
be translocated into cells and to the nucleus, and specific
binding proteins are also found to enter cells (186). Also
in this case, the evidence is only circumstantial and can
be interpreted in alternative ways.
12. Connective tissue growth factor
Connective tissue growth factor has mitogenic activity and is also involved in cell adhesion, angiogenesis, and
cell migration. It is a cysteine-rich glycoprotein with a
molecular mass of 36 –38 kDa. Recently, evidence was
presented that when given extracellularly it is able to
penetrate to the cytosol and to the nucleus (220). This
was demonstrated by the ability of the cells to phosphorylate the externally added growth factor, apparently by
PKC.
13. Lactoferrin and ␣-lactalbumin
Two unlikely proteins to enter the nucleus after having been applied to intact cells are multimeric lactoferrin
and ␣-lactalbumin. Externally added lactoferrin was reported to bind to specific sequences in DNA and to activate transcription (71). Hakansson et al. (69) found that
multimeric ␣-lactalbumin induces apoptosis in tumor
cells, and immunofluorescence data suggested that the
protein enters the nuclei. In both cases, more direct evidence is necessary before it can be established that these
two proteins really possess the ability to enter the nuclei
of living cells.
Physiol Rev • VOL
Angiogenin is a 14-kDa protein belonging to the pancreatic RNase superfamily. It induces growth of endothelial cells. A putative NLS is required for this activity.
Immunofluorescence data have indicated that this protein
is able to enter cells from the exterior and that it is
transported to the nucleus where it accumulates in the
nucleolus (110, 143, 144). The protein was found to accumulate in the nucleolus very rapidly. Unfortunately, evidence that the nucleolar localization was not due to a
redistribution during or after fixation was not provided.
A basic peptide derived from FGF-4 was used to
translocate Cre recombinase into cells (84, 157).
It may be concluded that many observations suggesting that a variety of cytokines and growth factors have
intracellular functions. Many of these proteins have sequence stretches rich in basic amino acids that resemble
NLSs. In some cases they have even been reported to
target passenger proteins to the nucleus. Removal or
mutation of these sequences has in several cases changed
or eliminated their biological activity or part of it (82, 83).
In some cases microinjection or expression of the growth
factor or cytokine in the cytosol has elicited the same
effect as external addition of the protein. Although these
are interesting observations compatible with intracellular
actions of the proteins, many of them can also be explained in alternative ways, and more direct evidence is
required before it can be established that the proteins act
intracellularly.
VI. TRANSPORT OF TRANSMEMBRANE
RECEPTORS TO THE NUCLEUS
Many growth factor and cytokine receptors have
been reported to be present in the nucleus (37, 117, 124,
216, 235).
Several groups have reported that FGF receptors are
found in the nucleus. Johnston et al. (86) found that a
splicing variant lacking the transmembrane domain of
FGFR-3 was found in the nuclei of mammary cancer cells.
When they expressed in cells a similar construct that also
lacked the signal sequence, it was efficiently transported
to the nucleus.
C-erbB-4/HER-4, a receptor related to the EGF receptor, was found by immunostaining with two different
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11. Insulin
15. Angiogenin
176
OLSNES, KLINGENBERG, AND WIE˛ DŁOCHA
Physiol Rev • VOL
factor from the membrane and transport it into the nucleus. Although this is mainly speculation at the present
time, it indicates that the idea that growth factor receptors in their intact form can be translocated to nucleus in
vivo should not be dismissed as biologically inconceivable.
VII. CONCLUDING REMARKS
A large amount of evidence suggests that many protein hormones and growth factors as well as their receptors may act intracellularly, preferentially in the nucleus.
In many cases the data are insufficient to conclude that
the necessary translocation across cellular membranes
has indeed taken place. However, the emerging picture is
that at least in some cases growth factors and cytokines
do translocate from the cell exterior to the cytosol and
nucleus. Particularly in the case of aFGF, bFGF, HIV-Tat,
and interferon-␥ is there reason to believe that translocation of biologically relevant amounts does occur. In several other cases, biological effects consistent with translocation were observed, but these effects could either be
explained in alternative ways or the test system was
extremely sensitive. It is known from work with toxins
that the enzymatic subunit, which is not able to efficiently
penetrate by itself, may be toxic if added at concentrations thousands to a million times higher than that required for the whole toxin. Apparently, a few molecules
may sicker through under these conditions. How this
occurs is at present unknown, but it could be by retrograde transport to the endoplasmic reticulum with subsequent translocation by the misfolded protein pathway. It
is unlikely that such translocation plays important physiological roles because the high extracellular concentrations required of the protein in question is unlikely to be
reached in vivo. At least in the case of aFGF, translocation
can be visualized at an extracellular concentration of 10
ng/ml, which is close to the expected physiological concentration that may occur in tissue. This translocation is
now partly characterized in that it requires the specific
receptors, it probably occurs from endocytic vesicles, it
requires a membrane potential across the vesicular membrane, and it is dependent on active phosphatidylinositol
3-kinase. It remains to be tested if the other proteins are
translocated by the same or by different mechanisms.
Should it turn out that translocation of protein hormones, growth factors, and cytokines to the cytosol and
nucleus is a widespread phenomenon, it could have considerable consequences for our understanding of the way
signal is transferred from the exterior to the interior of
cells.
This work was supported by The Norwegian Cancer Society, The Norwegian Research Council, Novo Nordic Foundation,
Blix Fund for the Promotion of Medical Research, Rachel and
Otto Kr. Bruun’s legat, Torsteds legat, and the Jahre Foundation.
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antibodies to be localized to the nucleus in some, but not
all, breast cancers tested (200). Recently, it was observed
that the cytoplasmic part of this receptor is cleaved off by
␥-secretase and enters the nucleus (150) in a similar fashion as Notch (89).
It has been reported that EGF receptor is transferred
to the nuclei upon stimulation with its ligand (73) and that
it has transactivating activity (113). The presence of the
receptor in the nuclei was studied by confocal microscopy (113), which indicated that it was located as an outer
rim around the nuclei. It is therefore possible that the
receptor is in the nuclear envelope rather than inside the
nucleus as such. If it is inserted into the inner nuclear
membrane, the cytoplasmic tail of the receptor could be
in contact with chromatin and thus have a transactivating
effect. Further studies are necessary to elucidate this
question.
It is mechanistically more difficult to understand how
FGFRs that are inserted into the membrane become translocated to the nucleus upon stimulation with bFGF as
demonstrated by confocal and electron microscopy and
by cell fractionation (123, 125, 159, 201–204). After binding of FGF to its receptors, the complex is internalized by
a process that at least partly involves coated pits (199).
The import of FGFR-1 to the nucleus induced by the
18-kDa form of bFGF involves importin-␤ (173). It appears that it is the full-length receptor that is transported
to the nucleus and not only the cytosolic part as in the
case of Notch (89). In bovine adrenal medullary cells, the
translocation of the FGFR to the nucleus results in transactivation of angiotensin responsive elements in the
bFGF promotor (158). This is important to keep in mind
as it indicates that addition of bFGF to cells may induce
synthesis of bFGF in the cells, which could erroneously
be interpreted as evidence for transport of the added
bFGF into the cells.
Recent studies on the ERAD of proteins (75, 102, 176,
224, 234, 236) could explain how such translocation could
take place. It was found that whole transmembrane proteins were translocated retrograde to the cytosol and then
degraded by the proteasomes. The proteasomes may even
participate in the translocation process (85, 176). When
proteins are overexpressed, the proteasomes may become overloaded, and some protein may then accumulate
in the cells as aggresomes (87). It is possible that this
system could be modified in such a way that the protein
could be released to the cytosol in an intact form and
carry out intracellular functions as was indeed found in
the case of ricin (226).
It is particularly interesting that some growth factor
receptors accumulate at the nuclear membrane (31, 113).
In the case that the receptor is inserted in the inner
nuclear membrane, the otherwise cytoplasmic tail could
interact directly with components in the nucleus. Structures in the nucleus could assist in releasing the growth
TRANSLOCATION OF GROWTH FACTORS
Address for reprint requests and other correspondence:
S. Olsnes, Institute for Cancer Research, The Norwegian
Radium Hospital, Montebello, Oslo 0310, Norway (E-mail:
[email protected]).
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