(Received for publication, September 30, 1996, and in revised form, October 14, 1996)
From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
The fibroblast growth factor (FGF) prototypes,
FGF-1 and FGF-2, lack a signal sequence, but both contain a nuclear
localization sequence. We prepared a series of FGF-1 deletion mutants
fused to the reporter gene, -galactosidase (
-gal) and determined
that a domain between residues 83 and 154 is responsible for FGF-1 cytosol retention in NIH 3T3 cells. Using a series of FGF-
-gal chimeric proteins prepared by the shuffling of cassette-formatted synthetic FGF prototype genes, we were able to demonstrate that the
nuclear localization sequence from the 5
-CUG region of FGF-2 is not
able to direct the nuclear association of FGF-1 due to its inability to
repress the function of the FGF-1 cytosol retention domain. We also
observed that while the FGF-1:
-gal chimera was released in response
to heat shock, the FGF-2:
-gal protein was not. Further, replacement
of the FGF-1 cytosol retention domain with the corresponding domain
from FGF-2 repressed the release of the chimeric protein. These data
suggest that the specificity of the stress-induced secretion pathway
for FGF-1 involves a carboxyl-terminal domain that is absent in FGF-2
and that the FGF-1 secretion pathway does not restrict the release of
high molecular weight forms of FGF-1.
The heparin-binding fibroblast growth factor (FGF)1 family currently consists of nine structurally related members with broad biological activities (1, 2), and the biological functions of the FGF gene family members are mediated through high affinity FGF receptors, which contain intrinsic tyrosine kinase activity (3). The FGF prototypes, FGF-1 (acidic) and FGF-2 (basic), lack a classical signal sequence for secretion through the conventional endoplasmic reticulum (ER)-Golgi apparatus, and the signal sequences present in other FGF gene family members contribute to their oncogenic potential (1, 2). Because the mitogenic potential of the FGF prototypes are mediated by the function of the FGF prototypes as exogenous proteins, it has been proposed that novel and unconventional secretory pathways may have evolved to regulate their activities as extracellular modifiers of biological responses (1, 2).
We have shown that FGF-1 is secreted from NIH 3T3 cell FGF-1
transfectants by a brefeldin A-insensitive, ER-Golgi-independent pathway in response to heat shock (4, 5). FGF-1 is released as a latent
homodimer with reduced affinity for immobilized heparin (4), and both
the mitogenic and heparin-binding activities of latent extracellular
FGF-1 can be activated by reducing agents (5). Mutagenesis of Cys
residues in FGF-1 enabled us to demonstrate that Cys30 is
solely responsible for the formation of the FGF-1 homodimer (6). In
addition, FGF-1 is a phosphatidylserine-binding protein (6), and FGF-1
at 42 °C is able to attain a molten globule character, which enables
it to associate with acidic phospholipid membranes (7). In contrast,
FGF-2 is also secreted by a brefeldin A-insensitive pathway (8), and,
although the Cys30 residue is conserved between FGF-1 and
FGF-2 (1, 2), FGF-2 neither forms a homodimer (9) nor is released in
response to temperature stress in vitro (8). Because it is
possible that the FGF prototypes are released by different
unconventional pathways, we sought to study the release of the FGF
prototypes using a strategy of molecular shuffling of four synthetic
cassettes, each encoding domains from FGF-1 (10) and FGF-2 (11). This
overlapping cassette shuffle strategy has previously enabled us to
construct and express a variety of novel recombinant FGF-1:FGF-2
chimeric proteins and determine that residues 65-81 from FGF-2
significantly contribute to the heparin-dependent character
of FGF-1 as an exogenous mitogen (11). We have utilized these
constructs to obtain stable NIH 3T3 cell FGF-1:FGF-2 cassette shuffle
transfectants and studied the intracellular locale and secretory
potential of these constructs as -galactosidase (
-gal) reporter
gene fusion proteins. We report that the carboxyl-terminal half of
FGF-1 (residues 84-154) is involved in cytosol retention and
contributes to the ability of FGF-1 to be released in response to heat
shock and that the corresponding domain in FGF-2 is responsible for the
inability of FGF-2 to be released in response to temperature stress
in vitro. Finally, we also report that there does not appear
to be a molecular weight restriction for the size of FGF-1 released in
response to temperature stress in vitro, and
nuclear-associated FGF-1 does not have access to the FGF-1 secretory
pathway.
The construction of plasmids pXZ45 and
pXZ55 were previously described (12). The plasmid pJS2, which contains
residues 21-78 of the human FGF-1 gene, was constructed by ligating
the 6673-base pair (bp) ScaI-EspI fragment of
pXZ55 and the 3011-bp XmaI-EspI fragment of pXZ55
together. The ScaI digestion was partial, and both the
ScaI and XmaI sites were filled in with Klenow
fragment (Boehringer Mannheim). The plasmid pJS123, which contains
residues 21-117 of the human FGF-1 gene, was constructed in four steps involving three intermediate constructs: pJS23temp, pJS23temp2, and
pJS23temp3. The plasmid pJS23temp was constructed by ligating the
BamHI fragment of pXZ55, which contains the -gal
fragment, into the HindIII site of pDS90 (10). The ligation
of the 5334-bp BamHI-SmaI fragment of pXZ55 and
the BamHI-SnaBI fragment of pJS23temp, which
contains residues 38-117 of the human FGF-1 gene, generated pJS23temp2. The ligation of the 2138-bp
BglII-EspI fragment of pXZ55 and the 7591-bp
BglII-EspI fragment of pJS23temp2 generated pJS23temp3. Finally, the pJS123 was generated by ligating the 7909-bp
ScaI fragment of pJS23temp3 with the 1905-bp ScaI
fragment of pXZ55.
The plasmid pJS5, which contains residues 28-154 of the human FGF-1
gene, was constructed by ligating the ScaI-NheI
fragment of pXZ31 (12) and the 5702-bp
ScaI-HindIII fragment of pXZ55 with the 3556-bp
HindIII-NheI fragment of pXZ55. The plasmids pSF25 and pSF26 were constructed in a multistep process involving intermediate plasmids pSF20 and pSF21. The plasmid pSF20 was
constructed by oligonucleotide mutagenesis. The resulting clone is
similar to hst:pMEXneo (13) except for five single base changes
resulting in Cys30
Ser (codon changes TGT
TCT),
Phe36
Ser (TTC
TCC), Glu63
Asp (GAA
GAT), Cys97
Ser (TGT
TCT), and
Cys131
Ser (TGC
TCC) mutations in the human FGF-1
open-reading frame (ORF). The plasmid pSF21 was constructed by filling
in the annealed oligonucleotides
5
-TACAAGGGATCCTCAGG(G/A)AGTGGCCCCCGTTGCTA(G/C)AGTAGAGGAGTTTGGGCTT-3
and
5
-GGGAAATACGTAGTCGACCTCGAGACCACCATGGCTAATTACAAGAAGCCCAAAC-3
with DNA polymerase, digestion with
SalI-BamHI, and ligation to the large
SalI-BamHI fragment of pSF20. The oligonucleotide
bases in parenthesis are 50% mixtures at C30S and F36S positions, and pSF21 contains the wild-type bases at these positions. The plasmid pSF25, which contains residues 21-39 and 77-154 of the human FGF-1 gene, was constructed by ligating annealed oligonucleotides sense, 5
-GATCCGCCAGTACTTGGC-3
, and antisense, 5
-CATGGCCAAGTACTGGCG-3
(BamHI-NcoI ends), with the 3329-bp
NcoI-KpnI fragment of pXZ55 and the large
BamHI-KpnI fragment of pSF21. The plasmid pSF26, which contains residues 21-40 and 112-154 of the human FGF-1 gene, was constructed by ligating the annealed oligonucleotides antisense, 5
-CTTCTTGGATATAAG-3
, and sense, 5
-GATCCTTATATCCAAGAAGCATG-3
(BamHI-SphI ends), with the 3222-bp
SphI-KpnI fragment of pXZ55 and the
BamHI-KpnI fragment of pSF21. The plasmid pJS9,
which contains residues 21-81 of the human FGF-1 gene and residues
85-155 of the FGF-2 gene, was constructed in two steps. The plasmid
pJS9temp was generated by ligating the PpuMI-AvaI
fragment of pT70405 (11) and the large
AvaI-NheI fragment of pT70405 with four annealed oligonucleotides: sense, 5
-GACCTGGGCAGAAAGCTATACTTTTTCTTC-3
; sense,
5
-CAATGTCTGCTAAGAGCAGTTTAAACCCCGGGAGTCTG-3
; antisense, 5
-CTAGCAGACTCCCGGGGTTTAAACTGCTCTTA-3
; and antisense,
5
-GCAGACATTGGAAGAAAAAGTATAGCTTTCTGCCCAG-3
(PpuMI-NheI ends). The plasmid pJS9 was made by
ligating the 343-bp BspEI-SmaI fragment of
pJS9temp and the 6663-bp BspEI-EspI
fragment of pXZ55 with the EspI-SmaI fragment of
pXZ55, which contains the
-gal gene. The plasmid pJS22temp
was constructed by ligating the 1038-bp
NdeI-BclI fragment of pSK(
)SVTC (SV40 large T
antigen ORF; a kind gift from Dr. Daniel Simmon) (14) and the 2900-bp BamHI-SacI fragment of pSK(
)SVTC with the
NdeI-SacI fragment of the polymerase chain
reaction product of pSK(
)SVTC generated with primers: sense,
5
-GCATAGAGCTCGGCGCGCCATGGATAAAGTTTTAA-3
, and antisense,
5
-ACAGCCTGTTGGCATATGGTTT-3
. The plasmid pJS22 was constructed by
ligating the 9775-bp AscI-BspEI fragment of pXZ55
and the AscI-BspHI fragment of pJS22temp, which
contains the SV40T gene, with four annealed oligonucleotides: sense,
5
-CATGATGCTAATTACAAGAAGCCCAAACTCCTCTA-3
; sense,
5
-CTGTAGCAACGGGGGCCACTTCCTGAGGATCCTT-3
; antisense,
5
-CCGGAAGGATCCTCAGGAAGTGGCCCCC-3
; and antisense,
5
-GTTGCTACAGTAGAGGAGTTTGGGCTTCTTGTAATTAGCAT-3
(BspEI-BspHI ends). The plasmid pJS8temp was
generated by ligation of the PpuMI-AvaI fragment
of pTI70407 (11) and the large AvaI-NheI fragment
of pTI70407 with four annealed oligonucleotides: sense, 5
-GACCTGGGCAGAAAGCTATACTTTTTCTTC-3
; sense,
5
-CAATGTCTGCTAAGAGCAGTTTAAACCCCGGGAGTCTG-3
; antisense,
5
-CTAGCAGACTCCCGGGGTTTAAACTGCTCTTA-3
; and antisense, 5
-GCAGACATTGGAAGAAAAAGTATAGCTTTCTGCCCAG-3
(PpuMI-NheI ends). The plasmid pJS8, which
contains residues 21-117 of the human FGF-1 gene and residues 120-155
of the human FGF-2 gene, was constructed by ligation of three DNA
fragments: the 343-bp BspEI-SmaI fragment of
pJS8temp, the 6563-bp BspEI-EspI fragment of
pXZ55, and the EspI-SmaI fragment of pXZ55. The
plasmid pJS7, which contains residues 21-81 and 118-154 of the human
FGF-1 gene and residues 85-120 of the human FGF-2 gene, was prepared
by ligating the 6563-bp EspI-BspEI fragment of
pXZ55 and the 3129-bp EspI-SphI fragment of pXZ55
with the 225-bp BspEI-SphI fragment of pTI70406
(11). The plasmid pJS14, which contains the NLS from the human FGF-2 gene, residues 21-81 from the human FGF-1 gene, and residues 85-155 from the human FGF-2 gene, was constructed by ligating the 4250-bp BamHI-HindIII fragment of pXZ104 (12), the 342-bp
BamHI-PmeI fragment of pJS9, and the 5477-bp
PmeI-HindIII fragment of pJS9. The plasmid pJS10
was constructed by ligating the NdeI-BamHI
fragment of pET3C (15) with the NdeI-BglII
fragment of the polymerase chain reaction product of pTI70301 (11)
generated with the following primers: sense,
5
-TAACATATGGCAGCCGGGAG-3
, and antisense,
5
-CTCAGATCTTCAGCTCTTAGCAGA3
. The plasmid pJS20temp was
constructed by ligating the 6570-bp XhoI-EspI
fragment of pJS14 and the NcoI-EspI fragment of
pXZ55, which contains the
-gal fragment, with the
XhoI-NcoI fragment of the polymerase chain
reaction product of pJS10 generated with the following primers: sense,
5
-GCAGCCTCGAGTTCCCACCGGTCCACT3
, and antisense,
5
-CCATCTTCCTCCATGGCCAGGTAAC-3
.
The plasmid pJS20, which contains the NLS from the human FGF-2 gene,
residues 1-85 from the human FGF-2 gene, and residues 82-154 from the
human FGF-1 gene, was constructed by ligating the 6570-bp
XhoI-EspI fragment of pJS20temp and the 3420-bp
AgeI-EspI fragment of pJS20temp with four
annealed oligonucleotides: sense, 5-TCGAGGATCAAGACCTGGCCCGGGTGCAGCCGGGA-3
; sense,
5
-GCATCACCACGCTGCCCGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCA-3
; antisense,
5
-CCGGTGGGAAGGCGCCGCTGCCGCCATCCTCGGGCAAGGCGGGCAGC-3
; antisense,
5
-GTGGTGATGCTCCCGGCTGCACCCGGGCCAGGTCTTGATCC-3
(AgeI-XhoI ends). The plasmid pJS30, which
contains the sequence from the human FGF-2 gene starting from the
upstream alternative CUG translational site, was constructed by
ligating the NcoI-EspI fragment of pJS14 and the
NcoI-XhoI fragment of pJS20 containing the
amino-terminal half of human FGF-2 with the large
XhoI-EspI fragment of pJS20. The plasmid pJS34,
which contains residues 1-155 from the human FGF-2 gene, was
constructed by ligating the 1699-bp ScaI-SalI fragment of pJS30 and the 8188-bp ScaI-AgeI
fragment of pJS30 with four annealed oligonucleotides: sense,
5
-TCGACGGGACCATGGCAGCCGGGAGCATCACCACGCTGCC-3
; sense,
5
-CGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCA-3
; antisense, 5
-CCGGTGGGAAGGCGCCGCTGCCGCCATCCTC-3
; and antisense,
5
-GGGCAAGGCGGGCAGCGTGGTGATGCTCCCGGCTGCCATGGTCCCG-3
(AgeI-SalI ends).
NIH 3T3 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% (v/v) bovine calf serum (Hyclone), and 1 × antibiotic-antimycotic (Life Technologies). Transfection was performed using the CaPO4 transfection protocol according to the manufacturer's instructions (Stratagene) as described previously (12). Individual transfected clones were selected and expanded by growth in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine calf serum, 1 × antibiotic-antimycotic, and 800 µg/ml geneticin (G418) (Life Technologies). Immunofluorescence was performed on NIH 3T3 cell transfectants grown on fibronectin-coated chamber slides (Nunc Inc.) as described previously (16). X-gal staining was performed on 70% confluent monolayers of NIH 3T3 cells transfected with the plasmid of interest as described previously (16).
Cell Fractionation, Immunoprecipitation, and Immunoblot AnalysisThe subcellular fractionation of NIH 3T3 cell transfectants was performed as described previously (12). Briefly, the cells were scraped, centrifuged, and resuspended in 1 ml of buffer A (15 mM HEPES, pH 7.5, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin). After incubation on ice for 10 min, the cells were centrifuged at 800 × g for 10 min at 4 °C. The supernatants were labeled as cytosol, and the nuclear pellets were resuspended in 1 ml of buffer A and centrifuged (800 × g, 10 min, 4 °C) through a 3-ml sucrose cushion (buffer A containing 350 mM sucrose). The nuclear pellets were resuspended in 1 ml of buffer A containing 0.4% (w/v) SDS, 8 mM MgCl2, and 10 units of DNase and incubated for 5 min at 37 °C followed by 15 min at room temperature. Both the nuclear and cytosol fractions were cleared by centrifugation (14,000 × g, 10 min, 4 °C).
Immunoprecipitation was performed by incubating either the cytosol or
nuclear fractions with 1 µl of anti--gal antibody (5 Prime
3 Prime, Inc., Boulder, CO) and rotating for 1 h at 4 °C, followed by the addition of 20 µl of protein A-Sepharose CL-4B (50%
(w/v); Pharmacia Biotech Inc.) and rotating for 1 h at 4 °C. The Sepharose beads were washed three times with radioimmune
precipitation buffer (10 mM Tris, pH 7.5, containing 150 mM NaCl, 1% Triton X-100 (v/v), 1 mM EDTA,
0.5% (w/v) deoxycholic acid, 0.1% (w/v) SDS) and extracted with 20 µl of 2 × SDS-sample buffer (6.25 mM Tris, 10%
(v/v) glycerol, 3 mM SDS, 1%
-mercaptoethanol, 0.75 mM bromphenol blue, pH 6.8) at 95 °C for 3 min. Samples
were resolved by 7.5% (w/v) SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred to a 0.45-µm Hybond C nitrocellulose
membrane (Amersham Corp.).
Immunoblot analysis was performed by incubating the membranes with TBS
buffer (50 mM Tris, pH 7.4, containing 150 mM
NaCl, 0.1% (v/v) Tween-20 containing 5% (w/v) BSA for 1 h at
room temperature followed by incubation with anti--gal antibody
(1:500 dilution in TBS buffer) for 1 h at room temperature. The
membranes were washed three times with TBS, incubated with horseradish
peroxidase-labeled goat anti-rabbit IgG (Bio-Rad) (1:30,000 dilution)
for 30 min, and washed three times with TBS, and the proteins were
detected using the ECL detection system (Amersham).
Heat shock experiments were performed
as described previously (4). Briefly, NIH 3T3 cell transfectants were
grown on fibronectin-coated plates. The medium was changed to
Dulbecco's modified Eagle's medium (serum-free) prior to heat shock,
and the cells were incubated at 42 °C for 2 h. The conditioned
medium was collected, filtered through a 0.22-µm filter (Corning),
activated with 0.1% (w/v) dithiothreitol for 2 h at 37 °C, and
processed over a 1-ml heparin-Sepharose-4B (Pharmacia) column
equilibrated with 50 mM Tris, pH 7.4, containing 10 mM EDTA (TEB). The column was washed with 50 ml of TEB and eluted with 2.5 ml of 1.5 M NaCl in TEB. The cells were
lysed in phosphate-buffered saline containing 1% (v/v) Triton X-100, sonicated for 15 s, clarified by centrifugation at 4 °C, and
processed over a heparin-Sepharose-4B column as described previously
(5). Both cell lysate and conditioned medium eluates were concentrated through a Centricon 10 (Amicon), resolved by 7.5% (w/v)
SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblot
analysis using anti--gal antibodies. NIH 3T3 cell transfectants
incubated for 2 h at 37 °C were used as controls.
The nomenclature for the human FGF-1 and FGF-2 plasmid discussion utilizes the subscript number to describe the FGF cassette number employed in the construction of the individual reporter gene sequence (10, 11). The residues used to describe the FGF-1 and FGF-2 sequences are based upon the translation of the FGF prototypes as AUG-initiated translation products (1).
We have previously prepared a synthetic human FGF-1 gene by the
ligation of four cassettes encoding the FGF-1 ORF (10) and have
reported that while the ligation of the FGF-1 NLS, NYKKPK (residues
21-26), to the reporter gene, -gal, efficiently translocates the
fusion protein, FGF-1(1):
-gal to the nucleus in
transfected NIH 3T3 cells, the FGF-1(1-2-3-4):
-gal
fusion product remains cytosol-associated (Ref. 12, Fig.
1A-D). Because these data suggest that FGF-1
may contain a domain that is able to repress the intracellular function
of the FGF-1 NLS, we (i) prepared a series of FGF-1:
-gal deletion
constructs (Fig. 1A), (ii) obtained stable NIH 3T3 cell
transfectants, and (iii) assessed the intracellular locale of the
reporter gene by immunofluorescence (Fig. 1B), X-gal staining (Fig. 1C), and subcellular fractionation followed
by anti-
-gal immunoblot analysis (Fig. 1D). While the
deletion of either cassettes 3 and 4 (residues 84-154) or the deletion
of cassette 4 (residues 118-154) does not repress the trafficking of
the reporter gene to the nucleus (Fig. 1, A-D), the
ligation of cassettes 3 and 4 to the FGF-1 NLS in cassette 1 does
repress the trafficking of the reporter gene to the nucleus (Fig. 1,
A-D). These data suggest that the carboxyl-terminal half of
FGF-1 is involved in the cytosol retention of FGF-1, and residues
84-154 may be important for this function.
In order to determine whether the temperature stress-induced FGF
secretion pathway is utilized by FGF-1 and FGF-2, NIH 3T3 cell
FGF-1(1-2-3-4):-gal and
FGF-2(1-2-3-4):
-gal transfectants (Fig.
2A) were examined for their ability to
release the growth factor:reporter gene chimera in response to heat
shock. As shown in Fig. 3, A and
B, the FGF-1(1-2-3-4):
-gal, but not the
FGF-2(1-2-3-4):
-gal chimeric protein, was released in
response to heat shock as a structurally intact polypeptide with an
apparent molecular mass of approximately 118 kDa. These data suggest
that the temperature stress-induced pathway is specific for FGF-1 and
that this pathway can release FGF-1 as a large molecular weight
reporter gene chimeric protein.
Because the temperature stress-induced secretion pathway appears to be
specific for FGF-1, we used a cassette shuffle strategy to assess
whether specific domains are involved in the release of FGF-1. As shown
in Fig. 3, A and B, the substitution of either cassette 3 or 4 from FGF-2 for the corresponding cassette in FGF-1 (Fig. 2A) inhibited the release of the
FGF-1(1-2-3):FGF-2(4):-gal and
FGF-1(1-2-4):FGF-2(3):
-gal chimeric proteins
in response to temperature stress from their respective NIH 3T3 cell
transfectants. In addition, the introduction of cassettes 3 and 4 from
FGF-2 as replacement for their corresponding cassettes in FGF-1 (Fig. 2A) also inhibited the release of
FGF-1(1-2):FGF-2(3-4):
-gal (Fig. 3,
A and B). In addition, the majority of stable NIH
3T3 cell transfectants exhibited similar staining as shown in Fig. 2B by X-gal staining and by
-gal immunofluorescence (data
not shown). These data suggest that the domain comprising residues 84-154 from FGF-1 may be involved in the regulation of FGF-1 secretion and that the corresponding domain in FGF-2 is able to repress the
release of the FGF reporter gene chimera in response to heat shock.
FGF-2 (17, 18) and FGF-3 (19, 20) are able to utilize alternative
5-CUG translation initiation start sites, which are able to direct the
intracellular traffic of these translation products to the nucleus.
Because FGF-1 does not contain this feature (21), we sought to
determine whether the FGF-1 cytosol retention domain would repress the
ability of the multiple NLS domain encoded by the FGF-2 alternative
5
-CUG translational initiation sequence to traffic FGF-1 to the
nucleus. As shown in Fig. 2, B and C, the
addition of this multiple NLS domain to the synthetic FGF-2 gene
inhibited the ability of the reporter gene to remain cytosol-associated and directed the FGF-2:
-gal chimeric protein to the nucleus in the
NIH 3T3 cell transfectants. Likewise, the replacement of cassettes 1 and 2 in FGF-2 with the corresponding cassettes from FGF-1 also enabled
the reporter gene to translocate to the nucleus (Fig. 2, B
and C). However, the replacement cassettes 3 and 4 in FGF-2 with the corresponding cassettes from FGF-1 not only inhibited the
nuclear traffic of the reporter gene (Fig. 2, B and
C) but enabled the chimera to be released from the NIH 3T3
cell transfectants in response to heat shock (Fig. 2D).
These data suggest that the domain in FGF-1 responsible for cytosol
retention is able to repress the nuclear trafficking properties of the
multiple NLS domain present in the alternative 5
-CUG-initiated high
molecular weight FGF-2 translation product.
In order to determine whether nuclear-associated FGF-1 is able to
access the FGF-1 secretion pathway, an additional construct was
prepared in which the entire SV40T ORF was ligated in-frame at the
5-end of the FGF-1:
-gal ORF, and the intracellular traffic and
stress-induced release of the translation product was assessed. As
shown in Figs. 2C and 3, A and B, the
SV40T:FGF-1:
-gal chimera was nucleus-associated and was not released
in response to heat shock following expression in NIH 3T3 cells. These
data suggest that the domain responsible for FGF-1 cytosol retention
may have evolved to ensure participation of FGF-1 in the temperature
stress-induced secretion pathway.
The FGF prototype cassette shuffle strategy has enabled us not only to
confirm the specificity of the FGF-1 secretion pathway induced by
temperature stress (4, 5, 6), but it has also enabled us to determine the
importance between the FGF prototype carboxyl-terminal domains as a
structural correlate for this specificity. Indeed, the similarities
between the FGF prototypes within this domain are accentuated by the
ability of these sequences to act as potential cytosol retention
domains that are able to repress the function of the NLS in their
respective AUG-initiated translation products. However, differences
within this domain between the FGF prototypes are revealed by the
ability of the FGF-1-derived domain to repress the function of the NLS
in the 5-CUG-initiated translation products and the ability of the
FGF-2 domain to repress the release of FGF-1 in response to heat shock.
It is also noteworthy that the carboxyl-terminal domain in FGF-1 is
involved in the association between FGF-1 and phosphatidylserine (6).
Further, our ability to study the secretion of the FGF prototype
homologs as chimeric proteins, as well as the inability of FGF-2 to be secreted under conditions where FGF-1 release is readily apparent, serves to reduce concerns relative to the potential artifactual nature
of this unconventional, brefeldin A-insensitive (5), stress-induced
FGF-1 secretion pathway.
Previous FGF-1 mutagenesis studies have identified the importance of Cys30 in the formation of the stress-induced FGF-1 homodimer (6), and we have proposed that temperature stress may enable Cys30 to gain access to solvent for homodimer formation. Indeed, Cys30 is conserved in FGF-2 (1); yet, interestingly, FGF chimeric proteins containing the carboxyl-terminal domain from FGF-2 also contain Cys30 but are not secreted in response to heat shock. Thus, we suggest that the carboxyl-terminal domain in FGF-2 may prevent solvent accessibility of Cys30 for FGF-2 homodimer formation under temperature stress conditions, and this feature may be involved in determining the specificity between the FGF prototypes for secretion in response to heat shock. This is consistent with the ability of FGF-1 but not FGF-2 to undergo Cu2+-induced homodimer formation (9) and heparin-induced conformational changes (11), although FGF-1 and FGF-2 are both Cu2+-and heparin-binding proteins (9).
The forced nuclear traffic of intracellular FGF-1 by SV40T is also
noteworthy, since it was difficult to obtain stable NIH 3T3 cell
transfectants using the NLS from either the SV40 large T antigen or
histone 2B genes to force the nuclear traffic of intracellular FGF-1 as
a result of their diminished proliferative capacity in vitro
(data not shown). Indeed, this is consistent with the reports that the
nuclear traffic of intracellular FGF-2 (22) and FGF-3 (23) as a result
of alternative 5-CUG translation initiation also results in a
reduction of proliferative capacity in vitro and may help
explain why the AUG-initiated FGF prototype translation products are
localized in the cytosol. Since FGF-1 gains access to the nuclear
compartment as a result of its receptor-dependent traffic
from the cell surface (12), we suggest that the nuclear traffic of
FGF-1 during the entire G1 period of the cell cycle (24)
may function, in part, to restrict endocytotic FGF-1 from participation
in the FGF-1 secretion pathway.