Vol. 11, No. 1
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1991, p. 573-577
0270-7306/91/010573-05$02.00/0
Alternative Initiation of Translation Determines Cytoplasmic or Nuclear Localization of Basic Fibroblast Growth Factor BEATRIX BUGLER,1 FRANCOIS AMALRIC,1* AND HERVE PRATS2 Centre de Recherche de Biochimie et de Genetique Cellulaires du Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse Cedex,1 and Laboratoire d'Endocrinologie Experimentale, C.H. U. Rangueil, Chemin du Vallon, 31054 Toulouse Cedex,2 France Received 20 April 1990/Accepted 1 October 1990 Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUGinitiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.
verted to GUG. The wild-type cDNA products showed cytoplasmic and nuclear staining (Fig. 1, la and b), while the point-mutated cDNA, allowing only synthesis of the CUGinitiated forms (12), gave a nuclear signal (Fig. 1, 2a and b). To discriminate between the different forms of bFGF and to identify the sequence involved in nuclearization, we used a chimeric gene constructed by fusion of complete bFGF and chloramphenicol acetyltransferase (CAT) open reading frames (pSC50; Fig. 1, 3a and b; Fig. 2a and b). In transfected cells, translation of this fused gene started at either one of the bFGF initiation codons and gave rise to three products, p47, p46, and p43, initiated at CUG-1 (C-1), CUG-2 (C-15), and AUG (A-55), respectively, as shown by Western immunoblot detection using a 1:500 dilution of serum against CAT protein (3'-5' Inc.) (Fig. 2d; Fig. 2e, lane 2). As a control, we used cells transfected with plasmid pSC expressing the native CAT protein p26 (Fig. 2d; Fig. 2e, lane 1). By indirect immunofluorescence using anti-CAT antibodies on transfected COS-7 cells, we showed that pSC50 chimeric proteins were both nuclear and cytoplasmic (Fig. 1, 3a and b), while the native CAT protein encoded by plasmid pSC was strictly cytoplasmic (Fig. 1, 4a and b). Taken together, the results of these two experiments strongly suggest that nuclear targeting observed with pSC50-encoded products or with the wild-type bFGF could be due to a signal localized in the NH2-terminal sequence between the CUG-2 and the AUG. A second set of experiments was designed to determine whether the NH2 part of the CUG-initiated forms is sufficient for nuclear targeting. The CAT gene was fused to the bFGF gene (156 residues) initiated at the AUG (pSC70; Fig. 2a and b) or to the 55-residue NH2 terminus upstream of the AUG (pSC3 and pSC6; Fig. 2a and c). These constructs resulted in the synthesis of chimeric proteins initiated at the AUG codon (p43) or either at both (p30 and p29) or only the second (p29) CUG start codon. For all constructs, the synthesized proteins were of the predicted sizes (Fig. 2d and e). The amount of p43 was at least 15 times lower with pSC50 than with pSC70. In pSC70, the 5'-terminal 455 nucleotides of pSC50 and of bFGF cDNA were deleted, suggesting the
Basic fibroblast growth factor (bFGF) promotes the proliferation and differentiation of a wide range of meso- and neuroectoderm-derived cells in vivo and in vitro and is involved in various fundamental processes, including angiogenesis and wound healing (9, 13). In most cases, bFGF is purified from tissues and cell lines as a 146-amino-acid molecule (8). However, this mature polypeptide must derive from longer native forms differing with respect to whether the N terminus initiates at an AUG codon (156 amino acids) or at one of the two CUG codons (195 and 210 amino acids) (12). All three native forms are synthesized with similar efficiencies in transfected COS-7 cells or in a cell-free system by an alternative translational mechanism (6, 12). The largest forms were also detected in various cell lines (14) and probably play a significant role in tissue regeneration (13). When used as exogenous factors, they exhibit identical abilities to promote aortic endothelial cell growth (12; unpublished data). Further insight into the functional significance of the heterogeneity of the bFGF NH2 terminus could be gained by determining the subcellular localization of the different bFGF forms. To examine the behavior of bFGF proteins, COS-7 cells grown on glass coverslips were transfected with bFGF cDNA containing plasmids by the DEAE-dextran method (7). After 70 h, the cells were fixed in 3% paraformaldehyde in phosphate-buffered saline and permeabilized in 0.05% Saponine (Fluka). After preincubation in goat nonimmune serum, the coverslips were incubated for 1 h at 37°C with a 1:50 dilution of serum against bFGF (Oncogene Science Inc.). After washing, immune complexes were detected with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Nordic Immunology) and visualized by fluorescence microscopy under a Leitz Ortholux II microscope after mounting in 50o glycerol. We used two kinds of constructs (12): the wild-type bFGF cDNA with the three putative initiation codons and a pointmutated cDNA in which the AUG initiation codon is con*
Corresponding author. 573
574
MOL. CELL. BIOL.
NOTES
CC n I1
S
CC cc
I
FGF
FGF
CCIR cc A
S
FGF
S
I__
CAT
S
R
CAT
FIG. 1. Subcellular location of bFGF and bFGF-CAT proteins. Shown are phase-contrast (la, 2a, 3a, and 4a) and corresponding fluorescent staining (lb, 2b, 3b, and 4b) micrographs of COS-7 cells transfected with wild-type cDNA (1), point-mutated bFGF cDNA (ATG-+GTG) (2), pSC50 (3), and pSC (4). Corresponding constructs inserted into the pSVL expression vector (Pharmacia) are represented below the micrographs.
existence of a translational control element within this sequence.
Immunodetection of the different proteins clearly established that the pSC70 product (p43) remained cytoplasmic (Fig. 3, la and b), while the pSC3 and pSC6 products (p29 and p30) accumulated in the nucleus of transfected cells (Fig. 3, 2atoc; Fig. 3, 3atoc). In this report, we present strong evidence that the cytoplasmic versus nuclear localization of the different bFGF wild-type or chimeric gene products is determined by the NH2-terminal sequence and is a consequence of an alternative translation mechanism. The protein initiated at the AUG is cytoplasmic, whereas those initiated at either CUG are translocated to the nucleus, suggesting that the N-terminal sequence contains a nuclear localization signal. This sequence is indeed sufficient for translocation of the reporter CAT protein to the nucleus. The amino acid sequence of the bFGF-CAT junction is not involved in a nuclear targeting
signal, since insertion of 20 additional residues at this position did not affect nuclear localization (data not shown). We conclude that residues 15 to 52 of the largest bFGF polypeptide are sufficient to act as a nuclear targeting signal. This shows no sequence similarity to nuclear signals identified so far (5, 10). It should be noted that the proteins initiated at the two CUG codons (positions 1 and 15) are similar in sequence within the first eight residues (underlined in Fig. 4), with only one amino acid replacement, D-3--- G-17. This sequence is present a third time (residues 28 to 35), again with only one change, A-9-*G-35. Acland et al. (2) have recently demonstrated that the subcellular localization of the Int-2 gene product is also controlled by an alternative translational mechanism. For this member of the bFGF family, the presence of an extra N-terminal domain localizes the CUG initiated Int-2 protein to the nucleus, while the AUG-initiated form remains cytoplasmic. We have noted a regular spacing of arginines in the repeated motif found at
NOTES
VOL. 11, 1991
575
a
b
c Xhol Xhol Bssh I Bsshil f
Sacl
.~~~A " Y,5
Apal
Aa TCC CGG CCG GGC CCT GAG Ser Arg Pro Gl0 Pro Glu -_l -_ FGt C AT
6zAT AGO C GCG GOC CCT GAG Al Ly.s Ser Ala Gly Pro GOu -_
-_l FGF
CAT
d
e
Plasmids FGF Initiation codon used
SC SC 70 SC 50
SC6 SC 3
AUG CUG 1 CUG2 AUG CUG2 CUG 1 CUG2
Numbw of FGF residues
Mr by SDS PAGE (10 3)
1 55 210 195 1 55
35 50 35
26 43 47 46 43 29 30 29
M 1 2 3 4 45-
30-
_ I
=:
-43
*_ -26
2014-
FIG. 2. Recombinant plasmid structure and immunodetection of bFGF-CAT fusion proteins. (a) The basic pSVL-derived plasmid, which contains a CAT-encoding sequence, lacking its AUG initiation codon, inserted into the polylinker between the XhoI and Sacl sites. Each bFGF fragment was inserted in frame with the CAT gene between the XhoI and ApaI sites. (b) pSC50, which contains the complete sequences of the bFGF and CAT genes in frame. The resulting fusion proteins are initiated at one of the three initiation codons (C-1, C-15, and A-55). The bold line indicates the 5' noncoding sequence adjacent to the bFGF and CAT gene sequences. pSC70 was derived from pSC50 by a deletion between the XhoI and ApaI sites (the latter is situated 10 nucleotides upstream of the bFGF AUG codon) and encoded only one fusion protein initiated at AUG (A-55). Three amino acids were added between the bFGF and CAT sequences to create an ApaI site. Arrows indicate the bFGF COOH-terminal codon and the first CAT NH2 codon. (c) pSC3, which includes the same noncoding sequence as pSC50 but only the first 52 bFGF NH2-terminal residues in frame with CAT. Fusion proteins are initiated at each of the two CUG codons (C-1 and C-15). pSC6 was derived from pSC3 by a deletion between the two BsshII sites. The resulting encoded protein is initiated only at C-15. (d) Summary of the different potential initiation codons, the number of bFGF residues contained in each fusion protein, and the sizes of the products deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (e) Western blots of fusion proteins. COS-7 cells were transfected with each recombinant plasmid. Expressed proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The fusion proteins were revealed by using antibodies directed against CAT, followed by 1251I-protein A detection and autoradiography. Lane 1 shows the product obtained with pSC encoding only CAT. Subsequent lanes correspond to fusion proteins encoded by pSC50 (lane 2), pSC70 (lane 3), and pSC3 (lane 4). Size standards (in kilodaltons) are shown on the left. Molecular sizes (in kilodaltons) of the six fusion proteins are shown on the right.
the bFGF N terminus; a similar motif is present in the extended N terminus of the Int-2 gene product. In this conserved RGR(X)5R consensus motif (Fig. 4, bold characters) two of the five variant amino acids (X) are always an alanine and a proline. This sequence, also conserved in bovine bFGF (only a partial extended N-terminal sequence is known [1]), is a potential candidate for a nuclear signal. Maher et al. (11) recently have shown that the nuclear targeting signal of the A chain of platelet-derived growth
factor is dependent on alternative splicing. Thus, bFGF represents another example of an endogenous growth factor with two possible subcellular locations regulated at the posttranscriptional level. Together, these data provide new insight into the autocrine regulation of growth factor-producing cells. Another interesting point is the difference in fates of endogenous and exogenous bFGF. We previously showed that bFGF (146 residues) provided to endothelial cells is translocated to the nucleus and accumulates in the nucleo-
576
MOL. CELL. BIOL.
NOTES
AM
I
2
___
FGF
CRT
cc FG
CAT
Iq
L
FGF
3
C
CRT
S
FGF
FIG. 3. Subcellular location of bFGF-CAT proteins. Shown are phase-contrast (la, 2a, and 3a), corresponding fluorescence (lb, 2b, and 3b), and 4,6-diamidino-2-phenylindole staining (2c and 3c) micrographs of COS-7 cells transfected with pSC70 (1), pSC3 (2), and pSC6 (3).
1 6 8 14 (L) G D R G R GR A L P G G R
15
HUMAN bFGF
18 20
(L) G
26
GRGR G R APE R
27 32 34 40 52 V G GRG RG RGT A APR A A PA AR G SR PG P
MOUSE INT-2
1
3
5
11
(L) AR G RV L PA PR L R ET
29
RAGAAA
A AG G RD AG (M)
FIG. 4. Alignment of human bFGF and mouse Int-2 NH2-terminal predicted amino acid sequences. The eight bFGF residues conserved with two modifications, D-3 and G-35, are underlined; the conserved motif between the bFGF and Int-2 gene products is in bold characters; corresponding amino acids in start codons are in
parentheses.
lus, specifically in Gl phase of the cell cycle, despite the absence of the N-terminal sequence containing the targeting signal (3, 4). This finding suggested the existence of different pathways for nuclear or nucleolar translocation, depending on whether bFGF is produced by or provided to the cells. On the other hand, the endogenous bFGF forms were translocated to the nucleus but did not show nucleolar accumulation. Although correlations have been established between nuclear localization of bFGF and cell proliferation (unpublished data), whether the subcellular location of different bFGF forms underlies different functions remains to be elucidated, as do the nuclear targets and their role in cell growth and differentiation. We thank C. Zanibellato for excellent technical assistance, N. Gas for advice on optical microscopy studies, and F. Bayard, M. Chandler, and A. Vincent for critical reading of the manuscript.
VOL . 1 l, 1991
This work was supported by grants from Institut National de la Sante et de la Recherche Medicale. REFERENCES 1. Abraham, J. A., A. Mergia, J. L. Whang, A. Tumolo, J. Friedman, K. A. Hjerrild, D. Gospodarowicz, and J. C. Fiddes.
2. 3.
4.
5. 6.
1986. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233: 545-548. Acland, P., M. Dixon, G. Peters, and C. Dickson. 1990. Subcellular fate of the Int-2 oncoprotein is determined by choice of initiation codon. Nature (London) 343:662-665. Baldin, V., A. M. Roman, I. Bosc-Bierne, F. Amalric, and G. Bouche. 1990. Translocation of bFGF to the nucleus is G1 phase specific in bovine aortic endothelial cells. EMBO. J. 9:15111517. Bouche, G., N. Gas, H. Prats, V. Baldin, J. P. Tauber, J. Teissie, and F. Amalric. 1987. Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing GO- G1 transition. Proc. Natl. Acad. Sci. USA 84:6770-6774. Dang, C. V., and W. M. F. Lee. 1988. Identification of the human c-myc protein nuclear translocation signal. Mol. Cell. Biol. 8:4048-4054. Florkiewiecz, R. Z., and A. Sommer. 1989. Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc. Natl. Acad. Sci. USA 86:3978-3981.
NOTES
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7. Gorman, C. 1985. DNA cloning, vol. 2, p. 143-164. IRL Press, Washington. 8. Gospodarowicz, D., J. Cheng, G. M. Lui, A. Baird, and P. Bohlen. 1984. Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography: identity with pituitary fibroblast growth factor. Proc. Natl. Acad. Sci. USA 81:6963-6967. 9. Gospodarowicz, D., G. Neufeld, and L. Schweigerer. 1986. Fibroblast growth factor. Mol. Cell. Endocrinol. 46:187-204. 10. Loewinger, L., and F. McKeon. 1988. Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm. EMBO. J. 7:2301-2309. 11. Maher, D. W., B. A. Lee, and D. J. Donoghue. 1989. An alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal. Mol. Cell. Biol. 9:2251-2253. 12. Prats, H., M. Kaghad, A. C. Prats, M. Klagsbrun, J. M. Lelias, P. Liauzun, P. Chalon, J. P. Tauber, F. Amalric, J. Smith, and D. Caput. 1989. High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc. Natl. Acad. Sci. USA 86:1836-1840. 13. Presta, M., M. Statuto, M. Rusnati, P. DeU'Era, and G. Ragnotti. 1989. Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver. Biochem. Biophys. Res. Commun. 164:1182-1189. 14. Rifkin, D. B., and D. Moscatelli. 1989. Recent developments in the cell biology of basic fibroblast growth factor. J. Cell Biol. 109:1-6.
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1991, p. 573-577
0270-7306/91/010573-05$02.00/0
Alternative Initiation of Translation Determines Cytoplasmic or Nuclear Localization of Basic Fibroblast Growth Factor BEATRIX BUGLER,1 FRANCOIS AMALRIC,1* AND HERVE PRATS2 Centre de Recherche de Biochimie et de Genetique Cellulaires du Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse Cedex,1 and Laboratoire d'Endocrinologie Experimentale, C.H. U. Rangueil, Chemin du Vallon, 31054 Toulouse Cedex,2 France Received 20 April 1990/Accepted 1 October 1990 Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUGinitiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.
verted to GUG. The wild-type cDNA products showed cytoplasmic and nuclear staining (Fig. 1, la and b), while the point-mutated cDNA, allowing only synthesis of the CUGinitiated forms (12), gave a nuclear signal (Fig. 1, 2a and b). To discriminate between the different forms of bFGF and to identify the sequence involved in nuclearization, we used a chimeric gene constructed by fusion of complete bFGF and chloramphenicol acetyltransferase (CAT) open reading frames (pSC50; Fig. 1, 3a and b; Fig. 2a and b). In transfected cells, translation of this fused gene started at either one of the bFGF initiation codons and gave rise to three products, p47, p46, and p43, initiated at CUG-1 (C-1), CUG-2 (C-15), and AUG (A-55), respectively, as shown by Western immunoblot detection using a 1:500 dilution of serum against CAT protein (3'-5' Inc.) (Fig. 2d; Fig. 2e, lane 2). As a control, we used cells transfected with plasmid pSC expressing the native CAT protein p26 (Fig. 2d; Fig. 2e, lane 1). By indirect immunofluorescence using anti-CAT antibodies on transfected COS-7 cells, we showed that pSC50 chimeric proteins were both nuclear and cytoplasmic (Fig. 1, 3a and b), while the native CAT protein encoded by plasmid pSC was strictly cytoplasmic (Fig. 1, 4a and b). Taken together, the results of these two experiments strongly suggest that nuclear targeting observed with pSC50-encoded products or with the wild-type bFGF could be due to a signal localized in the NH2-terminal sequence between the CUG-2 and the AUG. A second set of experiments was designed to determine whether the NH2 part of the CUG-initiated forms is sufficient for nuclear targeting. The CAT gene was fused to the bFGF gene (156 residues) initiated at the AUG (pSC70; Fig. 2a and b) or to the 55-residue NH2 terminus upstream of the AUG (pSC3 and pSC6; Fig. 2a and c). These constructs resulted in the synthesis of chimeric proteins initiated at the AUG codon (p43) or either at both (p30 and p29) or only the second (p29) CUG start codon. For all constructs, the synthesized proteins were of the predicted sizes (Fig. 2d and e). The amount of p43 was at least 15 times lower with pSC50 than with pSC70. In pSC70, the 5'-terminal 455 nucleotides of pSC50 and of bFGF cDNA were deleted, suggesting the
Basic fibroblast growth factor (bFGF) promotes the proliferation and differentiation of a wide range of meso- and neuroectoderm-derived cells in vivo and in vitro and is involved in various fundamental processes, including angiogenesis and wound healing (9, 13). In most cases, bFGF is purified from tissues and cell lines as a 146-amino-acid molecule (8). However, this mature polypeptide must derive from longer native forms differing with respect to whether the N terminus initiates at an AUG codon (156 amino acids) or at one of the two CUG codons (195 and 210 amino acids) (12). All three native forms are synthesized with similar efficiencies in transfected COS-7 cells or in a cell-free system by an alternative translational mechanism (6, 12). The largest forms were also detected in various cell lines (14) and probably play a significant role in tissue regeneration (13). When used as exogenous factors, they exhibit identical abilities to promote aortic endothelial cell growth (12; unpublished data). Further insight into the functional significance of the heterogeneity of the bFGF NH2 terminus could be gained by determining the subcellular localization of the different bFGF forms. To examine the behavior of bFGF proteins, COS-7 cells grown on glass coverslips were transfected with bFGF cDNA containing plasmids by the DEAE-dextran method (7). After 70 h, the cells were fixed in 3% paraformaldehyde in phosphate-buffered saline and permeabilized in 0.05% Saponine (Fluka). After preincubation in goat nonimmune serum, the coverslips were incubated for 1 h at 37°C with a 1:50 dilution of serum against bFGF (Oncogene Science Inc.). After washing, immune complexes were detected with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Nordic Immunology) and visualized by fluorescence microscopy under a Leitz Ortholux II microscope after mounting in 50o glycerol. We used two kinds of constructs (12): the wild-type bFGF cDNA with the three putative initiation codons and a pointmutated cDNA in which the AUG initiation codon is con*
Corresponding author. 573
574
MOL. CELL. BIOL.
NOTES
CC n I1
S
CC cc
I
FGF
FGF
CCIR cc A
S
FGF
S
I__
CAT
S
R
CAT
FIG. 1. Subcellular location of bFGF and bFGF-CAT proteins. Shown are phase-contrast (la, 2a, 3a, and 4a) and corresponding fluorescent staining (lb, 2b, 3b, and 4b) micrographs of COS-7 cells transfected with wild-type cDNA (1), point-mutated bFGF cDNA (ATG-+GTG) (2), pSC50 (3), and pSC (4). Corresponding constructs inserted into the pSVL expression vector (Pharmacia) are represented below the micrographs.
existence of a translational control element within this sequence.
Immunodetection of the different proteins clearly established that the pSC70 product (p43) remained cytoplasmic (Fig. 3, la and b), while the pSC3 and pSC6 products (p29 and p30) accumulated in the nucleus of transfected cells (Fig. 3, 2atoc; Fig. 3, 3atoc). In this report, we present strong evidence that the cytoplasmic versus nuclear localization of the different bFGF wild-type or chimeric gene products is determined by the NH2-terminal sequence and is a consequence of an alternative translation mechanism. The protein initiated at the AUG is cytoplasmic, whereas those initiated at either CUG are translocated to the nucleus, suggesting that the N-terminal sequence contains a nuclear localization signal. This sequence is indeed sufficient for translocation of the reporter CAT protein to the nucleus. The amino acid sequence of the bFGF-CAT junction is not involved in a nuclear targeting
signal, since insertion of 20 additional residues at this position did not affect nuclear localization (data not shown). We conclude that residues 15 to 52 of the largest bFGF polypeptide are sufficient to act as a nuclear targeting signal. This shows no sequence similarity to nuclear signals identified so far (5, 10). It should be noted that the proteins initiated at the two CUG codons (positions 1 and 15) are similar in sequence within the first eight residues (underlined in Fig. 4), with only one amino acid replacement, D-3--- G-17. This sequence is present a third time (residues 28 to 35), again with only one change, A-9-*G-35. Acland et al. (2) have recently demonstrated that the subcellular localization of the Int-2 gene product is also controlled by an alternative translational mechanism. For this member of the bFGF family, the presence of an extra N-terminal domain localizes the CUG initiated Int-2 protein to the nucleus, while the AUG-initiated form remains cytoplasmic. We have noted a regular spacing of arginines in the repeated motif found at
NOTES
VOL. 11, 1991
575
a
b
c Xhol Xhol Bssh I Bsshil f
Sacl
.~~~A " Y,5
Apal
Aa TCC CGG CCG GGC CCT GAG Ser Arg Pro Gl0 Pro Glu -_l -_ FGt C AT
6zAT AGO C GCG GOC CCT GAG Al Ly.s Ser Ala Gly Pro GOu -_
-_l FGF
CAT
d
e
Plasmids FGF Initiation codon used
SC SC 70 SC 50
SC6 SC 3
AUG CUG 1 CUG2 AUG CUG2 CUG 1 CUG2
Numbw of FGF residues
Mr by SDS PAGE (10 3)
1 55 210 195 1 55
35 50 35
26 43 47 46 43 29 30 29
M 1 2 3 4 45-
30-
_ I
=:
-43
*_ -26
2014-
FIG. 2. Recombinant plasmid structure and immunodetection of bFGF-CAT fusion proteins. (a) The basic pSVL-derived plasmid, which contains a CAT-encoding sequence, lacking its AUG initiation codon, inserted into the polylinker between the XhoI and Sacl sites. Each bFGF fragment was inserted in frame with the CAT gene between the XhoI and ApaI sites. (b) pSC50, which contains the complete sequences of the bFGF and CAT genes in frame. The resulting fusion proteins are initiated at one of the three initiation codons (C-1, C-15, and A-55). The bold line indicates the 5' noncoding sequence adjacent to the bFGF and CAT gene sequences. pSC70 was derived from pSC50 by a deletion between the XhoI and ApaI sites (the latter is situated 10 nucleotides upstream of the bFGF AUG codon) and encoded only one fusion protein initiated at AUG (A-55). Three amino acids were added between the bFGF and CAT sequences to create an ApaI site. Arrows indicate the bFGF COOH-terminal codon and the first CAT NH2 codon. (c) pSC3, which includes the same noncoding sequence as pSC50 but only the first 52 bFGF NH2-terminal residues in frame with CAT. Fusion proteins are initiated at each of the two CUG codons (C-1 and C-15). pSC6 was derived from pSC3 by a deletion between the two BsshII sites. The resulting encoded protein is initiated only at C-15. (d) Summary of the different potential initiation codons, the number of bFGF residues contained in each fusion protein, and the sizes of the products deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (e) Western blots of fusion proteins. COS-7 cells were transfected with each recombinant plasmid. Expressed proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The fusion proteins were revealed by using antibodies directed against CAT, followed by 1251I-protein A detection and autoradiography. Lane 1 shows the product obtained with pSC encoding only CAT. Subsequent lanes correspond to fusion proteins encoded by pSC50 (lane 2), pSC70 (lane 3), and pSC3 (lane 4). Size standards (in kilodaltons) are shown on the left. Molecular sizes (in kilodaltons) of the six fusion proteins are shown on the right.
the bFGF N terminus; a similar motif is present in the extended N terminus of the Int-2 gene product. In this conserved RGR(X)5R consensus motif (Fig. 4, bold characters) two of the five variant amino acids (X) are always an alanine and a proline. This sequence, also conserved in bovine bFGF (only a partial extended N-terminal sequence is known [1]), is a potential candidate for a nuclear signal. Maher et al. (11) recently have shown that the nuclear targeting signal of the A chain of platelet-derived growth
factor is dependent on alternative splicing. Thus, bFGF represents another example of an endogenous growth factor with two possible subcellular locations regulated at the posttranscriptional level. Together, these data provide new insight into the autocrine regulation of growth factor-producing cells. Another interesting point is the difference in fates of endogenous and exogenous bFGF. We previously showed that bFGF (146 residues) provided to endothelial cells is translocated to the nucleus and accumulates in the nucleo-
576
MOL. CELL. BIOL.
NOTES
AM
I
2
___
FGF
CRT
cc FG
CAT
Iq
L
FGF
3
C
CRT
S
FGF
FIG. 3. Subcellular location of bFGF-CAT proteins. Shown are phase-contrast (la, 2a, and 3a), corresponding fluorescence (lb, 2b, and 3b), and 4,6-diamidino-2-phenylindole staining (2c and 3c) micrographs of COS-7 cells transfected with pSC70 (1), pSC3 (2), and pSC6 (3).
1 6 8 14 (L) G D R G R GR A L P G G R
15
HUMAN bFGF
18 20
(L) G
26
GRGR G R APE R
27 32 34 40 52 V G GRG RG RGT A APR A A PA AR G SR PG P
MOUSE INT-2
1
3
5
11
(L) AR G RV L PA PR L R ET
29
RAGAAA
A AG G RD AG (M)
FIG. 4. Alignment of human bFGF and mouse Int-2 NH2-terminal predicted amino acid sequences. The eight bFGF residues conserved with two modifications, D-3 and G-35, are underlined; the conserved motif between the bFGF and Int-2 gene products is in bold characters; corresponding amino acids in start codons are in
parentheses.
lus, specifically in Gl phase of the cell cycle, despite the absence of the N-terminal sequence containing the targeting signal (3, 4). This finding suggested the existence of different pathways for nuclear or nucleolar translocation, depending on whether bFGF is produced by or provided to the cells. On the other hand, the endogenous bFGF forms were translocated to the nucleus but did not show nucleolar accumulation. Although correlations have been established between nuclear localization of bFGF and cell proliferation (unpublished data), whether the subcellular location of different bFGF forms underlies different functions remains to be elucidated, as do the nuclear targets and their role in cell growth and differentiation. We thank C. Zanibellato for excellent technical assistance, N. Gas for advice on optical microscopy studies, and F. Bayard, M. Chandler, and A. Vincent for critical reading of the manuscript.
VOL . 1 l, 1991
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