Vol. 10, No. 9
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4565-4573 0270-7306/90/094565-09$02.00/0 Copyright © 1990, American Society for Microbiology
Domain Swapping Reveals the Modular Nature of Fos, Jun, and CREB Proteins LYNN J. RANSONE, PENNY WAMSLEY, KIMBERLIN L. MORLEY, AND INDER M. VERMA* Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138 Received
5
April 1990/Accepted 4 June 1990
The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-l-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.
and DNA binding; this demonstrates that dimerization is required for DNA binding. Specific contact with DNA is mediated by a highly charged basic region just amino terminal of the leucine zipper (11, 13, 19, 28, 32a, 45). Because mutations in the basic domain affect DNA binding and can alter protein-protein affinities, we suggest that the leucine zipper and basic region are interdependent for optimal functional activity (L. Ransone, P. Wamsley, K. L. Morley, and I. M. Verma, submitted for publication). To assess the role of these two domains in the dimerization and DNA binding of three related proteins, Fos, Jun, and CREB, we carried out an extensive domain swap analysis. We show that (i) the specificity of dimer formation is determined by the leucine zipper, (ii) even though the Fos protein does not bind to a specific DNA sequence, a heterodimer containing two Fos basic regions can efficiently bind to an AP-1 site; (iii) chimeric proteins containing only Jun leucine zippers display weak binding to their cognate DNA sequence; (iv) residues outside the basic domain and leucine zipper contribute to DNA-binding affinities; and (v) Fos-Jun chimeric constructs cooperate in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.
The products of two cellular proto-oncogenes, Fos and Jun, appear to play a central role in the regulation of cell growth (7, 12, 21, 23, 31, 35, 36, 40). These proteins belong to a growing family of transcriptional regulators that include the cyclic AMP response element-binding protein (CREB) (14, 16), the yeast GCN4 protein (17), and C/EBP enhancerbinding protein (22). These transcription factors form dimeric complexes which recognize and bind to their specific cognate DNA sequences. Jun proteins will bind either as homodimers or as heterodimers with Fos to the three consensus sequences TGACATCA (an AP-1 site) (4, 20a), TGACTCA (a 12-O-tetradecanoylphorbol-13-acetate [TPA] response element [TRE]) (3, 12, 33), and TGACGTCA (a cyclic AMP response element) (25, 33). The CREB protein recognizes both the cyclic AMP response element and the AP-1 elements (16), while Fos alone does not bind to either site (15, 19, 25, 38). Fos-Jun heterodimers bind to AP-1 sites with a greater affinity than do Jun homodimers (5, 6, 25, 34, 38). This could be due to the increased stability of the Fos-Jun heterodimers compared with the Jun homodimer (13, 15, 34, 38) or to an increase in the affinity for DNA contributed by the Fos protein. Two domains of these proteins are required for the combined functions of proteinprotein and protein-DNA complex formation. The leucine zipper domain mediates the interaction between Fos-Jun heterodimers and between Jun and CREB homodimers; it consists of either four or five leucine residues separated at intervals of seven amino acids. The leucines are predicted to align along the hydrophobic face of an a-helix (22). Two helices are thought to form a bimolecular coiled-coil structure (29), and in the case of Fos and Jun they facilitate the formation of a very stable heterodimeric complex (11, 13, 19, 32, 38, 41, 45). Substitutions that change one or more of the residues in the leucine zipper can abolish dimer formation *
MATERIALS AND METHODS Construction and expression of chimeric plasmids. To generate Jun-Fos, CREB-Fos, and CREB-Jun chimeric cDNAs, we introduced restriction sites by site-specific mutagenesis in Fos, Jun, and CREB cDNA sequences flanking the basic region and leucine zipper domain as shown in Fig. 1 (32). The appropriate fragments were isolated, ligated, and back mutated to remove the introduced restriction site. The chimeric cDNAs were subcloned into pGem3 or pGem4 for in vitro RNA synthesis and into eucaryotic expression vectors (37, 38) for transient-transfection assays. Transcription and translation of the in vitro products were performed as previously described (32).
Corresponding author. 4565
4566
MOL. CELL. BIOL.
RANSONE ET AL.
A.
Jun Fos
CREB
Leucirn
NH2-Termius
Basic Region
J1.-2s
J256-282
PEP 2 Ab
PEP 1 Ab
FN-137
F138-164
NAb
MAb
C1-28
C284-310
C311-332
C333-341
341 aa
J F J C F
J J J J
J J J J F
212 aa
F
F
498 aa
F
F
526 aa
Zipper
J283-311
COOH-Termins
J312-333
333 aa 380 aa
W28 Ab
B. Chimeras
FJJJ FFJJ CJJJ
ccJJ JFFF JJFF CFFF
F F C C J J C
F
212 aa
358 aa 358 aa 498 aa
FIG. 1. Chimeric Fos, Jun, and CREB proteins. (A) Schematic diagram and nomenclature of protein domains used in the construction of chimeric proteins. The amino acids included in each domain are indicated (aa). The antibodies used in protein association are also indicated and are as follows: Jun, Pep 1, and Pep 2 (6); Fos and N and M antibodies (10); CREB and W28 antibody (14). (B) Chimeric protein constructs generated from fragments shown in panel A which contain either a Jun or Fos leucine zipper and carboxy terminus. The number of amino acids encoded by each chimeric construct is indicated.
Protein association and DNA-binding assays. Protein association, gel shift assays, and glutaraldehyde cross-linking assays were performed as previously described (9). The antibodies used in the protein association studies are indicated in Fig. 1. DNA transfection and transient-expression assay. NIH-3T3 cells were plated in Dulbecco modified Eagle medium-10%o calf serum, at 5 x 105 cells per 10-cm tissue culture dish, 24 h before DNA transfection. Cells were transfected by the calcium phosphate coprecipitation technique (40) and exposed to the precipitate for 12 h. After the cells had been washed with phosphate-buffered saline, fresh medium containing 0.5% calf serum was added, and the cells were harvested after 24 h. When less than 20 pug of specific DNA was used per 10-cm culture dish, pIBI31 plasmid DNA was added to give 20 ,ug of total DNA. Chloramphenicol acetyltransferase (CAT) activity was determined as described previously (40). RESULTS Dimer formation by chimeric proteins. To generate FosJun, Fos-CREB, and Jun-CREB chimeras, we constructed plasmids which contained restriction endonuclease cleavage sites introduced by site-directed mutagenesis at the junctions between the amino terminus, basic region, and leucine zipper regions of each protein as described in Materials and Methods. These insertions, which correspond to amino acids 255 and 282 in Jun, 137 and 164 in Fos, and 283 and 310 in CREB, allowed for the construction of the chimeric proteins
diagrammed in Fig. 1. The chimeric proteins were designed such that no additional amino acids were generated between the domains, and they were subsequently synthesized by translation of in vitro transcripts in rabbit reticulocyte lysates. The translation efficiency of each of the constructs was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methionine-labeled proteins before and after immunoprecipitation with the appropriate antibody (Fig. 1). To compare the dimerization properties of proteins containing either Jun or Fos leucine zipper domains, "S-labeled in vitro-translated chimeric proteins were cross-linked with glutaraldehyde and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results (Fig. 2A) demonstrate that all of the proteins which contained a Jun leucine zipper domain were able to form homodimers. Interestingly, proteins which contained either a Fos (FJJJ; lane 4) or CREB (CJJJ; lane 8) amino terminus juxtaposed to a Jun basic region or a Fos amino terminus and basic region adjacent to a Jun leucine zipper (FFJJ; lane 6) were less efficient at dimer formation than was either a chimeric construct containing a CREB amino terminus followed by a CREB basic region (CCJJ; lane 10) or a wild-type Jun protein (lane 2). In contrast, all constructs which contain a Fos leucine zipper (Fig. 2B) were unable to form homodimers, as previously observed (19, 20, 26, 30). Constructs containing either a Fos or Jun leucine zipper were able to form a heterodimeric protein complex with one another as predicted and could be immunoprecipitated with the appropriate antibody (data not shown). We conclude that although the
Fos, Jun, AND CREB
VOL. 10, 1990
A. h MW
kd 200-
FFJJ JJU-U FJJ + + - + FJJ
CCJJ CJJJ r-z- +11 - + Glutaraldehyde
9
-1
97-_
68-6845-
.A
-k4jjjb. Irv
29-
q"m
_
182
1
3
4
5
6
7
8
9
10
B. MW
kd 200
FOS
I_
+
JFFF
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4565-4573 0270-7306/90/094565-09$02.00/0 Copyright © 1990, American Society for Microbiology
Domain Swapping Reveals the Modular Nature of Fos, Jun, and CREB Proteins LYNN J. RANSONE, PENNY WAMSLEY, KIMBERLIN L. MORLEY, AND INDER M. VERMA* Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138 Received
5
April 1990/Accepted 4 June 1990
The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-l-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.
and DNA binding; this demonstrates that dimerization is required for DNA binding. Specific contact with DNA is mediated by a highly charged basic region just amino terminal of the leucine zipper (11, 13, 19, 28, 32a, 45). Because mutations in the basic domain affect DNA binding and can alter protein-protein affinities, we suggest that the leucine zipper and basic region are interdependent for optimal functional activity (L. Ransone, P. Wamsley, K. L. Morley, and I. M. Verma, submitted for publication). To assess the role of these two domains in the dimerization and DNA binding of three related proteins, Fos, Jun, and CREB, we carried out an extensive domain swap analysis. We show that (i) the specificity of dimer formation is determined by the leucine zipper, (ii) even though the Fos protein does not bind to a specific DNA sequence, a heterodimer containing two Fos basic regions can efficiently bind to an AP-1 site; (iii) chimeric proteins containing only Jun leucine zippers display weak binding to their cognate DNA sequence; (iv) residues outside the basic domain and leucine zipper contribute to DNA-binding affinities; and (v) Fos-Jun chimeric constructs cooperate in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.
The products of two cellular proto-oncogenes, Fos and Jun, appear to play a central role in the regulation of cell growth (7, 12, 21, 23, 31, 35, 36, 40). These proteins belong to a growing family of transcriptional regulators that include the cyclic AMP response element-binding protein (CREB) (14, 16), the yeast GCN4 protein (17), and C/EBP enhancerbinding protein (22). These transcription factors form dimeric complexes which recognize and bind to their specific cognate DNA sequences. Jun proteins will bind either as homodimers or as heterodimers with Fos to the three consensus sequences TGACATCA (an AP-1 site) (4, 20a), TGACTCA (a 12-O-tetradecanoylphorbol-13-acetate [TPA] response element [TRE]) (3, 12, 33), and TGACGTCA (a cyclic AMP response element) (25, 33). The CREB protein recognizes both the cyclic AMP response element and the AP-1 elements (16), while Fos alone does not bind to either site (15, 19, 25, 38). Fos-Jun heterodimers bind to AP-1 sites with a greater affinity than do Jun homodimers (5, 6, 25, 34, 38). This could be due to the increased stability of the Fos-Jun heterodimers compared with the Jun homodimer (13, 15, 34, 38) or to an increase in the affinity for DNA contributed by the Fos protein. Two domains of these proteins are required for the combined functions of proteinprotein and protein-DNA complex formation. The leucine zipper domain mediates the interaction between Fos-Jun heterodimers and between Jun and CREB homodimers; it consists of either four or five leucine residues separated at intervals of seven amino acids. The leucines are predicted to align along the hydrophobic face of an a-helix (22). Two helices are thought to form a bimolecular coiled-coil structure (29), and in the case of Fos and Jun they facilitate the formation of a very stable heterodimeric complex (11, 13, 19, 32, 38, 41, 45). Substitutions that change one or more of the residues in the leucine zipper can abolish dimer formation *
MATERIALS AND METHODS Construction and expression of chimeric plasmids. To generate Jun-Fos, CREB-Fos, and CREB-Jun chimeric cDNAs, we introduced restriction sites by site-specific mutagenesis in Fos, Jun, and CREB cDNA sequences flanking the basic region and leucine zipper domain as shown in Fig. 1 (32). The appropriate fragments were isolated, ligated, and back mutated to remove the introduced restriction site. The chimeric cDNAs were subcloned into pGem3 or pGem4 for in vitro RNA synthesis and into eucaryotic expression vectors (37, 38) for transient-transfection assays. Transcription and translation of the in vitro products were performed as previously described (32).
Corresponding author. 4565
4566
MOL. CELL. BIOL.
RANSONE ET AL.
A.
Jun Fos
CREB
Leucirn
NH2-Termius
Basic Region
J1.-2s
J256-282
PEP 2 Ab
PEP 1 Ab
FN-137
F138-164
NAb
MAb
C1-28
C284-310
C311-332
C333-341
341 aa
J F J C F
J J J J
J J J J F
212 aa
F
F
498 aa
F
F
526 aa
Zipper
J283-311
COOH-Termins
J312-333
333 aa 380 aa
W28 Ab
B. Chimeras
FJJJ FFJJ CJJJ
ccJJ JFFF JJFF CFFF
F F C C J J C
F
212 aa
358 aa 358 aa 498 aa
FIG. 1. Chimeric Fos, Jun, and CREB proteins. (A) Schematic diagram and nomenclature of protein domains used in the construction of chimeric proteins. The amino acids included in each domain are indicated (aa). The antibodies used in protein association are also indicated and are as follows: Jun, Pep 1, and Pep 2 (6); Fos and N and M antibodies (10); CREB and W28 antibody (14). (B) Chimeric protein constructs generated from fragments shown in panel A which contain either a Jun or Fos leucine zipper and carboxy terminus. The number of amino acids encoded by each chimeric construct is indicated.
Protein association and DNA-binding assays. Protein association, gel shift assays, and glutaraldehyde cross-linking assays were performed as previously described (9). The antibodies used in the protein association studies are indicated in Fig. 1. DNA transfection and transient-expression assay. NIH-3T3 cells were plated in Dulbecco modified Eagle medium-10%o calf serum, at 5 x 105 cells per 10-cm tissue culture dish, 24 h before DNA transfection. Cells were transfected by the calcium phosphate coprecipitation technique (40) and exposed to the precipitate for 12 h. After the cells had been washed with phosphate-buffered saline, fresh medium containing 0.5% calf serum was added, and the cells were harvested after 24 h. When less than 20 pug of specific DNA was used per 10-cm culture dish, pIBI31 plasmid DNA was added to give 20 ,ug of total DNA. Chloramphenicol acetyltransferase (CAT) activity was determined as described previously (40). RESULTS Dimer formation by chimeric proteins. To generate FosJun, Fos-CREB, and Jun-CREB chimeras, we constructed plasmids which contained restriction endonuclease cleavage sites introduced by site-directed mutagenesis at the junctions between the amino terminus, basic region, and leucine zipper regions of each protein as described in Materials and Methods. These insertions, which correspond to amino acids 255 and 282 in Jun, 137 and 164 in Fos, and 283 and 310 in CREB, allowed for the construction of the chimeric proteins
diagrammed in Fig. 1. The chimeric proteins were designed such that no additional amino acids were generated between the domains, and they were subsequently synthesized by translation of in vitro transcripts in rabbit reticulocyte lysates. The translation efficiency of each of the constructs was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methionine-labeled proteins before and after immunoprecipitation with the appropriate antibody (Fig. 1). To compare the dimerization properties of proteins containing either Jun or Fos leucine zipper domains, "S-labeled in vitro-translated chimeric proteins were cross-linked with glutaraldehyde and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results (Fig. 2A) demonstrate that all of the proteins which contained a Jun leucine zipper domain were able to form homodimers. Interestingly, proteins which contained either a Fos (FJJJ; lane 4) or CREB (CJJJ; lane 8) amino terminus juxtaposed to a Jun basic region or a Fos amino terminus and basic region adjacent to a Jun leucine zipper (FFJJ; lane 6) were less efficient at dimer formation than was either a chimeric construct containing a CREB amino terminus followed by a CREB basic region (CCJJ; lane 10) or a wild-type Jun protein (lane 2). In contrast, all constructs which contain a Fos leucine zipper (Fig. 2B) were unable to form homodimers, as previously observed (19, 20, 26, 30). Constructs containing either a Fos or Jun leucine zipper were able to form a heterodimeric protein complex with one another as predicted and could be immunoprecipitated with the appropriate antibody (data not shown). We conclude that although the
Fos, Jun, AND CREB
VOL. 10, 1990
A. h MW
kd 200-
FFJJ JJU-U FJJ + + - + FJJ
CCJJ CJJJ r-z- +11 - + Glutaraldehyde
9
-1
97-_
68-6845-
.A
-k4jjjb. Irv
29-
q"m
_
182
1
3
4
5
6
7
8
9
10
B. MW
kd 200
FOS
I_
+
JFFF
