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Fos and jun: The AP-1 connection
Cell, %I. 55, 395-397, November 4, 1988, Copyright 0 1988 by Cell Press
un: The AP-1 Connection
Tom Curran” and B. Robert Franza, Jr.? * Department of Molecular Oncology Roche Institute of Molecular Biology utley, New Jersey 07110 rCold Spring Harbor Laboratory Cold Spring Harbor, New York 11724
Oncogene research has led to the isolation of specific reagents that have allowed important insights in several disciplines. Recently, studies on oncogenes encoding nuclear proteins have united with those concerning the regulation of eukaryotic gene transcription. ed to Yeast Transcription Factor GCN4 gene products have been proposed to play a role in gene regulation. A solid link to a bona fide transcription factor was made by Vogt et al. (PNAS 84, 3316-3319, 1987) who reported a convincing similarity (44%) between a region of the protein encoded by the jun oncogene, which is responsible for induction of fibrosarcomas in chickens by avian sarcoma virus 17 (Maki et al., PNAS 84, 2848-2852, 1987), and the DNA-binding domain of the yeast transcriptional activator GCN4. GCN4 induces transcription of several genes involved in amino acid biosynthesis (Hope and Struhl, Cell 46, 885-894, 1986) and binds as a dimer to the target sequence ATGA(C/G)TCAT (Hill et al., Science 234, 451-457, 1986; Hope and Struhl, EMBO J. 6, 2781-2784, 1987). Recently, several other yeast proteins have been identified that recognize the same binding sites (Harshman et al., Cell 53, 321-330, 1988; Jones et al., Cell 53, 659-667, 1988). in addition, the DNA-binding domain of GCN4 can be replaced by the region of similarity in Jun to give a functional transcriptional activator (Struhl, Nature 332, 64950, 1988). un Is One of the Proteins Present in AP-7 Preparations The consensus binding site of GCN4 is closely related to that of the mammalian transcription factor AP-1 (Angel et al., Cell 49, 729-739, 1987; Lee et al., Cell 49, 741-752, 1987; Piette and Yaniv, EMBO J. 6, 1331-1337, 1987). Similarities between Jun and GCN4 stimulated an investigation of the relationship between Jun and AP-1 (Bohmann et al., Science 238, 1386-1392, 1987). Transcription factor AP-1 (activator protein-l) was described initially as a ~NA-binding activity in HeLa cell extracts that specifically recognizes the enhancer elements of SV40 and the human metallothionein IIA (hMIIA) gene (Lee et al., Nature 325, 368-372, 1987). AP-1 binding sites also occur in the control regions of viral and cellular genes that are stimulated by treatment of cells with phorbol ester (Angel et al., Lee etal., Cell, op, cit.). Indeed, tandemly linked API binding sites mediate the elevated transcription of heteroiogous genes after phorbol ester treatment. Antisera raised against two v-jun peptides, PEP1 (amino acids 209-225) and PEP2 (amino acids 73-87) (Bos et al., Cell 52, 705712! 1988) were used to examine affinity-
inireview
purified preparations of AP-1. in imm ot assays both antisera recognized a single major po tide (Bohmann et al., Science, op. cit.). Furthermore, sequencing of AP-1 tryptic peptides revealed that several were identical to portions of the predicted amino acid sequence of Jun. These data, together with the finding that a Jun polypepetide expressed in E. coli binds to the AP-1 siie (Bohmann et al., Science, op. cit.; Angel et al., Nature 332, 766-171, 1988), provided compelling evidence identifying Jun as AP-1. However, AP-1 preparations contain several proteins (Lee et al., Cell, op. cit.), and many of the AP-I peptides sequenced are unrelated to Jun. Indeed, some of the AP-1 peptides have now been identified in the predicted protein sequence of jun-S, a jun-related gene (Bohmann et al., Cold Spring Harbor Symp. Quant. Biol. 53, in press; Ryder et al., PNAS 85, 1487-1491, 1988). Thus, Jun is only one of the proteins that contribute to AP-3 activity and it should not be referred to as transcription factor API. In fact, it is unclear how many cellular proteins regulate transcription via AP-1 sequence elements. dun Is the Fos-associated Protein p3 Several other proteins covered by the umbrella term AP-1 have subsequently been identified as a result of studies on another oncogene product, Fos, which is present in complexes that recognize a specific nucleic acid regulatory element. The fos oncogene (v-fas) is responsible for the induction of osteogenic sarcomas by the FBJ murine sarcoma virus (Curran and Teich, J. Virol. 42, 114-122, 1982). Its normal cellular homolog, c-fos, encodes a nuclear protein (Fos) that undergoes extensive posttranslational modification and participates in protein complexes with a 39,000 dalton protein (p39) (Curran et al., Cell 36, 259-268, 1984; Curran et al., MCB 5, 167-172, 1985). Fos belongs to a family of related proteins whose expression is induced by a variety of extracellular stimuli (Franza et al., Oncogene 7,213-221, 1987; Cohen and Curran, MC6 8,2063-2069, 1988). Fos-p39 complexes are associated with chromatin in isolated nuclei and bind to DNA cellulose in vitro (Sambucetti and Curran, Science 234, 1417-1419, 1986). These features, together with the observation of a transactivation property associated with v-fos (Setoyama et al., PNAS 83, 3213-3217, 1986), led to the proposal that Fos is involved in gene regulation. The observation by Distel et al. (Cell 49,835~844, 1987) of a protein-DNA complex whose formation was inhibited by anti-Fos antibodies suggested that Fos interacts with a regulatory element of a gene specific for adipocyte differentiation. A combination of studies involving mutagenesis, competition analysis, and DNA-affinity precipitation (Franza et al., Nature 330, 393-395, 1987) revealed that Fos binds to the AP-1 consensus recognition sequence (Franza et al., Science 239,1150-i 153,1988; Rauscher et al., Cell 52, 471-480, 1988). Indeed, variations of the AP-1 site were found to bind several other proteins, including Fos-related antigens and ~39. While these studies estabiished a connection between Fos, Jun and A,P-1, they did lot establish the nature of the connection-that is,
Ceil 396
whether Fos and Fos-related proteins bind to the API site directly, indirectly by way of association with p39, or by interaction with Jun or other AP-1 polypeptides. Subsequent studies revealed that, at least in part, all of these possibilities are true. The availability of Jun-specific antibodies (80s et al., op. cit.) permitted immunological, gel-mobility and structural comparisons that identified p39, the Fosassociated protein, as the product of the jun protooncogene (Rauscher et al., Science 240, lOlO-1016, 1988). Similar studies by other investigators confirmed this identity (Chiu et al., Cell 54,541-552, 1988; SassoneCorsi et al., Cell 54, 553-560, 1988; Lamph et al., Nature 334, 629-631, 1988). OS and Several Fos-related Antigens Are Also P-7 Proteins p39 (now referred to as p3gc-jun)was described originally as a FBJ-MSV transformation-associated protein (Curran and Teich, Virology 776,221-2351982). It was later shown to form an extremely stable complex with v-Fos and c-Fos in the nucleus (Curran et al., op. cit.). The identification of p39 as the c-jun product implied that Fos might also be present in affinity-purified preparations of AP-1. Indeed, Fos and several Fos-related antigens (Fras) were detected in AP-1 preparations by immunoblot analysis (Rauscher et al., Science, op. cit.) and antibodies against the conserved domain of Fos reduced the DNA-binding and transcriptional stimulation properties of AP-1 (Bohmann et al., Cold Spring Harbor Symp. Quant. Biol., op. cit.). The amount of Fos detected in AP-1 preparations was quite low; however, these AP-1 proteins were purified from unstimulated HeLa cells that express low levels of Fos. In contrast, three Fras were present at relatively high levels (one was apparently more abundant than Jim). DNAaffinity assays (Miskimins et al., PNAS 82, 6741-6744, 1985) performed using radiolabelled AP-1 oligonucleotides, suggested that p39c-jon,one of the Fras, and an 80 kd protein bind to DNA directly and specifically (Rauscher et al., Science, op. cit.). Thus, more than one cellular protein can interact directly with AP-1 binding sites. The cooperative or competitive nature of these interactions remains to be determined. Role of Fos and Jun in Gene Regulation Both Fos and Jun accumulate to relatively high levels after cell stimulation (Franza et al., Science, op. cit.; Lamph et al., Nature, op. cit.; Rauscher et al., Science, op. cit.; Ryseek et al., Nature 334, 535-537, 1988; Quantin and Breathnach, Nature 334, 538-539, 1988). However, the levels of both proteins decline long before the major increase in AP-1 activity observed in cells treated with phorbol ester (Angel et al., Cell, op. cit.). Thus, these two proios-related gene fra-7 and the jun-related gene jun-B are fos-related gene fra-7 and the jun-related gene jum3 are also induced by serum stimulation and other treatments (Cohen and Curran, op. cit.; Ryder et al., op. cit.). In these cases, the kinetics of mRNA appearance are slower than that of c-fos. Similarly, several Fos-related proteins that are induced by serum persist in stimulated cells much longer than Fos (Cohen and Curran, op cit.). AP-1, therefore, corresponds to at least two groups of inducible genes encoding proteins whose function may be to mediate specific
alterations in gene transcription in response to environ mental cues. These proteins accumulate in a tissue-specific and temporally prescribed fashion and interact with AP-1 sites. Fos complexes may possess both transcriptional activation and repression properties according to transient transfection assays (Schonthai et al., Cell 54, 325-334, 1988; Chiu et al., op. cit.; Sassone-Corsi et al., Cell, op. cit.; Sassone-Corsi et al., Nature 334, 314-319, 1988). At present, it is not known whether particular AP-1 proteins and protein complexes recognize specific variants of the AP-1 site. Similarly, the role of posttranslational modification in determining specificity of Fos and Jun is not known. It is interesting to note that the CAMP response element (CRE) (Montminy et al., PNAS 83,6682-6686, 1986), also known as the ATF binding site (Lee et al., PNAS 84, 8355-8359,1987), is highly related to several AP-1 binding sites. Nuclear proteins called CREB or ATF, which are apparently neither Fos nor Jun, have been isolated by oligonucleotide affinity chromoatography using the CRE (Hurst and Jones, Genes Dev. 7, 1132-1146, 1987; Yamamoto et al., Nature 334, 494-498, 1988). These proteins can bind to AP-1 sites, but with a 5 to lo-fold lower affinity than to the CRE. Yamamoto et al. suggest that CREB binds to the CRE and stimulates transcription as a dimer and that phosphorylation promotes dimerization. Growing evidence from studies in both prokaryotic and eukaryotic systems supports a model in which transcription factors interact as dimers with half-sites of palindromic recognition sequences. Heterodimeric complexes may have altered affinities for one of these half-sites (although the consensus elements are usually palindromic, binding sites in the natural context are often imperfect palindromes). In this way a single recognition element could interact with a hierarchy of heterodimeric factors with differing activities. As described above, cell-surface stimulation results in increased synthesis of several Fosand Jun-related proteins that associate with AP-1 sites. These proteins appear and are turned over with different kinetics. Thus, the rate of transcriptional initiation of specific target genes may depend upon the concentrations and relative affinities of these proteins for homo- or heterodimeric protein complex formation. How Do Fos and Jun Physically Interact? Speculations such as these lead inevitably to a consideration of the physical nature of the Fos-Jun association. McKnight and his colleagues have recently proposed a general model to explain how a protein-in this case, a CCAAT box and enhancer-binding protein (CIEBP)-may form dimers (Landschulz et al., Science 240, 1759-1764, 1988). When a region of the sequence of ClEBP is arranged as an idealized a-helix, a periodic repetition of leutine residues, present at every seventh position over a distance of eight helical turns, aligns along one face. A similar array of five leucines was also noted in the sequences of Fos, Jun and GCN4. Landschulz et al. propose that these polypeptide segments exist in a helical configuration and that the leucine side chains extending from one a-helix interdigitate with those from a similar second a-heiix, forming a strong hydrophobic interaction. They sug-
y$ireview
gest that this interaction (evocatively termed the “leucine zipper”) facilitates formation of homodimers. In the case of Fos and Jun, a similar association could ‘ve rise to protein heterodimers or multimeric complexes. deed, theieucine-containing region of GCN4 is required for dimerization (Hope and Struhl, EMBC J., op. cit.). Interestingly, the putative zipper region, plus an adjacent highly charged domain containing stretches of acidic and basic amino acids, is similar in GCN4, Jun, Fos and Myc (Vogt et al., op. cit.). Furthermore, these domains are the most highly conserved regions between c-fos and the homologous gene fra-1 (Cohen and Curran, op. cit.). The basic amino acid regions of Jun and Fos are strong candidate DNA-binding domains. Thus, while the exact physical nature of the Fos-JunBP-1 site connection awaits resolution by X-ray crystallography, the leucine zipper hypothesis and studies on yeast transcriptional activators allow construction of testable models. In particular, fine-structure analyses of GAL4 and GCN4 suggest that in addition to dimerization and DNAinding domains, these proteins contain an acidic transcriptional activation region located within an amphipathic a-helix (Giniger and Ptashne, Nature 330, 670-672, 1987; Hope et al., Nature 333,635-640, 1988). This activation domain is proposed to interact with other transcription factors or with RNA polymerase II. Recent experiments indicate a direct interaction with the mammalian TATA factor (TFIID) in vitro (Horikoshi et al., Cell 54, 665-669, 1988). Indeed, portions of Jun and Fos can function as transcrip-
tional activators in yeast when fused to the DNA-binding domain of LexA (Lech et al., Ceil 52, 179-l op. cit.). Nucleoprotein Complexes as ~~t~a~~~ Receptors The association of Fos, Jun, and AP-I sites in the nucleus is analogous to cell-surface interactions of ligands and receptors. AP-1 elements may be regarded as nuclear receptors with different affinities depending upon exact nucleotide sequence, chromatin structure, associated proteins and, perhaps, structural modifications. Fos, Jun, and related proteins would constitute a class of ligands with multiple and variable subunits that interact with genomic receptors containing the Pap-1 site, thereby modulating gene transcription, The affinities, specificities, and activities of these ligands may depend upon subunit composition, posttranslational modifications, competing ligands, and flanking nucleotide sequences or overlapping binding sites. These studies have begun to reveal an unexpected level of complexity that may orchestrate genomic responses to inter- and intracellular signals. This signal transduction mechanism is not limited to the regulation of cellular proliferation; the expression of Fos and Jun has been associated with several cellular processes involved in differentiation, development, and neuronal function. Thus, Fos, Jun and related proteins may comprise a primordial signaling system adapted for use by selected target genes in different cell types.
un: The AP-1 Connection
Tom Curran” and B. Robert Franza, Jr.? * Department of Molecular Oncology Roche Institute of Molecular Biology utley, New Jersey 07110 rCold Spring Harbor Laboratory Cold Spring Harbor, New York 11724
Oncogene research has led to the isolation of specific reagents that have allowed important insights in several disciplines. Recently, studies on oncogenes encoding nuclear proteins have united with those concerning the regulation of eukaryotic gene transcription. ed to Yeast Transcription Factor GCN4 gene products have been proposed to play a role in gene regulation. A solid link to a bona fide transcription factor was made by Vogt et al. (PNAS 84, 3316-3319, 1987) who reported a convincing similarity (44%) between a region of the protein encoded by the jun oncogene, which is responsible for induction of fibrosarcomas in chickens by avian sarcoma virus 17 (Maki et al., PNAS 84, 2848-2852, 1987), and the DNA-binding domain of the yeast transcriptional activator GCN4. GCN4 induces transcription of several genes involved in amino acid biosynthesis (Hope and Struhl, Cell 46, 885-894, 1986) and binds as a dimer to the target sequence ATGA(C/G)TCAT (Hill et al., Science 234, 451-457, 1986; Hope and Struhl, EMBO J. 6, 2781-2784, 1987). Recently, several other yeast proteins have been identified that recognize the same binding sites (Harshman et al., Cell 53, 321-330, 1988; Jones et al., Cell 53, 659-667, 1988). in addition, the DNA-binding domain of GCN4 can be replaced by the region of similarity in Jun to give a functional transcriptional activator (Struhl, Nature 332, 64950, 1988). un Is One of the Proteins Present in AP-7 Preparations The consensus binding site of GCN4 is closely related to that of the mammalian transcription factor AP-1 (Angel et al., Cell 49, 729-739, 1987; Lee et al., Cell 49, 741-752, 1987; Piette and Yaniv, EMBO J. 6, 1331-1337, 1987). Similarities between Jun and GCN4 stimulated an investigation of the relationship between Jun and AP-1 (Bohmann et al., Science 238, 1386-1392, 1987). Transcription factor AP-1 (activator protein-l) was described initially as a ~NA-binding activity in HeLa cell extracts that specifically recognizes the enhancer elements of SV40 and the human metallothionein IIA (hMIIA) gene (Lee et al., Nature 325, 368-372, 1987). AP-1 binding sites also occur in the control regions of viral and cellular genes that are stimulated by treatment of cells with phorbol ester (Angel et al., Lee etal., Cell, op, cit.). Indeed, tandemly linked API binding sites mediate the elevated transcription of heteroiogous genes after phorbol ester treatment. Antisera raised against two v-jun peptides, PEP1 (amino acids 209-225) and PEP2 (amino acids 73-87) (Bos et al., Cell 52, 705712! 1988) were used to examine affinity-
inireview
purified preparations of AP-1. in imm ot assays both antisera recognized a single major po tide (Bohmann et al., Science, op. cit.). Furthermore, sequencing of AP-1 tryptic peptides revealed that several were identical to portions of the predicted amino acid sequence of Jun. These data, together with the finding that a Jun polypepetide expressed in E. coli binds to the AP-1 siie (Bohmann et al., Science, op. cit.; Angel et al., Nature 332, 766-171, 1988), provided compelling evidence identifying Jun as AP-1. However, AP-1 preparations contain several proteins (Lee et al., Cell, op. cit.), and many of the AP-I peptides sequenced are unrelated to Jun. Indeed, some of the AP-1 peptides have now been identified in the predicted protein sequence of jun-S, a jun-related gene (Bohmann et al., Cold Spring Harbor Symp. Quant. Biol. 53, in press; Ryder et al., PNAS 85, 1487-1491, 1988). Thus, Jun is only one of the proteins that contribute to AP-3 activity and it should not be referred to as transcription factor API. In fact, it is unclear how many cellular proteins regulate transcription via AP-1 sequence elements. dun Is the Fos-associated Protein p3 Several other proteins covered by the umbrella term AP-1 have subsequently been identified as a result of studies on another oncogene product, Fos, which is present in complexes that recognize a specific nucleic acid regulatory element. The fos oncogene (v-fas) is responsible for the induction of osteogenic sarcomas by the FBJ murine sarcoma virus (Curran and Teich, J. Virol. 42, 114-122, 1982). Its normal cellular homolog, c-fos, encodes a nuclear protein (Fos) that undergoes extensive posttranslational modification and participates in protein complexes with a 39,000 dalton protein (p39) (Curran et al., Cell 36, 259-268, 1984; Curran et al., MCB 5, 167-172, 1985). Fos belongs to a family of related proteins whose expression is induced by a variety of extracellular stimuli (Franza et al., Oncogene 7,213-221, 1987; Cohen and Curran, MC6 8,2063-2069, 1988). Fos-p39 complexes are associated with chromatin in isolated nuclei and bind to DNA cellulose in vitro (Sambucetti and Curran, Science 234, 1417-1419, 1986). These features, together with the observation of a transactivation property associated with v-fos (Setoyama et al., PNAS 83, 3213-3217, 1986), led to the proposal that Fos is involved in gene regulation. The observation by Distel et al. (Cell 49,835~844, 1987) of a protein-DNA complex whose formation was inhibited by anti-Fos antibodies suggested that Fos interacts with a regulatory element of a gene specific for adipocyte differentiation. A combination of studies involving mutagenesis, competition analysis, and DNA-affinity precipitation (Franza et al., Nature 330, 393-395, 1987) revealed that Fos binds to the AP-1 consensus recognition sequence (Franza et al., Science 239,1150-i 153,1988; Rauscher et al., Cell 52, 471-480, 1988). Indeed, variations of the AP-1 site were found to bind several other proteins, including Fos-related antigens and ~39. While these studies estabiished a connection between Fos, Jun and A,P-1, they did lot establish the nature of the connection-that is,
Ceil 396
whether Fos and Fos-related proteins bind to the API site directly, indirectly by way of association with p39, or by interaction with Jun or other AP-1 polypeptides. Subsequent studies revealed that, at least in part, all of these possibilities are true. The availability of Jun-specific antibodies (80s et al., op. cit.) permitted immunological, gel-mobility and structural comparisons that identified p39, the Fosassociated protein, as the product of the jun protooncogene (Rauscher et al., Science 240, lOlO-1016, 1988). Similar studies by other investigators confirmed this identity (Chiu et al., Cell 54,541-552, 1988; SassoneCorsi et al., Cell 54, 553-560, 1988; Lamph et al., Nature 334, 629-631, 1988). OS and Several Fos-related Antigens Are Also P-7 Proteins p39 (now referred to as p3gc-jun)was described originally as a FBJ-MSV transformation-associated protein (Curran and Teich, Virology 776,221-2351982). It was later shown to form an extremely stable complex with v-Fos and c-Fos in the nucleus (Curran et al., op. cit.). The identification of p39 as the c-jun product implied that Fos might also be present in affinity-purified preparations of AP-1. Indeed, Fos and several Fos-related antigens (Fras) were detected in AP-1 preparations by immunoblot analysis (Rauscher et al., Science, op. cit.) and antibodies against the conserved domain of Fos reduced the DNA-binding and transcriptional stimulation properties of AP-1 (Bohmann et al., Cold Spring Harbor Symp. Quant. Biol., op. cit.). The amount of Fos detected in AP-1 preparations was quite low; however, these AP-1 proteins were purified from unstimulated HeLa cells that express low levels of Fos. In contrast, three Fras were present at relatively high levels (one was apparently more abundant than Jim). DNAaffinity assays (Miskimins et al., PNAS 82, 6741-6744, 1985) performed using radiolabelled AP-1 oligonucleotides, suggested that p39c-jon,one of the Fras, and an 80 kd protein bind to DNA directly and specifically (Rauscher et al., Science, op. cit.). Thus, more than one cellular protein can interact directly with AP-1 binding sites. The cooperative or competitive nature of these interactions remains to be determined. Role of Fos and Jun in Gene Regulation Both Fos and Jun accumulate to relatively high levels after cell stimulation (Franza et al., Science, op. cit.; Lamph et al., Nature, op. cit.; Rauscher et al., Science, op. cit.; Ryseek et al., Nature 334, 535-537, 1988; Quantin and Breathnach, Nature 334, 538-539, 1988). However, the levels of both proteins decline long before the major increase in AP-1 activity observed in cells treated with phorbol ester (Angel et al., Cell, op. cit.). Thus, these two proios-related gene fra-7 and the jun-related gene jun-B are fos-related gene fra-7 and the jun-related gene jum3 are also induced by serum stimulation and other treatments (Cohen and Curran, op. cit.; Ryder et al., op. cit.). In these cases, the kinetics of mRNA appearance are slower than that of c-fos. Similarly, several Fos-related proteins that are induced by serum persist in stimulated cells much longer than Fos (Cohen and Curran, op cit.). AP-1, therefore, corresponds to at least two groups of inducible genes encoding proteins whose function may be to mediate specific
alterations in gene transcription in response to environ mental cues. These proteins accumulate in a tissue-specific and temporally prescribed fashion and interact with AP-1 sites. Fos complexes may possess both transcriptional activation and repression properties according to transient transfection assays (Schonthai et al., Cell 54, 325-334, 1988; Chiu et al., op. cit.; Sassone-Corsi et al., Cell, op. cit.; Sassone-Corsi et al., Nature 334, 314-319, 1988). At present, it is not known whether particular AP-1 proteins and protein complexes recognize specific variants of the AP-1 site. Similarly, the role of posttranslational modification in determining specificity of Fos and Jun is not known. It is interesting to note that the CAMP response element (CRE) (Montminy et al., PNAS 83,6682-6686, 1986), also known as the ATF binding site (Lee et al., PNAS 84, 8355-8359,1987), is highly related to several AP-1 binding sites. Nuclear proteins called CREB or ATF, which are apparently neither Fos nor Jun, have been isolated by oligonucleotide affinity chromoatography using the CRE (Hurst and Jones, Genes Dev. 7, 1132-1146, 1987; Yamamoto et al., Nature 334, 494-498, 1988). These proteins can bind to AP-1 sites, but with a 5 to lo-fold lower affinity than to the CRE. Yamamoto et al. suggest that CREB binds to the CRE and stimulates transcription as a dimer and that phosphorylation promotes dimerization. Growing evidence from studies in both prokaryotic and eukaryotic systems supports a model in which transcription factors interact as dimers with half-sites of palindromic recognition sequences. Heterodimeric complexes may have altered affinities for one of these half-sites (although the consensus elements are usually palindromic, binding sites in the natural context are often imperfect palindromes). In this way a single recognition element could interact with a hierarchy of heterodimeric factors with differing activities. As described above, cell-surface stimulation results in increased synthesis of several Fosand Jun-related proteins that associate with AP-1 sites. These proteins appear and are turned over with different kinetics. Thus, the rate of transcriptional initiation of specific target genes may depend upon the concentrations and relative affinities of these proteins for homo- or heterodimeric protein complex formation. How Do Fos and Jun Physically Interact? Speculations such as these lead inevitably to a consideration of the physical nature of the Fos-Jun association. McKnight and his colleagues have recently proposed a general model to explain how a protein-in this case, a CCAAT box and enhancer-binding protein (CIEBP)-may form dimers (Landschulz et al., Science 240, 1759-1764, 1988). When a region of the sequence of ClEBP is arranged as an idealized a-helix, a periodic repetition of leutine residues, present at every seventh position over a distance of eight helical turns, aligns along one face. A similar array of five leucines was also noted in the sequences of Fos, Jun and GCN4. Landschulz et al. propose that these polypeptide segments exist in a helical configuration and that the leucine side chains extending from one a-helix interdigitate with those from a similar second a-heiix, forming a strong hydrophobic interaction. They sug-
y$ireview
gest that this interaction (evocatively termed the “leucine zipper”) facilitates formation of homodimers. In the case of Fos and Jun, a similar association could ‘ve rise to protein heterodimers or multimeric complexes. deed, theieucine-containing region of GCN4 is required for dimerization (Hope and Struhl, EMBC J., op. cit.). Interestingly, the putative zipper region, plus an adjacent highly charged domain containing stretches of acidic and basic amino acids, is similar in GCN4, Jun, Fos and Myc (Vogt et al., op. cit.). Furthermore, these domains are the most highly conserved regions between c-fos and the homologous gene fra-1 (Cohen and Curran, op. cit.). The basic amino acid regions of Jun and Fos are strong candidate DNA-binding domains. Thus, while the exact physical nature of the Fos-JunBP-1 site connection awaits resolution by X-ray crystallography, the leucine zipper hypothesis and studies on yeast transcriptional activators allow construction of testable models. In particular, fine-structure analyses of GAL4 and GCN4 suggest that in addition to dimerization and DNAinding domains, these proteins contain an acidic transcriptional activation region located within an amphipathic a-helix (Giniger and Ptashne, Nature 330, 670-672, 1987; Hope et al., Nature 333,635-640, 1988). This activation domain is proposed to interact with other transcription factors or with RNA polymerase II. Recent experiments indicate a direct interaction with the mammalian TATA factor (TFIID) in vitro (Horikoshi et al., Cell 54, 665-669, 1988). Indeed, portions of Jun and Fos can function as transcrip-
tional activators in yeast when fused to the DNA-binding domain of LexA (Lech et al., Ceil 52, 179-l op. cit.). Nucleoprotein Complexes as ~~t~a~~~ Receptors The association of Fos, Jun, and AP-I sites in the nucleus is analogous to cell-surface interactions of ligands and receptors. AP-1 elements may be regarded as nuclear receptors with different affinities depending upon exact nucleotide sequence, chromatin structure, associated proteins and, perhaps, structural modifications. Fos, Jun, and related proteins would constitute a class of ligands with multiple and variable subunits that interact with genomic receptors containing the Pap-1 site, thereby modulating gene transcription, The affinities, specificities, and activities of these ligands may depend upon subunit composition, posttranslational modifications, competing ligands, and flanking nucleotide sequences or overlapping binding sites. These studies have begun to reveal an unexpected level of complexity that may orchestrate genomic responses to inter- and intracellular signals. This signal transduction mechanism is not limited to the regulation of cellular proliferation; the expression of Fos and Jun has been associated with several cellular processes involved in differentiation, development, and neuronal function. Thus, Fos, Jun and related proteins may comprise a primordial signaling system adapted for use by selected target genes in different cell types.