Nuclear proto-oncogenes fos and jun.

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NUCLEAR PROTO-ONCOGENES FOS AND JUN Annu. Rev. Cell. Biol. 1990.6:539-557. Downloaded from www.annualreviews.org by University of Illinois - Urbana Champaign on 04/16/13. For personal use only.

Lynn J. Ransone and Inder M. Verma Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138 KEY WORDS:

proto-oncogenes, nuclear oncoproteins Fos and Jun, transcrip tional regulators, leucine zipper domain, transformation

CONTENTS INTRODUCTION

.................................... . .................................... .....................................

BIOLOGY AND PATHOLOGY OF THE FBJ-VIRUS, FBR-VIRUS AND AVIAN RETROVIRUS ASV 17

The fos Gene of Murine Osteosarcoma Viruses......................................................... The v-jun Oncogene of Avian Sarcoma Virus 17....................................................... STRUCTURE OF THE FOS GENE AND PROTEIN ................. ............ ....................................... STRUCTURE OF THE JUNGENE AND PROTEIN ...................................................................

539 541 541 542 542

CHARACTERIZATION OF THE FOS-JUN COMPLEX ..

. ..... ... ........................................... ..... ....

545 546 546

REGULATION AND EXPRESSION OF PROTO-ONCOGENES FOS AND JUN.................................

550

TRANSFORMAnON BY FOS AND fUN................................................................................

Exploration of the cancer cell is akin to archaeology: we must infer the past from its remnants in the present, and the remnants are often cryptic. J. Michael Bishop: Les Prix Nobel, 1989

INTRODUCTION

Acutely oncogenic retrovirus cause a broad spectrum of malignancies (for review see Graf & Beug 1978; Teich et al 1982). The genomes of these viruses harbor cellular sequences that are responsible for the induction of cellular transformation in vitro and neoplasia in vivo. These acquired sequences, termed viral oncogenes, have their origin in the normal cellular genome (Bishop & Varmus 1982). Proto-oncogenes, the cellular cognates 539 0743-4634/90/1115-0539$02.00

Annu. Rev. Cell. Biol. 1990.6:539-557. Downloaded from www.annualreviews.org by University of Illinois - Urbana Champaign on 04/16/13. For personal use only.

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RANSONE & VERMA

of nearly two scores of different retroviraI oncogenes have now been identified and extensively analyzed (Stehelin et a1 1976; Bishop & Varmus 1982; Bishop 1983, 1987; van Beveren & Verma 1985). In recent years, major research efforts have focused on elucidating the physiologic role of proto- oncogenes in normal cells. The ability of acutely oncogenic retro viruses to interfere with the control of normal cell proliferation and differ entiation has led to the postulate that the cellular homologues of viral oncogene products may fulfill certain physiologic functions in these pro cesses (Graf & Beug 1978; Ellis et al 1981; Bishop 1981, 1983; Bishop & Varmus 1982; Goyette et aI 1983). Proto-oncogenes can now be classified functionally into three broad categories: (a) growth factors and receptors; (b) mediators of intracellular signal transduction pathways; and (e) regu lators of gene expression (Bishop 1987). It is likely that interaction and cooperation among the products of the different classes of proto-oncogenes play a cardinal role during cell growth, differentiation, and development. Growth factors bind, presumably, to their cognate receptors and activate a cascade of intracellular events culminating in modulation of gene expression. Thus the final impact of the external stimulus is on the nucleus. Since the products of a number of proto-oncogenes have been found to reside in the nucleus, we have elected to study these proto-oncogenes with the hope of understanding their role in gene expression. A partial list of the oncogenes that encode nuclear proteins include mye, myb, ios, jun, erbA, ski, rel, EIA, SV40large T antigen, and polyoma large T antigen. Additionally, the product of the well-studied anti-oncogene rbl (retino blastoma) is also a nuclear protein. Products of nuclear oncogenes like fun and erbA have been identified as transcription factors, the former being the phorbol-ester-responsive factor AP- l , while the latter is the thyroid hormone receptor (Sap et a1 1986; Weinberger et a11986; Bohmann et al 1987; Angel et al 1 988a) . Other nuclear oncoproteins like Fos, myc, and ReI may act as transregulators of transcription by interacting with other nuclear factors (Setoyama et al 1986; Distel et al 1987; Verma & Sassone-Corsi 1987; Rauscher et aI1988a ) . Nuclear oncogenes share many common features including (a) a rapid and often transient induction in response to numerous agents capable of promoting either growth and development, or inducing differentiation; (b) a short half-life of messenger RNA, which may in part be due to the presence of adenine-thymine (AT) rich destabilizing sequences in the 3' untranslated region; (e) the proteins encoded by many nuclear oncogenes have a short half-life of 20-90min; (d) the nuclear oncoproteins are invariably post-translationally modified, usually by serine phospho-esterification; and (e) a number of nuclear oncoproteins bind DNA and in some cases the DNA binding is sequence specific. Overall, the nuclear oncoproteins appear to have a deliberate

FOS

AND fUN

541


transient function presumably because their sustained expression has the potential to induce cellular transformation. We have been interested in studying the regulation of the proto-onco gene fos and its association with another nuclear oncoprotein, Jun. The purpose of this article is to review our present knowledge of the regulation of Fos and Jun oncoproteins in normal and transformed cells. We shall attempt to summarize the available information on the biology and path ology of tumors induced by the fos and jun oncogenes, the structure of both oncogenes, and the regulation of their expression. BIOLOGY AND PATHOLOGY OF THE FBJ- AND FBR-VIRUS AND AVIAN RETROVIRUS ASV 17

The [os Gene of Murine Osteosarcoma Viruses Oncogene fos is the resident transforming gene of both the FBI-murine osteosarcoma virus (FBJ-MSV) and FBR-MSV (Finkel et a1 1966; Finkel & Biskis 1968). The FBJ-MSV was isolated from a spontaneous bone tumor in a CFI mouse, whereas FBR-MSV was isolated from a radiation induced bone tumor (for review, see Verma & Graham 1987). Both viruses, however, cause transformation of fibroblasts in vitro and induced osteo sarcomas in vivo. Over 90% of mice infected with the fos viruses develop tumors associated with bone. These tumors often arise on several bones and sometimes in the peritoneum. This indicates multiple sites of viral tumor formation, although metastases are not seen (Ward & Young 1976). A 55K phosphoprotein encoded by the oncogene (v-fos) was identified by immunoprecipitation using sera from rats that had been injected with FBJ MSV-transformed cells (Curran & Teich 1982; Curran et al 1982). Such rats developed tumors. In addition their sera (tumor-bearing rat sera; TBRS) precipitated a 39K protein of host origin (Curran & Teich 1982; Curran et al 198 4), which was later shown to be structurally, and func tionally related to nuclear factor AP- I (Jun) (Rauscher et al 1988a,b; Sassone-Corsi et al 1988c; Angel et al 1988a). The transforming protein encoded by FBR-MSV is a 75K gag-fos fusion phosphoprotein that con tains a region homologous to c-fos (Curran & Verma 198 4; van Beveren et al 1984). Mice injected with the FBR-viral complex develop a disease that is identical to that obtained with the FBJ viral complex, i.e. parosteal tumors. As with FBJ-MSV, when molecularly cloned FBR-MSV DNA is transfected onto cells, they exhibit a transformed morphology. FBR-MSV DNA, however, also immortalized primary cells in culture, and these cells produced tumors when injected into syngeneic or nude mice (Jenuwein et al 1985). In certain respects then, the FBR-viral complex appears to be a more potent transforming agent as compared with the FBI viral complex.

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Nevertheless, these findings again place v-los sequences in a pivotal role for tumor formation (Verma & Graham 1987).


The v-jun Oncogene of Avian Sarcoma Virus

17

The v-jun oncogene was isolated from avian sarcoma virus 17 (ASV 17) found in a spontaneous sarcoma in an adult chicken (Cavalieri et al 1985; Maki et aI1987). It is derived from the proto-oncogene c-jun that encodes transcription activator protein AP- l (Vogt et a11987; Bohmann et al 1987; Angel et al 1988a). ASV 17 induces progressively growing fibrosarcomas in chickens and transforms cultured chicken embryo fibroblasts (CEF) into elongated spindle-shaped neoplastic cells (Cavalieri et al 1985; Maki et al 1987). The ASV 17 transformation specific protein p65gag.jun is a fusion protein. Immunofluorescence studies have revealed that the v-jun protein is localized in the nucleus of CEF transfected with ASV 17 (Bos et al 1988). Nucleotide sequence analysis has indicated that the carboxy terminus of v-jun is similar to the carboxy terminus of the yeast transcriptional activator GCN4 (Vogt et al 1987; Struhl 1987), which regulates the expression of genes involved in amino acid biosynthesis (Hope & Struhl 1987). The conserved region is restricted to the carboxy terminal portion of v-jun, which has 44% homology with the DNA-binding domain of GCN4. Thus it was proposed that v-jun might encode a sequence-specific DNA-binding protein (Vogt et al 1987). An important clue that v-jun might have a normal cellular counterpart encoding a sequence specific DNA-binding factor came with the discovery that the core consensus DNA sequence, ATGACTCA T, recognized by GCN4, is very similar to the binding site of the human transactivator protein AP- I(Lee et aI1987a,b; Bohmann et al 1987). STRUCTURE OF THE

FOS

GENE AND PROTEIN

The complete nucleotide sequences of the FBJ-MSV and FBR-MSV pro viral DNAs and the cellular progenitor of the los gene were determined (van Beveren et a1 1983, 1984; van Straatten et aI1983). FBJ-MSV proviral DNA contains 4026 nucleotides, including two long terminal repeats (LTRs) of 617 nucleotides each, 1639 nucleotides of acquired cellular sequence (v-los), and a portion of the envelope (env) gene (Figure la). The viral Fos protein encoded by the FBJ-MSV is 381 amino acids long while the normal Fos protein contains 380 amino acids, with an apparent molecular weight of 55K on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The two proteins are identical in the first 332 amino acids (with five single amino acid substitutions), while the remaining sequences in the carboxy terminal region are in a different

FOS AND fUN

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Figure 1 A: Structure of the c-fos (mouse) gene and FBJ- and FBR-MSV proviral DNAs. (Top and bottom) S' and 3' long terminal repeats (LTR) sequences of FBJ- and PBR-MSV are depicted by open boxes, other noncoding regions are indicated by a solid line. The portions of the v-fos and gag-fos genes encoding the viral homologue to c-fos are indicated by stippled boxes. (Middle) Exons are depicted by stippled boxes, introns and 5' and 3' untranslated regions are indicated by a solid line. Indicated below each exon is the length (in amino acids) of the segment of the fos protein it encodes. Also indicated in the figure are the TGA termination codon used by c-fos, the TAG termination codon used by v-fos, and the location of the TATA box, 5'-cap, CRE, AP-l, DSE, SCM, and poly(A)-addition signal (compiled from van Deveren et al 1 983, 1 984). B: Structure of ASV 1 7 proviral DNA (top) and mouse c-jun gene (bottom). The partial gag sequences fused to the v-jun sequence are indicated (Ll gag); other noncoding sequences are depicted by a solid line. For the genomic c-jun clone DNA, the location of the AP-l site, the TATA box, and the start site for tran scription are indicated. The 27 amino acid insertion present in c-jun, but not in v-jun, is also depicted. The data in this figure were compiled from Maki et a1 1987; Hattori et a1 1 988; W. W. Lamph (personal communication).

reading frame because of an out of frame deletion of 1 04 base pairs during the biogenesis of the v-fos protein (van Beveren et al 1983). Despite their different carboxy termini, both v-fos and c-fos proteins are nuclear phosphoproteins (Curran et aI1984). Cellular Fos protein undergoes much more extensive modification than v-Fos, mostly because of phospho-esteri fication at the serine residues in the carboxy terminus of the protein (Barber & Verma 1987). In addition to Fos, other fos-related proteins, Fra- l and FosB, have been identified (Cohen et al 1989; Zerial et al 1989). Both


544

RANSONE & VERMA

proteins contain several regions of homology to Fos. The Fra-l protein is post-translationally modified at its carboxy terminus and is found in both the nucleus and the cytoplasm of serum-induced COS cells (Cohen et al 1 98 9). The c-fos gene has also been identified in chicken and Xenopus. In both of these organisms, amino acid sequences reveal extensive identities with the mammalian Fos protein (Kindy & Verma 1 9 90). The c-fos gene is inducible, with a wide variety of agents capable of initiating cell growth, differentiation, and development (Verma 1 986). The c-fos promoter is responsive to agents that activate either the intracellular protein kinase C, or adenylate cyclase pathways (Fisch et al 1 987; Sassone Corsi et al 1 988c). The c-fos protein contains a region of periodic repeats of leucine residues every seven amino acids (the leucine zipper) adjacent to a highly basic domain (Figure 2). This structure, which is found in a variety of transcription factors, is necessary for heterodimer formation with the product of another nuclear oncogene,jun (see characterization of the Fos-lun complex below) (Gentz et al 1 98 9; Turner & Tjian 1 98 9; Ransone et al 1 98 9). Association with lun occurs via the leucine zipper domain and is necessary for the cooperative transcriptional transactivation of promoters containing TPA (12-0-tetradecanoyl-phorbol-13-acetate) responsive promoter elements (TRE) sequences (Chiu et a11988; Sassone Corsi et aI1 988a,b).

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Conservation of residues in leucine zipper-containing proteins. The amino acid

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STRUCTURE OF THE JUN GENE AND PROTEIN

The jun gene is a cell- derived sequence that has been identified as the transforming gene of ASV 17 (Maki et al 1987; Vogt et al 1987). Although v-jun and the cellular counterpart c-jun are closely related, they differ in several respects. In ASV 17, the 5' end of jun is fused in frame to a portion of the viral gag sequences. The viral jun protein has also suffered a 27 amino acid deletion in the amino-terminal half of the molecule (Figure lb). Additionally, in the DNA-binding, carboxy terminal portion of v-jun there are two non-conservative amino acid substitutions (Bohmann et al 1987). It is possible that one or more of these differences between v-jun and c-jun account for the jun oncogenic activation. The v-jun protein is expressed as a 65K protein (p65gag.jun) that contains partial gag sequences at its amino terminus fused to jun sequences that make up the carboxy terminal two thirds of the molecule (Bos et al 1988). The mouse c-jun gene product is 334 amino acids long and, like the avian and human counterparts, its gene is unusual in being devoid of introns (Hattori et al 1988; W. W. Lamph, personal communication). Expression of cloned c-jun in bacteria produced a 39-kd protein with sequence-specific DNA-binding properties identical to the phorbol ester inducible enhancer binding protein, AP-l (Bohmann et a1 1987). Anti bodies raised against two distinct peptides derived from v-jun react specifi cally with AP-l (Bohmann et al 1987). Transcription factor AP-1 was described initially as a DNA-binding activity in HeLa cell extracts that specifically recognizes the enhancer elements of SV40 and the human metallothionein IIA gene (Lee et a1 1987a,b). AP-l binding sites (or TREs) have also been identified in the control regions of viral and cellular genes that are stimulated by the treatment with phorbol ester (Angel et al 1987; Lee et al 1987a,b). lun belongs to a multi-gene family of which junB (Ryder et al 1988; Schutte et a1 1989a) and junD (Ryder et a1 1989; Hirai et al 1989) are other members. Both junB and junD code for proteins that are structurally similar to Jun, but appear to have different and distinct functions as suggested by their temporal and spatial patterns of expression. All mem bers of the jun gene family identified so far have leucine zipper and DNA-binding domains, and associate with Fos protein to form Fos-Iun heterodimers (Landschultz et al 1988; Halazonetis et al 1988; Kouzarides & Ziff 1988; Gentz et a1 1989; Nakabeppu et al 1988; Ransone et al 1989; Sassone-Corsi et al 1989 ; Schuermann et al 1989; Turner & Tjian 1989). The affinity of the lun protein for TRE sequences is vastly increased by the presence of the Fos protein, probably a result of increased stability of

546

RANSONE & VERMA

the Fos-Jun heterodimer as opposed to the Jun homodimer (Kouzarides & Ziff 1 988; Sassone-Corsi et al 1 988b).


TRANSFORMATION BY FOS AND JUN Both FBJ-MSV and FBR-MSV containing the Fos gene can transform established fibroblast cell lines (Curran et a1 1 982; Curran & Verma 1 984). Additionally, it was reported that these viruses induce foci in primary fibroblasts, myoblasts, and osteoblasts (Jenuwein et al 1 985). The cellular fos gene can also induce transformation, but requires at least two manipu lations: (a) addition of LTR sequences, presumably to increase tran scription by providing enhancer sequences, and (b) removal of sequences downstream of the coding domain (Miller et al 1 984). A number of recom binant constructs were generated that contained various portions of viral and cellular fos genes (called v/c-fos recombinant). Briefly, the v-fos and c-fos genes were split into three parts, namely (a) the promoter region and the first 316 amino acids originating from either the v-fos or c-fos gene; (b) the carboxyl terminus, 64 or 65 amino acids of the coding domain of either the v-fos or c-fos gene; and (c) the 3' non-coding domain [including the poly(A) addition signal] originating from either the v-fos or c-fos gene. Transformation by the c-fos gene requires removal of an AT-rich 67-bp region located some 500 nucleotides downstream from the end of the coding domain and about 120 nucleotides upstream of the poly(A) addition signal sequence (Meijlink et al 1985). The precise nature of the 67-bp region in influencing the transforming potential of the c-fos gene is not understood, but two possible mechanisms can be envisaged: (a) autoregulation of c-fos protein synthesis by interaction of the c-fos protein with the 67-bp region, or (b) the presence of the 67-bp region may influence the stability or translational efficiency of the c-fos RNA. In v/c-fos recombinants, the removal of the 67-bp region (shown to be an mRNA destabilizing element) increases the half-life of v/c-fos mRNA (Lee et al 1 988; Raymond et al 1 989) and thereby allows sustained synthesis of the Fos protein. The minimum region in the chicken c-fos protein capable of transformation has been identified between residues 111-206 (Yoshida et aI1 98 9). Interestingly, this region contains the basic region and the leucine zipper. Transgenic mice containing the c-fos gene under the regulation of the metallothionein promoter display bone deformities (Ruther et aI1 985). In contrast, no tumors were observed in transgenic animals expressing Fos in the pancreas (E. Sandgreen, I. Verma, unpublished results). Similarly, transgenic animals expressing c-fos mRNA in their thymus showed hyper plasia, but no tumors (Wagner et al 1989). It remains a puzzle as to why Fos induces only bone tumors, even though sustained amounts of protein


FOS AND

fUN

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can be expressed in other cell types. Perhaps, osteoblastic cells supply some additional factor that can collaborate with the fos gene product to induce neoplasia. Alternatively, in bone cells, Fos is able to induce other gene products whose activation leads to osteosarcomas. While the ability of the retroviral oncogene v-jun to transform chicken cells led to its discovery, the oncogenic potential of its cellular homologue, c-jun, is relatively unknown. The structure of v-jun reveals several deletions when compared to c-jun, thus raising the possibility that the oncogenic activity of v-jun results from these mutations. Studies by Schutte et al (1989a,b) demonstrated that deregulated expression of normal human c jun can transform primary rat embryo fibroblasts in collaboration with the c-Ha-ras gene. In these experiments the malignant cell transformation could be due either to constitutively increased AP-l activity, or to decreased AP-I activity that may be caused by the formation of an excess of biologically less active Jun homodimers (compared with Fos-Jun hetero dimers) (Schutte et al 1989a). In fact, work by Schuermann et al (1989) demonstrated that the leucine zipper motif in Fos, which is required for association with Jun, is also required for transformation. A prediction of the constitutive transcription model for c-jun mediated transformation is that a combination of deregulated c-fos and c-jun expression would further enhance transformation. Cotransfection experiments using either c-jun or junB and c-fos demonstrated that there is a specific enhancement of transformation in the presence of c-fos which is consistent with the hy pothesis that Jun and Fos proteins participate in malignant transformation through constitutive activation of their normal transcriptional functions. In contrast, cotransfection of both c-jun and junB with c-fos leads to a marked decrease in transformation and transactivation (Schutte et al 1989b). Thus junB is a negative regulator of c-jun function. These results suggest that different members of the Jun family can participate in both transactivation and transformation; however, they also appear to be able to modulate each other's action. CHARACTERIZATION OF THE FOS-JUN COMPLEX

The c-fos gene is a member of a family of related genes sharing extensive identities in specific regions of the encoded proteins (Figure 2). Both v-fos and c-fos can be a part of a nuclear protein complex that can bind to DNA in the presence of nuclear extracts and can associate with chromatin (Curran et al 1985; Sambrucetti & Curran 1986; Renz et al 1986; Franza et al 1987). While the Jun oncoprotein binds directly to sequence-specific motifs on DNA (Bohmann et aI 1987; Bos et al 1 988; Angel et al 1 988a); the Fos protein does not appear to bind any specific DNA sequence.


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The finding of sequence-specific interaction between DNA and the Fos complex, using regulatory sequences (FSE2) from the 5'-flanking region of an adipocyte-specific gene, aP2 (Distel et al 1987), was the catalyst to search for the nucleotide sequence motif recognized by the Fos complex. Several approaches, involving mutagenesis, competition studies, and DNA-affinity assays, eventually identified the binding site as the consensus sequence of transcription factor AP-I. These studies established a con nection between Fos, Jun, and AP- l ; however, it was not clear whether Fos and Fos-related proteins bind to the AP-I site directly, or indirectly by way of association with p39 or other polypeptides. A combination of structural and immunological comparisons eventually identified p39 [which was first identified as an FBJ-MSV transformation-associated pro tein (Curran & Teich 1982)] as the protein product of the jun proto oncogene (Sassone-Corsi et a11988a; Rauscher et al 1988a). Furthermore, cooperation between these two nuclear oncoproteins was shown to be required for full activation of transcription from a TPA-responsive element in transfected mammalian cells (Chiu et a11988; Sassone-Corsi et aI 1988a). The stable Fos and Jun complex can be reproduced in vitro (Sassone Corsi et al 1988b). Using in vitro translation products of ios and jun, several groups have demonstrated that Fos directly modulates lun function by forming a heterodimer of Fos and Jun proteins (Sassone-Corsi et al 1988c; Kouzarides & Ziff 1988; Nakabeppu et al 1988; Halazonetis et al 1988; Rauscher et al 1988b). J un protein alone can form a homodimer that binds to TRE or AP-I sites, but it is an inefficient transcriptional transactivator. Fos, on the other hand, does not form homodimers or bind alone to DNA, or activate transcription; yet in a Fos-lun heterodimer, Fos not only contributes to DNA binding specificity, it also cooperates in transactivation. Landschultz et al ( 1988) proposed a model to explain how specific DNA-binding proteins form dimers. When the leucine zipper region of a protein is arranged on an idealized a-helix, a periodic repetition of leucine residues, present at every seventh position over a distance of eight helical turns, aligns along one face. The leucine side chains extending from one a-helix interact with those displayed from a similar a helix of a second polypeptide, thereby facilitating dimerization through strong hydrophobic interactions. Thus, this hypothetical structure, referred to as the leucine zipper, facilitates the formation of homodimers. Such a sequence is present in a number of known or probable transcription factors such as nuclear oncoproteins Fos, Myc, and Jun; the yeast transcription factors GCN4 and yAP-I; enhancer binding protein (CjEBP); and cAMP responsive element-binding protein (CREB) (Hope & Struh11987; Harsh man et al 1988; Kouzarides & Ziff 1988; Sassone-Corsi et al 1988b; Naka beppu et al 1988; Halazonetis et al 1988; Schuermann et al 1989; Turner


FOS AND

JUN

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& Tjian 1989; Gentz et al 1989; Ransone et al 1989; Dang et al 1989; Landschultz et al 1988; Gonzalez et al 1989; Figure 2). The conserved region is actually composed of two structures: the basic motif, an arginine and lysine-rich region, and a leucine zipper. Many groups (Kouzarides & Ziff 1988; Sassone-Corsi et al 1988b; Bos et al 1989; Gentz et al 1989; Neuberg et al 1989b; Ransone et al 1989; Schuermann et al 1989; Turner & Tjian 1989) demonstrated that the leucine zipper domain of both Fos and lun are necessary for heterodimer formation and thus transcriptional transactivation. The mere presence of a leucine zipper is, however, not sufficient for dimer formation. Fos does not form homodimers, and neither lun nor Fos can dimerize with CREB (Dwarki et al 1990) or GCN4 (O'Shea et al 1989a). Deletions extending into the amino or carboxy terminus of the lun or Fos leucine zipper abolish dimerization. Insertions or deletions within this region also affect protein protein association, presumably by disrupting the a-helical structure of the leucine repeats and therefore altering the hydrophobic interactions. Mutation of a single leucine in either Fos or lun has no effect on protein complex formation, although a mutation in the first leucine residue in either Fos or lun reduces the ability of the heterodimer to bind to DNA. Mutations of two consecutive leucine residues in lun have no effect on either homodimer or heterodimer formation; however, in the case of Fos, mutation of two consecutive leucine residues results in an inability to form a heterodimer. Domain swapping experiments have demonstrated that the major factors determining the specificity of dimer formation lie within the leucine zipper domain itself. Fos protein containing either a lun or GCN4 leucine zipper is capable of forming homodimers and can bind to DNA (Kouzarides & Ziff 1989; Neuberg et al 1989a; Sellers & Struhl 1989). Synthetic leucine zipper peptides have also been used to demonstrate that the leucine zipper domains of Fos and lun correspond to autonomous helical dimerization sites. Using these peptides, O'Shea et al (l989b) demonstrated that there was preferential heterodimer formation over homodimers by at least lOOO -fold. Both homodimers and heterodimers were shown to be parallel a-helices, which most likely correspond to coiled coils containing a characteristic hydrophobic repeat (the 4-3 repeat) interspersed within the leucine heptad repeat. Interactions within this repeat are likely to be important for determining the specificity of inter action between F os and lun (O'Shea et aI1989b). Results with site-directed mutagenesis suggest that swapping of these residues within the dimeriza tion domain of either protein has no effect on protein-protein interaction, but can alter the ability of a mutant lun protein to bind cooperatively with wild-type Fos to DNA (L. l. Ransone, unpublished results). A com pensatory mutation in the 4-3 repeat of Fos restores DNA-binding activity


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of the mutant lun protein. Thus the leuzine zipper domain contributes to the DNA-binding potential of a protein, in addition to playing a role as the dimerization domain. The region rich in basic amino acids mediates specific DNA binding, since insertions and deletions in this region still allow protein-protein interaction, but diminish or abolish DNA binding (Kouzarides & Ziff 1 988 ; et al 1 98 9b; Ransone et al 1 98 9; 1 98 9). Site-specific mutagenesis has identified functionally crucial amino acids in the basic region of Fos and lun, and the conservation of these residues in both proteins suggests that there is a parallel symmetry to the specific TRE binding (Ransone et al 1 990). However, sequential muta genesis of the cognate DNA recognition element, the TRE, shows that the interaction between Fos-Iun and DNA is not completely symmetrical (Risse et a1 1 98 9; Hirai et aI 1 98 9). Furthermore, mutations within the basic region of the Jun protein can alter its affinity for Fos protein (Ransone et aI 1 990). Therefore, the DNA-binding domain also contributes to protein dimerization affinities. These DNA-binding defective lun polypeptides are capable of suppressing wild-type Fos-Iun DNA binding and tran scriptional transactivation in transiently transfected F 9 embryonal car cinoma cells and, therefore, behave as transdominant negative mutants. They will be valuable tools in the study of Fos and lun functions. REGULATION AND EXPRESSION OF PROTO-ONCOGENES FOS AND JUN

Expression of proto-oncogenes fos and jun is induced by a wide variety of agents, such as mitogens, differentiation factors, specific pharmacological agents, stress and heat shock (Greenberg & Ziff 1 98 4; Kruijer et al 1 98 4; Muller et al 1 98 4; Lamph et a11 988; Ryder et aI1 988). Induction is rapid and transient and occurs at the level of transcription. Expression of the fos gene appears within minutes after addition of the inducer, reaches maximal levels by 30 -60 min, and is essentially undetectable by 120 min. This rapid induction is likely to involve post-translational modifications of Fos protein. In the presence of inhibitors of protein synthesis, the c-fos mRNA is induced at the normal rate, but remains detectable for up to 46 hr. This suggests that, following mitogen treatment, pre-existing factors are utilized for fos transcriptional activation (Treisman 1 986; Sassone Corsi & Verma 1 987). A detailed description of the mechanism of induction of fos and jun genes is not within the scope of this review. Briefly, induction of the fos


POS

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gene is carried out through the mediation of adenylate cyclase and protein kinase C pathways, two major signal transduction pathways in the cell. Inspection of the human fos promoter reveals the presence of two cAMP dependent response elements (CREs) at position -60 and -350 required for induction by agonists of the adenylate cyclase pathway (Sassone-Corsi et al 1988d; Gilman et al 1986) (Figure la). Located between positions - 299 and - 320 is the dyad symmetry element (DSE) essential for induc tion by serum [also referred to as serum response element (SRE)] (Des champs et a11985; Treisman 1985, 1986; Gilman et a1 1986) (Figure Ib). This element is also required for induction by EGF (Prywes & Roeder 1986), phorbol esters (Mitchell et al 1985), oncogenes src (Fujii et al 1989), ras (Sassone-Corsi et al 1989), Tax-l of HTLV- 1 (Fujii et al 1988), fibroblast growth factor (FGF), and nerve growth factor (NGF) (Sheng et al 1988; Visvader et aI 1988). A 67-kd protein, which binds to SRE and is referred to as serum response factor (SRF), has been identified and its DNA cloned (Norman et al 1988). This DNA-binding protein acts as a dimer and requires phosphorylation for transcriptional activation of heterogenous genes linked to DSE/SRE (Norman et al 1988). Another 62-kd protein has also been implicated in binding to DSE that forms a tertiary complex with the 67-kd serum response factor (Shaw et al 1989). Intriguingly, immediately downstream of the DSE is a bonafide AP-l site capable of binding Fos/Jun complex, which is dispensable for transcription of the Fos gene, since DSE linked to heterologous promoter is sufficient for induction. The Saccharomyces cervisiae MCM l gene product [also referred to as pheromone/receptor transcription factor (PRTF)] shares striking homology to the DNA-binding domain of human SRF (Passmore et al 1989; Jarvis et al 1989). It can bind to the human fos DSE in vivo and, like other MCMl control elements, can mediate upstream activating sequence (VAS) activity in vivo. In the mammalianjos promoter, another element has been identified at position - 340, which is required for induc tion by v-Sis conditioned media (SCM), produced from a NRK-Simian sarcoma virus non-producer cell line; but the precise nature of this factor remains unknown (Hayes et al 1987). In contrast to the extensive delineation of the regulatory elements in the fos promoter, relatively little is known about the mechanism of induction of the c-jun gene. It appears that agents that induce the fos gene usually also induce c-jun gene transcription, yet no DSE element (CArG box CA rich) can be identified in a region containing 300 nucleotides upstream of the transcription start site (W. W. Lamph, personal communication). The immediate upstream region of the c-jun promoter is also devoid of CRE, which might explain its inability to be induced by forskolin. Interestingly, the c-jun promoter has an AP-l binding site (TGACATAC) that is recog-


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nized by the Fos-Jun complex (Figure lb). The promoter regions of other Jun-related genes ( junB, junD) have not yet been identified. One of the interesting questions in modern biology is the regulation of regulator! Induction offos gene transcription requires either activation of SRF, or phosphorylation of CREB protein by a catalytic subunit of protein kinase A. The induction is transient and independent of protein synthesis. De novo transcription of the c-fos gene, however, continues in the presence of protein synthesis inhibitors, which suggests the requirement for some repressor-like protein whose synthesis is required to shut off transcription of the c-fos gene. Several researchers have shown that the c-fos protein can repress the transcription of the c-fos gene (Lucibello et a1 1 989; Sassone Corsi et al 1 988c; Schonthal et al 1 988; Wilson & Treisman 1 988). While the precise mechanism of repression remains unknown, some general izations can be advanced: (a) Repression of transcription requires the C terminal region of the Fos protein since no repression is observed with the v-fos protein, and C-terminal deletions of the c-Fos protein block repression (Sassone-Corsi et a1 1 988b; Wilson & Treisman 1988); (b) phos phorylation of the C-terminus of Fos is essential because mutations that block phosphorylation of C-terminal serine resid,ues Ofir, personal communication); (c) the leucine zipper domain of the Fos protein is required for repression, thus suggesting its requirement to form a heterodimer; (d) repression of the c-fos gene is mediated through DSE; and (e) the AP-l site immediately 3' of the DSE (Figure 1) is dispensable for repression by the c-fos protein. It is not yet known if FRA-l or FosB also repress transcription of the c-fos gene or their own genes, but based on the identities in the C-terminal domain, they are likely to regulate fos gene expression. Unlikefos, the jun gene is positively regulated by its own product. The Fos-Jun complex can efficiently bind to the AP-l site in the c-jun promoter (Angel et a1 1988b; Lamph et aI 1990). Cotransfection of a c-jun expression vector with the c-jun promoter linked to a heterologous reporter gene into mouse F9 embryonal carcinoma cells results in transcriptional activation of the reporter gene that is further augmented by the addition of a c-fos expression vector. Regulation of c-jun, however, is complex because its expression is negatively regulated by JunB (Angel et al 1988b; Schutte et a1 1 989b) and CREB protein (Lamph et aI 1 990). Suppression of c-jun by CREB can, however, be overcome by phosphorylation of CREB with the catalytic subunit of cAMP-dependent protein kinase A (Lamph et aI 1 990). Thus the regulation of nuc1ear oncoproteins Fos-Jun is regulated not only by their own products, but also by related transcription factors. The Fos-Jun paradigm exemplifies the intricate regulation of early response genes upon external stimulus. Since there are multiple siblings of

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these transcriptional regulators, it is likely they function in a combinatorial manner during temporal and spatial regulation of gene expression in cell growth, differentiation, and development. The challenge in the future is to delineate the precise cascade of events involved in their induction and to identify genes whose expression is governed by the hierarchy of the Fos Jun complex.


ACKNOWLEDGMENTS

We thank Drs. W. W. Lamph and J. Atwater for critical reading of the review and Ashley Geist for help in the preparation of this manuscript. L. J. Ransone is supported by post-doctoral training grant CA 08585-01. This work was supported by grants from the National Institutes of Health and American Cancer Societ y to 1. M. Verma.

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The nuclear protooncogenes c-jun and c-fos as regulators of DNA replication.

Nuclear factor of activated T cells contains Fos and Jun.

Transcriptional activation of c-fos and c-jun protooncogenes by serum growth factors in osteoblast-like MC3T3-E1 cells.

Fos and Jun: oncogenic transcription factors.

Cell-specific inhibitory and stimulatory effects of Fos and Jun on transcription activation by nuclear receptors.

AP-1 oncoprotein-mediated transcription.

Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity.

Trans-dominant negative mutants of Fos and Jun.

Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus.

Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells.

Altered protein conformation on DNA binding by Fos and Jun.

Expression of c-fos, c-jun, jun-B, metallothionein and metalloproteinase genes in human chondrocyte.

Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly.

Mechanism of specificity in the Fos-Jun oncoprotein heterodimer.

Estrogen regulates expression of the jun family of protooncogenes in the uterus.

Transcriptional activation of a conserved sequence element by ras requires a nuclear factor distinct from c-fos or c-jun.

c-Jun Is Required for Nuclear Factor-κB-Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages.

Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.

Expression of c-fos, c-jun and jun B in peripheral blood lymphocytes from young and elderly adults.

Expression of c-jun, jun-B, and c-fos proto-oncogenes in human primary melanocytes and metastatic melanomas.

Fos and jun in rat central amygdaloid nucleus and paraventricular nucleus after stress.

The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains.

Targeted degradation of c-Fos, but not v-Fos, by a phosphorylation-dependent signal on c-Jun.