ONCOGENES AND PROTO-ONCOGENES | jun Oncogenes

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by EphA-type receptors, while ephrin B ligands contain a transmembrane domain and a short cytoplasmic region and are bound by EphB-type receptors (Figure 4). Eph receptor stimulation by their ephrin ligands mediates angiogenesis, cell segregation, and cell attachment, shape, and motility. Recent studies have demonstrated upregulation/downregulation of specific Eph receptors and their ephrin ligands in lung cancer, as well as correlation between levels of expression and stage of disease. High expression of EphA2 in NSCLC appears to favor the development of metastasis to the brain while low expression serves as a predictor of disease-free survival or contralateral lung metastasis. The Eph1B receptor is believed to affect SCLC behavior through ephrin-B ligand expression. Recent development of targeted therapies includes antibody targeting of EphA2 as well as monoclonal antibodies to mimic ephrin A1. Animal model studies have been successful in demonstrating ability of these antibodies to inhibit tumor angiogenesis and cell growth; however, to date this effect has not been demonstrated in human models. See also: Hepatocyte Growth (Scatter) Factor. Oncogenes and Proto-Oncogenes: jun Oncogenes; MYC; RAS. Oxidants and Antioxidants: Oxidants. Signal Transduction. Transcription Factors: ATF; NF-kB and Ikb. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Lymphoma.

Further Reading ASCO (2005) Annual meeting proceedings. Journal of clinical Oncology (supplement) 23: 165. Fong KM, Sekido Y, Gazdar AF, and Minna JD (2003) Lung cancer 9: Molecular biology of lung cancer: clinical implications. Thorax 58(10): 892–900. Gazdar AF and Carbone DP (1994) The Biology and Molecular Genetics of Lung Cancer. Austin, TX: RG Landes (Medical Intelligence Unit). Haura EB, Sotomayor E, and Antonia SJ (2003) Gene therapy for lung cancer. Molecular Biotechnology 25(2): 139–148. Sanchez-Cespedes M (2003) Dissecting the genetic alterations involved in lung carcinogenesis. Lung Cancer 40(2): 111–121. Sattler M and Salgia R (2003) Molecular and cellular biology of small cell lung cancer. Seminars in Oncology 30(1): 57–71. Talbot SG, O-Charoenrat P, Sarkaria IS, et al. (2004) Squamous cell carcinoma related oncogene regulates angiogenesis through vascular endothelial growth factor-A. Annals of Surgical Oncology 11(5): 530–534. Valle RP, Chavany C, Zhukov TA, and Jendoubi M (2003) New approaches for biomarker discovery in lung cancer. Expert Review of Molecular Diagnostics 3(1): 55–67. Wistuba II and Gazdar AF (2003) Characteristic genetic alterations in lung cancer. Molecular Medicine 74: 3–28.

jun Oncogenes P K Vogt and A G Bader, The Scripps Research Institute, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The discovery of oncogenic transcription factors brought into focus an important aspect of oncogenesis, deregulated transcription. Transcriptional controls are the principal determinant of gene expression profiles that characterize specific cellular phenotypes in development and differentiation. Jun is a transcriptional regulator with strong oncogenic potential. It plays an important role in the cell cycle and promotes cell proliferation. Jun activity is tightly controlled in the normal cellular context. With that control absent, Jun can induce cancer in the animal and likely contributes to the development of malignancies in man, including pulmonary disease.

The jun Oncogene The jun gene was originally identified as the oncogenic element of the avian sarcoma virus 17, a retrovirus that readily causes fibrosarcomas in chickens (ASV17; ‘jun’ is a truncated form of ju-nana, Japanese for 17). This viral jun (v-jun) is derived from the genome of the avian host and inserted into the retroviral genome as a result of recombination between virus and cell. The N-terminus of the v-Jun protein is joined to viral sequences, and in the infected cell vJun is expressed as a hybrid Gag–Jun fusion protein (Figure 1). Besides being fused to Gag, the v-Jun protein differs from the cellular c-Jun protein by (1) the N-terminal addition of nine amino acids encoded by the 50 untranslated region of c-Jun, (2) an internal deletion of 27 amino acids defining the delta domain (d), and (3) two point mutations, altering serine 222 to phenylalanine and cysteine 248 to serine. These structural distinctions of v-Jun and its high expression level that is controlled by the active retroviral promoter are the underlying cause for the potent tumorigenicity of v-Jun.

Jun is a Transcription Factor The structural and biochemical properties of Jun are that of a transcription factor. Jun is primarily an activator of transcription, but in certain cellular and genomic contexts can also function as a transcriptional repressor. There are two other members of the Jun protein family, JunB and JunD. Their roles of transcription are highly dependent on the cellular environment. The domain structure of Jun includes two N-terminal activation regions (A1 and A2) and a C-terminal bZIP structure consisting of a basic region and a leucine zipper (Figure 1). The basic region

242 ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 222 248

1


A1

bZIP A2 Ser Cys

A1

A2

220

1 ∆Gag

310 c-Jun 512 v-Jun

bZIP

Phe Ser Figure 1 The primary structure of the chicken c-Jun and v-Jun proteins. Positions of amino acids are shown in the figure. Red bars indicate structural differences between c-Jun and v-Jun. Various protein domains are color-coded: yellow: bZIP, basic region and leucine zipper; blue: transactivation domains A1 and A2; dark gray: nine additional amino acids encoded by the 50 untranslated region of chicken c-jun; green: ASV17 viral Gag protein sequences.

Table 1 bZIP protein families and their members Protein family

Name

Protein members Homodimers

Jun

Fos ATF CREB C/EBP Maf PAR

v-jun avian sarcoma virus 17 oncogene homolog FBJ murine osteosarcoma viral oncogene homolog Activating transcription factor cAMP responsive element binding protein CCAAT/enhancer binding protein Avian musculoaponeurotic fibrosarcoma virus homolog Proline and acidic rich

Homo- and Heterodimers

Heterodimers

c-Jun, JunD, JunB

ATF1, ATF6

ATF2, ATF4, ATF7

c-Fos, FosB, Fra-1, Fra-2, JDP1, JDP2 ATF3

CEBPa, CEBPb, CEBPd, CEBPe, CEPBg MafF, MafG, MafK

c-Maf, MafB, NRL

CREB1, CREB3, CREM, CREB-H, CREB-L1

DBP, TEF, HLF

Jun

Leucine zipper

Fos

sic

Ba ion

reg

of the bZIP domain forms the DNA contact surface, and the leucine zipper structure mediates dimerization of the Jun and related proteins. The bZIP region is emblematic for a large class of transcriptional regulators encompassing the families of Jun, Fos, activating transcription factor (ATF), cAMP responsive element binding protein (CREB), CCAAT enhancer binding protein (C/EBP), avian musculoaponeurotic fibrosarcoma virus homolog (Maf), and proline and acidic rich (PAR) proteins (Table 1). For these proteins, dimerization is a prerequisite for DNA binding and hence for transcriptional activity. Some bZIP proteins form only homodimers, others require heterodimerization, and a third group is able to homo- and heterodimerize. These dimerization specificities are encoded in the amino acid sequences of the leucine zippers. The crystal structure of the bZIP domains of the Jun ortholog in yeast, GCN4, and of the Jun–Fos heterodimer bound to DNA shows a pair of continuous a-helices that form a Y (Figure 2). The branches mark the N-terminal region of the structure and contain the basic regions that reach into the major grove of the target DNA whereby arginine and lysine residues interact with the DNA backbone. Further

DNA

TGACTCA Figure 2 Schematic of AP-1 bZIP structure on DNA. The leucine zipper domains of the Jun (blue) and Fos (green) proteins facilitate dimerization, the basic regions make direct contacts with the DNA duplex. The classic DNA-binding site recognized by the Jun–Fos dimer is TGACTCA.

N-terminally positioned residues, one asparagine, alanines and other amino acids, make specific contacts with DNA base pairs and are responsible for the recognition of the DNA target sequence. The stem of the Y consists largely of the leucine zipper dimerization region that extends away from the DNA at an angle of almost 901. In the leucine zipper, every seventh position is a leucine or an alternative

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hydrophobic residue (heptad repeat). Since the ahelix has a periodicity of 3.5 residues per turn, every seventh amino acid is in the same structural environment. Leucine zipper helices are amphipathic with opposing hydrophobic and hydrophilic faces oriented along the long axis of the helix. In the dimer structure, the hydrophobic faces containing the leucines form the contact surface. Nonleucine residues within the proximity of the hydrophobic core determine the stability of the dimer and the compatibility of dimerization partners. The heterodimer of Jun and Fos is the classic form of the functional Jun. It is also known as activator protein 1 (AP-1). AP-1 preferentially binds to the pseudopalindromic DNA sequence TGA C/G TCA. Transcription of genes regulated by AP-1-binding sites is stimulated by 12-O-tetradecanoyl 13-phorbol acetate (TPA), an activator of protein kinase C that works through AP-1. The AP-1-binding sequence is therefore also known as the TPA response element (TRE). Since Jun proteins are able to dimerize with various bZIP proteins, the definition of AP-1 proteins has been extended to any dimer that involves at last one Jun protein. The dimerization partners of Jun can modulate the specificity of DNA-binding, and some Jun heterodimers interact preferentially with variant binding sites. Jun-ATF2 for instance binds to the cAMP response element TGACGTCA or TTACGTCA.

Regulation of AP-1 Activity The regulation of AP-1 activity occurs at several levels. The first level is transcriptional. The jun genes and genes encoding proteins of the AP-1 complex are regulated individually. Whereas some are expressed constitutively, others become activated only upon certain stimuli. Some are tissue specific, others are found ubiquitously. Members of the jun and fos gene families belong to the class of ‘immediate-early’ genes that are highly activated in response to mitogenic signals during the cell cycle in the transition from G1 into the S phase. Yet, the expression profiles

CKII

JNK T91 T93

1


A1 S63 S73 JNK ERK

of these genes are distinct. c-fos mRNAs are rapidly elevated within 30 min after growth stimulation, followed by c-jun, junB, fra-1, and fra-2. In contrast, junD mRNAs are expressed at steady-state levels during the cell cycle. The second level of regulation occurs at the stage of mRNA and protein turnover. Specific protein domains or sequences within the mRNA determine the stability and half-life of these molecules. c-jun and c-fos mRNAs contain a series of AU-rich elements (AREs) with the sequence AUUUA or variations thereof in the 30 untranslated region. AREs facilitate the interaction with the AU-rich binding protein AUF-1 that induces rapid degradation of the target mRNA. Similar to mRNAs, Jun and Fos proteins are short-lived and rapidly degraded by the 26 S proteasome. c-Jun is a moderately labile protein with a half-life of 2 h; JunD is much more stable. Protein stability is regulated by N-terminal Jun protein sequences and the degree of phosphorylation mediated by various kinases. Transient transcriptional stimulation followed by rapid mRNA and protein turnover generates a distinct peak of Jun expression in the first hour after induction that gives way to basal levels shortly thereafter. A third level of control is achieved by posttranslational modification. Jun activity is regulated by protein kinases. c-Jun becomes phosphorylated by extracellular-signal-regulated kinases (ERKs) and Jun amino-terminal kinases (JNKs, also referred to as stress-activated protein kinases or SAPKs). A map of c-Jun phosphorylation sites is shown in Figure 3. ERKs and JNKs belong to the class of mitogen-activated protein kinases (MAPKs) that represent the terminal members of a series of kinases transducing signals from the plasma membrane to intracellular effectors. Phosphorylation of human c-Jun occurs in the transactivation domain A1 at serines 63 and 73, as well as threonines 91 and 93. The phosphorylation of residues 63 and 73 is mediated by JNKs or ERKs; phosphorylation of amino acids at positions 91 and 93 is responsive to JNKs only. The C-terminally located transactivation domain A2 is targeted ERK

T231 

S243

A2

331 bZIP

T239 GSK3

Figure 3 Map of phosphorylation sites of human c-Jun. Positions of amino acids are indicated by numbers. See text for explanations. JNK, Jun N-terminal kinase; ERK, extracellular regulated kinase; CKII, casein kinase II; GSK3, glycogen synthase kinase 3.

244 ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes

by casein kinase II (CKII), glycogen synthase kinase 3 (GSK3), and ERKs at three major phosphorylation sites: threonine 231, threonine 239 and serine 243. The phosphorylation of A1 and A2 has opposite effects on Jun activity. Phosphorylation of A1 increases the transcriptional activity and the half-life of c-Jun, phosphorylation of A2 negatively controls Jun by decreasing DNA binding. The identity of the phosphatases responsible for dephosphorylation of sites in A2 remains to be determined. In a simplified model, phosphorylation of c-Jun in A1 precedes and stimulates the dephosphorylation of sites in A2 which activates DNA binding. The phosphorylation sites in A1 are functionally linked to the two domains d and e (Figure 3). The d domain is a docking platform to recruit JNKs for subsequent phosphorylation. In addition, the d domain has an inhibitory function, as does the e domain. Both domains interact with histone deacetylases (HDACs) and other transcriptional co-repressors that dissociate from c-Jun upon phosphorylation of A1. At the final stage of control, AP-1 activity is regulated by the composition of the protein dimer and AP-1 interacting proteins. Jun forms homo- or heterodimers with other members of the bZIP family. The identity of the dimer depends on the availability of and the preference for the individual dimerization partners. The composition of the dimer determines the preference for the particular DNA-binding site, and as a consequence the regulation of an individual set of target genes. In addition, the AP-1 dimer together with DNA is able to form a quarterny complex with ancillary proteins. Proteins known to interact with AP-1 include members of the Ets and Smad transcription factor families, the octamer binding proteins (Oct), nuclear factor of activated T cells (NFAT), and nuclear factor kappa B (NF-kB) proteins. Binding to these cofactors alters DNA-binding specificity of the AP-1 protein complex.

Oncogenicity of Jun Jun and its partner proteins affect numerous biological processes in embryonic development, programmed cell death, proliferation, and tumorigenesis. Genetic knockout of the c-jun or junB genes in mice results in early embryonic death, demonstrating the importance of Jun in cellular biology (Table 2). It induces proliferation by stimulating the transition from the G1 into the S phase of the cell cycle. c-Jun transcriptionally activates the expression of cyclin D1, a positive regulator of proliferation, and represses expression of p53, a cell-cycle inhibitor. Most members of the Jun and Fos families are inherently oncogenic. The viral proteins, v-Jun and v-Fos, as well as c-Jun, c-Fos, FosB,

Table 2 Analysis of jun and fos knockout mice Gene

Phenotype

Affected organ

c-jun junB junD c-fos fosB fra-1

Embryonic lethality Embryonic lethality Male sterlity Osteopetrosis Nurturing defect Embryonic lethality

Liver Extra-embryonic tissue, placenta Testis Bone Brain Extra-embryonic tissue, placenta

Fra-1, and Fra-2 are transforming in cell culture; vJun, v-Fos, c-Fos, and Fra-1 cause tumors in the animal. In contrast, JunB and JunD have antioncogenic activity. The effect of structure–function changes on oncogenicity is illustrated by a comparison of v-Jun and c-Jun (Figure 1). Mutational analyses show that the lack of the d domain and the two point mutations within the A2 and bZIP domains in v-Jun enhance oncogenicity of Jun. N-terminal Gag sequences of vJun are dispensable for oncogenic activity. In human cJun, phosphorylation of serine 243 negatively affects DNA binding. The corresponding residue in chicken c-Jun is serine 222 that is altered to phenylalanine in v-Jun. This mutated residue can no longer be phosphorylated and, hence, contributes to enhanced DNAbinding activity in v-Jun. Deletion of the d domain enhances the transcriptional activity and oncogenic potential of c-Jun. Jun lacking the d domain is free of transcriptional co-repressors and bypasses intracellular controls by JNKs and ERKs. The present understanding of Jun views oncogenesis as a deregulation of transcriptional control. Several studies have attempted to identify target genes that are either up- or downregulated by oncogenic Jun and whose differential expression is an essential feature of oncogenic transformation. The relevance for the oncogenic process is judged by the partial transforming activities of upregulated targets and by the antioncogenic activities of downregulated targets (Table 3). Cells with the characteristics of Jun-transformed cells cannot be generated by the differential expression of any single target. Jun-induced oncogenicity is therefore likely the result of a combined effect originating in the differential expression of several targets.

Jun in Human Cancer The identification of Jun as the tumorigenic component of a chicken retrovirus established a fundamental role of transcription in cancer. Yet, there is no genetic evidence that Jun is directly involved in human cancer. There are no activating Jun mutations, nor have any gene amplifications or chromosomal translocations involving jun been found. Nevertheless, indirect

ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 245 Table 3 v-Jun target genes that affect the cellular phenotype Gene

Name

Function

Upregulated genes contributing to oncogenic cell transformation when overexpressed HB-EGF Heparin-binding epidermal growth factor-like Growth factor growth factor Jac Jun-activated gene in chicken Unknown Toj3 Target of Jun Nucleolar protein of unknown function Downregulated genes interfering with oncogenic cell transformation when overexpressed Sparc Secreted protein, acidic and rich in cysteine Extracellular matrix protein Marcks Akap12 a

Myristylated alanine-rich protein C kinase substrate A-kinase anchor protein 12

Cellular phenotype Focus formation and colony formationa Colony formation Colony formation

Scaffolding protein

Reduced tumor growth induced by v-Jun Reduced colony formation

Scaffolding protein

Reduced colony formation

Focus and colony formation assays are tools to measure anchorage-independent growth, a hallmark of cancer cells.

evidence suggests that Jun may participate as an essential component in human tumors. Jun is able to transform mammalian cells in cooperation with another activated oncoprotein, such as Ras or Src. Exposure to cigarette smoke and asbestos, and more specifically reactive oxygen species (ROS), upregulate AP-1 expression and AP-1 DNA-binding activity which contributes to the progression of pulmonary disease. Transgenic expression of Fra-1 induces lung tumors in mice. Deletion of c-jun in mouse hepatocytes does not interfere with normal cellular function, but prevents the occurrence of chemically induced hepatocellular carcinomas. Similarly, dominant negative Jun mutants attenuate the growth of various human tumor cell lines and interfere with oncogenic cell transformation linked to the Ras pathway. c-Jun levels are elevated in tumor cells from patients with pancreatic cancer, acute myeloid leukemia, Hodgkin lymphomas, or anaplastic large cell lymphomas.

Acknowledgments This is TSRI manuscript number 16993-MEM. This work was supported by grants from the National Cancer Institute, National Institutes of Health. See also: Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. DNA: Structure and Function. Gene Regulation. Oncogenes and Proto-Oncogenes: Overview; MYC; RAS. Signal Transduction. Transcription Factors: Overview; AP-1; ATF; PU.1.

Further Reading Dunn C, Wiltshire C, MacLaren A, and Gillespie DA (2002) Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signalling 14: 585–593.

Eferl R and Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nature Reviews: Cancer 3: 859–868. Ellenberger TE, Brandl CJ, Struhl K, and Harrison SC (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the proteinDNA complex. Cell 71: 1223–1237. Glover JN and Harrison SC (1995) Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature 373: 257–261. Hartl M, Bader AG, and Bister K (2003) Molecular targets of the oncogenic transcription factor jun. Current Cancer Drug Targets 3: 41–55. Maki Y, Bos TJ, Davis C, Starbuck M, and Vogt PK (1987) Avian sarcoma virus 17 carries the jun oncogene. Proceedings of the National Academy of Sciences, USA 84: 2848–2852. Morton S, Davis RJ, McLaren A, and Cohen P (2003) A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO Journal 22: 3876–3886. O’Shea EK, Klemm JD, Kim PS, and Alber T (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254: 539–544. Reddy SPM and Mossman BT (2002) Role and regulation of activator protein-1 in toxicant-induced responses of the lung. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L1161–L1178. Shaulian E and Karin M (2002) AP-1 as a regulator of cell life and death. Nature Cell Biology 4: E131–E136. Sprowles A and Wisdom R (2003) Oncogenic effect of delta deletion in v-Jun does not result from uncoupling Jun from JNK signaling. Oncogene 22: 498–506. Vinson C, Myakishev M, Acharya A, et al. (2002) Classification of human B-ZIP proteins based on dimerization properties. Molecular Cell Biology 22: 6321–6335. Vogt PK (2002) Fortuitous convergences: the beginnings of JUN. Nature Reviews: Cancer 2: 465–469. Wagner EF (ed.) (2001) AP-1. Oncogene 20: 2333–2497. Weiss C and Bohmann D (2004) Deregulated repression of c-Jun provides a potential link to its role in tumorigenesis. Cell Cycle 3: 111–113. Weiss C, Schneider S, Wagner EF, et al. (2003) JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO Journal 22: 3686– 3695. Wisdom R (1999) AP-1: one switch for many signals. Experimental Cell Research 253: 180–185.