The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells.

Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit-erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady-state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation.

Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1.

Nuclear oncogene products have the potential to induce alterations in gene regulation leading to the genesis of cancer. The biochemical mechanisms by which nuclear oncoproteins act remain unknown. Recently, an oncogene, v-jun, was found to share homology with the DNA binding domain of a yeast transcription factor, GCN4. Furthermore, GCN4 and the phorbol ester-inducible enhancer binding protein, AP-1, recognize very similar DNA sequences. The human proto-oncogene c-jun has now been isolated, and the deduced amino acid sequence indicates more than 80 percent identity with v-jun. Expression of cloned c-jun in bacteria produced a protein with sequence-specific DNA binding properties identical to AP-1. Antibodies raised against two distinct peptides derived from v-jun reacted specifically with human AP-1. In addition, partial amino acid sequence of purified AP-1 revealed tryptic peptides in common with the c-jun protein. The structural and functional similarities between the c-jun product and the enhancer binding protein suggest that AP-1 may be encoded by c-jun. These findings demonstrate that the proto-oncogene product of c-jun interacts directly with specific target DNA sequences to regulate gene expression, and therefore it may now be possible to identify genes under the control of c-jun that affect cell growth and neoplasia.

Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase.

The avian sarcoma virus 16 (ASV 16) is a retrovirus that induces hemangiosarcomas in chickens. Analysis of the ASV 16 genome revealed that it encodes an oncogene that is derived from the cellular gene for the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase). The gene is referred to as v-p3k, and like its cellular counterpart c-p3k, it is a potent transforming gene in cultured chicken embryo fibroblasts (CEFs). The products of the viral and cellular p3k genes have PI 3-kinase activity. CEFs transformed with either gene showed elevated levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate and activation of Akt kinase.

Fos-associated protein p39 is the product of the jun proto-oncogene.

The Fos protein complex and several Fos-related antigens (FRA) bind specifically to a sequence element referred to as the HeLa cell activator protein 1 (AP-1) binding site. A combination of structural and immunological comparisons has identified the Fos-associated protein (p39) as the protein product of the jun proto-oncogene (c-Jun). The p39/Jun protein is one of the major polypeptides identified in AP-1 oligonucleotide affinity chromatography extracts of cellular proteins. These preparations of AP-1 also contain Fos and several FRA's. Some of these proteins bind to the AP-1 site directly whereas others, like Fos, appear to bind indirectly via protein-protein interactions. Cell-surface stimulation results in an increase in c-fos and c-jun products. Thus, the products of two protooncogenes (and several related proteins), induced by extracellular stimuli, form a complex that associates with transcriptional control elements containing AP-1 sites, thereby potentially mediating the long-term responses to signals that regulate growth control and development.

The oncogene jun and nuclear signalling.

Jun is a transcription factor that can also induce oncogenic transformation. Its DNA-binding domain is conserved from yeast to man and shows homology to several other transcriptional regulators. Jun dimerizes with the fos protein through an alpha-helical domain termed the leucine zipper, and the jun-fos heterodimers bind to DNA and regulate transcription of numerous specific unlinked genes.

A short N-terminal sequence of PTEN controls cytoplasmic localization and is required for suppression of cell growth.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.

Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta.

v-Jun overrides the mitogen dependence of S-phase entry by deregulating retinoblastoma protein phosphorylation and E2F-pocket protein interactions as a consequence of enhanced cyclin E-cdk2 catalytic activity.

v-Jun accelerates G(1) progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G(1)/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic "clock" is reset. D-cyclin-cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16(INK4A) inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin-cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun-estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin-cdk, is likely to be the critical Rb kinase target of v-Jun.

Avian oncovirus Mill Hill No. 2: pathogenicity in chickens.

Mill Hill No. 2 (MH2), an avian tumor virus, was studied for its transforming and oncogenic effects. In tissue culture it induced transformation of chicken fibroblasts and yolk sac macrophages. When injected into the chicken, the main feature of this virus was its ability to cause liver and kidney carcinoma, in addition to sarcoma. MH2-associated viruses did not transform cell cultures but were able to cause only lymphoma in the birds. Light and electron microscopy were used in a detailed histologic study of the tumors induced by MH2 virus. An unclassified round cell sarcoma was produced in soft tissues at sites of injection; there was no evidence of origin from endothelium. In the kidney, carcinomas mixed with a malignant stroma were found. Hepatocarcinomas were the dominant tumors found in the liver. The lymphomas produced by the associated virus were poorly differentiated and highly malignant. The study illustrated the highly oncogenic potential of this virus and offered a model for the analysis of the carcinogenic events in a more specific way.

Differential and antagonistic effects of v-Jun and c-Jun.

We compared the ability of cellular and viral Jun (c-Jun and v-Jun) to transactivate target genes. c-Jun and v-Jun bind specifically to 12-O-tetradecanoylphorbol-13-acetate responsive elements [TREs, also called activator protein 1 (AP-1) motifs]. However, whereas c-Jun activates TRE-controlled promoters, v-Jun represses them. Cotransfection of the two Jun proteins reduces c-Jun-dependent transactivation. The expression of the endogenous c-jun gene, regulated through a promoter-proximal AP-1-binding site, is repressed in v-Jun-transformed chicken embryo fibroblasts. It is suggested that an M(r) 18,000 v-Jun peptide prominent in v-Jun-transformed cells acts as a transdominant-negative regulator of AP-1 activity and of c-jun expression. In contrast to the results with TRE sites, both v-Jun and c-Jun activate transcription through the human T-cell leukemia virus type I 21-bp repeat which contains a sequence homologous to the cyclic AMP responsive element. However, full-length Jun proteins bind to this site only with low affinity, and binding of the truncated v-Jun was barely detectable. These observations show that the oncogenic viral form of Jun differs from the cellular version in promoter preference and on certain promoters acts as an antagonist to c-Jun.

The oncogenic potential of the high mobility group box protein Sox3.

Sox proteins belong to the superfamily of high mobility group (HMG) proteins. Sox3 is expressed predominantly in the immature neuroepithelium. Ectopic expression of Sox3 causes oncogenic transformation of chicken embryo fibroblasts (CEFs). The oncogenicity of Sox3 is correlated with nuclear localization and transcriptional regulatory activity; mutants containing deletions in the HMG box or the transactivation domain fail to induce foci of transformation. These observations suggest that Sox proteins can induce aberrant cell growth and strengthen the link of HMG proteins to oncogenesis.

Aberrant cell growth induced by avian winged helix proteins.

Winged helix transcription factors act as important regulators of embryonal development and tissue differentiation in vertebrates and invertebrates. Identification of the retroviral oncogene v-qin as a member of the winged helix family showed that these developmental regulators also have oncogenic potential. We used low-stringency hybridization of a chicken embryonic cDNA library to isolate cDNA clones coding for the three chicken winged helix (CWH) proteins, CWH-1, CWH-2, and CWH-3. The CWH genes are transcribed in a tissue-restricted pattern in adult and embryonic chicken tissues. The CWH proteins bind to conserved DNA binding sites for winged helix proteins in a sequence-specific manner. Expression of the CWH proteins from replication-competent retroviral RCAS vectors induces changes in morphology and growth pattern of chicken embryo fibroblasts. CWH-1 and CWH-3 also induce anchorage-independent growth in agar. Chicken embryo fibroblasts expressing the RCAS constructs release replication-competent viruses that are able to elicit the same cellular changes as the parental plasmid DNA. Our results suggest that winged helix transcription factors not only function as regulators of development and differentiation but also have the potential to stimulate abnormal cell proliferation.

Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in v-jun transgenic murine cells.

The oncogene jun encodes a transcription factor of the AP-1 family. In mice carrying viral jun (v-jun) as a transgene, wounding is a prerequisite for tumorigenesis, suggesting collaboration between the transgene and a wound-related event. To define possible candidates for this collaborative process, we examined the effect of several wound-related polypeptide growth factors on cells from transgenic mice. Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in embryo fibroblasts and tumor cell revertants from these mice. This effect was specific for the two cytokines and was restricted to cells from v-jun transgenic mice. Anchorage independence required the continued presence of the cytokines. Transfection of transgenic cells with a v-jun expression plasmid also induced anchorage independence and a tumorigenic phenotype in transgenic tumor cell revertants. However, there was no correlation between anchorage independence, expression of Jun, and AP-1 activity. These results suggest that while increased transgene expression can enhance the growth properties of v-jun transgenic cells, there exist other cytokine-dependent mechanisms that have a similar effect. Retinoic acid, dexamethasone, or forskolin inhibits induction of anchorage independence by tumor necrosis factor alpha, interleukin 1 alpha, and transfected v-jun. Although these agents affect both AP-1 transactivation potential and DNA binding in the transgenic cells, the changes are not correlated with the inhibition of growth.

The oncogene qin codes for a transcriptional repressor.

The retroviral oncogene qin codes for a protein that belongs to the winged helix family of transcriptional regulators. The Qin protein is localized in the nucleus and binds to the same DNA consensus sequence as rat brain factor 1 (BF-1). Cellular Qin shows greater affinity to DNA than does viral Qin. Alone or fused to the DNA-binding domain of the yeast GAL4 protein, both Qin proteins act as transcriptional repressors. The major transcriptional repression domain maps to the region of amino acids 252-395 of viral Qin.

Jun, the oncoprotein.

Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.

The avian cellular homolog of the oncogene jun.

We have isolated chicken genomic and cDNA clones representing the jun oncogene of avian sarcoma virus 17 (ASV17). The genomic clone lacks intron sequences within its protein coding domain, contains a CAAT box, seven SP-1 consensus sequences and TATA box-like elements upstream and two poly(A) addition signals downstream of the coding domain. The cellular jun protein is 310 amino acids in length. Cellular and viral jun proteins differ by three nonconservative amino acid substitutions of which two are located in the DNA-binding domain, by a 27-amino-acid deletion in the amino terminal third of the viral jun protein, by eleven cell-coded amino acids that link the cellular jun coding domain to the viral gag domain and by the partial gag sequences constituting the amino terminal of the viral gag-jun fusion protein. The availability of a cellular jun cDNA now allows the construction of reciprocal recombinants between the viral and the cellular gene which will define the structural features required for the oncogenicity of v-jun.

The chicken junD gene and its product.

We have isolated and characterized the chicken junD gene. It does not contain an intron; its upstream regulatory sequences lack the AP-1-binding site seen in c-jun but include two CRE elements. Downstream untranslated sequences do not show the destabilizing signal ATTTA. The amino acid sequence of the chicken JunD protein is closely related to that of mouse JunD in the dimerization and DNA contact surfaces of the carboxy-terminal region; additional homologies to mouse JunD are seen in acidic and amphipathic amino-terminal domains. Chicken JunD contains stretches of oligoglycines, alanines and prolines, possibly acting as hinges that connect functionally distinct domains of the protein. Chicken junD is broadly expressed at low basal levels in differentiated tissues and at somewhat higher levels in cultured fibroblasts. The cDNA clone of junD was transcribed and translated in vitro. The resulting JunD protein migrates in between 40 and 50 kDa in an SDS gel and can be precipitated with an antibody prepared against a polypeptide consisting of the carboxy-terminal 100 amino acids of mouse c-Jun.

The growth-promoting activity of the Bad protein in chicken embryo fibroblasts requires binding to protein 14-3-3.

Phosphorylation of the Bad protein is a key regulatory event in the prevention of apoptosis by survival factors. Phosphorylated Bad binds to the cytosolic 14-3-3 protein and is sequestered from the apoptotic machinery of the mitochondrial membrane. To examine the role of Bad in cell growth and apoptosis in primary cultures, we produced stable Bad transfectants of chicken embryo fibroblasts (CEF). As expected, serum starvation of Bad transfectants promoted apoptosis. However, Bad-transfected CEF maintained in media with a high serum concentration were capable of anchorage-independent growth and grew to a higher saturation density than control CEF transfected with the empty vector. High dilutions of the infectious retroviral vector RCAS expressing Bad led to the formation of multilayered cell foci. The growth-promoting effects of Bad were dependent on the serine 136 phosphorylation site and correlated directly with binding of Bad to 14-3-3. These results suggest that phosphorylated Bad promotes cell growth and in oncogenic transformation may contribute to the neoplastic phenotype of the cell.

Domains of the qin protein required for oncogenic transformation.

The qin oncogene is a cell-derived insert in the genome of avian sarcoma virus 31 (ASV 31) and functions as the oncogenic determinant of that virus. Overexpression of the viral and cellular versions of the Qin protein (v-Qin and c-Qin) induces oncogenic transformation of chicken embryo fibroblasts (CEF); v-Qin also rapidly induces fibrosarcomas in chickens. Qin proteins can bind to specific DNA sequences and act as transcriptional repressors. In this study, mutants of Qin were constructed in order to determine the molecular domains required for transformation of chicken embryo fibroblasts. Our data indicate that three regions required for transforming activity are located (i) between residues 74-141 at the amino terminus, (ii) in the winged helix domain and (iii) between residues 383-395 at the carboxyl terminus. A Qin mutant with 12 amino acids deleted from the carboxyl terminus (383-395) showed transforming activity that was lower than that of wild type Qin for CEF. Compare to wild type Qin transformants, the mutant transformed cells had a reduced ability for multilayered and for anchorage independent growth. Deletion of 48 amino acids from the carboxyl terminus of the Qin protein (347-395) completely abolished transforming activity. In contrast, deletion of 74 amino acids from the amino terminus did not affect transformation of CEF. However, further deletion of 68 amino acids (74-141) reduced but did not abolish transforming activity. Finally, deletion in the winged helix domain (218-295) completely abrogated oncogenic capacity in CEF. These results suggest that DNA binding and transcriptional repression may be important in Qin-induced oncogenic transformation.