Search

Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.

Helical peptide segments that change their conformation due to external stimuli have often been employed in peptide-based molecular devices and materials. Using helices containing a pair of the iminodiacetic acid derivatives of lysine (Ida), we show that metal-induced helix destabilization is a promising approach to functional switching, especially for helices that are intrinsically stable. By i and i + 2 positioning of the Ida residues in a 17-residue model peptide, a significant decrease in the helical content was observed by the addition of Fe(III), whereas Fe(II) had no influence on the stability of the helix. The possibility of redox control of the helical structure was exemplified by the reduction of Fe(III) to Fe(II) using Na(2)S(2)O(4) followed by the subsequent reoxidation. Mutual recognition of the transcription factor Jun-derived leucine-zipper peptide segment with the Fos leucine-zipper segment containing Ida residues was also modulated in the presence of Fe(III).

Overexpression of v-Jun in chicken embryo fibroblasts (CEF) leads to oncogenic transformation phenotypically characterized by anchorage independent growth and release from contact inhibition (focus formation). The mechanisms involved in this oncogenic conversion however, are not yet clear. Because Jun is a transcription factor, it has been assumed that oncogenic transformation results directly from deregulated AP-1 target gene expression. However, a number of experimental observations in avian cell culture models fail to correlate oncogenesis with AP-1 activity suggesting that transformation induced by v-Jun may occur through an indirect mechanism. To test this possibility, we introduced point mutations into the basic DNA binding domain of v-Jun and created mutants that exhibit altered binding specificity. When expressed in CEF, these mutants fail to deregulate three known v-Jun target genes (JTAP-1, apolipoprotein A1, c-Jun) thus demonstrating in vivo specificity changes. Each of the binding specificity mutants was also tested for its ability to induce oncogenic transformation. Interestingly, expression of these mutants in CEF results in a phenotype indistinguishable from the vector control with respect to growth rate, focus formation and the ability to form colonies in soft agar. These results are consistent with a model requiring direct AP-1 target deregulation as a prerequisite of v-Jun induced cell transformation. With this in mind, we generated a series of additional mutants that retain the ability to bind AP-1 sequence elements, but vary in their oncogenic potential. We demonstrate the use of these mutants to screen v-Jun induced gene targets for a functional role in cell transformation.

1alpha,25(OH)2D3 and its analogs are potent mediators of keratinocyte differentiation in vitro. The precise mechanism of this action is still unknown. The nuclear transcription factor activator protein 1 seems to play an important role in keratinocyte differentiation. The purpose of this study was to investigate the effect of 1alpha,25(OH)2D3 on activator protein 1 DNA binding activity in cultured human keratinocytes. In a time-course study of human keratinocytes incubated with 1alpha,25(OH)2D3 (10-7-10-11 M) a significant dose-dependent increase in activator protein 1 DNA binding activity as determined by electrophoretic mobility shift assay was seen after 36 h. This increase was followed by a significant dose-dependent decrease in activator protein 1 DNA binding activity after 72 h. When differentiation was induced by raising the calcium concentration in the culture medium from 0.09 to 0.3 mM a similar increase in activator protein 1 DNA binding activity was observed after incubation for 48 h. Pharmacologic down-modulation of the protein kinase C activity with GF 109203X reversed the calcium-induced increase in activator protein 1 DNA binding activity and abolished keratinocyte differentiation as determined by a transglutaminase assay. In contrast, activator protein 1 DNA binding activity and keratinocyte differentiation were not affected when protein kinase C activity was down-modulated in the experiments with 1alpha,25(OH)2D3. The activator protein 1 complex in human keratinocytes consists of dimers of Fra-1, Fra-2, c-Jun, JunD, and c-Fos. Our results demonstrate that 1alpha, 25(OH)2D3- and calcium-induced keratinocyte differentiation are accompanied by changes in activator protein 1 DNA binding activity. Protein kinase C activation appears to be essential for the calcium-dependent induction of keratinocyte differentiation, whereas a protein-kinase-C-independent activation of activator protein 1 DNA binding and keratinocyte differentiation is responsible for the 1alpha,25(OH)2D3-induced effects.

GKH-Fos(139-211)/Jun(248-334) (GKH: glycine-lysine-histidine) is a modified Fos/Jun heterodimer designed to contain a metal binding motif in the form of a GKH tripeptide at the amino terminus of Fos bZIP domain dimerized with the Jun basic region leucine zipper (bZIP) domain. We examined the effect of the addition of positively charged GKH motif to the N-terminus of Fos(139-211) on the DNA binding characteristics of the Fos(139-211)/Jun(248-334) heterodimer. Binding studies indicate that while the nonspecific DNA binding affinity of the GKH modified heterodimer increases 4-fold, it specifically binds the activating protein-1 (AP-1) site 6-fold less tightly than the control unmodified counterpart. Furthermore, helical phasing analysis indicates that GKH-Fos(139-211)/Jun(248-334) and control Fos(139-211)/Jun(248-334) both bend the DNA at the AP-1 site toward the minor groove. However, due to the presence of the positively charged GKH motif on Fos, the degree of the induced bend by GKH- Fos(139-211)/Jun(248-334) is greater than that induced by the unmodified Fos/Jun heterodimer. Our results suggest that the unfavorable energetic cost of the increased DNA bending by GKH-Fos(139-211)/Jun(248-334) results in a decrease in both specificity and affinity of binding of the heterodimer to the AP-1 site. These findings may have important implications in protein design as well in our understanding of DNA bending and factors responsible for the functional specificity of different members of the bZIP family of transcription factors., (Copyright 1999 John Wiley & Sons, Inc.)

Studies of DNA bending by Fos and Jun using different methods have yielded contradictory results. Whereas gel electrophoretic phasing analysis indicates that Fos and Jun bend DNA, results obtained through X-ray crystallography and ligase-catalyzed cyclization suggest that they do not. To test the assumptions underlying phasing analysis and to examine DNA bending by Fos and Jun, a multifactorial phasing analysis approach based on the distinct electrophoretic mobilities of DNA fragments of diverse shapes was developed. In this approach, the spacing between the bends, the length of sequences flanking the bends, and the acrylamide concentration in the gel are varied. Two closely spaced intrinsic bends with long flanking sequences had the same effect on electrophoretic mobility as a single bend corresponding to the sum of the bends when they were arranged in phase, and the difference between the bends when they were arranged out of phase. Based on the phase-dependent electrophoretic mobility variation of fragments containing intrinsic DNA bends of different magnitudes, three criteria for determination whether the phase-dependent mobility variation of protein-DNA complexes is caused by DNA bending were adopted. Complexes formed by the bZIP domains of Fos and Jun fulfilled each of these criteria. First, the electrophoretic mobility variation induced by Fos and Jun was proportional to that caused by an intrinsic bend over a broad range of acrylamide concentrations. Second, the mobility difference between fragments containing in phase and out of phase bends was reduced by an increase in the separation between the bends. The separation between the bends had the same effect on the electrophoretic mobility variation caused by Fos and Jun as well as intrinsic bends on long DNA fragments at low acrylamide concentrations. Third, on short DNA fragments analyzed at high acrylamide concentrations, two intrinsic bends separated by long spacers caused a larger decrease in electrophoretic mobility when they were out of phase than when they were in phase. This reversal of the phase dependence of the electrophoretic mobility variation was also observed for complexes formed by truncated Fos and Jun. Thus, the phase-dependent mobility variation of Fos and Jun complexes is due to DNA bending.

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36 --> E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., & Easterbrook-Smith, S.B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.

Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.

To study the contribution of v-Jun homodimers to oncogenesis, we constructed artificial v-Jun derivatives in which the natural dimerization domain of v-Jun was replaced by an heterologous homodimerization domain from either the viral EB1 or the yeast GCN4 transcription factor. The resulting v-Jun chimeric proteins, called v-Juneb1 and v-Jungcn4, which can no longer dimerize with Jun or Fos, should only form homodimers in the cell. Helper-independent retroviruses expressing v-Jun, v-Juneb1 and v-Jungcn4 were generated. All three viruses transformed primary cultures of chick embryo cells with the same high efficiency and promoted local tumor growth after subcutaneous injection of infected cells in young animals. In contrast, after intravenous injection of viral suspensions into chick embryos, only the chimeric proteins produced internal tumors that were lethal. These tumors were leiomyosarcomas located within the liver and along the digestive tract. Thus, in vivo, v-Juneb1 and v-Jungcn4 are more potent oncoproteins than v-Jun. These data demonstrate that when forced to accumulate, v-Jun homodimers can induce tumors efficiently. They also show that the oncogenic potential of v-Jun can be regulated through the properties of its dimerization domain.

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.

Synthesis and DNA-binding activity of the synthetic 26-residue peptide, containing in two copies a part of the DNA-binding domain of the transcription activator v-Jun, are reported. Using CD spectroscopy, it has been shown that the peptide exists in a random coil conformation in aqueous solution, but assumes partially an alpha-helical conformation in the presence of 20% trifluoroethanol. The percentage of alpha-helix is increased in the presence of 40% trifluoroethanol up to approximately 80%. It has been shown that the peptide forms two types of complexes with DNA. The first type of complexes saturates when one peptide molecule occupies six base pairs. At further increase of molar peptide to DNA ratio the binding became a cooperative process. The binding approaches saturation when one peptide molecule is bound approximately to four DNA base pairs. The binding constant of the monomer peptide complex with DNA has been estimated to be approximately 1.10(5) M-1 in the presence of 0.2 M NaCl. The peptide binds more strongly to poly(dG).poly(dC) and poly(dA).poly(dT) than to poly[d(GC)].poly[d(GC)]. We found that the DNA minor groove-binding antibiotic distamycin A competes effectively with the peptide for binding to poly(dA).poly(dT).

Synthesis and DNA-binding activities of the two synthetic 26-residue peptides, containing in two copies a part of the DNA-binding region of the transcription activator v-Jun, are reported. Aminoacid sequences of the two peptides are identical, but in one of them the structure of the DNA-binding region is stabilized by S-S-bond between the two cysteine residues. Using CD spectroscopy, it is shown that the two peptides exist in a random coil conformation in aqueous solution, but assume partially an alpha-helical conformation in the presence of 20% trifluoroethanol. The percentage of alpha-helix is increased in the presence of 40% trifluoroethanol up to approximately 65% and 40% in the absence and presence of S-S-bond between the two cysteine residues, respectively. Evidently, formation of S-S-bond prevents a coil to alpha-helix transition in one of the two DNA-binding regions of the peptide, whereas the formation of alpha-helix in another DNA-binding region is allowed. It is shown that the two peptides bind to DNA. We found that the DNA minor groove-binding antibiotic distamycin A competes with the two peptides for binding to poly(dA).poly(dT). The binding of the two peptides to DNA is accompanied by conformational transitions in the peptide molecules, whereas the structure of DNA does not undergo a marked change. The difference CD spectrum obtained by subtracting the spectrum of DNA from the spectrum of a peptide-DNA mixture differs from the CD spectrum of the free peptide. The shapes of the difference CD spectra are consistent with alpha-beta and coil-beta transitions induced upon binding of the two peptides to DNA. DNase I footprinting diagrams show that peptides mediated cleavage protection of DNA takes place at regions containing 5'-TGA-3' and 5'-TGC-3' nucleotide sequences.

Growth factors, phorbol esters, and oncogenes such as ras, src, and sis are believed to stimulate c-Jun transcriptional activation by inducing increased phosphorylation at two serine residues (S63 and S73) within the N-terminal transactivation domain. Although S63 and S73 are conserved in the mutant v-Jun oncoprotein, they are not phosphorylated by two enzymes which target the corresponding residues in c-Jun in vitro; namely a partially purified c-Jun kinase from TPA-stimulated U937 cells and purified p54 mitogen activated protein (MAP) kinase. In addition, v-Jun activates transcription more strongly than c-Jun when fused to the Gal4 DNA-binding domain, and transcriptional activation by Gal4-v-Jun is unaffected when S63, S73, or both, are replaced with non-phosphorylatable alanine residues, amino acid substitutions which severely impair transcriptional activation by Gal4-c-Jun. The novel biochemical and transcriptional properties of v-Jun result from deletion of a 27 amino acid segment, termed delta, which is important for transforming activity. On the basis of these results we propose that unlike c-Jun, v-Jun transcriptional activation is independent of positive regulatory phosphorylation and that this may contribute to oncogenesis by v-Jun.

The preferential assembly of Jun and Fos into heterodimers has been shown to be mainly driven by 16 amino acids (8 from each protein) situated in positions e and g of the leucine zipper coiled-coil structures of the two proteins (O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1992) Cell 68, 699-708). Using a similar approach, we show that among these residues two pairs of oppositely charged amino acids account in fact for most of the additional free energy of heterodimerization in this system. These residues are 2 glutamic acid side chains in positions g1 and e2 of the Fos leucine zipper and 2 lysine residues in the equivalent positions of the Jun zipper. These amino acids were placed in the context of a GCN4 leucine zipper using peptide synthesis. These peptides contain unique cysteine residues enabling the formation of covalent dimers. The gain in heterodimer free energy has been determined both by cysteine-linked dimer formation under redox conditions and by thermal melting experiments of covalent dimers using circular dichroism experiments. The two pairs of oppositely charged residues (Glu,Glu and Lys,Lys) in positions g1 and e2 contribute at least -1.9 kcal/mol of additional free energy, accounting for a 50-fold excess of the heterodimer with respect to one of the homodimers. Thermal denaturation studies as a function of pH and ionic strength suggest that electrostatic effects should indeed be a major driving force for heterodimerization. On the contrary, peptides harboring the 12 amino acids from Jun and Fos in the other e and g positions (i.e. in e1, g2, e3, g3, e4, and g4) show only a moderate tendency to form heterodimers.

The retroviral oncogene v-jun and its cellular counterpart code for proteins that function as major components of the transcription factor complex AP-1. Jun proteins bind to the AP-1 consensus sequence as homodimers or heterodimers with members of the Fos protein family. This report compares the ability of viral and cellular Jun proteins (v-Jun and c-Jun) to activate transcription and to stimulate DNA synthesis. The effect of amino acid substitutions on cellular transformation is also described. In F9 cells c-Jun is a more effective transactivator than v-Jun, which carries two amino acid substitutions in the carboxy-terminal region that together down-regulate transactivation. The delta deletion, present in the amino-terminal region of v-Jun, does not affect transactivation in F9 cells; however, it does modulate the stimulation of DNA synthesis. When delta is deleted, the amino acid substitutions are without consequence on DNA synthesis. In the presence of delta the amino acid substitutions down-regulate DNA synthesis. Deletion of the Jun transactivation domain, which is required for cellular transformation, abolishes both transactivation and stimulation of DNA synthesis. We conclude that transformation, transactivation and stimulation of DNA synthesis all depend on the presence of the transactivation domain. The three functions are, however, not tightly correlated, and further work is needed to define the role of the biochemical activities of Jun in oncogenesis.

We present a general strategy for designing proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to establish that the dimer binds specifically to the DNA sequence TCATCGATGA but does not bind to the wild-type DNA sequences, nor to oligonucleotides in which the recognition half-site is modified by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.