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Jun and DNA bending

The transcription activation domains of both Fos and Jun induce DNA bending. In chimeric proteins, these same domains induce DNA bending independent of the DNA-binding domains. DNA bending by the chimeric proteins is directed diametrically away from the transcription activation domains. Therefore, the opposite directions in which the DNAs bend are caused, in part, by the opposite locations of the Fos and Jun transcription activation domains, relative to the DNA-binding domains in these proteins. DNA bending is reduced in the presence of multivalent cations, indicating that electrostatic interactions contribute to DNA bending by Fos and Jun (Kerppola, 1997).

Interactions among transcription factors that bind to separate promoter elements depend on the distortion of DNA structure and the appropriate orientation of transcription factor binding to allow juxtaposition of complementary structural motifs. Fos and Jun induce distinct DNA bends at different binding sites, and heterodimers bind to AP-1 sites in a preferred orientation. Sequences on each side of the consensus AP-1 recognition element have independent effects on DNA bending. A single base pair substitution outside the sequences contacted in the X-ray crystal structure alters DNA bending. Substitution of sequences flanking the AP-1 site has converse effects on DNA bending in the opposite direction, suggesting that the extent of DNA bending by Fos and Jun is determined in part by the anisotropic bendability of sequences flanking the AP-1 site. DNA bending by Fos and Jun, and the orientation of heterodimer binding are interrelated. Reversal of the orientation of heterodimer binding causes a shift in the direction of DNA bending. The preferred orientation of heterodimer binding is determined both by contacts between a conserved arginine in the basic region of Fos and the central asymmetric guanine as well as the structure of sequences flanking the AP-1 site. Consequently, the structural adaptability of the Fos-Jun-AP1 complex may contribute to its functional versatility at different promoters (Rajaram, 1997).

The use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, was used to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos-Jun do not bend the AP-1 target site. The DNA constructs contain an AP-1 site phased by 1-4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos-Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos-Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos-Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos-Jun-DNA complexes, in which the A-tract bend is positioned close (1-2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos-Jun-DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos-Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos-Jun-DNA complexes are not indicative of (dbd)Fos-Jun-induced DNA bending (upper limit 5 degrees ) in the low salt conditions of gel electrophoresis. Instead, it is proposed that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend (Sitlani, 1998).

Jun, JNK and activation of Jun as a transcription factor

Jun kinases (See Drosophila Basket of Jun N-terminal kinase) phosphorylate c-Jun very efficiently, JunD less efficiently, but they do not phosphorylate JunB. No other Map Kinases are involved in c-Jun N-terminal phosphorylation in mammalian cells. Effective JNK substrates require a separate docking site and specification-conferring residues flanking the phosphoacceptor. The docking site increases the efficiency and specificity of the phosphorylation reaction. JunB has functional JNK docking site but lacks specificity-conferring residues. Insertion of such residues brings JunB under JNK control. JunD, by contrast, lacks a JNK docking site, but its phosphoacceptor peptide is identical to that of c-Jun. Efficient N-terminal c-Jun phosphorylation requires dimerization. Substrates such as JunD can be phosphorylated by JNK through heterodimerization with docking competent partners. Therefore, heterodimerization can affect the recognition of transcription factors by signal-related protein kinases (Kallunki, 1996).

c-Jun is a major component of the heterodimeric transcription factor AP-1 and is essential for embryonic development, since fetuses lacking Jun die at mid-gestation with impaired hepatogenesis and primary Jun-/- fibroblasts have a severe proliferation defect and undergo premature senescence in vitro. c-Jun and AP-1 activities are regulated by c-Jun N-terminal phosphorylation (JNP) at serines 63 and 73 through Jun N-terminal kinases (JNKs). JNP is thought to be required for the anti-apoptotic function of c-Jun during hepatogenesis, because mice lacking the JNK kinase SEK1 exhibit liver defects similar to those seen in Jun-/- fetuses. To investigate the physiological relevance of JNP, endogenous Jun was replaced by a mutant Jun allele with serines 63 and 73 mutated to alanines [Jun(tm1wag); hereafter referred to as JunAA]. Primary JunAA fibroblasts have proliferation- and stress-induced apoptotic defects, accompanied by reduced AP-1 activity. JunAA mice are viable and fertile, smaller than controls and resistant to epileptic seizures and neuronal apoptosis induced by the excitatory amino acid kainate. Primary mutant neurons are also protected from apoptosis and exhibit unaltered JNK activity. These results provide evidence that JNP is dispensable for mouse development, and identify c-Jun as the essential substrate of JNK signaling during kainate-induced neuronal apoptosis (Behrens, 1999).

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73, and consequently, AP-1 activation as well, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. It is shown here that dexamethasone, a glucocorticoid receptor agonist, and two other nuclear hormone receptors, the retinoic acid receptor and the thyroid hormone receptor, also block c-Jun activation by a mechanism that is (1) independent of the c-Jun DNA binding domain and is one which (2) relies specifically on the c-Jun amino-terminal phosphorylation step. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite for interaction with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents. Nuclear receptor interference would rely on the inhibition of MEKK activity or a downstream step in the pathway. Dexamethasone can also inhibit a constitutively active MAPK pathway (Caelles, 1997).

Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun. This mutation markedly enhances v-Jun oncogenicity; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation. Using a two-hybrid approach, it is shown that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonize SAPK/JNK-dependent gene expression. This conclusion challenges the long-standing assumption that v-Jun represents a 'super-activated' form of c-Jun, and predicts that V-Jun acts as a 'dominant negative' mutant that will block or antagonize SAPK/JNK-regulated gene expression. It is therefore suggested that repression of growth-inhibitory or pro-apoptotic genes may be more closely linked to v-Jun-mediated oncogenesis than is activation of growth-stimulatory genes, as previously supposed (May, 1998).

Electrical stimulation of contractions (pacing) of primary neonatal rat ventricular myocytes increases intracellular calcium and activates a hypertrophic growth program that includes expression of the cardiac-specific gene, atrial natriuretic factor (ANF). To investigate the mechanism whereby pacing increases ANF, pacing was tested for its ability to regulate mitogen-activated protein kinase family members, ANF promoter activity, and the trans-activation domain of the transcription factor, Sp1. Pacing and the calcium channel agonist BAYK 8644 activate c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. Pacing stimulates ANF-promoter activity approximately 10-fold. Transfection with an expression vector for c-Jun, a substrate for JNK, also activates the ANF promoter; the combination of pacing and c-Jun is synergistic, consistent with roles for JNK and c-Jun in calcium-activated ANF expression. Proximal serum response factor and Sp1 binding sites are required for the effects of pacing or c-Jun on the ANF promoter. Pacing and c-Jun activate a GAL4-Sp1 fusion protein by 3- and 12-fold, respectively, whereas the two stimuli together activate GAL4-Sp1 synergistically, similar to their effect on the ANF promoter. Transfection with an expression vector for c-Fos inhibits the effects of c-Jun, suggesting that c-Jun acts independently of AP-1. These results demonstrate an interaction between c-Jun and Sp1 and are consistent with a novel mechanism of calcium-mediated transcriptional activation involving the collaborative actions of JNK, c-Jun, serum response factor, and Sp1 (McDonough, 1997).

Lysophosphatidylcholine (lyso-PC), a natural lipid generated through the action of phospholipase A2 on membrane phosphatidylcholine, has been implicated in atherogenesis and the inflammatory process. In vitro studies have established a role for lyso-PC in modulation of gene expression and other cellular responses including differentiation and proliferation. There is also evidence that lyso-PC may act as an intracellular second messenger transducing signals elicited from membrane-associated receptors. The mechanisms underlying the diverse activities of lyso-PC are poorly understood. Treatment of cultured cells with exogenous lyso-PC, at nontoxic concentrations, potently induces activator protein-1 (AP-1) DNA binding and transcriptional activity independent of well known AP-1 activators, either protein kinase C or mitogen-activated protein kinases ERK1 and ERK2. Lyso-PC also activates the c-Jun N-terminal kinase (JNK/SAPK). The stimulated JNK and AP-1 activities probably mediate or contribute to some bioactive effects of lyso-PC 9 (Fang, 1997).

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1-dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, a dominant inhibitory mutant of the regulatory subunit of protein kinase A was transfected. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. Thus BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons (Gaiddon, 1996).

Adenovirus E1B proteins (19,000-molecular-weight [19K] and 55K proteins) inhibit apoptosis and cooperate with adenovirus E1A to induce full oncogenic transformation of primary cells. The E1B 19K protein has previously been shown to be capable of activating transcription; however, the underlying mechanisms are unclear. Adenovirus infection is shown to activate the c-Jun N-terminal kinase (JNK) and the E1B gene products are shown to be necessary for adenovirus to activate JNK. The E1B 19K protein is sufficient to activate JNK and can strongly induce c-Jun-dependent transcription. Mapping studies show that the C-terminal portion of E1B 19K is necessary for the induction of c-Jun-mediated transcription. Using dominant-negative mutants of several kinases upstream of JNK, it has been shown that MEKK1 and MKK4, but not Ras, are involved in the induction of JNK activity by adenovirus infection. The same dominant-negative kinase mutants also block the ability of E1B 19K to induce c-Jun-mediated transcription. Taken together, these results suggest that E1B 19K may utilize the MEKK1-MKK4-JNK signaling pathway to activate c-Jun-dependent transcription and demonstrate a novel, kinase-activating activity of E1B 19K that may underlie its ability to regulate transcription (See, 1998).

Table of contents

DJun: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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