DJun

EVOLUTIONARY HOMOLOGS

Table of contents

Jun activation by ERK

The baculovirus/Sf9 cell system can be used to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus. Different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 are coexpressed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos are assayed. Activation of ERK-1 via Raf-1 and p21ras (See Drosophila Ras) dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v-ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. The observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. These data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors (Agarwal, 1995).

The two MAP kinases JNK and ERK direct distinct cellular activities, even though they share a number of common substrates, including several transcription factors. JNK and ERK signaling were compared during PC12 cell differentiation; an investigation was carried out to determine how activation of c-Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1 -- two treatments that cause differentiation of PC12 cells into a neuronal phenotype -- results in activation of ERK-type MAP kinases and phosphorylation of c-Jun on several sites, including Ser63 and Ser73. Constitutively activated c-Jun, which mimics the MAPK-phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independent of upstream signals. Conversely, expression of dominant-negative c-Jun bZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c-Jun is co-expressed with activated MEKK1 or SEK1. Consistently, MEK-induced ERK activation in PC12 cells induces c-Jun expression, while JNK signaling does not. Therefore, dual input of expression and phosphorylation of c-Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells (Leppa, 1998).

The expression of the c-jun proto-oncogene is rapidly induced in response to mitogens acting on a large variety of cell surface receptors. The resulting functional activity of c-Jun proteins appears to be critical for cell proliferation. A large family of G protein-coupled receptors (GPCRs), represented by the m1 muscarinic receptor, can initiate intracellular signaling cascades that result in the activation of mitogen-activated protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK). The activation of JNK but not of MAPK correlates with a remarkable increase in the expression of c-jun mRNA. GPCRs can potently stimulate the activity of the c-jun promoter through MEF2 transcription factors, which do not act downstream from JNK. In view of this, the nature of the signaling pathway linking GPCRs to the c-jun promoter was investigated. Utilizing NIH 3T3 cells, it was found that GPCRs can activate the c-jun promoter in a JNK-independent manner. These GPCRs can elevate the activity of novel members of the MAPK family, including ERK5, p38alpha, p38gamma, and p38delta. The activation of certain kinases acting downstream from MEK5 (ERK5) and MKK6 (p38alpha and p38gamma) is necessary to fully activate the c-jun promoter. Moreover, in addition to JNK, three other signaling proteins, ERK5, p38alpha, and p38gamma, were found to stimulate the c-jun promoter by acting on distinct responsive elements. Taken together, these results suggest that the pathway linking GPCRs to the c-jun promoter involves the integration of numerous signals transduced by a highly complex network of MAPK, rather than resulting from the stimulation of a single linear protein kinase cascade. These findings suggest that each signaling pathway affects one or more regulatory elements on the c-jun promoter and that the transcriptional response most likely results from the temporal integration of each of these biochemical routes (Marinissen, 1999).

Other Jun protein interactions

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, a constitutively active and nuclear form of the protein was constructed. Active nuclear Abl efficiently phosphorylates c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, c-Jun and Abl interact via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activate nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signaling models (Barila, 2000).

These data can be summarized in a model, according to which elevated c-Jun levels can activate Abl in the nucleus. Activation requires the central part of the c-Jun protein and the presence of Tyr170 in order to be efficient. In turn, and consistent with previous reports, activation of nuclear Abl results in an increase in JNK activity. This may occur through a mechanism that involves a positive effect on a JNK activator, a negative effect on a JNK repressor, or it could even be direct. Once activated, JNK may then phosphorylate c-Jun at the N-terminal sites and increase c-Jun stability, DNA binding and transcriptional activity. If JNK then dissociates from Jun after phosphorylation, as proposed, this would allow more JNK-free Jun to activate Abl and result in a positive feedback loop (Barila, 2000).

The mechanism by which c-Jun could achieve activation of nuclear Abl is not clear. One possibility is that c-Jun activates Abl allosterically, by direct binding to Abl. The data show that nuclear Abl is regulated by intramolecular interactions involving the SH2-catalytic domain linker, as occurs for the bulk cellular Abl protein and for Abl synthesized in reticulocyte lysate. It is assumed that c-Jun binding to Abl may interfere with the regulatory intramolecular interactions. Initially, tyrosine phosphorylation of c-Jun may not be required for this activation and could occur, for example, via interaction of the proline-rich sequences in c-Jun with the Abl SH3 domain. Alternatively, basal phosphorylation of c-Jun by Abl may suffice to initiate the positive feedback loop. Although Abl and/or Arg seem to be required for c-Jun activation of JNK, the possibility cannot be excluded that c-Jun, or other AP-1 family members, can activate JNK by a mechanism that does not involve Abl in other cell types or under different physiological circumstances. It will be interesting to test whether some of the severe defects in mice lacking c-Jun can be attributed to defects in activating JNK or related kinases (Barila, 2000 and references therein).

Jun-activation-domain-binding protein 1 (JAB1) interacts with c-Jun and JunD, but not with JunB or v-Jun. As a result, JAB1 selectively potentiates transactivation by only c-Jun and JunD. JAB1 specifically stabilizes complexes of c-Jun or JunD with AP-1 sites. The amino-terminal half of JAB1 is very similar to the amino terminal region of Pad1 from fission yeast, which has been identified genetically as a coactivator of a subset of AP-1 target genes. JAB1 and Pad1 are also functionally interchangeable. They define a new group of coactivators that increase the specificity of target gene activation by AP-1 proteins (Claret, 1996).

The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator; loss of Rb function is associated with a variety of human cancer types. Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. The complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation. Transient transfection assays were performed to determine what effect expression of Rb has on Jun-mediated transcription driven by a single AP-1 consensus binding site from the collagenase gene promoter. In both primary human keratinocytes and CV-1P cells, addition of exogenously expressed c-Jun causes, on average, a 10-fold increase in luciferase activity over endogenous levels with the vector alone. When c-Jun and Rb are co-transfected, transactivation increases another 5- to 6-fold when compared with the participation of c-Jun alone. Wild-type Rb as well as Rb small protein (RbSP) with and without a mutation at amino acid 706 are able to activate transcription, while the N-terminal domain (pRbNT amino acids 1-329), which does not bind c-Jun, is unable to activate c-Jun. c-Jun is auto-regulated through an AP-1 site; similar activation by Rb was observed using a region of the c-Jun promoter containing the AP-1 site (Nead, 1998).

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of parathyroid, enteropancreatic neuroendocrine tissue, and anterior pituitary. Recently, the MEN1 gene was identified by positional cloning. MEN1 encodes menin, a 67 kDa protein. Germline mutations in the MEN1 gene cause familial MEN1 and sporadic MEN1. Tumors in affected individuals have lost the wild-type MEN1 allele at chromosome 11q13, suggesting that MEN1 is a tumor suppressor gene. Loss of heterozygosity at 11q13 is found not only in MEN1 tumors, but also in sporadic endocrine tumors of patients without MEN1. Somatic MEN1 mutations are the most common mutations in several types of non-MEN1 tumors, including parathyroid adenomas (MEN1 mutation in 21%), gastrinomas (33%), insulinomas (17%), and bronchial carcinoids (36%). Germline and somatic mutations in MEN1 occur throughout the predicted protein coding region with no obvious hot spots or correlation between genotype and phenotype. The majority of mutations predict premature protein truncation due to either nonsense or frame-shifting type mutations, but missense mutations have also been identified in about 30% of cases. The frequent protein truncation mutations likely cause gene inactivation, consistent with the first hit to a tumor suppressor gene. Few other clues about the function of menin can be deciphered from the predicted amino acid sequence or the pattern of mutations. However, menin recently was found to localize to the nucleus, with two independent nuclear localization signals (Agarwal, 1999 and references).

To begin to ascertain the molecular function of menin, an attempt was made to identify menin-interacting proteins. A yeast two-hybrid screen was performed with full-length menin fused to the Gal4 DNA-binding domain (Gal4DBD). The AP1 (activator protein 1) transcription factor JunD was identified as an interacting partner of menin. Menin does not interact directly with other Jun and Fos family members. The menin-JunD interaction was confirmed in vitro and in vivo. Menin represses transcriptional activation mediated by JunD fused to the Gal4 DNA-binding domain from a Gal4 responsive reporter, or by JunD from an AP1-responsive reporter. Several naturally occurring and clustered MEN1 missense mutations disrupt menin interaction with JunD. These observations suggest that menin's tumor suppressor function involves direct binding to JunD and inhibition of JunD activated transcription (Agarwal, 1999).

Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNA-binding sites in response to transforming growth factor beta (TGFbeta) and thereby activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites, which do not interact with Smads directly. Smad3 and Smad4 are shown to be able to physically interact with AP-1 family members. In vitro binding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGFbeta. Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. This result provides evidence for the importance of this interaction between Smad and Jun proteins. Together, these data suggest that TGFbeta-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors (Liberati, 1999).

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing has shown that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfers with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells (Westermarck, 2002).

The basic region-leucine zipper transcription factor c-Jun regulates gene expression and cell function. It participates in the formation of homo- or hetero-dimeric complexes that specifically bind to DNA sequences called activating protein 1 (AP-1) sites. The stability and activity of c-Jun is regulated by phosphorylation within the N-terminal activation domain. Mitogen- and stress-activated c-Jun N-terminal kinases (JNKs) were previously the only described enzymes phosphorylating c-Jun at the N terminus in vivo. A JNK-independent activation of c-Jun in vivo directed by the constitutive photomorphogenesis (COP9: see COP9 complex homolog subunit 5) signalosome has been demonstrated. The overexpression of signalosome subunit 2 (Sgn2), a subunit of the COP9 signalosome, leads to de novo assembly of the complex with a partial incorporation of the overexpressed subunit. The de novo formation of COP9 signalosome parallels an increase of c-Jun protein resulting in elevated AP-1 transcriptional activity. The c-Jun activation caused by Sgn2 overexpression is independent of JNK and mitogen-activated protein kinase kinase 4. The data demonstrate the existence of a novel COP9 signalosome-directed c-Jun activation pathway (Naumann, 1999).

The bZIP transcription factor ATF2 regulates gene expression in response to environmental changes. ATF2 binds its target promoter/enhancers as a homodimer or as a heterodimer with a restricted group of other bZip proteins, the most well known of which is the c-jun oncogene product. Upon exposure to cellular stresses, the mitogen-activated protein kinase (MAPK) cascades including SAPK/JNK and p38 can enhance ATF2's transactivating function through phosphorylation of Thr69 and Thr71. However, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. In fibroblasts, insulin, epidermal growth factor (EGF) and serum each activate ATF2 via a so far unknown two-step mechanism involving two distinct Ras effector pathways: the Raf-MEK-ERK pathway induces phosphorylation of ATF2 Thr71, whereas subsequent ATF2 Thr69 phosphorylation requires the Ral-RalGDS-Src-p38 pathway. Cooperation between ERK and p38 was found to be essential for ATF2 activation by these mitogens; the activity of p38 and JNK/SAPK in growth factor-stimulated fibroblasts is insufficient to phosphorylate ATF2 Thr71 or Thr69 + 71 significantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 efficiently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor (Ouwens, 2002).

c-Jun binds directly to the N-terminal 163 amino acids of Homo sapiens TATA-binding protein-associated factor-1 (hsTAF1), causing a derepression of transcription factor IID (TFIID)-driven transcription. This region of hsTAF1 binds TATA-binding protein to repress TFIID DNA binding and transcription. The basic leucine zipper domain of c-Jun, which allows for DNA binding and homodimerization, is necessary and sufficient for interaction with hsTAF1. Interestingly, the isolated basic leucine zipper domain of c-Jun is able to derepress TFIID-directed basal transcription in vitro. Moreover, when the N-terminal region of hsTAF1 is added to in vitro transcription reactions and overexpressed in cells, it blocks c-Jun activation. c-Fos, another basic leucine zipper protein, does not interact with hsTAF1, but c-Fos/c-Jun heterodimers bind the N terminus of hsTAF1. These studies show that, in addition to dimerization and DNA binding, the well characterized basic leucine zipper domain of c-Jun functions in transcriptional activation by binding to the N terminus of hsTAF1 to derepress transcription (Lively, 2004).

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. Lysine 265 of c-Fos is shown to be conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 (see Drosophila SUMO), and c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, it has been shown that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. It also is shown to inhibit c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally it is reported that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation (Bossis, 2005).

Acquisition of epidermal barrier function, that serves to prevent water loss, occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. The molecular pathways active during epidermal barrier formation were investigated. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. These data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. This study therefore describes a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development (O'Shaughnessy, 2009).

The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. This study identified a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interactes with c-Jun independently of amino-terminal phosphorylation, and is both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 is attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promotes Lys 63-linked ubiquitylation of RACO-1, which antagonizes Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduces cellular proliferation and decreases expression of several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. Moreover, transgenic overexpression of RACO-1 augments intestinal tumour formation triggered by aberrant Wnt signalling and cooperates with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation (Davies, 2010).

Activation of Jun expression

Serum induction of c-jun expression in HeLa cells requires a MEF2 site (see Drosophila MEF2) at -59 in the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are bound by a family of transcription factors, MEF2A through -D, which are related to serum response factor in their DNA binding domains. MEF2D is the predominant protein in HeLa cells that binds to the c-jun MEF2 site. Serum induction of a MEF2 reporter gene is not observed in a line of NIH 3T3 cells that contains low MEF2 site binding activity. Transfection of MEF2D into NIH 3T3 cells reconstitutes serum induction, demonstrating that MEF2D is required for the serum response. Deletion analysis of MEF2D shows that its DNA binding domain, when fused to a heterologous transcriptional activation domain, is sufficient for serum induction of a MEF2 reporter gene. This is the domain homologous to that in the serum response factor that is required for serum induction of the c-fos serum response element, suggesting that serum regulation of c-fos and c-jun may share a common mechanism (Han, 1995).

The expression of the high mobility group I (HMGI)-C chromatin component is essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, the AP-1 complex was analyzed in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. Neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation (Vallone, 1997).

The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens both transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The cellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated. Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are required in HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI, Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).

In Rat-1 fibroblasts, nonmitogenic doses of lysophosphatidic acid (LPA) stimulate a transient activation of mitogen-activated protein kinase (MAPK), whereas mitogenic doses elicit a sustained response. This sustained phase of MAPK activation regulates cell fate decisions such as proliferation or differentiation, presumably by inducing a program of gene expression that is not observed in response to transient MAPK activation. The expression of members of the AP-1 transcription factor complex has been examined in response to stimulation with different doses of LPA. c-Fos, c-Jun, and JunB are induced rapidly in response to LPA stimulation, whereas Fra-1 and Fra-2 are induced after a significant lag. The expression of c-Fos is transient, whereas the expression of c-Jun, JunB, Fra-1, and Fra-2 is sustained. The early expression of c-Fos can be reconstituted with nonmitogenic doses of LPA, but the response is transient when compared to that observed with mitogenic doses. In contrast, expression of Fra-1, Fra-2, and JunB and optimal expression of c-Jun are observed only with doses of LPA, which induce sustained MAPK activation and DNA synthesis. LPA-stimulated expression of c-Fos, Fra-1, Fra-2, c-Jun, and JunB is inhibited by the MEK1 inhibitor PD098059, indicating that the Raf-MEK-MAPK cascade is required for their expression. In cells expressing a conditionally active form of Raf-1 (DeltaRaf-1:ER), selective, sustained activation of Raf-MEK-MAPK is sufficient to induce expression of Fra-1, Fra-2, and JunB but, interestingly, such activation induces little or no c-Fos or c-Jun. The induction of c-Fos observed in response to LPA is strongly inhibited by buffering the intracellular [Ca2+]. Moreover, although Raf activation or calcium ionophores induce little c-Fos expression, a synergistic induction in response to the combination of DeltaRaf-1:ER and ionomycin is observed. These results suggest that kinetically distinct phases of MAPK activation serve to regulate the expression of distinct AP-1 components, such that sustained MAPK activation is required for the induced expression of Fra-1, Fra-2, c-Jun, and JunB. However, in contrast to the case for Fra-1, Fra-2, and JunB, activation of the MAPK cascade alone is not sufficient to induce c-Fos expression, which rather requires cooperation with other signals such as Ca2+ mobilization. Finally, the identification of the Fra-1, Fra-2, c-Jun, and JunB genes as those that are selectively regulated by sustained MAPK activation, or in response to activated Raf, suggests that these genes are candidates to mediate certain effects of Ras proteins in oncogenic transformation (Cook, 1999).

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, with the phosphorylation of histone H3 and is HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Direct evidence is provided that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes become doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance (Clayton, 2000).

Transcriptional activation of the c-jun gene is a critical event in the differentiation of F9 cells. An element [differentiation response element (DRE)] in the c-jun promoter is both necessary and sufficient to confer the capacity for differentiation-dependent up-regulation. This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus E1A-associated protein p300, whose sequence is closely related to Creb binding protein. Activation transcription factor-2 (ATF-2) is identified as a DNA-binding subunit of the DRF complex. ATF-2 is a member of the ATF/CREB family of basic region-leucine zipper (bZip domain) transcription factors (see Drosophila CrebB-17A). p300 and ATF-2 interact with each other in vivo and in vitro. The bromodomain and the C/H2 domain of p300 mediate the binding to ATF-2, which in turn requires a proline-rich region between amino acids 112 and 350 for its interaction with p300. The phosphorylation of the serine residue at position 121 of ATF-2 appears to be induced by protein kinase Calpha (PKCalpha) after treatment of cells with retinoic acid (RA) or induction with E1A. In cotransfection assays, wild-type ATF-2 enhances the transcription of an E2/tk-luciferase construct, in conjunction with p300-E2. However, this is not the case for a mutant form of ATF-2, with a mutation at position 121 (pCMVATF-2Ser121-Ala). These results suggest that ATF-2 and p300 cooperate in the control of transcription by forming a protein complex that is responsive to differentiation-inducing signals, such as RA or E1A, and moreover, that the phosphorylation of ATF-2 by PKC is probably a signaling event in the pathway that leads to the transactivation of the c-jun gene in F9 cells (Kawasaki, 1998).

The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Muller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. These results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus (Oren, 1999).

Glucocorticoid receptor (GR)-mediated transrepression of the transcription factors AP-1 and NF-kappaB, responsible for most of the anti-inflammatory effects of glucocorticoids, is initiated by the tethering of GR to the promoters of target genes. This tethering is mediated by a nuclear isoform of the focal adhesion LIM domain protein Trip6. Trip6 functions as a coactivator for both AP-1 and NF-kappaB. As shown by chromatin immunoprecipitation, Trip6 is recruited to the promoters of target genes together with AP-1 or NF-kappaB. In the presence of glucocorticoids, GR joins the Trip6 complex. Reducing the level of Trip6 by RNA interference or abolishing its interaction with GR by dominant-negative mutation eliminates transrepression. It is proposed that GR tethering to the target promoter through Trip6 forms the basis of transrepression, and that Trip6 exerts its nuclear functions by acting as a molecular platform, enabling target promoters to integrate activating or repressing signals (Kassel, 2004).

Cell-type-selective expression of the TFIID subunit TAFII105 (renamed TAF4b) in the murine ovary is essential for proper follicle development. Although a multitude of signaling pathways required for folliculogenesis have been identified, downstream transcriptional integrators of these signals remain largely unknown. This study shows that TAF4b controls the granulosa-cell-specific expression of the proto-oncogene c-jun, and together they regulate transcription of ovary-selective promoters. Instead of using cell-type-specific activators, these findings suggest that the coactivator TAF4b regulates the expression of tissue-specific genes, at least in part, through the cell-type-specific induction of c-jun, a ubiquitous activator. Importantly, the loss of TAF4b in ovarian granulosa cells disrupts cellular morphologies and interactions during follicle growth that likely contribute to the infertility observed in TAF4b-null female mice. These data highlight a mechanism for potentiating tissue-selective functions of the basal transcription machinery and reveal intricate networks of gene expression that orchestrate ovarian-specific functions and cell morphology (Geles, 2006: full text of article).

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