Fos-related antigen/kayak


Transcriptional regulation

Fra/kayak expression is induced by Dpp signaling and is unchanged in labial mutants. In dpp mutants, the band of elevated Fra expression in the second gut lobe is no longer visable. Conversely, when Dpp is expressed ubiquitously, strong Fra staining is observed throughout the endoderm. Fra is expressed in the absence of wingless and does not require schnurri (as does labial expression). Ectopic Wg does not induce Fra expression ectopically in the endoderm (Riese, 1997)

Drosophila JNK is activated by endotoxic lipopolysaccharide (LPS). LPS is a component of bacterial cell walls, and is known to be a stimulant for the immune response in both insects and mammals. The response of Drosophila to bacterial infection can be studied by examining the effect of LPS on cultured cell lines. Addition of LPS to cultured cell lines, including mbn-2 hemocytes and Schneider S2 embryonic cells, causes marked induction of cecropin and diptercin genes. basket is activated within 5 minutes of LPS addition. However, basket activation is transient and returns to basal levels after 1 hour. The activation of DJun by DJNK in LPS-treated cells may lead to increased AP-1 (a heterodimer of DFos and DJun) transcriptional activity. Targets of Drosophila AP-1 may include the DJun promoter (Sluss, 1996).

Raw mediates antagonism of AP-1 activity in Drosophila

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. This study shows that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. These studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, evidence is provided that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor (Bates, 2008).

The initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (Byars, 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but this study reports use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular (1) the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components was assessed, (2) genes, which have designated the raw group, have been assessed that share an array of loss-of-function phenotypes, (3) the interaction phenotypes among the raw-group loci were determined, and (4) raw transgenics were generated, that were utilized to probe sites of Raw function. These analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. The characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing (Bates, 2008).

The current study shows that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, raw is required broadly in the epidermis to effect normal dorsal closure. Overall, these studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead to an extension of existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells. In particular, the data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis. AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Bates, 2008).

The AP-1 abnormality in raw-group mutant embryos has not yet been molecularly defined. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally. It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, it is speculated that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is the observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis. Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (Bates, 2008).

The discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity, but this analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways (Bates, 2008).

The genetic interaction that was documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, the observation that raw and rib hypomorphs interact genetically during dorsal closure is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (Blake, 1998; Blake, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (Byars, 1999). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system (Bates, 2008).

In addition to providing evidence for raw-mediated global silencing of AP-1, this study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was the observation that overexpression of raw+ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in the LE-gal4/UAS-raw+ transgenics, this interpretation is not favored since the LE-GAL4 driver used in this study has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (Bates, 2008).

The finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules. Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins. Thus, despite the broad epidermal competence for AP-1 signaling that has been shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE (Bates, 2008).

dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects. In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle was tested. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (Bates, 2008).

This study found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. It was also found that although brk+ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis (Bates, 2008).

What cannot be discerned from these studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, a previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary. Even more significant is the finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (BYARS, 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, these data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo (Bates, 2008).

Having established a dependence upon Jun for raw-dependent ventral cuticular defects, it is postulated that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. It is suspected that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by this strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation (Bates, 2008).

In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. This study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. These data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. The suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal). In addition, and as was suggested is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1) (Bates, 2008).

Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation (Bates, 2008).

Targets of Activity

The endoderm of Drosophila is patterned during embryogenesis by an inductive cascade emanating from the adhering mesoderm. An immediate-early endodermal target gene of this induction is Dfos whose expression is upregulated in the middle midgut by Dpp signaling. Previous evidence based on a dominant-negative Dfos construct has indicated that Dfos may cooperate with Dpp signaling to induce the HOX gene labial, the ultimate target gene of the inductive cascade. Here, kayak mutants that lack Dfos were examined to establish that Dfos is indeed required for labial induction. Evidence is provided that Dfos acts on labial through a CRE (cyclic AMP response element)-like sequence, previously identified to be a target for signaling by Dpp and by the Epidermal growth factor receptor (Egfr) in the embryonic midgut. Dfos expression is stimulated by Egfr signaling. Dominant negative Egfr-expressing embryos were stained with antibody against Dfos. These embryos never show high levels of Dfos protein in the endodermal cells of the parasegment (ps) 6/7 region in the midgut as normally seen in the wild type. Finally, Dfos function is found to be required for its own upregulation. Thus, endoderm induction is based on at least four tiers of positive autoregulatory feedback loops (Szuts, 2000b).

These results show that Dfos acts through a CRE-like sequence. It is possible that Dfos binds directly to this sequence, but needs a partner protein to do so, explaining why efficient binding in vitro of Dfos to this sequence could not be detected. In support of this, Dfos is found to be a highly context-dependent transcriptional activator that requires additional DNA-binding partners to stimulate transcription. Of note, a CRE reporter without any CRE context sequence does not confer any expression in transgenic embryos, again indicating the need for DNA-binding partners. Alternatively, and perhaps less likely, Dfos may act indirectly through the CRE-like sequence, by upregulating locally the expression of an unknown CRE-binding protein (Szuts, 2000b).

Dfos expression is upregulated locally only in the inner cell layer of the midgut, the endoderm, but not in the outer cell layer, the visceral mesoderm. Consistent with this, the current results indicate that Dfos is only required in the former but not in the latter. Nevertheless, CRE- like sequences are present in the midgut enhancers of both Ubx (expressed in the visceral mesoderm) and labial (expressed in the endoderm), and function in both enhancers in the response to Dpp. Therefore, the stimulatory effects of Dpp signaling may be mediated by Dfos only in the endoderm, whereas an unknown protein may substitute for this role of Dfos in the visceral mesoderm. This explains perhaps why the minimal CRE reporters function robustly only in the endoderm but not in the visceral mesoderm, despite the fact that they are derived from the mesodermally expressed Ubx gene. For these reporters to function in the visceral mesoderm, they need additional context, one of these being the TCF binding site (Szuts, 2000b).

The CRE-like sequence appears to be a target for Egfr signaling. Furthermore, the embryonic gut phenotype of kay1 mutants in the endoderm is similar to that due to loss of Egfr signaling. In particular, both mutant conditions seem to cause some degree of cell death in the midgut epithelium. Although this cell death may contribute to, it does not account completely for, the mutant phenotypes observed. Finally, it has been shown that Dfos upregulation in the ps6/7 region of the endoderm depends on Egfr function. Taken together, these observations indicate that Dfos, or its DNA-binding partner(s), may be a critical target of Egfr signaling during endoderm induction, and that the effects of Egfr signaling in the endoderm may be partly if not largely mediated by Dfos (Szuts, 2000b).

Dfos is a context-dependent transcriptional activator whose function in the embryo requires a dimerization partner, such as Djun, as well as combined Jun N-terminal kinase (JNK) and Dpp signaling. This dimerization partner rather than Dfos itself is likely to be the target factor that is directly modified and activated by JNK signaling. In some embryonic tissues, for example the dorsal epidermal cells, this dimerization partner is probably Djun, a transcription factor known to be targeted directly by JNK and Rolled MAP kinase signaling. However, Djun is not a good candidate for a MAP kinase-activated dimerization partner of Dfos in the midgut since Djun neither detectably affects labial nor CRE-mediated expression in the midgut. This putative signal-activated dimerization partner of Dfos in the midgut thus remains elusive (Szuts, 2000b).

The data indicate that Dfos autostimulates its own upregulation in the middle midgut. This parallels labial induction which is autoregulatory in the same endodermal region. The autoinductive function of Labial is probably provided by the low levels of Labial protein expressed in the endodermal primordia. Similarly, the low basal levels of Dfos expression in the endoderm may provide the autoregulatory function of Dfos in this tissue. Thus, the process of endoderm induction in Drosophila involves at least four tiers of positive autoregulatory feedback loops, two in the visceral mesoderm (Ubx and dpp) and two in the subjacent endoderm (Dfos and labial). In each case, the autoregulatory capacity of the transcription factors involved depends on simultaneous stimulation by extracellular signals (Dpp, Wingless or Vein). Thus, each of these positive feedback loops is conditional on cell communication. This design may serve to safeguard against fluctuation in the genetic activity of individual cells and may ensure the co-ordinated pursuit of a given developmental pathway within a group of cells (Szuts, 2000b).

Fra plays a critical role during Drosophila endoderm induction. Fra is required downstream of Dpp signaling for the regulation of labial. Expression of labial in the midgut coincides with copper cells. Labial has a role not only in determination and differentiation of copper cells, but also in the maintenance of their differentiated state. A truncated version of Fra (Fbz)(Eresh, 1997) acts in a dominant negative fashion to interfere with Fra function. Fra acting through labial, by stimulating its expression, to promote copper cell development. The number of labial expressing cells is much reduced in Fbz-expressing larvae. It follows that Fbz must have an effect on labial induction as early as this induction can be detected. These results suggest an early function of Fra during labial induction. Although the Dpp response sequence of a labial midgut enhancer is a cyclic AMP response element (CRE), thought to be the target of Creb-17A, and no interference of Fbz can be detected with the CRE-mediated response, this does not rule out the possibility that Fra acts through the CREs of the labial enhancer, especially if Fra interacts with another DNA-binding protein to do so (Riese, 1997).

The Drosophila homolog of c-Jun regulates epithelial cell shape changes during the process of dorsal closure in mid-embryogenesis. Here, mutations in the DFos gene are described. A mutation in DFos shows defects in dorsal closure and also interacts with mutations in DJun. Like Djun mutations, DFos null alleles completely block shape changes that normally occur in the leading edge of the lateral epithelium during dorsal closure. In dorsal closure, DFos cooperates with DJun by regulating the expression of dpp; Dpp acts as a relay signal that triggers cell shape changes and DFos expression in neighboring cells (Riesgo-Escovar, 1997).

In vertebrates, c-Jun and c-Fos activities are regulated at various levels. Whereas c-Jun is widely expressed at low levels and activated primarily by NH2-terminal phosphorylation by JNKs, c-fos expression is dynamic and activated in response to various extracellular stimuli. A similar dichotomy of DJun and Dfos regulation occurs in Drosophila: DJun is widely expressed during embryogenesis and is phosphorylated by DJNK (Basket), whereas DFos expression is dynamic. There is strong expression of DFos in leading edge cells and cells of the lateral epithelium. Expression of DFos in the lateral epithelium is reduced in thick veins and punt mutant embryos but is still detectable in the leading edge. Therefore, not only does Dfos control expression of dpp in the leading edge, but in a reciprocal manner, DFos expression is dependent on Dpp function in cells of the lateral epithelium. Similarly, in late embryos, DFos expression in the endoderm depends on dpp expression in the overlying visceral mesoderm. Activation of the Dpp signaling pathway is indeed sufficient to activate DFos expression. Ectopic expression of an activated Dpp receptor (Thickveins) in a segmental pattern results in a corresponding pattern for DFos expression. It is concluded that DFos may be required in all ectodermal cells in order to activate target genes required for cell shape changes (Riesgo-Escovar, 1997).

In addition to the joint requirement of DFos and DJun during dorsal closure, DFos functions independently of DJun during embryogenesis. Early dpp expression on the dorsal side of embryos induces expression of several genes, including race, which encodes a protein with homology to angiotensin-converting enzyme in the amnioserosa. The race cis-acting sequences required for dpp-mediated expression contain AP-1 binding sites. Consistent with DFos-mediated direct activation of race through these AP-1 sites, race expression in the amnioserosa is abolished in DFos mutants. In contrast, race expression is normal in DJun or basket mutant embryos. This early DJun-independent function of DFos may be mediated by a DFos homodimer. During wound healing in vertebrates (a process that exhibits parallels with dorsal closure), TGF-beta induces c-fos expression and AP-1 activity. The reciprocal regulatory relation between DFos and dpp in Drosophila appears to be conserved in mammalian cells. In mammalian myeloid cells, induction of c-jun and c-fos by serum or oncogenic v-src results in expression of TGF-beta1 by direction activation of TGF-beta1 transcription by AP1. TGF-beta induces AP-1 activity in keratinocytes during wound healing. These findings demonstrate common and distinct roles for DFos and DJun during embryogenesis and suggest a conserved link between AP-1 (activating protein-1) and TGF-beta (transforming growth factor-beta) signaling during epithelial cell shape changes (Riesgo-Escovar, 1997).

IkappaB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. Crosstalk occurs between the Drosophila Jnk and IKK pathways; this leads to downregulation of each other's activity. The inhibitory action of Jnk is mediated by binding of Drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding leads to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes (Kim, 2005).

Control of G2/M transition by Drosophila Fos

The transcription factors of the Fos family have long been associated with the control of cell proliferation, although the molecular and cellular mechanisms that mediate this function are poorly understood. This study investigated the contributions of Fos to the cell cycle and cell growth control using Drosophila imaginal discs as a genetically accessible system. The RNA interference-mediated inhibition of Fos in proliferating cells of the wing and eye discs resulted in a specific defect in the G2-to-M-phase transition, while cell growth remained unimpaired, resulting in a marked reduction in organ size. Consistent with the conclusion that Fos is required for mitosis, cyclin B was identified as a direct transcriptional target of Fos in Drosophila, with Fos binding to a region upstream of the cyclin B gene in vivo and cyclin B mRNA being specifically reduced under Fos loss-of-function conditions (Hyun, 2006; full text of article).

A loss-of-function analysis relying mostly on RNAi technology was performed in the Drosophila imaginal-disc system to dissect the function of Fos in the growth control of an intact developing tissue. The data obtained in these studies suggest the following model. In continuously cycling cells, D-Fos is required for the propagation of the cell cycle. If D-Fos function is reduced, components that are limiting for the successful transition from the G2 phase to mitosis are not supplied in sufficient amounts. Consequently, cells cannot leave G2/M, which causes the accumulation of cells of 4N DNA content and an overall increase of cell size, as determined by forward scatter measurements. Such a block of cell cycle function in primary cells is expected to cause the activation of checkpoints leading ultimately to the apoptotic removal of the affected cells. The conclusion that the observed increased frequency of cell death results from defects in the cell cycle is supported by experiments with eye imaginal discs. The removal of D-Fos function from cells slated for transit through a developmentally defined mitotic wave results in apoptosis at a relatively sharp time point after cell division would normally have been completed. This indicates that apoptosis is a consequence of rather than a cause for the reported cell cycle defects. Analysis of eye discs in which Fos function has been depleted but apoptosis is inhibited by the expression of p35 supports this view: in such a genotype, defects of eye development are enhanced rather than suppressed, indicating that Fos is not required just for survival signaling. The identification of cyclin B as a transcriptional target of D-Fos in imaginal discs offers a molecular explanation for the G2/M phenotype observed under D-Fos loss-of-function conditions (Hyun, 2006).

It is possible that Fos has functions in other stages of the cell cycle that do not become phenotypically apparent at the levels of Fos suppression achieved by the RNAi-based approach employed in these studies. It has, for example, been suggested that Fos has a function during the G1/S transition and regulates cyclin D transcription. It is important to note, however, that, in contrast to studies of continuously growing imaginal-disc cells, these experiments were conducted on cultured cells that entered the cell cycle from a quiescent state upon serum stimulation. For this G0-to-G1 transition, the de novo synthesis of cyclin D can be expected to be limiting and require higher levels of Fos activity than in asynchronously cycling cells (Hyun, 2006).

The studies presented here show that Fos can control specific aspects of cell cycle progression, at least in Drosophila imaginal-disc cells. This observation, if it can be extended to higher organisms, might explain the oncogenic activities of Fos proteins. However, it is important to keep in mind that Fos is a protein with complex and pleiotropic functions that can interact with multiple other transcription factors and signaling pathways. Thus, efforts to unravel the contributions of Fos proteins to cancer and other pathologies will have to consider this complexity and integrate the contribution of Fos to processes other than growth control, such as the differentiation and control of cell mobility (Hyun, 2006).

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. This study shows that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, it was shown that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. These results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized (Brock, 2012).

The traditional model of the actin cytoskeleton in cell migration, based on in vitro cell culture and biochemical assays, provides a useful framework for the mechanics of how cell migration is regulated. However, there is need for in vivo studies in order to answer important questions that are not addressed by the current model: 1. Is there a role for Profilin-mediated recycling during wound-induced migration of differentiated cells in vivo? 2. Is there a role for transcriptional regulation of actin regulators during such migrations? This latter question emerges because the basic model generally assumes that migratory cells have an intact actin-regulatory apparatus that needs only to be activated to initiate and sustain migration. While this assumption may be correct for migrating cells in developmental contexts one could imagine that initially non-migratory differentiated cells may need more than their resting complement of actin regulators in order to effect long-distance migration (Brock, 2012).

Unwounded larval epidermal cells have an even distribution of actin and Profilin throughout the cytoplasm and are thought to be non-migratory. These fully differentiated epithelial cells secrete an apical cuticle and a basal lamina. They respond to the physiological signal of tissue damage by partially dedifferentiating and becoming migratory. This study shows that the leading edge cells form multiple actin-based structures including a discontinuous cable, filopodia, and lamellipodia. A working model is proposed where the basal levels of Profilin are sufficient to make actin-based structures, but wound-induced transcription of chic is required for the cells to efficiently migrate. The lack of actin-based structures at the wound edge in cells lacking Profilin would indicate that if Formin-mediated actin nucleation is involved in wound healing, it likely requires Profilin. An epidermal sheet lacking detectable Profilin fails to close wounds or form any actin-based structures at the wound edge whereas a sheet containing only a basal level of Profilin (i.e., one that is lacking proteins that transcriptionally regulate Profilin after wounding, such as JNK, Fos, or Jun) forms actin structures at the wound edge, but is ultimately unable to efficiently migrate and close the wound. This model is complicated by the fact that cells lacking JNK, Fos, or Jun also have defects in dedifferentiation, as these cells do not stop secreting cuticle following wounding. Thus, the possibility cannot completely excluded that the lack of wound closure is due to defects in dedifferentiation. However, it is entirely possible that upregulation of actin-binding regulators is an important component of the dedifferentiation process, as this involves returning to a state during which these cells were competent to migrate (Brock, 2012).

Current wound closure models have identified two signaling pathways that are important for healing. One is Pvr signaling, where the secreted VEGF-like ligand Pvf1 activates the Pvr receptor. Currently, only a few proteins are suspected of being downstream of Pvr signaling, but Profilin is not among them. Given that epidermal cells lacking Pvr are unable to mobilize actin to the wound edge, Pvr is likely upstream of actin regulatory proteins that initiate actin polymerization at the leading edge of migrating cells. The second pathway is JNK signaling, which is required for closure but not for actin polymerization at the wound edge. Naively, it was anticipated that wound-induced chic expression would be regulated by Pvr since epidermal expression of UAS-chicRNAi also blocks actin accumulation at the wound edge. Surprisingly, this is not the case. chiclacZ expression is instead regulated by JNK signaling, as it is in the developing embryo during DC . This data reveals that although the JNK signaling pathway is not required for actin nucleation at the wound edge it contributes to actin dynamics through regulating expression of chic and perhaps other genes important for migration (Brock, 2012).

How does JNK signaling activate chic transcription after wounding? Although the upstream signal for the JNK signaling pathway is still unknown, the kinase cascade is well-defined and is thought to culminate with the phosphorylation of the transcription factors, DJun and DFos. These two proteins are commonly thought to act as a dimer (AP-1) to mediate transcriptional activation of target genes. In the early DC studies chickadee expression was shown to depend on the JNK signaling pathway. This study did not address the roles of DJun and DFos in particular, although these transcription factors are required for DC. In wound healing contexts, however, it appears that DFos can act without DJun to activate a ddc-wound reporter and a msn-lacZ wound reporter. This study found that both DJun and DFos are required to activate chic. Additionally, two consensus binding sequences for the AP-1 transcription factor (TGANTCA) are located upstream of the chic start codon (depending on the message isoform the sites are located in the 5’UTR, the first intron, or the promoter region), indicating that it is at least possible that the upregulation of chic transcription is directly accomplished by Jun and Fos. The consensus sequence is also located upstream of the human Pfn1, indicating that there is potential for this regulation to be conserved. This suggests that in the migrating cells at the wound edge, DFos can act either as a homodimer, with unidentified binding partners, or with DJun to regulate the necessary transcriptional targets (Brock, 2012).

In Drosophila embryonic models of wound closure both the contractile actin cable and filopodial processes are important for wound closure, but their relative contributions are still unclear. There has been debate over whether the cable mediates closure through contraction, through serving as a platform for extension of processes into the wound gap, or through a combinaton of these functions. From the data shown in this study it seems that actin-based contraction is not a major contributor to larval wound closure. First, the actin concentrations that appear at larval wound edges are discontinuous. Second they do not appear to be locally contractile given that the cells behind prominent concentrations do not obviously taper toward the wound. This is similar to what has been observed in the embryonic Xenopus epithelium where actin cables form but differently shaped wounds do not round up as would be expected from cable contraction. Thus it would appear that in larvae the actin concentrated at the wound edge primarily facilitates process extension into the wound gap (Brock, 2012).

This study has establish a connection between a known wound-induced signaling pathway, JNK signaling, and Profilin-mediated regulation of the actin cytoskeleton. It is speculated that transcriptional induction of actin-regulators may be a general feature of cell migration in differentiated cells as suggested by a recent study of cells undergoing EMT. By connecting upstream signaling pathways to downstream actin dynamics, this work begins to unravel the logic of how the cellular movements required for wound closure are orchestrated (Brock, 2012).

Impaired Hippo signaling promotes Rho1-JNK-dependent growth

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).

Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).

Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).

Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).

Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).

Protein Interactions

Fra, in contrast to the mammalian Fos proteins, recognizes the AP-1 site on its own and activates transcription in vitro in the absence of either Drosophila Jra or vertebrate Jun. Heteromeric complexes formed between Fra and Jra bind the AP-1 site better than either protein alone. The two proteins activate transcription synergistically in vitro (Perkins, 1990).

Drosophila Jun-related antigen, in cooperation with mouse c-FOS, can trans-activate an activator protein 1(AP-1) DNA binding site when introduced into mammalian cells. These data suggest that DJUN, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. The identification of DJUN allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving DJUN and thus vertebrate c-jun (Zhang, 1990).

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. This approach was applied to assay the transactivation potential of proteins in transgenic Drosophila embryos. A chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaves as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) requires an unexpected degree of context to function as a transcriptional activator. Evidence suggests that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, these data suggest that context-dependence of transcription factors may be more prevalent than previously thought (Szuts, 2000a).

Which factors provide the context for Dfos function? Several lines of evidence implicate JNK and Dpp signaling and their transcriptional target factors Djun and Mad as the essential context. (1) The only embryonic cells in which LexFos functions reliably and robustly to stimulate transcription are the dorsal leading edge cells which experience both of these signals. (2) Neither of the LexFos derivatives (LexFosN, LexFosC) function in these cells, strongly implicating the basic leucine zipper domain of LexFos (the only domain absent from both derivatives) in its function. As this domain mediates dimerization with Djun, the only known dimerization partner of Dfos in Drosophila, this indicates that the activity of LexFos depends on Djun. Recall that Djun is present and activated by JNK signaling in the leading edge cells. (3) JNK signaling as mimicked by overexpression of constitutively acitive Drac* or Dcdc42*, potently synergizes with LexFos to mediate widespread transactivation in the embryo. A very similar widespread synergy has also been seen between LexFos and Jun*, a mutant form of c-Jun that mimics signal-activation of this protein. The embryonic territories in which these synergies are observed appear to correspond to sites of Dpp stimulation. Consistent with this, a limited synergy between Dpp and LexFos has also been observed in some embryonic cells. These synergies strongly implicate JNK and Dpp as necessary context signals for LexFos function. (4) LexFos activity strictly depends on the context sequence in the MadL target reporter; under no conditions does it transactivate a reporter that contains four tandem LexA binding sites (albeit LexGAD very efficiently does so). The context sequence in MadL essentially consists of a binding site for the Dpp response factor Mad, which is thus a likely partner for the putative LexFos/Djun* dimer (Szuts, 2000a).

These results indicate that JNK-activated Djun and Dpp-activated Mad may be critical and widespread context partners of Dfos. Consistent with this, Dfos function is required for dorsal closure of the embryo and, by implication, functions normally in cells that experience JNK and Dpp signaling. In the embryonic midgut, Dfos functions in cells that experience Dpp and Egfr signaling. Because the LexFos/JNK synergy in the mesoderm implies that JNK signaling is normally absent from this tissue, this suggests that the normal partner of Dfos in the midgut visceral mesoderm may be a factor, as yet unidentified, that is activated by Egfr signaling. Interestingly, synergy between the c-Jun/c-Fos dimer and TGF-beta activated Smad has also been observed in mammalian cells. Furthermore, Jun proteins have recently been shown to bind directly to Smad3/4. Thus, the partnership between signal-activated Jun/Fos dimers and Smads may be fairly widespread and fundamental (Szuts, 2000a).

Phosphorylation of Fos by ERK and JNK signals

During Drosophila development Fos acts downstream from the JNK pathway. It can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos reveals that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses (Ciapponi, 2001).

In addition to its embryonic expression pattern, D-Fos can be detected at later stages of development, for example, in wing and eye imaginal discs. To gain insight into late developmental functions of D-Fos, its contribution to wing development was analyzed. The early lethality of Drosophila fos mutants ruled out a direct assessment of imaginal disc development in flies lacking D-fos. Generation of somatic mutant clones of cells deficient for D-fos was not feasible because the only extant alleles, kay1 and kay2, are not suitable for such experiments. kay2 represents a hypomorphic mutation of unknown nature. Therefore, the occasional viable homozygous flies (escapers) that are recovered from this stock might not display the full range of phenotypes that can arise from defects in D-fos. The null allele kay1, in contrast, causes cell lethality in imaginal disc cell clones homozygous for this mutation. It is not clear, however, whether this drastic phenotype is caused by a loss of D-Fos function, since it cannot be rescued by transgenic D-Fos expression. This may be due to the fact that the kay1 mutation consists of a deletion that removes more than one lethal complementation group. Therefore, fly strains expressing a dominant-negative mutant form of D-Fos (subsequently referred to as D-FosbZIP) were examined. This truncated version of D-Fos consists of the isolated bZIP domain. Thus, it can dimerize with endogenous Fos partner proteins such as D-Jun and bind to DNA, but it lacks the ability to stimulate transcription (Ciapponi, 2001).

A number of observations have shown that the expression of D-FosbZIP causes phenotypes specifically resembling those elicited by D-fos loss-of-function alleles (kay1 and kay2): (1) when expressed in epidermal cells during embryogenesis, D-FosbZIP causes a dorsal anterior hole, which phenocopies the dorsal closure defect of fos-deficient embryos; (2) expression of D-FosbZIP in the dorsal part of the wing imaginal disc epithelium, from which the adult thoracic body wall derives, results in a cleft along the dorsal midline, as do D-fos mutant alleles; (3) expression of D-FosbZIP in the embryonic endoderm elicits a midgut phenotype and reduces Labial expression, reminiscent of kay mutants. D-FosbZIP, therefore, is a suitable tool with which to analyze the function and the regulation of D-Fos in later developmental processes such as imaginal disc morphogenesis (Ciapponi, 2001).

Using the UAS/Gal4 system, D-FosbZIP was expressed in regions of the wing imaginal disc that give rise to the entire adult wing structure or parts of it. Expression under the control of the three Gal4 drivers used, dpp Gal4, en Gal4, and sd Gal4, results in loss of vein material in the respective expression domains. The expression of D-FosbZIP under dpp control, between the longitudinal veins II and III, causes disappearance of the anterior crossvein. When driven in the posterior compartment by en Gal4, D-FosbZIP causes partial loss of the longitudinal vein V. Finally, if the expression of D-FosbZIP is driven by sd Gal4 in the entire wing pouch, the phenotype is most striking, displaying a partial loss of veins II and IV and of almost the entire vein V. Significantly, the D-FosbZIP-dependent loss-of-vein phenotype is enhanced in a heterozygous D-fos loss-of-function (kay1) background, that is, when the endogenous D-fos gene dose is reduced. This confirms that the observed effect of D-FosbZIP is specific and caused by interference with endogenous D-Fos (Ciapponi, 2001).

The loss of wing vein tissue on expression of D-FosbZIP resembles phenotypes resulting from defects in the Drosophila epidermal growth factor receptor (DER) pathway, caused, for example, by loss-of-function alleles of DER itself or of other genes required for DER signaling, such as rhomboid, vein, ras, and ERK/rolled. Therefore, the above results might indicate that D-Fos acts as a mediator of the DER/ERK signaling pathway during wing vein differentiation. The artificial activation of this RTK pathway, by gain-of-function alleles or by overexpression of downstream effectors, gives rise to ectopic veins in the wing. To establish whether D-Fos might act epistatically to DER, and whether D-FosbZIP or a reduced D-fos gene dose (kayak alleles) might suppress such a phenotype was examined (Ciapponi, 2001).

The EllipseB1 (ElpB1) allele of DER represents an activated component of the DER/ERK signaling pathway. In addition to other phenotypes, for example, in the eye, ElpB1 animals consistently develop wings with ectopic wing vein material. Strikingly, both D-FosbZIP expression in the wing imaginal disc (using the 32B Gal4 driver) or the removal of one copy of D-fos in a kay heterozygote, suppresses this phenotype almost completely. To confirm that the observed effect is specific and caused by a reduction of endogenous D-Fos function, add-back experiments were performed in which this reduction was compensated by supplying extra wild-type D-Fos from a transgene, driven by the heat shock promoter (hs D-fos. Significantly, the presence of the D-fos transgene abrogates the suppression of ElpB1 by kay and reinstates the extra vein phenotype caused by elevated DER activity. This result confirms that the suppression of the activated DER allele is due to a loss of D-fos activity. Hence, D-Fos mediates wing vein patterning downstream from or in parallel with DER (Ciapponi, 2001).

Next, whether D-Fos mediates ERK signaling also during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles ElpB1 and sevS11. The ElpB1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sevS11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sevS11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sevS11 in a heterozygous kay2 background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sevS11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).

Considering that D-Fos is a transcription factor and based on the precedents of D-Jun and c-Fos, the most obvious role for D-Fos in DER and Sev signal transduction would be that of an effector of the Drosophila MAP kinase Rolled. Therefore, the effect of reducing D-fos activity in animals expressing the gain-of-function allele rlSem was examined. Expression of RlSem under UAS control in the wing imaginal disc results in an extra-vein phenotype, markedly when the flies are reared at 25°C and milder at 18°C. Simultaneous expression of D-FosbZIP along with RlSem causes a striking suppression of ectopic vein formation, whereas additional expression of full-length D-Fos leads to a strong enhancement of the phenotype. To confirm that the observed suppression of the rlSem phenotype was not due to an effect of D-Fos on the transgene promoters, a similar genetic interaction experiment was performed using the endogenous rlSem gain-of-function allele and kay2 allele. kay2 heterozygosity suppresses the rlSem-induced extra-vein phenotype. This effect is reverted by ubiquitous expression of D-Fos, indicating that the suppression is due specifically to the decreased activity of D-Fos. These genetic interactions confirm that the role of D-Fos in RTK signal is that of an effector of Rolled (Ciapponi, 2001).

The results described above reveal D-Fos as a downstream component of the ERK signal transduction pathway, yet previous genetic analyses have shown that the transcription factor serves as an effector of JNK. This raises the question of whether the function of D-Fos as recipient of ERK or JNK is mutually exclusive and determined by the cellular context, or whether the transcription factor may mediate both JNK and ERK responses in one tissue or one cell. The developing eye provides a system to approach such a question. Biochemical and genetic studies have indicated that the planar polarity pathway downstream from Frizzled (Fz) and Dishevelled (Dsh) leads to the activation of a JNK-type MAPK module. During retinal morphogenesis, this pathway controls the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline. Thus, in the developing eye the activity of JNK and ERK signal transduction can be monitored separately in vivo (by planar polarity and R-cell recruitment, respectively) (Ciapponi, 2001).

To determine whether D-Fos is involved in planar polarity signaling, the effect of D-FosbZIP expressed under the control of Gal4 drivers in the developing eye was examined. When D-Fos function is thus reduced, a striking combined phenocopy of defects in ERK and JNK signal transduction ensues. Sections of eyes of the hairy Gal4/UAS D-fosbZIP or of the sev Gal4/UAS D-fosbZIP genotypes display both a lack of photoreceptor cells, diagnostic of inadequate ERK signal transduction, and misoriented ommatidia, indicating defects in planar polarity signaling. This mutant phenotype makes it plausible that D-Fos, in addition to its role in photoreceptor cell recruitment downstream from ERK, acts as an effector of JNK signaling in planar polarity determination. Therefore, D-Fos mediates both JNK and ERK responses in a defined group of cells of the developing retina (Ciapponi, 2001).

To investigate the potential regulation of D-Fos by protein phosphorylation, in vitro kinase assays were performed in which recombinant JNK/Bsk and ERK/Rl were used as kinases and different bacterially expressed versions of D-Fos were used as substrates. In this in vitro setting, both Bsk and Rl could phosphorylate full-length D-Fos. To get an initial indication as to which residues might serve as target sites for ERK or/and JNK, the D-Fos amino acid sequence was compared with that of mammalian Jun and Fos proteins. The sequence alignments identified several sequences in D-Fos with similarity to confirmed JNK or ERK phosphorylation sites in the mammalian molecules. T89 and T93 of D-Fos correspond in their sequence context and relative location to established JNK phosphorylation sites in c-Jun. Alignment of the C-terminal parts of D-Fos and c-Fos show a conserved residue (T584) that corresponds to a previously described MAPK phosphorylation site in c-Fos. Moreover, several serine or threonine residues were identified that might serve as target sites for the proline-directed MAPKs. Mutant derivatives of D-Fos were generated in which one or more of these candidate phosphorylation sites was substituted by alanine. JNK/Bsk, but not ERK/Rl, can efficiently phosphorylate a fragment spanning the 170 N-terminal amino acids of D-Fos. When alanine substitutions are introduced in positions T89 and T93, the N-terminal D-Fos fragment is no longer an efficient substrate for JNK phosphorylation (Ciapponi, 2001).

Additional N-terminal residues that conform to the S/TP consensus (T234, S235, T237, and T254) are phosphorylated by neither JNK nor ERK. Similar to the 170-amino-acid N-terminal fragment, a D-Fos fragment covering the N-terminal 285 amino acids is not a substrate for ERK. This indicates that the N-terminal part of D-Fos is a good substrate for JNK/Bsk but not for ERK/Rl, and that the residues T89 and T93 serve as the main N-terminal JNK target sites (Ciapponi, 2001).

Interestingly, a small deletion that removes a sequence with remote similarity to the c-Jun delta-domain (amino acids 28-56), but that does not span the phosphorylation sites T89 and T93, completely abrogates phosphorylation of the D-Fos N-terminal fragment by JNK/Bsk. It is possible that this deletion destroys a delta-domain-like JNK docking site present in D-Fos (Ciapponi, 2001).

Next, the C-terminal part of D-Fos was analyzed; it contains seven potential phosphorylation sites for MAPKs. A fragment spanning the C-terminal 280 amino acids of D-Fos is, in contrast to the N terminus, phosphorylated efficiently by both Bsk and Rl. Alanine substitutions in all seven putative MAPKs target sites causes complete loss of phosphorylation. However, mutating subsets of the seven putative phosphorylation sites does not result in a complete loss of phosphorylation by either Rl or Bsk, indicating the presence of multiple phosphorylation sites in the C-terminal part of D-Fos. These results indicate that, at least in vitro, D-Fos is a direct substrate of both Drosophila JNK and ERK and that it contains overlapping, but distinct, sets of phosphorylation sites for the two kinases (Ciapponi, 2001).

After establishing which residues serve as substrates for JNK and/or ERK in vitro, it was important to determine the regulatory relevance of these sites in vivo. Transgenic fly strains expressing mutant forms of full-length D-Fos were generated in which either the putative N-terminal, JNK-specific phosphorylation sites, or the C-terminally located ERK and JNK substrate sites were replaced by alanine (D-FosN-Ala and D-FosC-Ala. In the D-Fospan Ala mutant, all the putative MAPK phosphorylation sites, both N- and C-terminal, were substituted by alanine. When the nonphosphorylatable D-Fospan Ala was expressed from a UAS-driven transgene under the control of the epidermal driver 69B Gal4, it gave rise to a strong thoracic cleft at the dorsal midline, resembling kay or hep mutants, or phenotypes that result from D-FosbZIP expression. Evidently, D-Fospan Ala represents a dominant-negative mutant that can interfere with JNK-dependent thorax closure (Ciapponi, 2001).

The relevance was investigated of subgroups of the D-Fos MAPK phosphorylation sites during thorax closure. Expression of D-FosN-Ala results in a distinctive thorax cleft, although not quite as pronounced as in the case of D-Fospan Ala. Importantly, this result shows that the N-terminal, JNK-specific phsosphorylation sites in D-Fos are required for a well-defined JNK-dependent developmental mechanism. Expression of D-FosC-Ala, which lacks the C-terminal phosphorylation sites, or of D-Foswt has no discernible effect. These results indicate that either the C-terminal sites play only an ancillary role and are not essential for JNK signaling in the signal transduction pathway controlling thorax, or that this mutant does not compete well with endogenous D-Fos and therefore has no dominant-negative effect in this context (Ciapponi, 2001).

Next, whether expression of the different phosphorylation mutants of D-Fos might also interfere with ERK responses was investigated. Expression of D-Fospan Ala in the posterior compartment of wing imaginal disc (using en Gal4) causes loss of wing vein material typical of mutants defective in DER to Rolled signaling. Thus, consistent with the observation that D-Fospan Ala has lost all substrate sites for both Rolled and Bsk, it acts as a dominant-negative form that interferes with D-Fos function in both the ERK and the JNK pathways (Ciapponi, 2001).

Interestingly, however, the D-FosN-Ala and D-FosC-Ala mutants influence the ERK and JNK response differently. D-FosN-Ala, which interfers dominantly with JNK-mediated thorax closure, has no effect on ERK-dependent wing vein formation. This is consistent with these sites not being substrates for Rolled. The D-FosC-Ala mutant, however, which is neutral in thorax development, causes loss-of-vein phenotype (Ciapponi, 2001).

To examine whether differential phosphorylation of D-Fos might also be used in the developing eye to distinguish between ERK and JNK signaling, the effect of the D-FosAla mutants on ERK-dependent photoreceptor cell recruitment and JNK-mediated ommatidial rotation was examined. Different D-FosAla mutants were expressed along with sevS11 in the eye imaginal disc under the control of the sevenless enhancer. As in the case of wing vein formation, D-FosN-Ala does not alter the sevS11 phenotype, whereas the expression of D-FosC-Ala or of D-Fospan Ala causes a significant suppression of the extra R7-cell recruitment. Thus, ERK signaling, whether it is triggered by DER or by Sev, appears to require only the C-terminal phosphorylation sites of D-Fos (Ciapponi, 2001).

Next, whether the D-FosAla mutants could suppress the ommatidial misrotation phenotype that is elicited by overexpression of Fz and the ensuing activation of JNK was tested. Coexpression of all Ala mutants and Fz under the control of sev Gal4 leads to a significant suppression of the misrotation phenotype observed in flies expressing Fz alone. Wild-type D-Fos does not have this effect. These results indicate that the JNK-phosphorylation sites of D-Fos are required for the manifestation of the Fz gain-of-function phenotype. The Fz-JNK response in the eye is also affected by mutation of the C-terminal sites that do not visibly disturb the thorax closure response. This might be explained by the higher sensitivity of the ommatidial rotation paradigm or a higher relative expression of the FosAla mutant transgene in the photoreceptor cells (Ciapponi, 2001).

The dominant phenotypic effects of D-FosAla expression support the interpretation that Fos is regulated by protein phosphorylation to mediate developmental decisions and indicates that the residues identified by mutagenesis and in vitro kinase assay are required for in vivo function. Moreover, these findings indicate clearly that the function of D-Fos as a mediator of JNK/Bsk and ERK/Rl cascades is in both cases that of a direct kinase substrate (Ciapponi, 2001).

An important question raised by the finding that D-Fos can mediate signaling by both ERK and JNK is how the decision between these distinct cellular responses is made, that is, how the cell 'knows' which program to execute when D-Fos becomes phosphorylated. Several mechanisms have been suggested to contribute to signal specificity in such situations, in which one protein mediates different cellular responses. One model proposes a combinatorial mechanism by which several factors with overlapping broad responsiveness have to cooperate to mediate a defined specific cellular behavior. However, the observation that D-Fos participates in ERK as well as JNK signal transduction in the same group of cells of the developing Drosophila eye, by regulating photoreceptor differentiation and ommatidial rotation, respectively, argues against cell type-specific cofactors that modulate the response to D-Fos phosphorylation. The distinct substrate sites in D-Fos phosphorylated by ERK and JNK raise a novel possibility to explain the signal-specific D-Fos response. It is suggested that D-Fos exists in two different activated forms, depending on whether it is phosphorylated by ERK or JNK. These differentially phosphorylated forms might then selectively trigger either the ERK or the JNK response. This idea is supported by in vivo experiments in which phosphorylation site-specific point mutants of D-Fos were expressed in the developing fly. A mutant that lacks all phosphorylation sites interferes dominantly with both ERK and JNK signaling in thorax closure, the wing, and the eye imaginal disc, supporting further the general relevance of D-Fos phosphorylation in developmental decisions. D-Fos mutants lacking subsets of phosphorylation sites, however, affected JNK and ERK signal responses differentially. An N-terminal cluster of JNK sites that is not phosphorylated by ERK is critical for the JNK response in vivo. A mutant lacking these sites interfers with thorax closure and planar polarity regulations, both bona fide JNK responses, but not with wing vein formation or photoreceptor differentiation, which are regulated by ERK. Conversely, a mutant that removes all ERK substrate sites dominantly suppresses processes normally controlled by this MAP kinase. These data indicate that signal-responsive transcription factors, such as D-Fos, may have different signal-specific functions. It is tempting to speculate that such a mechanism might be used by other signaling proteins that are receptive to different upstream signals (Ciapponi, 2001).

Factors acting upstream of Fos

In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depends on the amount of Bsk activity, and the amount of Bsk activity depends on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrate a physiological link between Src and JNK (Tateno, 2000).

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis. Mutations in genes such as hemipterous (hep) and basket (bsk, also known as DJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft. Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade. Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, a screen was carried out for mutants showing the dorsal midline cleft phenotype, like a mild hep mutant. The mutant for Src42A shows this phenotype and Src42A regulates Bsk during Drosophila development (Tateno, 2000).

Furthermore, the Tec29 Src42A double mutant shows complete embryonic lethality, and a certain fraction of the dead embryos show the dorsal open phenotype. Activated DJun, a transcription factor downstream of Bsk, partially rescues the dorsal open phenotype in the Tec29 Src42A double mutant. Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. The double mutant for Src64 and Src42A manifests a mild but clear dorsal open phenotype, which suggests a functional redundancy between Src64 and Src42A (Tateno, 2000).

Expression of puc is known to be induced by the Bsk signal. In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum. In the wing disc of the Src42AJp45 mutant, puc expression is reduced. In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) shows ectopic expression of puc. Further, introduction of a hep null mutation reduces the amount of ectopic puc expression. It is known that Bsk induces expression of puc and decapentaplegic (dpp) during embryonic dorsal closure. The embryos of the Tec29 Src42A double mutant do not show any puc or dpp expression in the leading edge cells. These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure (Tateno, 2000).

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells. In the absence of the Bsk signal, these cells do not fully elongate. The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells. Accumulation of these substances is disturbed in the DJun and the puc mutants. In the double mutant for Tec29 and Src42A, the leading edge cells contain reduced quantities of F-actin and P-Tyr, and these cells are only partially elongated. Thus, the defect in embryonic dorsal closure in the Tec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant (Tateno, 2000).

A model is proposed in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK. As in Drosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis. Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved (Tateno, 2000).

It can be also interpreted that Src42A acts upstream of DFos, a dimerization partner of DJun. Although the Src42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype, the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice have a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity is milder in c-src than in c-fos knockouts; this can be explained by the functional overlap in multiple Src-family tyrosine kinases. Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex (Tateno, 2000).

Transrepression of AP-1 by nuclear receptors

Mammalian cell culture studies have shown that several members of the nuclear receptor super family such as glucocorticoid receptor, retinoic acid receptor and thyroid hormone receptor can repress the activity of AP-1 proteins (referring to Drosophila Kayak and Jun) by a mechanism that does not require the nuclear receptor to bind to DNA directly, but that is otherwise poorly understood. Several aspects of nuclear receptor function are believed to rely on this inhibitory mechanism, which is referred to as transrepression. This study presents evidence that nuclear receptor-mediated transrepression of AP-1 occurs in Drosophila melanogaster. In two different developmental situations, embryonic dorsal closure and wing development, several nuclear receptors (Seven up, Tailless, and Eagle) antagonize AP-1. The inhibitory interactions with nuclear receptors are integrated with other modes of AP-1 regulation, such as mitogen-activated protein kinase signaling. Discussed here is a potential role of nuclear receptors in setting a threshold of AP-1 activity required for the manifestation of a cellular response (Gritzan, 2002).

The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure. During DC, lateral epidermal cells migrate dorsally and close the epidermis on the dorsal side of the embryo. Failure to undergo DC results in a characteristic dorsal open phenotype, the cuticle of affected embryos displays a dorsal hole. Mutations in genes encoding the Drosophila homologs of JNKK, (JNK, Jun and Fos) all give rise to similar dorsal open phenotypes. Thus, it is thought that DC requires activation of Jun/Fos heterodimers by a JNK-type MAPK cascade. Embryos homozygous for kay1, a fos null allele are devoid of zygotic Fos activity and DC fails. A large dorsal hole forms and the cuticle collapses. In an embryo homozygous kay2, a hypomorphic fos-allele, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorso-anterior hole (Gritzan, 2002).

To test whether Drosophila NRs can antagonize AP-1, a variety of AP-1 constructs were in the embryonic epidermis. Interestingly, expression of some, but not all, NRs tested result in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 results in a DC phenotype reminiscent of that of kay2 homozygotes. This finding is consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter causes a weak dorsal open phenotype. The differentiation of ventral cells does not seem to be disturbed by Tll expression since the pattern of denticles in this part of the epidermis appears grossly normal. Thus, Tll expression specifically affects the dorsal epidermis where AP-1 activity is required. The expression of Knrl under the control of pnrGal4 elicits stronger DC phenotypes with the dorsal hole frequently extending over several segments (Gritzan, 2002).

If the DC defects caused by NR expression reflect a negative effect on AP-1, the defects should be sensitive to changes in Fos or Jun activity. In genetic interaction experiments, the dorsal open phenotypes caused by NR expression were compared in a wild-type background and in embryos with altered levels of AP-1 activity. Embryos heterozygous for kay1 carry only one copy of the fos gene. While these embryos are phenotypically normal, their levels of AP-1 are reduced and they might therefore be more susceptible to a further decrease of this activity. If expression of NRs causes DC defects by antagonizing AP-1, it should have stronger phenotypic consequences in embryos heterozygous for kay1 than in wild type embryos. Indeed, while expression of Eagle (Eg) in a wild type background mostly results in DC phenotypes of intermediate strength, NR expression in embryos heterozygous for kay1 typically elicits complete failure of DC, indicative of a severe reduction of AP-1 activity. Since embryos of both genotypes display somewhat variable phenotypes, the effect of kay1 heterozygosity is best appreciated by quantitative analysis. A clear reduction in size, a collapsed folded cuticle and a dorsal hole extending over at least half of the body length are described as characteristics of a strong DC phenotype. Embryos with a smaller dorso-anterior hole and normal body size were scored as showing weak DC phenotypes. This analysis confirms that Eg expression has more severe phenotypic consequences in kay1 heterozygotes than in wild type embryos and supports the suggestion that NRs cause defective DC by suppressing AP-1 activity (Gritzan, 2002).

In a complementary experiment, the effect of Eg expression was examined in embryos with increased AP-1 activity. In embryos heterozygous for pucE69, the levels of the CL100 phosphatase Puckered (Puc) which specifically inactivates JNK are reduced. Thus, in contrast to kay1 heterozygotes, embryos of this genotype have elevated levels of JNK, and consequently AP-1, activity. If the phenotypic outcome of NR expression in the embryo is mediated by transrepression of AP-1, the DC defects should be weaker in puc heterozygotes than in a wild type background. Quantitative analysis reveals that the frequency of strong DC phenotypes is indeed greatly reduced in pucE69 heterozygotes expressing Eg compared to Eg expression in a wild type background. Expression of the NR Knrl in the various backgrounds yields essentially identical results. These data support the hypothesis that several Drosophila NRs can antagonize AP-1 as has been shown for mammalian NRs (Gritzan, 2002).

Based on the results of the DC assays, it cannot be determined whether the antagonism between AP-1 and NRs is caused by the downregulation of direct AP-1 target genes by NRs or whether the effect is more indirect. To address this issue, the effect of Drosophila NRs on bona fide AP-1 target genes was monitored. However, direct AP-1 target genes have not yet been clearly defined in Drosophila. While it is known that dpp and puc expression in DC requires AP-1, it cannot be excluded that this effect is indirect. To circumvent this problem, a mammalian cell culture system was used. Transrepression of AP-1 by mammalian NRs was first described in the context of collagenase transcription. Extensive studies of the collagenase promoter have identified AP-1 as one of its primary regulators. Activation of the GR down-regulates AP-1-mediated transcription of collagenase. It was asked whether Drosophila NRs behave similarly in this well-defined assay. Transcriptional activation by AP-1 was measured using a reporter construct in which transcription of the firefly luciferase gene is controlled by the upstream region of the human collagenase gene. Comparing luciferase activity in the presence and absence of Drosophila NRs, it was found that both Eg and Tll efficiently antagonized AP-1 activity in a dose-dependent manner. The observed effects are quantitatively comparable to those reported for the GR. Taken together, these data strongly suggest that Drosophila NRs are competent for AP-1 transrepression. Furthermore, the cell culture assay demonstrates that Drosophila NRs can antagonize mammalian AP-1 and implies that the mechanism of transrepression is conserved between Drosophila and mammals (Gritzan, 2002).

Does modulation of AP-1 activity by NRs occur only in situations where AP-1 is regulated by JNK or does this type of regulation also operate in different contexts? A function for Fos downstream of ERK has been demonstrated in the differentiation of wing veins. Extra vein material can result from elevated levels of ERK, as in flies carrying a gain-of-function allele of the rolled gene, which encodes Drosophila ERK. This allele, called rolledSevenmaker (rlSem), encodes a form of ERK with increased resistance to inactivation by dephosphorylation. Expression of a dominant-negative form of Fos in the wings of rlSem flies results in loss of ectopic vein material. Conversely, overexpression of Fos enhances the extra-vein phenotype caused by rlSem (Gritzan, 2002).

32B Gal4, UAS Sem flies express the RlSem form of ERK in the wing from a UAS-driven transgene. As a consequence of elevated levels of ERK activity, these animals develop ectopic wing vein material. Reducing fos gene dosage in this system strongly suppresses the vein phenotype, consistent with the proposed role of Fos as an ERK effector. Thus, 32B Gal4 UAS Sem flies provide a suitable system to examine how genetic manipulations of AP-1 activity affect vein differentiation. To investigate a potential role of the Drosophila NRs in this process, one copy of kni, eg, tll or svp was removed in 32B Gal4, UAS Sem flies. Reducing kni function does not influence the vein phenotype. However, heterozygosity for any of the other three receptors tested reproducibly leads to a mild enhancement of the ectopic vein differentiation. As an unambiguously scoreable criterion to statistically evaluate phenotypic effects, the presence of ectopic vein material posterior to L5 was chosen. This area of the wing is relatively resistant to the formation of extra vein material. Quantitative analysis clearly shows that whereas the formation of extra vein material posterior to L5 in 32B Gal4 UAS Sem flies is suppressed by reducing fos activity, it is enhanced by a reduction of eg, svp or tll function. These data suggest that all three NRs antagonize AP-1 activity in wing vein differentiation, conceivably in a redundant manner (Gritzan, 2002).

It is speculated that the modulation of AP-1 activity by NRs contributes to what has recently been termed signal consolidation. Cells have to place a value on incoming signals (e.g. EGF-induced ERK activity) such that they are either answered by a biological response (e.g. the execution of a transcriptional program) or disregarded as noise. It is proposed that the modulation of AP-1 activity by NRs facilitates the interpretation of the EGF signal in wing vein differentiation by defining a threshold of ERK activity. Cells in which ERK activity does not reach this threshold do not mount an AP-1-dependent transcriptional response to the EGF signal. When transrepressional control is impaired (as in the svp, tll double mutant clones) the threshold is lowered and more cells than appropriate interpret EGF-induced ERK activity as a consolidated signal. This leads to the formation of ectopic vein material. This model is supported by the finding that the ectopic vein tissue observed in clones of tll and svp mutant tissue did arise close to the position of the endogenous veins and not randomly throughout the clonal area. Thus, the regulation of AP-1 by NRs appears to convey cell-intrinsic information (Gritzan, 2002).

Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila

Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. This study shows that the conserved coactivator multiprotein bridging factor 1 (MBF1) is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. It is concluded that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress (Jindra, 2004).

Sensitivity of AP-1 to oxidation requires a mechanism to protect AP-1 activity. This study introduces MBF1 as a new player that allows cells to maintain adequate AP-1 activity under oxidative stress. Drosophila AP-1 components D-Jun and D-Fos undergo oxidative inactivation via the same cysteine residues as the human orthologs. MBF1 prevents this oxidation and preserves the DNA-binding activity. In mbf1 mutants, an AP-1-dependent developmental process becomes hypersensitive to oxidative stress, suggesting that MBF1 also protects D-Jun from an oxidative modification in vivo. The protection is unlikely to be complete because it relies on the binding of MBF1 to Jun. Thus, the AP-1 action may be in an equilibrium between acceleration by the MBF1 protection of Jun and brake by the oxidative inactivation of Jun (Jindra, 2004).

The mechanism by which MBF1 ensures the activity of AP-1 is different from that of the nuclear protein Ref-1, which reactivates oxidized AP-1 by reduction. MBF1 was a much stronger enhancer of AP-1 activity when coexpressed and copurified with D-Jun from E. coli than when it was added to the DNA-binding assay separately. Unlike Ref-1, MBF1 was unable to restore AP-1 activity once lost. Thus, rather than reactivating AP-1, MBF1 protects it from oxidation in a preventive manner. Protection from oxidation is however one of several stabilizing effects that MBF1 exerts on AP-1, because MBF1 can stimulate DNA binding even of mutant AP-1 proteins, possessing serine instead of the redox-sensitive cysteine residues (Jindra, 2004).

MBF1 enhanced the DNA-binding activity of AP-1 selectively through D-Jun. Since MBF1 bound D-Jun but not D-Fos in a direct interaction assay, it is proposed that the selectivity is based on an exclusive contact between MBF1 and D-Jun. This was unexpected as human MBF1 was shown to bind a GST-c-Fos fusion. On the other hand, D-Jun and c-Jun share more similarity than the Fos orthologs; in particular, the critical cysteine context KCR reads RCR in D-Fos (Jindra, 2004).

Although AP-1 regulation via the redox-sensitive cysteine residues was postulated more than a decade ago, the nature of the cysteine modification remained unknown. The prediction is that a regulatory oxidation may involve a reversible formation of sulfenic acid or a disulfide bond. To examine how the critical cysteine is modified, the molecular mass was determined of the bacterially expressed D-Jun used in DNA-binding assays. The E. coli system allowed expression of D-Jun in the absence of endogenous MBF1. Surprisingly, a previously undescribed modification was identified of the critical cysteine, S-cystenyl cystenylation. In a striking contrast, no such modification occurred in D-Jun coexpressed with MBF1 or in D-Jun lacking the critical cysteine residue. S-glutathiolation of the cysteine, a similar modification that was known to prevent binding of c-Jun to an AP-1 site, was not observed in D-Jun despite GSH:GSSG is an abundant redox system in E.coli. Whether S-cystenyl cystenylation is only a product of the prokaryotic expression system or whether it represents true physiological regulation of AP-1 activity remains to be tested. However, the aim of this study was to disclose the role for MBF1, and the ability of MBF1 to avert S-cystenyl cystenylation shows that this role is to protect D-Jun (Jindra, 2004).

While the data illuminate the role of MBF1 in the protection of the redox-sensitive cysteine in D-Jun, MBF1 also stimulated DNA binding of the serine mutant. Thus the effect of MBF1 on D-Jun is not limited to protecting the critical cysteine but includes a more general stabilizing effect on the basic region. This is consistent with the observation that yeast MBF1 enhanced DNA binding of GCN4, which harbors a serine in the position of the oxidation-sensitive cysteine. Analysis of yeast MBF1 and GCN4 indicates that this serine resides within the region contacted by MBF1. It is speculated that it is this evolutionarily ancient function of MBF1 to support the activity of bZIP proteins that permitted the acquisition of the redox regulation of AP-1 by oxidation of the critical cysteine; in the absence of MBF1, such mutation (serine to oxidation-sensitive cysteine) would be prone to the total destruction of the AP-1 activity even under mild oxidative conditions. Interestingly, the yeast counterpart of AP-1 (yAP-1) is also required for antioxidant defense and is accordingly regulated by the redox state, albeit at the level of nuclear export. The metazoan AP-1 may have introduced redox sensing at the DNA-binding step since it is directly involved in transcriptional regulation compared with the nuclear export (Jindra, 2004).

Despite the fact that evolutionary conservation of MBF1 suggests an essential role for the protein, null mutants lacking MBF1 proved to be viable in Drosophila and yeast under laboratory conditions. Strikingly, however, in both organisms, MBF1 is essential during stress situations encountered in the real world: Drosophila mbf1 mutants are sensitive to oxidative stress induced by H2O2, and yeast MBF1 mutants are unable to overcome nutritional stress due to their inability to maintain the activity of GCN4, a regulator of amino-acid synthesis. A comparative advantage provided by MBF1 under stress conditions is thus the likely cause of its evolutionary conservation. It is proposed that in both yeast and Drosophila, MBF1 achieves these functions via the same mechanism, through binding a bZIP transcription factor (Jindra, 2004).

The interaction between MBF1 and D-Jun, documented in this study, provides a molecular basis of the H2O2 sensitivity of mbf1 mutants. This is supported by the recently published evidence that JNK signaling is indeed required for oxidant resistance in Drosophila. A developmental defect that can occur in mbf1 mutants under oxidative stress is the failure to form a continuous cuticle at the dorsal midline. The cell shape changes of epithelia that occur at the dorsal closure during embryogenesis and adult morphogenesis are regulated by the JNK signaling pathway, culminating in the phosphorylation of D-Jun. Using a knockdown experiment, this study shows that also D-Jun is directly involved in the adult thorax closure. Because MBF1 exhibits a genetic interaction with AP-1 subunits under H2O2 challenge, it is likely that D-Jun requires its partner MBF1 to be protected from oxidation during its function in thorax closure. Necrotic wounds in mbf1 D-fos/mbf1 flies are a newly observed phenomenon, which may be connected with the exposure to H2O2 and may reflect a specific requirement for Fos in wound healing. Another phenotype that mbf1 mutants display is the reduced longevity when challenged with H2O2. Since AP-1 is known to trigger antioxidant defense, the idea is favored that H2O2 hypersensitivity of mbf1 mutants is also due to their failure to protect Drosophila AP-1 activity during oxidative condition. For either phenotype function, the possibility remains that MBF1 also supports functions of other transcription factors (Jindra, 2004).

MBF1 was first described as a coactivator that bridges bZIP transcription factors and the basal transcriptional machinery. Yeast MBF1 supports GCN4-dependent activation of the HIS3 gene and Drosophila MBF1 serves as a coactivator of a bZIP protein Tracheae defective/Apontic during morphogenesis of the tracheal and nervous systems (Liu, 2003). In either case, MBF1 facilitates the formation of a ternary complex consisting of the bZIP protein, MBF1 and the general transcription factor TBP. MBF1 has been recently shown to interact also with human AP-1 proteins and function as a novel transcriptional coactivator of c-Jun in a human cell line (Jindra, 2004).

Results presented in this study suggest a new function for coactivators. This study demonstrates that MBF1 can prevent an oxidative modification of D-Jun produced in bacteria, and that MBF1 activity becomes important under oxidative environmental conditions in vivo. Association of MBF1 with D-Jun in Drosophila cells and the D-Jun-dependent nuclear localization of MBF1 suggest that endogenous Jun, once synthesized, is quickly bound by MBF1. Thus it is possible that transcriptional coactivators may exert a stabilizing or protective effect on their partner transcription factors even before they engage in transcription, and that the formation of the ternary complex is a two-step phenomenon involving a preformed complex and TBP (Jindra, 2004).

Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis

Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. The histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during pupal thorax metamorphosis and JNK-induced apoptosis in Drosophila. It has been demonstrated, in cultured cells, that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism (Miotto, 2006).

Whether Chm directly binds to DFos and/or DJun was investigated using GST pull-down assays. The C-terminal half of Chm (amino acids 494-812), which contains the MYST domain, displays strong in vitro affinity for an N-terminal fragment of DFos (including the N terminus and the basic DNA-binding domain), and binds DJun as well, although less efficiently. Conversely, the cytoplasmic kinase Basket (Bsk)/DJNK does not bind to the His-Chm fusion protein. The Chm N terminus (amino acids 20-400) does not associate with DFos or DJun. Similar experiments with GST-fused DFos deletion mutants identified the basic region of DFos as the predominant Chm-interacting domain, although significant association with the C-terminal part of DFos was also observed. Immunoprecipation assays followed by Western analyses confirmed that these interactions occur in vivo. Both DFos and DJun coprecipitate with Myc-Chm from nuclear extracts of larvae expressing a Myc-tagged version of the protein. DFos is eluted from the immunoprecipitate at higher salt concentrations than DJun; this indicates a more stable association with Chm and that Chm to DFos interaction can occur in the absence of DJun. Confirming the specificity of the assay, nuclear proteins unrelated to JNK signaling, the chromatin-associated protein Modulo (Mod), the homeodomain transcription factors Ultrabithorax (Ubx) and Engrailed (En), as well as the basic helix-loop-helix (bHLH) factor DMyc are not precipitated by Myc-Chm. In reciprocal experiments, an anti-TAP antibody coprecipitates Myc-Chm from nuclear extracts of larvae ubiquitously expressing Myc-Chm and TAP-DFos or TAP-DJun. The results of the in vitro and the in vivo experiments, taken together, show that Chm, DFos and DJun can directly interact and form multimeric protein complexes in larvae (Miotto, 2006).

Fos-related antigen/kayak: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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