Biomedical subjects

D Bohmann

Publications and source records attributed to D Bohmann.

At least 19 recordsLinked to original sources

PubMed2001

Drosophila Fos mediates ERK and JNK signals via distinct phosphorylation sites.

During Drosophila development Fos acts downstream from the JNK pathway. Here we show that 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 revealed 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.

Animals↗

PubMed2001

Drosophila AP-1: lessons from an invertebrate.

In recent years, studies in the model organism Drosophila melanogaster have contributed significant insights into the molecular and developmental biology of the AP-1 transcription factors Jun and Fos. Powerful genetic and biochemical approaches uncovered a baffling complexity and variability of the signaling connections to and from AP-1. The range of biological processes that Jun and Fos regulate in this organism is equally multi-faceted. Regulatory interactions between AP-1 and JNK, ERK, TGFbeta, Notch or other signaling systems have been implicated in the control of a multitude of embryonic and adult events, including tissue closure processes, patterning of eye, gut and wing, as well as apoptosis. Here we review the information that has been gathered on Drosophila AP-1 in signal transduction and on the developmental and cellular functions controlled by AP-1-mediated signals in the fly. Lessons learned from the studies on AP-1 in Drosophila may contribute to our general understanding, beyond species boundaries, of this fundamental class of transcriptional regulators.

Animals↗

PubMed2001

The role of the Drosophila TAK homologue dTAK during development.

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGF-beta activated kinase (TAK) it has, however, subsequently been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. Here we report the isolation and genetic characterization of the Drosophila TAK homologue (dTAK). By employing overexpression and double-stranded RNA interference (RNAi) techniques we have analyzed its function during embryogenesis and larval development. Overexpression of dTAK in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of dTAK in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-dTAK expression studies in the embryo argue for a differential requirement of dTAK during developmental processes controlled by JNK signaling, and a redundant or minor role of dTAK in dorsal closure. In addition, dTAK-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that dTAK can also act through the p38 and Nemo kinases in imaginal discs. Our results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure.

Amino Acid Sequence↗

PubMed2001

The genomic response of the Drosophila embryo to JNK signaling.

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. We analyzed the genome-wide transcriptional response to activation of the JNK pathway in the Drosophila embryo by serial analysis of gene expression (SAGE) and identified loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly.

Actins↗

PubMed2001

Complex functions of AP-1 transcription factors in differentiation and survival of PC12 cells.

c-Jun activation by mitogen-activated protein kinases has been implicated in various cellular signal responses. We investigated how JNK and c-Jun contribute to neuronal differentiation, cell survival, and apoptosis. In differentiated PC12 cells, JNK signaling can induce apoptosis and c-Jun mediates this response. In contrast, we show that in PC12 cells that are not yet differentiated, the AP-1 family member ATF-2 and not c-Jun acts as an executor of apoptosis. In this context c-Jun expression protects against apoptosis and triggers neurite formation. Thus, c-Jun has opposite functions before and after neuronal differentiation. These findings suggest a model in which the balance between ATF-2 and Jun activity in PC12 cells governs the choice between differentiation towards a neuronal fate and an apoptotic program. Further analysis of c-Jun mutants showed that the differentiation response requires functional dimerization and DNA-binding domains and that it is stimulated by phosphorylation in the transactivation domain. In contrast, c-Jun mutants incompetent for DNA binding or dimerization and also mutants lacking JNK binding and phosphorylation sites that cannot elicit neuronal differentiation efficiently protect PC12 cells from apoptosis. Hence, the protective role of c-Jun appears to be mediated by an unconventional mechanism that is separable from its function as a classical AP-1 transcription factor.

Activating Transcription Factor 2↗

PubMed2000

A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK.

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.

Amino Acid Sequence↗

PubMed1999

Diverse functions of JNK signaling and c-Jun in stress response and apoptosis.

c-Jun/AP-1 activation has been implicated in various, often opposing cellular responses. For example, although there is considerable evidence that c-Jun activation can be a positive step in the events leading a cell towards apoptosis, there are also many reports stating the opposite: that under certain circumstances c-Jun can inhibit apoptosis and promote proliferation or differentiation instead - and that these responses are important for normal mammalian development. It is clear that the effects of c-Jun on cellular responses depend strongly on the cell type and the context of other regulatory influences that the cell is receiving. This review focuses on recent developments in understanding how activation of JNK and c-Jun contributes to different cellular responses.

Animals↗

PubMed1999

Thorax closure in Drosophila: involvement of Fos and the JNK pathway.

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, we studied another cell sheet movement that we term thorax closure, the joining of the parts of the wing imaginal discs which give rise to the adult thorax during metamorphosis. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibres during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localises with elevated levels of D-Fos, is reduced in a JNK or D-Fos loss-of-function background and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well.

Animals↗

PubMed1999

Sec61beta, a subunit of the protein translocation channel, is required during Drosophila development.

We have identified and isolated mutations in the first Drosophila gene encoding a subunit of the Sec61 protein translocation channel, DSec61beta. While neither the Saccharomyces cerevisiae Sec61beta nor its functional Escherichia coli homologue are essential for viability or for protein translocation, we show that DSec61beta is essential for embryonic development. Homozygous mutant embryos die at the end of embryogenesis and are impaired in the secretion of cuticle proteins from the epidermis. DSec61beta germ line clones, result in defects in dorso-ventral patterning of the egg and are consistent with affected secretion of the protein Gurken from the oocyte to the follicle cells. Clonal analyses in the imaginal discs reveal defects in adult structures, including rhabdomere morphogenesis and a reduction of the size of tarsal segments in the leg. This is the first in vivo study of a component of the protein translocation machinery in higher eukaryotes, and illustrates how a protein that has an inessential, kinetic function in single-cell organisms can become critical for the complex development of a multicellular organism.

Alleles↗

PubMed1998

Differential regulation of c-Jun by ERK and JNK during PC12 cell differentiation.

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

Animals↗

PubMed1997

Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants.

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp.

Animals↗

PubMed1997

Jun in Drosophila development: redundant and nonredundant functions and regulation by two MAPK signal transduction pathways.

Drosophila Jun is shown to be involved in different signal transduction pathways and developmental decisions. Dorsal closure, a morphogenetic process occurring during Drosophila embryogenesis, is regulated by Hemipterous (Hep) and Basket (Bsk), homologs of JNKK and JNK, respectively. Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of bsk and hep mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway that is comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly.

Amino Acid Sequence↗

PubMed1997

Requirement for Drosophila 14-3-3 zeta in Raf-dependent photoreceptor development.

Based on biochemical and functional data obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated in a number of different signal transduction processes, in particular in the signal-dependent activation of protein kinases. We performed a functional analysis of 14-3-3 in a multicellular organism, initiated by the cloning of a 14-3-3 zeta homolog of Drosophila melanogaster, termed D14-3-3 zeta. D14-3-3 zeta transcripts are strongly enriched in the developing central nervous system. In addition, they are predominantly expressed in the region posterior to the morphogenetic furrow of the eye imaginal disc where cells differentiate as photoreceptors. In these cells D14-3-3 zeta is localized apically. Both the expression pattern and the subcellular localization are consistent with the proposed function of 14-3-3 proteins in Ras/Raf/MAPK signaling. D14-3-3 zeta mutant analysis combined with rescue experiments involving gain-of-function alleles of Raf and Ras indicate that D14-3-3 zeta is an essential component of the Raf/Ras signaling pathway and necessary for photoreceptor differentiation. It acts upstream of Raf and downstream of Ras.

14-3-3 Proteins↗

PubMed1997

A CREB-binding site as a target for decapentaplegic signalling during Drosophila endoderm induction.

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-beta family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. Here, we show that a cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and that endoderm expression from a labial enhancer depends on multiple CREs. Furthermore, the Drosophila CRE-binding protein dCREB-B binds to the Ultrabithorax CRE, and ubiquitous expression of a dominant-negative form of dCREB-B suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a CREB protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signalling in the embryonic midgut.

Activating Transcription Factor 1↗

PubMed1997

Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases.

The proto-oncogene-encoded transcription factor c-Jun activates genes in response to a number of inducers that act through mitogen-activated protein kinase (MAPK) signal transduction pathways. The activation of c-Jun after phosphorylation by MAPK is accompanied by a reduction in c-Jun ubiquitination and consequent stabilization of the protein. These results illustrate the relevance of regulated protein degradation in the signal-dependent control of gene expression.

3T3 Cells↗

PubMed1997

The deubiquitination enzyme fat facets negatively regulates RTK/Ras/MAPK signalling during Drosophila eye development.

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. Here we show that faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. The results indicate that RTK/Ras signalling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf eye discs at the time when the ectopic photoreceptors are induced. We propose that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism.

Animals↗

PubMed1996

Ubiquitin in signal transduction and cell transformation.

Since the discovery of ubiquitin-dependent protein degradation almost two decades ago, great strides have been made towards a detailed understanding of the biochemistry of this process (reviewed in [1-3]). It was, however, only in recent years that the physiological role of the ubiquitin system in signal transduction and the regulation of several cell functions started to be appreciated and experimentally addressed. As with other principal mechanisms of signal transduction, such as phosphorylation or GTP hydrolysis, much of the information regarding the role of the ubiquitin system as a component of cell regulation and signaling cascades, was gained in studies of transformation and the control of cell growth. It seems, however, that ubiquitin-dependent proteolysis, and possibly other processes that are controlled by protein ubiquitination, play a role in many aspects of cellular function from the control of differentiation to intracellular trafficking [1,3,4]. Here we will review some of the results that implicate ubiquitin-dependent proteolysis in the control of cell growth and that indicate how perturbations of ubiquitin-dependent degradation of oncogene and tumor suppressor gene products may contribute to cell transformation and oncogenesis.

Animals↗

PubMed1996

Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation.

Drosophila Jun (D-Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D-Jun is a substrate for the ERK-related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D-Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate-mimetic Asp residues, as well as a VP16-D-Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non-neuronal fate during Drosophila eye development.

Amino Acid Sequence↗