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E Hafen

Publications and source records attributed to E Hafen.

At least 19 recordsLinked to original sources
PubMed1999

Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells.

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.

Amino Acid Sequence↗
PubMed1999

Drosophila S6 kinase: a regulator of cell size.

Cell proliferation requires cell growth; that is, cells only divide after they reach a critical size. However, the mechanisms by which cells grow and maintain their appropriate size have remained elusive. Drosophila deficient in the S6 kinase gene (dS6K) exhibited an extreme delay in development and a severe reduction in body size. These flies had smaller cells rather than fewer cells. The effect was cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minutes). Thus, the dS6K gene product regulates cell size in a cell-autonomous manner without impinging on cell number.

Animals↗
PubMed1999

Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4.

The control of growth is fundamental to the developing metazoan. Here, we show that CHICO, a Drosophila homolog of vertebrate IRS1-4, plays an essential role in the control of cell size and growth. Animals mutant for chico are less than half the size of wild-type flies, owing to fewer and smaller cells. In mosaic animals, chico homozygous cells grow slower than their heterozygous siblings, show an autonomous reduction in cell size, and form organs of reduced size. Although chico flies are smaller, they show an almost 2-fold increase in lipid levels. The similarities of the growth defects caused by mutations in chico and the insulin receptor gene in Drosophila and by perturbations of the insulin/IGF1 signaling pathway in vertebrates suggest that this pathway plays a conserved role in the regulation of overall growth by controling cell size, cell number, and metabolism.

Amino Acid Sequence↗
PubMed1999

The dominant mutation Glazed is a gain-of-function allele of wingless that, similar to loss of APC, interferes with normal eye development.

Dominant mutations have served as invaluable tools for Drosophila geneticists. Here we analyze the dominant eye mutation Glazed (Gla) that was described by T. H. Morgan more than 50 years ago. We show that Gla causes the loss of photoreceptor cells during pupal stages, in a process reminiscent of apoptosis, with a concomitant overproduction of eye pigment. This phenotype is very similar to that caused by the loss of D-APC, a negative regulator of Wingless (Wg) signal transduction. Genetic analyses reveal however that the Gla gain-of-function phenotype can be reverted to wild-type. By generating a P-element-induced revertant of Gla we demonstrate that Gla is allelic to wg. The molecular lesion in Gla indicates that the insertion of a roo retrotransposon leads to ectopic expression of wg during pupal stages. We show that the Gla phenotype is similar to that caused by ectopic expression of Wg driven by the sevenless (sev) enhancer. In both cases Wg exerts its effect, at least in part, by negatively regulating the expression of the Pax2 homolog sparkling (spa). Gla represents not only the first dominant allele of wg, but it may also be the first allele ever described for wg.

Alleles↗
PubMed1999

Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein.

Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.

Amino Acid Sequence↗
PubMed1999

PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development.

Mutations in the tumor suppressor gene PTEN (MMAC1/TEP1) are associated with a large number of human cancers and several autosomal-dominant disorders. Mice mutant for PTEN die at early embryonic stages and the mutant embryonic fibroblasts display decreased sensitivity to cell death. Overexpression of PTEN in different mammalian tissue culture cells affects various processes including cell proliferation, cell death and cell migration. We have characterized the Drosophila PTEN gene and present evidence that both inactivation and overexpression of PTEN affect cell size, while overexpression of PTEN also inhibits cell cycle progression at early mitosis and promotes cell death during eye development in a context-dependent manner. Furthermore, we have shown that PTEN acts in the insulin signaling pathway and all signals from the insulin receptor can be antagonized by either Drosophila or human PTEN, suggesting a potential means for alleviating symptoms associated with altered insulin signaling.

Amino Acid Sequence↗
PubMed1998

Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis.

The "cysteine string protein" (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a "J" domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the "tall cells" of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.

Age Factors↗
PubMed1998

Ras--a versatile cellular switch.

With the number of known roles played by Ras proteins increasing rapidly, finding answers to how the diverse cellular responses are triggered is becoming increasingly pertinent. Although our understanding of the control of specificity of signal transduction is still small, the combination of biochemical, structural and genetic analyses is starting to reveal how the cell-specific responses to Ras activation are controlled.

Animals↗
PubMed1997

Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye.

Patterning of the compound eye begins at the posterior edge of the eye imaginal disc and progresses anteriorly toward the disc margin. The advancing front of ommatidial differentiation is marked by the morphogenetic furrow (MF). Here we show by clonal analysis that Hedgehog (Hh), secreted from two distinct populations of cells has two distinct functions: It was well documented that Hh expression in the differentiating photoreceptor cells drives the morphogenetic furrow. Now we show that, in addition, Hh, secreted from cells at the posterior disc margin, is absolutely required for the initiation of patterning and predisposes ommatidial precursor cells to enter ommatidial assembly later. These two functions of Hh in eye patterning are similar to the biphasic requirement for Sonic Hh in patterning of the ventral neural tube in vertebrates. We show further that Hh induces ommatidial development in the absence of its secondary signals Wingless (Wg) and Dpp and that the primary function of Dpp in MF initiation is the repression of wg, which prevents ommatidial differentiation. Our results show that the regulatory relationships between Hh, Dpp, and Wg in the eye are similar to those found in other imaginal discs such as the leg disc despite obvious differences in their modes of development.

Animals↗
PubMed1997

Common and distinct roles of DFos and DJun during Drosophila development.

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. 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. In addition to the joint requirement of DFos and DJun during dorsal closure, DFos functions independently of DJun during early stages of embryogenesis. These findings demonstrate common and distinct roles of 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.

Animals↗
PubMed1997

Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure.

During Drosophila embryogenesis, ectodermal cells of the lateral epithelium stretch in a coordinated fashion to internalize the amnioserosa cells and close the embryo dorsally. This process, dorsal closure, requires two signaling pathways: the Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Dpp pathway. We have identified mutations in DJun and show that DJNK controls dorsal closure by activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. DJun and Aop regulate dpp expression in the most dorsal row of cells. Secreted Dpp then instructs more ventrally located cells to stretch. Our results provide a causal link between the DJNK and Dpp pathways during dorsal closure. Interestingly, in vertebrates, transforming growth factor-beta and c-Jun regulate collagenase gene expression during wound healing, a process that also involves the closing of an epithelial sheath.

Animals↗
PubMed1997

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.

Amino Acid Sequence↗
PubMed1997

Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo.

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression of wild-type D-Raf inhibits the formation of R7 cells in a dose-dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to negatively regulate Raf kinase activity in vivo.

14-3-3 Proteins↗
PubMed1996

The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth.

Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium. They are activated by a multitude of extracellular signals and implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape. Here we describe the molecular and functional analysis of Drosophila p110 (Dp110). A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta. Overexpression of Dp110 in wing or eye imaginal discs resulted in flies with enlarged wings or eyes respectively. In contrast, overexpression of Dp110 containing a mutation predicted to result in the loss of catalytic activity resulted in smaller wings and eyes. The alterations in wing size result from changes in both cell size and cell number, whereas in the eye only differences in cell size were detected. These data imply a role for Dp110 in growth control during Drosophila development and have implications for the function of class I PI3Ks in other organisms.

Amino Acid Sequence↗
PubMed1996

The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye.

We cloned and characterized the Drosophila homolog of mammalian Jun-N-terminal kinases (DJNK). We show that DJNK is encoded by basket (bsk). Like hemipterous (hep), which encodes the Drosophila JNK kinase, bsk is required in the embryo for dorsal closure, a process involving coordinate cell shape changes of ectodermal cells. Dorsal closure can also be blocked by dominant negative Drosophila cdc42, which has been shown to act upstream of JNKK in vertebrates. Therefore it appears that the JNK pathway is conserved and that it is involved in controlling cell morphogenesis in Drosophila. Although DJNK efficiently phosphorylates DJun in vitro, bsk function is not required for the specification of cell fate in the developing eye, a process that requires MAP kinase and DJun function.

Amino Acid Sequence↗
PubMed1996

Biochemical characterization of rolledSem, an activated form of Drosophila mitogen-activated protein kinase.

The rolled (rl) gene of Drosophila encodes a homologue of vertebrate mitogen-activated protein kinases. Genetic analyses have shown that the gain-of-function mutation rolledSevenmaker (rlSem) is sufficient to activate developmental pathways controlled by distinct receptor tyrosine kinases, such as Sevenless, Torso, and the Drosophila epidermal growth factor receptor homologue. Here we show that mutant RlSem protein, immunoprecipitated from transiently transfected COS cells, exhibits a moderate increase in kinase activity compared with wild-type Rl protein. Time course studies revealed that RlSem is more active than Rl following short term as well as prolonged treatment with epidermal growth factor. Interestingly, a more pronounced difference in kinase activity is observed when the proteins are immunoprecipitated from extracts of Drosophila rl and rlSem larvae. In fact, the kinase activity of RlSem from larvae extracts is comparable to the kinase activity of larvae expressing either an activated Sevenless receptor or an activated Raf kinase. We also demonstrate that Dsor1, which has been placed upstream of rl genetically, is able to phosphorylate and activate Rl in vitro.

Amino Acid Sequence↗
PubMed1996

DOS, a novel pleckstrin homology domain-containing protein required for signal transduction between sevenless and Ras1 in Drosophila.

The specification of the R7 photoreceptor cell in the developing eye of Drosophila is dependent upon activation of the Sevenless (SEV) receptor tyrosine kinase. By screening for mutations that suppress signaling via a constitutively activated SEV protein, we have identified a novel gene, daughter of sevenless (dos). DOS is required not only for signal transduction via SEV but also in other receptor tyrosine kinase signaling pathways throughout development. The presence of an amino-terminally located pleckstrin homology domain and many potential tyrosine phosphorylation sites suggests that DOS functions as an adaptor protein able to interact with multiple signaling molecules. Our genetic analysis demonstrates that DOS functions upstream of Ras1 and defines a signaling pathway that is independent of direct binding of the DRK SH2/SH3 adaptor protein to the SEV receptor tyrosine kinase.

Amino Acid Sequence↗