Dynamic phosphorylation of the kinesin Costal-2 in vivo reveals requirement of fused kinase activity for all levels of hedgehog signalling.

Hedgehog (Hh) proteins are secreted molecules that play an essential role in development and tumorigenesis. In Drosophila cultured cells, phosphorylation of the kinesin-like Costal2 (Cos2) protein at Ser572 is triggered by the kinase fused (Fu) upon Hh pathway activation. Here, we validate the first phospho-antibody for one of the Hh pathway components, Cos2, as a universal in situ readout of Hh signal transduction. For the first time, this tool allows the visualisation of a gradient of signalling activity and therefore the range of the activating Hh ligand in different tissues. We also show that, in vivo, Fu kinase is activated by and necessary to transduce all levels of intracellular Hh signalling. Our study fills a gap in the understanding of the Hh pathway by showing that the molecular cascade leading to Cos2 phosphorylation is conserved in all cells activated by Hh. Therefore, we propose that the extracellular Hh information is conveyed to an intracellular signal through graded Fu kinase activity.

The conserved PI3'K/PTEN/Akt signaling pathway regulates both cell size and survival in Drosophila.

Akt (or PKB) is an oncogene involved in the regulation of cell survival. Akt is regulated by phosphatidylinositol 3-OH kinase (PI3'K) signaling and has shown to be hyperactivated through the loss of the PTEN tumor suppressor. In Drosophila, insulin signaling as studied using the Drosophila IRS-4 homolog (Chico) has been shown to be a crucial regulator of cell size. We have studied Drosophila Akt (Dakt1) and have shown that it is also involved in the regulation of cell size. Furthermore we have performed genetic epistasis tests to demonstrate that in Drosophila, PI3'K, PTEN and Akt comprise a signaling cassette that is utilized during multiple stages of development. In addition, we show that this signaling cassette is also involved in the regulation of cell survival during embryogenesis. This study therefore establishes the evolutionary conservation of this signaling pathway in Drosophila. Oncogene (2000) 19, 3971 - 3977.

Regulation of the protein kinase activity of Shaggy(Zeste-white3) by components of the wingless pathway in Drosophila cells and embryos.

The protein-serine kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins.

Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex.

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.

Genetic analysis of protein kinase B (AKT) in Drosophila.

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.

Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells.

BACKGROUND: Exposing eukaryotic cells to lithium ions (Li+) during development has marked effects on cell fate and organization. The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases. RESULTS: We investigated the effect of Li+ on the activity of the GSK-3 family. At physiological doses, Li+ inhibits the activity of human GSK-3 beta and Drosophila Zw3Sgg, but has no effect on other protein kinases. The effect of Li+ on GSK-3 is reversible in vitro. Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/beta-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3. CONCLUSIONS: Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li+ action on development and differentiation.

Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila.

Like the neuroblasts of the central nervous system, sensory organ precursors of the peripheral nervous system of the Drosophila thorax arise as single spaced cells. However, groups of cells initially have neural potential as visualized by the expression of the proneural genes achaete and scute. A class of genes, known as the 'neurogenic genes', function to restrict the proportion of cells that differentiate as sensory organ precursors. They mediate cell communication between the competent cells by means of an inhibitory signal, Delta, that is transduced through the Notch receptor and results in a cessation of achaete-scute activity. Here we show that mutation of either the bHLH-encoding genes of the Enhancer of split complex (E(spl)-C) or groucho, like Notch or Delta mutants, cause an overproduction of sensory organ precursors at the expense of epidermis. The mutant cells behave antonomously suggesting that the corresponding gene products are required for reception of the inhibitory signal. Epistasis experiments place both E(spl)-C bHLH-encoding genes and groucho downstream of Notch and upstream of achaete and scute, consistent with the idea that they are part of the Notch signalling cascade. Since all competent cells produce both the receptor and its ligand, it was postulated that Notch and Delta are linked within each cell by a feedback loop. We show, that, like mutant Notch cells, cells mutant for E(spl)-C bHLH-encoding genes or groucho inhibit neighbouring wild-type cells causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex (AS-C) which must therefore regulate the signal Delta. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(spl)-C and AS-C genes.

Evaluation of Creatine Phosphokinase Activity as a Means of Determining Cooking End-Point Temperature.

The influence of cooking end-point temperatures (EPTs) of 62.8, 66.7, 67.8, 68.9, 70.0, 71.1, 73.9, and 76.7°C on residual creatine phosphokinase (CPK) activity in laboratory prepared model systems of ground chicken and turkey breast meat was determined. CPK activity was also assayed in commercially prepared chicken, turkey, and meat products using a Sigma #661 CPK test kit. Three tenths milliliter of 0.9% saline extracts obtained from the chicken, turkey, and meat products was substituted for 0.3 ml serum specified in the test kit procedure. For the model samples, there was a marked decrease in CPK activity as EPT increased from 66.7 to 76.7°C; however, model samples heated to 76.7°C did retain low amounts of CPK activity. In general, very low levels of CPK were found in commercially prepared chicken and turkey products (0 to 10.6 Sigma units/ml). Results of CPK activity in commercially prepared meat products would indicate that the test is product dependent, with values ranging from zero for beef franks to 258 Sigma units/ml for hard salami. Thus, while CPK activity may be useful for detecting cooking EPT for quality control purposes, it should not be used as a regulatory procedure where experience with the specific product is not available.

Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch.

During neurogenesis in Drosophila, groups of equipotential, neurally competent cells choose between epidermal and neural fates. Notch, a phylogenetically conserved transmembrane protein, may act as a receptor in a lateral signalling pathway in which a single neural precursor is chosen from each group and the neural fate of the other cells is inhibited, causing them to differentiate into epidermis. Possible intracellular transduction events mediating signals from Notch are, however, unknown. shaggy is also required for the lateral signal and encodes serine/threonine protein kinases with homology to the glycogen synthase kinase-3 (GSK-3) enzymes that act in signal transduction pathways in vertebrates. We report here that, in transgenic flies, GSK-3 beta can substitute for shaggy, and we also present a study of epistatic relationships between shaggy and gain and loss of function alleles of Notch. The results indicate that shaggy/GSK-3 is part of a signalling pathway downstream of Notch.

Functional significance of a family of protein kinases encoded at the shaggy locus in Drosophila.

The characterization of the structurally complex gene shaggy is presented. This gene encodes multiple proteins with putative serine/threonine kinase activity thought to be involved in signal transduction mechanisms that take place during several patterning events throughout Drosophila development. The gene comprises two transcription units that give rise to 10 transcripts and five different proteins with a common kinase catalytic domain and overlapping patterns of expression during development. Mutational analysis of shaggy defines a single complementation group, lethality of which is associated with the loss of two major shaggy proteins. These studies allow the first definition of a true null allele. Two proteins may fulfill maternal requirements. Phenotypes of flies expressing individual shaggy proteins revealed that although there is some redundancy between the different forms they do not all carry out identical functions in vivo. However, under experimental conditions, a single form of the protein was able to carry out all known requirements. This protein probably also functions as part of a signal transduction cascade in the imaginal neuroepithelium, where cells have to choose between epidermal and neural fates.

A dual role for the protein kinase shaggy in the repression of achaete-scute.

achaete and scute are expressed in a spatially restricted pattern and provide neural potential to cells. The domains of expression depend partly on extramacrochaetae whose product is itself spatially restricted and acts as a negative post-translational regulator of achaete and scute. The protein kinase shaggy also represses achaete and scute at many sites but may act via intermediate transcription factors. However shaggy and extramacrochaetae act synergistically and molecular studies suggest that they may be part of the same pathway. shaggy is functionally homologous to the mammalian glycogen synthase kinase-3 and analogy with the known physiology of this enzyme, suggests that this function of shaggy may result from the "constitutive" activity. At the site where a single neural precursor will develop, achaete and scute are initially expressed in a group of equivalent cells. The genes Notch and Delta are part of a lateral signal required to single out one precursor cell and to silence achaete and scute expression in the other cells. shaggy is required downstream of Notch for transduction of the inhibitory signal. This second role of shaggy may be due to modulation of enzymatic activity during signalling.

An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily.

The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

Effect of Air Movement During Fermentation on Certain Properties of Natural Flora and Starter Culture-Fermented Sausage.

Effects of air movement (0, 5, 20 and 35 changes/min) during fermentation on certain chemical, physical and microbiological properties of a fermented and cooked summer sausage were determined. Four batches of summer sausage were prepared. Half of each batch was fermented by natural flora and the other half by a Pediococcus cerevisiae starter culture. Sausages were fermented in chambers at 38°C with 94% RH, and samples were taken at 0, 6, 12, 18 and 24 h during fermentation. Samples were also taken after heat processing and overnight chilling. Air movement during fermentation had no significant effect on pH, lactic acid content, cured color development or proximate composition regardless of method of fermentation. Removal of sausage casing was very difficult for all natural flora sausage chubs that were fermented at 5, 20 and 35 air changes/min; however, ease of casing removal improved somewhat at 18 and 24 h of fermentation for sausages made with natural flora and fermented at 0 air change/min. Regardless of air movement treatment, removal of casing from sausages made with starter culture was poor at 6 h of fermentation, but was much improved at 12 h of fermentation and thereafter. Microbial growth was fastest and highest among the natural flora sausage fermented without air flow. An undesirable surface film which developed on the natural flora sausage fermented without air flow consisted of gram negative rods and gram positive cocci.