The Drosophila Sterile-20 Kinase Slik Controls Cell Proliferation and Apoptosis during Imaginal Disc Development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development

Bicaudal C mutation causes myc and TOR pathway up-regulation and polycystic kidney disease-like phenotypes in Drosophila

Progressive cystic kidney degeneration underlies diverse renal diseases, including the most common cause of kidney failure, autosomal dominant Polycystic Kidney Disease (PKD). Genetic analyses of patients and animal models have identified several key drivers of this disease. The precise molecular and cellular changes underlying cystogenesis remain, however, elusive. Drosophila mutants lacking the translational regulator Bicaudal C (BicC, the fly ortholog of vertebrate BICC1 implicated in renal cystogenesis) exhibited progressive cystic degeneration of the renal tubules (so called ªMalpighianº tubules) and reduced renal function. The BicC protein was shown to bind to Drosophila (d-) myc mRNA in tubules. Elevation of d-Myc protein levels was a cause of tubular degeneration in BicC mutants. Activation of the Target of Rapamycin (TOR) kinase pathway, another common feature of PKD, was found in BicC mutant flies. Rapamycin administration substantially reduced the cystic phenotype in flies. We present new mechanistic insight on BicC function and propose that Drosophila may serve as a genetically tractable model for dissecting the evolutionarily-conserved molecular mechanisms of renal cystogenesis

Patterns of nucleotide diversity at the regions encompassing the Drosophila insulin-like peptide (dilp) genes: demography vs positive selection in Drosophila melanogaster.

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events

Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth

The Drosophila Sterile-20 kinase Slik promotes tissue growth during development by stimulating cell proliferation and by preventing apoptosis. Proliferation within an epithelial sheet requires dynamic control of cellular architecture. Epithelial integrity fails in slik mutant imaginal discs. Cells leave the epithelium and undergo apoptosis. The abnormal behavior of slik mutant cells is due to failure to phosphorylate and activate Moesin, which leads to excess Rho1 activity. This is distinct from Slik's effects on cell proliferation, which are mediated by Raf. Thus Slik acts via distinct pathways to coordinate cell proliferation with epithelial cell behavior during tissue growth

A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls <i>Drosophila</i> Smoothened Activity

<div><p>Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of <i>Drosophila</i> Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of <i>Drosophila</i> Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.</p></div

bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila

AbstractCell proliferation, cell death, and pattern formation are coordinated in animal development. Although many proteins that control cell proliferation and apoptosis have been identified, the means by which these effectors are linked to the patterning machinery remain poorly understood. Here, we report that the bantam gene of Drosophila encodes a 21 nucleotide microRNA that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. We identify the pro-apoptotic gene hid as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity

Fold downregulation of Smo^SD peptide phosphorylation in Gprk2-depleted cells.

<p>**, signal in Gprk2-depleted cells too low to measure. T, trypsin; AN, AspN; C, chymotrypsin.</p

Gprk2 phosphorylation is required for maximal Smo activity <i>in vivo</i>.

<p>Confocal micrographs of wing discs, oriented with anterior [A] compartment to left, dorsal [D] compartment up. (<i>A</i>–<i>F</i>) Endogenous Smo was depleted from the D compartment by <i>ap-GAL4</i>-driven expression of <i>smo 3′UTR</i> dsRNA and replaced with GFP (<i>A</i> and <i>B</i>), Smo^WT-GFP (<i>C</i> and <i>D</i>), or Smo^c1-4A-GFP (<i>E</i> and <i>F</i>). Immunostaining: Ptc and <i>dpp-LacZ</i> (<i>A</i>, <i>C</i>, and <i>E</i>); En (<i>B</i>, <i>D</i>, and <i>F</i>). <i>Dotted lines</i>: anterior/posterior (A/P) compartment boundary (based on Ci immunostaining, not shown). Smo^c1-4A rescued Dpp and Ptc but not En expression. (<i>G</i>–<i>J</i>) En expression in wing discs expressing Smo variants in D compartment: Smo^SD (<i>G</i> and <i>I</i>), Smo^SD.c1-4A (<i>H</i>), or Smo^SD.c12D (<i>J</i>). Discs are wild-type (<i>G</i> and <i>H</i>) or <i>gprk2</i> mutant (<i>I</i> and <i>J</i>) background. <i>Dotted lines</i>: A/P compartment boundaries. Ala substitution of all four Gprk2 phosphorylation clusters modestly impairs Smo^SD activity <i>in vivo</i>, whereas phosphomimetic substitutions restore full Smo^SD activity in the absence of Gprk2. (<i>K–L</i>) GFP fluorescence in D compartment of wing discs with endogenous Smo depleted and replaced with Smo^WT-GFP (K) or Smo^c1-4A-GFP (<i>L</i>). <i>Brackets</i>: Hh-responding cells (identified by increased Ci¹⁵⁵ immunostaining). The Gprk2 non-phosphorylatable form of Smo accumulates ectopically in Hh-responding cells.</p