Wingless (Wg) is an important signaling molecule in the development of Drosophila, but little is known about its signal transduction pathway. Genetic evidence indicates that another segment polarity gene, dishevelled (dsh) is required for Wg signaling. We have recently developed a cell culture system for Wg protein activity, and using this in vitro system as well as intact Drosophila embryos, we have analyzed biochemical changes in the Dsh protein as a consequence of Wg signaling. We find that Dsh is a phosphoprotein, normally present in the cytoplasm. Wg signaling generates a hyperphosphorylated form of Dsh, which is associated with a membrane fraction. Overexpressed Dsh becomes hyperphosphorylated in the absence of extracellular Wg and increases levels of the Armadillo protein, thereby mimicking the Wg signal. A deletional analysis of Dsh identifies several conserved domains essential for activity, among which is a so-called GLGF/DHR motif. We conclude that dsh, a highly conserved gene, is not merely a permissive factor in Wg signaling but encodes a novel signal transduction molecule, which may function between the Wg receptor and more downstream signaling molecules.

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair [5]. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.

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Casein kinase I (CKI) was recently reported as a positive regulator of Wnt signaling in vertebrates and Caenorhabditis elegans. To elucidate the function of Drosophila CKI in the wingless (Wg) pathway, we have disrupted its function by double-stranded RNA-mediated interference (RNAi). While previous findings were mainly based on CKI overexpression, this is the first convincing loss-of-function analysis of CKI. Surprisingly, CKIalpha- or CKIepsilon-RNAi markedly elevated the Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting its mRNA levels. Pulse-chase analysis showed that CKI-RNAi stabilizes Arm protein. Moreover, Drosophila embryos injected with CKIalpha double-stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKIalpha induced hyper-phosphorylation of both Arm and Dishevelled in S2R+ cells and, conversely, CKIalpha-RNAi reduced the amount of hyper-modified forms. His-tagged Arm was phosphorylated by CKIalpha in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste-white 3. Thus, we propose that CKI phosphorylates Arm and stimulates its degradation.

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In an attempt to devise a method for collecting large volumes of thermally induced sweat with less epidermal contamination and evaporative water loss, we developed an anaerobic sweat collector by using a sheet of polyethylene film placed over a thin layer of Vaseline and paraffin oil on the skin. To test the validity of the new method, sweat samples collected every 5 min from the new collector (sweat A) were compared with those obtained from a second collector using no oil (sweat B) and scraped sweat for concentrations of adenosine 3',5'-cyclic monophosphate (cAMP), protein, glucose, urea, lactic acid, calcium, sodium, potassium, and cholesterol. The concentration of sweat ingredients in scraped sweat was often far greater than could be expected from evaporative water loss alone. When compared with sweat A, sweat B also had higher concentrations of these ingredients in the initial samples, indicating epidermal contamination, which was especially marked in cAMP, protein, urea, cholesterol, and calcium. Concomitant with a rise in plasma glucose following the administration of a glucose bolus, the sweat glucose significantly increased, indicating the plasma as a major source of sweat glucose. We conclude that the new sweat collector is instrumental in collecting large volumes of the cleanest possible human sweat.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ), the viral gene transcribed from the antisense strand, is consistently expressed in ATL cells and promotes their proliferation. In this study, we found that a Wnt pathway-related protein, disheveled-associating protein with a high frequency of leucine residues (DAPLE), interacts with both HTLV-1 Tax and HBZ. In the presence of DAPLE, Tax activated canonical Wnt signaling. Conversely, HBZ markedly suppressed canonical Wnt activation induced by either Tax/DAPLE or β-catenin. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets lymphoid enhancer-binding factor 1, one of the key transcription factors of the pathway, and impairs its DNA-binding ability. We also observed that the canonical Wnt pathway was not activated in HTLV-1-infected cells, whereas the representative of noncanonical Wnt ligand, Wnt5a, which antagonizes canonical Wnt signaling, was overexpressed. HBZ was able to induce Wnt5a transcription by enhancing its promoter activity through the TGF-β pathway. Importantly, knocking down of Wnt5a in ATL cells repressed cellular proliferation and migration. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulates arm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in the Drosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wildtype and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an aminoterminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.