Academic journal article Genetics

An Unconventional Nuclear Localization Motif Is Crucial for Function of the Drosophila Wnt/Wingless Antagonist Naked Cuticle

Academic journal article Genetics

An Unconventional Nuclear Localization Motif Is Crucial for Function of the Drosophila Wnt/Wingless Antagonist Naked Cuticle

Article excerpt

ABSTRACT

Wnt/β-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/β-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the β-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.

PATTERN formation in multicellular animals is governed by the intensity, duration, and combination of signals received by each developing cell. Wnts are a family of highly potent and potentially oncogenic protein signals that specify cell fate and behavior throughout the animal kingdom, and, in the vertebrate, renew stem cells (LOGAN and NUSSE 2004; REYA and CLEVERS 2005). Abnormal Wnt signaling perturbs development and can cause human diseases (MOON et al. 2004). Feedbackregulation-thesignal-dependentinduction of genes that control signal flux-is a prominent mechanism by which responses to Wnt and other signals are kept within a physiological range, thereby ensuring accurate patterning in the face of environmental perturbation or altered gene dosage (FREEMAN 2000).

Many Wnts manifest activity via accumulation of β-catenin, a bifunctional, intracellular adaptor protein that regulates cell adhesion at the plasma membrane and transmits Wnt signals into the nucleus (BIENZ 2005; HARRIS and PEIFER 2005). Indeed, in a variety of contexts and animals, loss or gain of β-catenin activity mimics absent or maximal Wnt signaling, respectively (e.g., PAI et al. 1997; GAT et al. 1998; HUELSKEN et al. 2001; ZECHNER et al.2003). Inthecanonical "Wnt/β-catenin"pathway (recently reviewed by CADIGAN and LIU 2006; WILLERT and JONES 2006), Wnt engages Fz/LRP receptors to activate Dishevelled (Dsh), which inactivates a β-catenin "destruction complex" composed of the tumor suppressors Axin/Apc and kinases CK1/GSK3β, leading to intracellular β-catenin accumulation and activation of Wnt target genes via binding to Lef/TCF and other transcriptional regulatory proteins (see http://www.stanford.edu/ ∼rnusse/wntwindow.html). Dsh also relays a parallel, LRP-dependent signal that culminates in Axin/LRP association and Axin degradation (CLIFFE et al. 2003; TOLWINSKI et al. 2003; DAVIDSON et al. 2005; ZENG et al. 2005). Fz and Dsh, but not LRP or downstream proteins that regulate β-catenin turnover, participate in noncanonical pathways, the best understood of which executes planar cell polarity (PCP) (VEEMAN et al. 2003).

Although often dubbed a "scaffolding protein" by virtue of its ability to bind a multitude of proteins, Dsh has been likened to a network hub or node because it links distinct signaling inputs to pathway-specific effectors. However, Dsh's dynamic localization to several subcellular compartments, its tendency to aggregate, and its apparent lack of catalytic activity have rendered accurate comprehension of its molecular and cell biological mechanisms an unexpectedly daunting prospect (TORRES and NELSON 2000; CAPELLUTO et al. …

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