Abstract
BackgroundWnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes.ResultsUsing totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3β, and β-catenin. Cellular content of β-catenin and Axin, and phospho-glycogen synthase kinase-3β, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas β-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3β initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase-3β, and with β-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two.ConclusionThis study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.
Highlights
Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes
In the fly as in the mouse, Wnt3a binds Frizzled-1 (Fz1) and co-receptor LRP5/6, activates Dsh/ Dvl which, in turn, suppresses the activity of a key protein kinase, glycogen synthase kinase-3β (GSK3β) [9]. β-catenin, Dishevelleds, and GSK3β appear to interact in a multiprotein Axin based complex which includes the product of the adenomatous polyposis coli (APC) tumor suppressor gene [16,17]
We intentionally focused our analysis on several key elements of the Wnt canonical pathway, namely Axin, Dvl2, β-catenin, and GSK3β, whose trafficking has been reported to be regulated by the Wnt canonical pathway [21]
Summary
Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes. Upon binding of Wnt to Frizzled-1 and its co-receptor LRP5/6, a signal is transduced to the cytoplasmic phosphoprotein, Dishevelled (Dsh/Dvl) [12]. The canonical Wnt/β-catenin pathway stimulates stabilization and accumulation of cytosolic, and later of nuclear β-catenin, which binds to Lef/Tcf-sensitive transcription factors and activates genes necessary for early development [15]. In the fly as in the mouse, Wnt3a binds Frizzled-1 (Fz1) and co-receptor LRP5/6, activates Dsh/ Dvl which, in turn, suppresses the activity of a key protein kinase, glycogen synthase kinase-3β (GSK3β) [9]. Wnt stimulates changes in the abundance as well as the nucleo-cytoplasmic shuttling of β-catenin and perhaps other key signaling elements in the canonical pathway [18,19]. Developing a quantified measurement of the abundance, localization, and complexation of key elements in response to Wnt is an essential goal
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