Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

Abstract

BackgroundDishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called “signalsomes”, which propagate signals from the Wnt receptor Frizzled to downstream elements.ResultsVery large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a.ConclusionThese data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.

Dvl3 translocates IPMK to the cell membrane in response to Wnt

“…3A). Dvl3 is a phosphoprotein, herein displaying M r = 78 and 80 kDa, reflecting the multiply phosphorylated nature of this scaffold protein [12,15]. Wnt3a treatment stimulated increased content of Myc-tagged IPMK in pull-downs of Dvl3 from lysates of F9 cells treated with Wnt3a over a 30-min time course (Fig.…”

“…The unique need for a mobile scaffold like Dvl3 to organize docking molecules at the cell membrane is reinforced herein by studies of IPMK. The dynamic character of Dvl-based signalsome formation and translocation to the cell membrane is well documented, established biochemically by steric-exclusion chromatography on large-bore matrices, optically, by fluorescence correlation spectroscopy in live cells challenged with Wnt3a, and by simple fluorescence microscopy that first revealed very large Dvl-based punctae behaving as we now know signalsome do [11,12,21]. Each of these distinctly different methodologies of interrogation discern Dvl3-based complexes to be large, dynamic in character, functioning and changing locale in response to Wnt signaling.…”

“…The consequence is accumulation of cytosolic β-catenin and subsequent activation of lymphoid enhancer factor/T-cell factor protein (Lef/Tcf)-dependent transcription [10]. Equally essential is the role of Dishevelled (Dvl), in particular Dvl3, in organizing large supermolecular complexes (i.e., signalsomes) necessary for signaling downstream from Frizzled-1 [11,12]. Dvl supermolecular complexes operate as mobile scaffolds, intracellular “toolboxes” that organize signaling partners temporally and spatially.…”

Wnt and β-Catenin Signaling and Skeletal Muscle Myogenesis in Response to Muscle Damage and Resistance Exercise and Training

“…Once the Fzd receptor has been activated, it then switches on an intracellular signaling cascade by activating the disheveled proteins (Dvl) and heterotrimeric G-proteins (α, β and γ subunits), which are required for downstream signaling. In conjunction with the phosphorylation of LRP5/6 proteins, this cascade results in the inactivation of the 'degredation complex' of proteinsm which consists of adenomatosis polyposis coli (APC), axin, serine-threonine kinase of glycogen synthase kinase-3β (GSK-3β), and casein kinase-1 (CK-1) (Li et al, 2012;Tanneberger et al, 2011;Yokoyama, Markova, Wang, & Malbon, 2012). Inactivation of this complex liberates unphosphorylated β-catenin to accumulate in the cytosol and sequentially translocate into the nucleus for gene transcription.…”

“…The downstream effectors of Wnt signaling have been purported to activate of c-Jun-N-terminal kinase (JNK)-dependent or calcium-dependent signaling pathways. Similarly, this non-canonical pathway has also been suggested to activate of PCP (planar-cell-polarity), Ras-related protein 1 (rap1), atypical protein kinase C (aPKC), mechanistic target of rapamycin (mTOR), protein kinase A (PKA), and phosphatidylinositide 3-kinase (PI3K) (Baarsma et al, 2013;Gomez-Orte, Saenz-Narciso, Moreno, & Cabello, 2013;Logan & Nusse, 2004;Semenov, Habas, Macdonald, & He, 2007;Yokoyama et al, 2012).…”