If so , the disinhibition of WWP2 by Dvl2 should depend on its ability to polymerize. To test this, we co-expressed the polymerization-defective mutants M2 and M4 [9] with WWP2, and monitored their autoubiquitylation in His pull-down assays. Notch signalling. These regulatory interactions are conserved inDrosophilawhose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch Genz-123346 free base signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state. Keywords: Dishevelled polymerization, NEDD4 family HECT E3 ubiquitin ligases, DEP domain, Notch, Wnt == 1 . Introduction == Wnt/-catenin signalling controls normal animal development and tissue homeostasis, and aberrant activation of the pathway leads to many cancers, most notably colorectal cancer [1]. The OFF state of this signalling pathway is determined largely by modification of its key effector -catenin with canonical lysine 48-linked ubiquitin Rabbit Polyclonal to DUSP6 (K48-Ub) [2]: in the absence of Wnt stimulation, -catenin is continuously targeted for proteasomal degradation by an SCF E3 ubiquitin ligase, whose -TrCP adaptor recognizes a phosphodegron in its N-terminus. This phosphorylation mark is imparted by the serine/threonine kinases of the Axin degradasome (i. e. glycogen synthase kinase 3 and casein kinase 1) whose assembly and function depends on the APC tumour suppressor [35]. Upon binding of Wnt ligand to its Frizzled receptor and LRP5/6 co-receptor, the Axin degradasome is recruited into a signalosome assembled by Dishevelled (Dvl, or Dsh inDrosophila) at the cytoplasmic face of Frizzled and LRP5/6, which blocks its activity. Thus, -catenin is stabilized and accumulates, to co-activate a Wnt-specific transcriptional programme in the nucleus [6, 7]. However , Dishevelled can also signal through non-nuclear (-catenin-independent) Wnt signalling branches, to control cytoplasmic events such as planar cell polarity (PCP) and cell movement [8]. Nuclear and non-nuclear Wnt signalling branches are mutually exclusive, owing to Dishevelled which acts as a pivot between them. To assemble a signalosome, Dishevelled undergoes dynamic head-to-tail polymerization through its DIX domain which can be triggered by Wnt binding to its Genz-123346 free base receptors, or by high expression levels [7, 9]. To avoid inadvertent signalling, its levels must therefore be kept low, which is achieved by ubiquitylation by various E3 ligases, including a Cullin 3 ubiquitin ligase complex which targets it for proteasomal degradation [10]. Similarly, Dvl can be targeted for proteasomal degradation by the anaphase-promoting complex (APC/C) which recognizes a D-box in its DEP domain [11] and, notably, by several members of the NEDD4 family of HECT ubiquitin E3 ligases (including Itch, NEDD4L, NEDD4 and Smurf2) which bind to a highly conserved PPxY motif in Dvl via their WW domains [1216]. Itch- and Smurf2-dependent ubiquitylation of Dvl correlates with its phosphorylation [12, 13], which is acquired during signalling [1719], suggesting that Dvl is downregulated by these ligases during or after signalling. Indeed, some of these ligases seem to specifically target the PCP signalling function of Dvl [11, 12], underscoring the notion that the levels of Dvl need to be tightly regulated in post-mitotic cells. In addition to targeting Dvl for proteasomal degradation, ubiquitylation of its DIX domain, for example by the HECT E3 ligase HUWE1 [20], blocks its polymerization [21] and thus attenuates Dvl’s signalling function without destabilizing it. The ubiquitylation imparted Genz-123346 free base on Dvl by HUWE1 involves predominantly K11-Ub and K63-Ub linkages [20], similarly to ubiquitylation by the NEDD4 family ligases which also generate these non-canonical ubiquitin linkages [22]. Consistent with this, the de-ubiquitinase (DUB) USP14 appears to stimulate the signalling activity of Dvl by trimming its K63-linked ubiquitin chains [23]. By contrast, another K63-specific DUB called CYLD attenuates its signalling activity [24], which implies that ubiquitylation can also have a positive impact on Dvl. Examining the ubiquitylation of Dvl2 by mass spectroscopy, we found K11-Ub in the absence of Wnt, and additionally K63-Ub and K48-Ub after Wnt stimulation, consistent with previous reports [20, 24]. Because each of these ubiquitin linkages can be generated by NEDD4 family E3 ligases [22], they could be the enzymes imparting these ubiquitylations. Here, we test the interaction between Dvl2 and members of the human NEDD4 family, and identify WWP2 as a preferred binding partner. Like its relatives, WWP2 binds to the conserved PPxY motif of Dvl2, but also interacts with the DEP domain, thereby ubiquitylating and destabilizing Dvl2. Modification by WWP2.