Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism

Abstract

Previously, we have shown that Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). Here, we identify additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, we reveal that casein kinase 1 (CK1) δ and CK1ϵ are crucial for Dvl phosphorylation by non-canonical Wnts. We show that in response to Wnt-5a, CK1ϵ binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1ϵ, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1ϵ kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, we show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, our data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/ϵ-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling.

Activation of the non-canonical Dvl–Rac1–JNK pathway by Frizzled homologue 10 in human synovial sarcoma

“…In addition, CK1 inhibitor, D4476, blocked the differentiation of primary dopaminergic neuronal precursor cells (Bryja et al, 2007a). Phosphorylated Dvl protein was reported to be involved in the full activation of b-catenin indicated by active b-catenin-specific antibody (Bryja et al, 2007b). In our results, phosphorylated Dvl3 was pulled down with an active Rac1 in the presence of FZD10 (Supplementary Figure S2).…”

Sequential Activation and Inactivation of Dishevelled in the Wnt/β-Catenin Pathway by Casein Kinases

“…Immunoblotting and sample preparations were performed as previously described (14). For solubility assays, cells were seeded out on 12-well plates and transfected according to the scheme.…”

“…However, these predictions have not been supported by experimental data. First, overexpression of CK1 dissolved (and not promoted) Dvl aggregates in all tested cell types (14,20,22). Second, endogenous PS-Dvl formed irrespective of whether or not the activating Wnt triggered the Wnt/␤-catenin pathway (14,15,23).…”

“…Wnt-induced activation of Dvl is visible as a Wnt-induced shift in the electrophoretic mobility of all three Dvl isoforms, forming the so called phosphorylated and shifted (PS) Dvl (14 -16). It has been clearly demonstrated that both the activation of Dvl in the Wnt/␤-catenin pathway (17)(18)(19)(20)(21) and Wnt-induced PS-Dvl formation are dependent on casein kinase 1 (CK1) ␦/⑀ activity (14,16). It is, thus, conceivable that CK1␦/⑀ phosphorylation of Dvl promotes the formation of signalosomes/Dvl aggregates, which is unique for the Wnt/␤-catenin pathway.…”

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

“…Samples were then subjected to SDS-PAGE followed by western blot analysis using anti-HA (Abcam), anti-Vangl2 (R&D Systems), anti-Rac1 (Upstate) or antiRhoA (Santa Cruz Biotechnology) antibodies. Western blotting was performed as described previously (Bryja et al, 2007).…”