Differential mediation of the Wnt canonical pathway by mammalian Dishevelleds-1, -2, and -3.

In the Drosophila, a single copy of the phosphoprotein Dishevelled (Dsh) is found. In the genomes of higher organism (including mammals), three genes encoding isoforms of Dishevelled (Dvl1, Dvl2, and Dvl3) are present. In the fly, Dsh functions in the Wnt-sensitive stabilization of intracellular beta-catenin and activation of the Lef/Tcf-sensitive transcriptional response known as the Wnt "canonical" pathway. In the current work we explore the expression of Dishevelleds in mammalian cells and provide an estimate of the relative cellular abundance of each Dvl. In mouse F9 cells, all three Dvls are expressed. Dvl2 constitutes more than 95% of the total pool, the sum of Dvl1 and Dvl3 constituting the remainder. Similarly, Dvl2 constitutes more than 80% of the Dvl1-3 pool in mouse P19 and human HEK 293 cells. siRNA-induced knock-down of individual Dvls was performed using Wnt3a-sensitive canonical pathway in F9 cells as the read-out. Activation of the canonical signaling pathway by Wnt3a was dependent upon the presence of Dvl1, Dvl2, and Dvl3, but to a variable extent. Wnt3a-sensitive canonical transcription was suppressible, by knock-down of Dvl1, Dvl2, or Dvl3. Conversely, the overexpression of any one of the three Dvls individually was found to be capable of promoting Lef/Tcf-sensitive transcriptional activation, in the absence of Wnt3a, i.e., overexpression of Dvl1, Dvl2, or Dvl3 is Wnt3a-mimetic. Graded suppression of individual Dvl isoforms by siRNA was employed to test if the three Dvls could be distinguished from one another with regard to mediation of the canonical pathway. Canonical signaling was most sensitive to changes in the abundance of either Dvl3 or Dvl1. Changes in expression of Dvl2, the most abundant of the three isoforms, resulted in the least effect on canonical signaling. Dvl-based complexes were isolated by pull-downs from whole-cell extracts with isoform-specific antibodies and found to include all three Dvl isoforms. Rescue experiments were conducted in which depletion of either Dvl3 or Dvl1 suppresses Wnt3a activation of the canonical pathway and the ability of a Dvl isoform to rescue the response evaluated. Rescue of Wnt3a-stimulated transcriptional activation in these siRNA-treated cells occurred only by the expression of the very same Dvl isoform depleted by the siRNA. Thus, Dvls appear to function cooperatively as well as uniquely with respect to mediation of Wnt3a-stimulated canonical signaling. The least abundant (Dvl1, 3) plays the most obvious role, whereas the most abundant (Dvl2) plays the least obvious role, suggesting that individual Dvl isoforms in mammals may operate as a network with some features in common and others rather unique.