Activation of the low-density lipoprotein–related protein 6 (LRP6) by Wnt (called Wingless or Wg in Drosophila) is thought to cause GSK-3 (glycogen synthase kinase 3)–mediated phosphorylation of one of the five Pro-Pro-Pro-Ser/Thr-Pro (PPPS/TP) motifs in the LRP6 intracellular domain. This phosphorylation event may lead to the stabilization of β-catenin and subsequent downstream Wnt signaling events. There is controversy, however, about whether phosphorylation of the PPPS/TP motifs in LRP6 occurs constitutively or only after activation with Wnt. To identify other kinases that target these motifs in LRP6, Davidson et al. (see also Kaldis and Pagano) performed an RNA interference (RNAi) screen targeting kinases in Drosophila S2R+ cells. The authors found that transfection of double-stranded RNA (dsRNA) in S2R+ cells to knock down the cyclin-dependent kinase (CDK)–like protein L63 decreased phosphorylation at Ser1490 and Thr1479 of LRP6 (detected by immunoblotting with phosphorylation site–specific antibodies) and reduced Wg signaling due to overexpression of Wg or LRP6 (detected with a reporter gene assay). Furthermore, RNAi against the DCyclin Y, a Drosophila cyclin, also decreased LRP6 phosphorylation at Ser1490 and Wg transcriptional activity due to overexpression of Wg or LRP6, suggesting that DCyclin Y interacted with and regulated L63. In vitro assays confirmed that DCyclin Y was required for L63 to phosphorylate the intracellular domain of LRP6. The vertebrate homologs of L63 (PFTK1) and of DCyclin Y (cyclin Y) rescued the reductions in LRP6 phosphorylation and Wg transcriptional activity in S2R+ cells transfected with dsRNAs against L63 or DCyclin Y, respectively. In synchronized human embryonic kidney (HEK) 293T or HeLa cells, cyclin Y activity, LRP6 phosphorylation at Ser1490, and Wnt transcriptional activity in response to exogenous Wnt3a were highest at G2/M. Injection of morpholino antisense oligonucleotides (morpholinos) against lrp6 or cycY into Xenopus embryos caused anteriorization, a phenotype consistent with inhibition of Wnt signaling; in addition, depletion of cyclin Y in Xenopus oocytes decreased Wnt signaling, an effect that was rescued by injection of the mRNA for β-catenin. In S2R+ cells, RNAi against the phosphatase Cdc25A, which promotes cell cycle progression, increased Wg signaling and LRP6 phosphorylation, effects that were blocked by RNAi against DCyclin Y. Thus, LRP6-mediated Wnt signaling is regulated in a cell cycle–dependent manner.
G. Davidson, J. Shen, Y.-L. Huang, Y. Su, E. Karaulanov, K. Bartscherer, C. Hassler, P. Stannek, M. Boutros, C. Niehrs, Cell cycle control of Wnt receptor activation. Dev. Cell 17, 788–799 (2009). [Online Journal]
P. Kaldis, M. Pagano, Wnt signaling in mitosis. Dev. Cell 17, 749–750 (2009). [Online Journal]