Dual Phosphorylation of LRP6

Abstract

Wnt stimulates the stabilization of β-catenin through interactions with a receptor complex composed of the seven-transmembrane Frizzled and the single-transmembrane low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). In the absence of Wnt, dual phosphorylation of β-catenin by two kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), leads to the destruction of β-catenin. Two groups (Davidson et al. and Zeng et al. ) report the identification of the kinases responsible for the dual phosphorylation of LRP5/6 in the presence of Wnt, which leads to sequestration of axin away from β-catenin, thereby allowing β-catenin to accumulate, enter the nucleus, and regulate transcription. In an expression screen, Davidson et al. identified CK1γ, which is a family of palmitoylated and membrane-associated isoforms of CK1, as a kinase that decreased the mobility of LRP6, indicative of phosphorylation. The interaction of LRP6 and CK1γ and the phosphorylation of LRP6 by CK1γ were confirmed. A positive role for CK1γ-mediated LRP6 phosphorylation in Wnt signaling was provided by gain-of-function and loss-of-function assays in cultured cells (Wnt reporter assays) and in Xenopus embryos. The cytoplasmic domain of LRP5/6 contains five PPPSP potential phosphorylation motifs; however, Wnt signaling can be observed in receptors with only one PPPSP site, which is flanked by CK1 sites. Point mutagenesis of a receptor with a CK site 1, a PPPSP, and a CK site 2 (1/S/2) cytoplasmic domain showed that in coimmunoprecipitation assays, all three phosphorylation sites synergized to promote axin binding, and in reporter assays, all three sites were required for maximal activity. Zeng et al. demonstrated that the PPPSP sites of the LRP6 were phosphorylated by GSK3 and that a second phosphorylation site that resembled a CK1 phosphorylation consensus motif followed each PPPSP site. Mutation of each of the CK1 sites inhibited activation of a Wnt reporter. In vitro binding of axin to LRP6 was maximal in the presence of GSK3 and CK1. GSK3 is generally associated with inhibition of Wnt signaling (β-catenin phosphorylation), and phosphorylation of LRP6 has a stimulatory effect on Wnt signaling. Thus, spatial localization may allow GSK3 to exert these differential effects. Zeng et al. found that a membrane-localized GSK3 activated a Wnt reporter gene and in Xenopus embryos promoted axis duplication, consistent with a positive effect on Wnt signaling. How Wnt signaling controls the dual phosphorylation of the LRP5/6 to promote axin binding and β-catenin stabilization remains to be clearly established (see Nusse for commentary). G. Davidson, W. Wu, J. Shen, J. Bilic, U. Fenger, P. Stannek, A. Glinka, C. Niehrs, Casein kinase 1 γ couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438 , 867-872 (2005). [PubMed] X. Zeng, K. Tamai, B. Doble, S. Li, H. Huang, R. Habas, H. Okamura, J. Woodgett, X. He, A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438 , 873-877 (2005). [PubMed] R. Nusse, Relays at the membrane. Nature 438 , 747-749 (2005). [PubMed]