Regulation of Lrp6 phosphorylation

Abstract

The Wnt/beta-catenin signaling pathway plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Wnts transduce signals via transmembrane receptors of the Frizzled (Fzd/Fz) family and the low density lipoprotein receptor-related protein 5/6 (Lrp5/6). A key mechanism in their signal transduction is that Wnts induce Lrp6 signalosomes, which become phosphorylated at multiple conserved sites, notably at PPSPXS motifs. Lrp6 phosphorylation is crucial to beta-catenin stabilization and pathway activation by promoting Axin and Gsk3 recruitment to phosphorylated sites. Here, we summarize how proline-directed kinases (Gsk3, PKA, Pftk1, Grk5/6) and non-proline-directed kinases (CK1 family) act upon Lrp6, how the phosphorylation is regulated by ligand binding and mitosis, and how Lrp6 phosphorylation leads to beta-catenin stabilization.

Fig. 1

Wnt/β-catenin signaling pathway. a In the absence of Wnt, β-catenin (β-cat) is sequentially phosphorylated by Axin-Apc-bound CK1α and Gsk3, targeting β-catenin for degradation. b Wnt binds to transmembrane receptors Frizzled (Fzd/Fz) and Lrp6 (or Lrp5), and recruits Dishevelled (Dvl) polymers to the plasma membrane. In turn, Dvl recruits an Axin–Gsk3 complex to the plasma membrane, promoting the formation of Lrp6 signalosomes. c Polymerized Lrp6 is then phosphorylated by CK1γ, in particular, at T1479 in the S/T cluster (site I). The phosphorylation of PPSP motif A by Gsk3 is stimulated by Wnt and Dvl, and serves to prime the CK1-mediated phosphorylation of S in PPSPXS (site II), whose phosphorylation is also induced by Wnt. Doubly phosphorylated PPSPXS motifs then recruit more Axin and Gsk3, and inhibit Gsk3 by acting as direct inhibitors of this kinase. Multiple phospho-PPSPXS repeats cooperate in amplifying this signal. For simplicity only a single PPSPXS repeat is shown. β-catenin then accumulates and enters the nucleus, binds to Tcf/Lef family of transcriptional factors (Tcf) and activates gene transcription

Fig. 2

Lrp6 structure and phosphorylation sites. a Lrp6 is a single-pass transmembrane protein. It has a modular extracellular domain (ECD), consisting of YWTD domains, EGF-like repeats, and LDL repeats. The intracellular domain (ICD) consists of PPSP repeats (A–E), each juxtaposed with CK1 sites. Protein kinases promoting Lrp6 signal transduction are indicated. Note that N-terminal phosphorylation by CK1ε is inhibitory to Wnt/Lrp6 signaling. b Alignment of the ICD sequences from human (h) and Xenopus (x) Lrp6, Lrp5, and Drosophila Arrow. The S/T cluster, PPSPXS motifs, and CK1ε sites are highlighted in red. The target sites and corresponding protein kinases are indicated by arrows. Residues are numbered according to the human Lrp6 sequence

Fig. 3

Hierarchy of Lrp6 phosphorylation at S/T Cluster and PPSPXS motif A. a S1490 primes T1493 phosphorylation. b Phosphorylation of the S/T cluster promotes S1490 phosphorylation. c Sequential phosphorylation (“ping-pong” mode): S/T cluster (including T1479) phosphorylation by CK1γ occurs independently of S1490 and T1493 phosphorylation (vertical arrow) and may promote S1490 phosphorylation by Gsk3 (dashed arrow), which primes CK1 phosphorylation at T1493