Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.
The Wnt/beta-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated beta-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, beta-catenin phosphorylation by GSK3 is inhibited and beta-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of beta-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent beta-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit beta-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of beta-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of beta-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin. This model provides a possible mechanism to account, in part, for inhibition of beta-catenin phosphorylation by Wnt-activated LRP6.
Wu
Geng
G
F M Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA. geng.wu@sjtu.edu.cn
Huang
He
H
Garcia Abreu
Jose
J
He
Xi
X
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000737:chemistry / Q000378:metabolism*; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014981:Xenopus; D051176:beta Catenin / Q000378:metabolism*
D026502:LDL-Receptor Related Proteins; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051176:beta Catenin; D038362:Glycogen Synthase Kinase 3