MacDonald Bryan T, Yokota Chika, Tamai Keiko, Zeng Xin, He Xi
F. M. Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2008 Jun 6;283(23):16115-23. doi: 10.1074/jbc.M800327200. Epub 2008 Mar 24.
Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.
低密度脂蛋白受体相关蛋白6（LRP6）及其同源物LRP5作为Wnt共受体，对Wnt/β-连环蛋白信号通路至关重要。Wnt激活LRP6会导致支架蛋白Axin的募集，并抑制Axin介导的β-连环蛋白磷酸化/降解。我们发现LRP6胞内结构域中的五个保守PPPSP基序是LRP6功能所必需的，这些基序一起突变会消除LRP6信号活性。我们进一步表明，Wnt诱导一个典型PPPSP基序的磷酸化，该基序为Axin提供了一个对接位点，并且足以将信号活性传递给异源受体。然而，LRP6中多个PPPSP基序的活性、调节和功能尚未得到表征。在此，我们对LRP6中的所有五个PPPSP基序进行了全面分析。我们通过丙氨酸扫描诱变定义了一个典型PPPSP基序的核心氨基酸残基，并证明五个PPPSP基序中的每一个单独都表现出信号传导和Axin结合活性。我们针对额外的PPPSP基序生成了两种新型磷酸化特异性抗体，并表明Wnt通过糖原合酶激酶3在内源性LRP6中诱导这些基序的磷酸化。最后，我们通过证明缺乏单个PPPSP基序的LRP6突变体功能受损，而缺乏五个PPPSP基序中的两个的LRP6突变体大多无活性，揭示了全长LRP6中PPPSP基序的关键协同作用。这种协同作用似乎反映了PPPSP基序促进彼此磷酸化并与Axin协同相互作用的能力。这些结果确立了PPPSP基序在LRP6中的关键作用和共同的磷酸化/激活机制，并表明PPPSP基序保守的多重性和协同作用代表了LRP6家族受体对Wnt信号的一种内置放大器。
