Ser33 and Ser37 doubly-phosphorylated -catenin is specifically identified by -TrCP [16]C[22], a subunit of the SCF-TrCP E3 ubiquitin ligase complex. GSK3 inside a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is definitely SAV1 direct and specific for GSK3 phosphorylation of -catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of -catenin at Ser45, and is self-employed of Axin function. We also display that a phosphorylated PPPSPXS peptide is able to activate Wnt/-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of Rafoxanide GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 locations GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 Rafoxanide phosphorylation of -catenin. This model provides a possible mechanism to account, in part, for inhibition of -catenin phosphorylation by Wnt-activated LRP6. Intro The Wnt/-catenin transmission transduction pathway takes on central functions in many aspects of cell proliferation and differentiation, such as segment polarity determination in (APC), phosphorylates -catenin at Thr41, Ser37, and Ser33 [5]C[15]. Ser33 and Ser37 doubly-phosphorylated -catenin is usually specifically recognized by -TrCP [16]C[22], a subunit of the SCF-TrCP E3 ubiquitin ligase complex. The SCF-TrCP ubiquitin ligase poly-ubiquitinates -catenin, leading to -catenin degradation via the proteosome pathway [23], [24]. In the presence of Wnt ligands, the activation of the Wnt pathway results in inhibition of -catenin phosphorylation at Ser33 and Ser37 (and Thr41) by GSK3, thereby preventing -catenin ubiquitination and degradation. Stabilized -catenin translocates into the nucleus and complexes with users of the T cell factor (TCF)/lymphoid enhancer factor Rafoxanide (LEF) family of transcription factors [25]C[27], leading to the activation of Wnt/-catenin responsive genes such as c-myc and cyclin D1 [28], [29]. Therefore, inhibition of amino-terminal phosphorylation of -catenin by GSK3 is usually a central step in Wnt/-catenin signaling. Wnt activates the -catenin pathway via two unique classes of receptors around the cell surface: one is a member of the Frizzled family of seven-transmembrane receptors, and the other is usually a single transmembrane receptor referred to as LDL receptor related protein 6 (LRP6), or its relative LRP5. Wnt may induce a Frizzled-LRP6 coreceptor complex [30]C[33], which in turn triggers Rafoxanide the phosphorylation of LRP6 intracellular domain name at five conserved PPP(S/T)PX(S/T) motifs (referred to as PPPSPXS for simplicity) [34], [35]. The phosphorylated PPPSPXS motif provides an optimal binding site for Axin [34], [35], thereby recruiting Axin and likely associated proteins to the Frizzled-LRP6 receptor complex [33], [36] and leading to the inhibition of -catenin phosphorylation. Importantly the phosphorylated PPPSPXS motif represents a key and minimal functional module of the Wnt receptor complex, since it is sufficient to trigger -catenin signaling when transferred to a heterologous receptor [34], [35], [37]. PPPSPXS phosphorylation is usually carried out sequentially by GSK3 and CK1 [35], [37], [38] and is under the control Rafoxanide by Frizzled and its downstream partner Dishevelled protein [39], [40]. How PPPSPXS phosphorylation and its recruitment of Axin result in inhibition of -catenin phosphorylation remains a critical question. To address this issue we established an in vitro -catenin phosphorylation system using recombinant Axin, GSK3 and CK1. We found that each of the multiple phosphorylated PPPSPXS peptides inhibits the phosphorylation of -catenin at Ser33/Ser37/Thr41 by GSK3 in a sequence and phosphorylation-dependent manner. This inhibition is usually specific for GSK3, as these phospho-peptides do not impact -catenin Ser45 phosphorylation by CK1, and occurs regardless of the presence or absence of Axin. We also found that a phosphorylated PPPSPXS peptide is able to activate Wnt/-catenin signaling and to induce axis duplication in Xenopus embryos, presumably via inhibition of GSK3 in vivo. These results suggest a potential mechanism to account, in part, for the inhibition GSK3 phosphorylation of -catenin by the activated LRP6. While this manuscript was in previous review processes, Cselenyi reported that this LRP6 intracellular domain name directly inhibits GSK3 phosphorylation of -catenin in a PPPSPXS-dependent manner [41]. Our results based on studying individual phospho-PPPSPXS peptides are consistent with their main conclusion. However, while Cselenyi suggested that LRP6 specifically inhibits GSK3 phosphorylation of -catenin but not of other substrates [41], our data suggest that the phosphorylated PPPSPXS peptide behaves as a general GSK3 inhibitor. Results.
