The Wnt family is composed of 19 secreted glycoproteins that act as ligands for the Frizzled family of receptors. Frizzled receptors are members of the G protein coupled receptor family and are found in multiprotein complexes. The best characterized example of Wnt signaling is the Wnt/β-Catenin pathway. β-Catenin regulates the transcription of genes involved in development, stem cell fate, proliferation, and renewal of stem and progenitor cells. These critical roles in development require tight regulation of this signaling pathway with over or under expression of target genes resulting in disease and cancer (1).
In the absence of Wnt, a destruction complex composed of GSK3, casein kinase 1a, Axin, and APC, targets β-Catenin for proteasomal degradation. Specifically, phosphorylation of β-Catenin by GSK3 promotes recruitment of the SCF βTRCP E3 ubiquitin ligase. The SCF βTRCP E3 ubiquitin ligase induces the polyubiquitination of β-Catenin, resulting in proteasomal degradation. Destabilization of β-Catenin prevents the transcription factor from entering the nucleus, thereby preventing expression of Wnt responsive genes (see figure
β-Catenin destruction) (1).
In the presence of Wnt, the degradation complex is inhibited, allowing accumulation of β-Catenin. Wnt binds to a receptor complex comprised of Frizzled and LRP5/6 and promotes recruitment of activated Dvl phosphoproteins, forming an endocytic signalosome. Subsequent phosphorylation of LRP6 promotes recruitment and inhibition of the β-Catenin destruction complex. This allows β-Catenin to accumulate in the cytoplasm and translocate to the nucleus to stimulate transcription (1).
Back to top Receptor downregulation through endocytosis
Receptor endocytosis is a common mechanism for modulation of receptor induced signal transduction. Endocytosis is stimulated by ligand induced receptor activation resulting in post translational modification of the cytoplasmic tail of the receptor. Modification by ubiquitination or phosphorylation mediates endocytosis as well as sorting to different endosomal pathways (2).
Internalized receptor complexes are sorted to either recycling or endosomal degradation pathways. Many receptors remain ligand bound and actively signaling within the endosome. In some cases endocytosis is required for complete signal transduction activation. The small volume of the endosome favors ligand receptor interaction. Endosomes are able to move towards the nucleus using microtubules and they contain resident proteins that are responsible for assembly of signaling scaffold complexes. In addition, the acidic pH of late endosomes favors the activity of proteolytic enzymes that participate in signaling (2).
Back to top The ESCRT pathway mediates multivesicular body formation
The ability for signaling to occur in endosomes suggests that there must be a mechanism for modulation. Signaling can be terminated through endosome-lysosome fusion or through sorting to ILV of MVB. This insulates the ligand bound receptors from their cytoplasmic effectors and promotes receptor proteolysis through delivery of ILV to the lysosome. Sorting to ILVs and trafficking to the lysosome is regulated by receptor ubiquitination. Inhibition of deubiquitination enzymes has been shown to increase ubiquitination and degradation of receptor tyrosine kinases, therefore downregulating signal transduction (2).
The first example of receptor downregulation through MVB sequestration was shown in epidermal growth factor (EGF) signaling. EGF stimulates cell growth and division by binding and activating the receptor tyrosine kinase EGFR. Negative regulation of EGF signaling occurs through sorting to MVB. Sorted receptors are already associated with components of the cytoplasmic signal transduction machinery and are actively signaling. The whole complex is sorted to MVB where it is segregated from the cytoplasmic targets. Mutations that interfere with the MVB sorting pathway lead to tumorigenesis in mice and defects in embryonic patterning in Drosophila, highlighting the importance of this mechanism of signal modulation (2).
The ESCRT machinery is responsible for MVB sorting. The ESCRT machinery consists of five different ESCRT complexes and their related proteins; ESCRT-0, I, II, III, and Vps4-Vtal. Ubiquitination signals destination of cargo to MVBs. The ESCRT machinery contains a number of ubiquitin interaction motifs that signal recruitment of ubiquitinated cargo and assembly of ESCRT components. The ESCRT proteins are targeted to endosomes through specific and nonspecific interactions with membrane lipids. For example, the FYVE domain in the Hrs component of ESCRT-0 specifically binds to phosphatidylinositol 3-phosphate and the GLUE domain in the Vps36 subunit in ESCRT-III binds to phosphatidylinositol 3-phosphate as well as other lipids (3).
Vps4 and the ESCRT-III complex are highly conserved and central to membrane cleavage. The ESCRT-III complex is recruited from the cytosol to the membrane where components undergo a conformational change and recruit additional effector proteins that function in membrane fission. Vps4 is a AAA type ATPase that functions in the dissociation and recycling of ESCRT components from the endosomes (3).
Back to top Wnt signaling is promoted through sequestration of GSK3 in MVB
Receptor endocytosis is a well characterized mechanism for signal downregulation. In the case of Wnt signaling, however, receptor endocytosis encourages gene expression by allowing for the accumulation of β-Catenin. Until recently, it was known that GSK3 inhibition was required for Wnt signal transduction; however the exact mechanism for GSK3 inhibition was unclear. Now Taelman et al provide evidence of GSK3 sequestration in MVB as a mechanism of inhibition (4).
Wnt binds to Frizzled and LRP6, resulting in the recruitment of Dvl and Axin and GSK3, forming the LRP6 signalosome. In the absence of Wnt, phosphorylation of β-Catenin by the GSK3 containing destruction complex targets β-Catenin for proteasomal degradation. In the presence of Wnt GSK3 activity is inhibited. One possible mechanism of GSK3 inhibition was interaction with the LRP6 cytoplasmic tail. LRP6 contains PPPSP repeats that act as substrate for GSK3 and may decrease GSK3 activity through competitive inhibition. The low affinity of GSK3 binding, suggested that an additional mechanism of inhibition was at play (4).
Taelman et al evaluated localization of GSK3 and found that Wnt induced GSK3 relocalization to vesicles that colocalized with endocytosed Wnt, the MVB marker Rab7, and the lysosome marker lysotracker. Inhibition of MVB formation by depleting ESCRT components HRS/VPS27 or a dominant negative Vps4 blocked Wnt signal transduction. These data suggest that Wnt induces localization of GSK3 to MVB and that relocalization is required for transcription of Wnt responsive genes (see figure
GSK3 sequestration during Wnt signaling) (4).
Interestingly, addition of Wnt resulted in decreased cytosolic GSK3 activity, however full enzyme activity was recovered after solubilization of membranes in TritonX-100. This data suggests that GSK3 activity was being sequestered in membrane bound vesicles. This membrane bound GSK3 was also protected from proteinase K treatment (4).
Phosphorylation by GSK3 frequently targets the substrate for ubiquitination and proteasomal degradation. An estimated 20% of the proteome contains GSK3 phosphorylation sites. Taelman et al demonstrate an increase in total cellular protein half-life with Wnt treatment or GSK3 inhibition. In a proof of principle experiment, the author added a GSK3 phosphorylation site to GFP and demonstrated the effect of Wnt treatment on GFP stability. This observation suggests that GSK3 functions not only in the Wnt signaling pathway but in control of global protein stability (4).
The role of sequestration in MVB in GSK3 regulation has broad implications in how we think about signal transduction, however many questions remain. Wnt treatment results in immediate stabilization of β-Catenin, however GSK3 was observed in MVB 6 hours post Wnt treatment. How soon does GSK3 become relocalized to MVB? Is this a secondary mechanism to inhibition through interaction with phospho-LRP6? Further research is required to resolve the relative contributions of phospho-LRP6 and MVB sequestration. Another interesting observation is the localization of β-Catenin to MVB. The function of Wnt signaling and GSK3 inhibition is to allow β-Catenin to accumulate, enter the nucleus and promote transcription. Sequestration of β-Catenin to MVB seems counterintuitive. What is the role of β-Catenin in MVB? Does this β-Catenin escape from the MVB and travel to the nucleus or does newly translated β-Catenin participate in transcription? If that is the case does β-Catenin play a functional role in MVB? Is there a structural function or is it purely just a bystander effect? Finally the authors demonstrate a profound role for GSK3 in control of protein stability. This changes the way we think of GSK3 as a transcriptional regulator and opens up a whole area of research into GSK3 mediated protein turnover as a mechanism of gene regulation (5).
- Angers, Stephane, and Randall T. Moon, (2009) Proximal events in Wnt signal transduction. Nat Rev Mol. Cell. Biol 10, 468-477.
- Sorkin, Alexander, and Mark von Zastrow, (2009) Endocytosis and signalling: intertwining molecular networks. Nat. Rev. Mol. Cell. Biol 10, 609-622.
- Wollert, Thomas, Dong Yang, Xuefeng Ren, H. H. Lee, Y. J. Im, and James H. Hurley, (2009) The ESCRT machinery at a glance. Journal of Cell Science 122, 2163-2166.
- Taelman, Vincent F., et al., (2010) Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes. Cell 143, 1136-1148.
- Niehrs, Christof, and Sergio P. Acebron, (2010) Wnt Signaling: Multivesicular Bodies Hold GSK3 Captive. Cell 143, 1044-1046.
