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Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling.

Toruń A, Szymańska E, Castanon I, Wolińska-Nizioł L, Bartosik A, Jastrzębski K, Miętkowska M, González-Gaitán M, Miaczynska M - PLoS ONE (2015)

Bottom Line: These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip.In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans.Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109, Warsaw, Poland.

ABSTRACT
Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

No MeSH data available.


Related in: MedlinePlus

Tollip requires an intact ubiquitin-binding domain to function in canonical Wnt signaling.(A) Schematic representation of deletion and point mutants of human Tollip. (B-D) myc-tagged Tollip wild-type (wt) and mutants were tested in the Super8xTOPFlash reporter assay (upper panels; B, deletion mutants; C, ubiquitin-binding deficient M240A/F241A mutant; D, phosphoinositide-binding deficient point mutants). Increasing amounts of mutant-encoding plasmids were transfected. Ctrl, cells transfected with an empty pcDNA plasmid instead of a Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 (C) or 4 (B, D) independent experiments; *P≤0.05, **P<0.01, ***P<0.001 (Mann-Whitney U test). Expression of mutated Tollip proteins was verified by Western blotting (WB) using anti-myc antibodies, with α-tubulin as a loading control (lower panels). (E) Expression of FGF9 and NRP1 genes upon overexpression of Tollip wt, K150E and M240A/F241A mutants in Wnt3a-stimulated HEK293 cells (Ctrl), measured by qPCR. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). Data are mean ± SEM from 3 independent experiments; *P≤0.05 (Mann-Whitney U test). (F) Immunoprecipitation of β-catenin from lysates of HEK293 cells transfected with an empty plasmid (Ctrl) or with constructs expressing wild-type Tollip (wt), M240A/F241A mutant or deletion 1 (del.1) mutant. Cells were either untreated or incubated with MG132 for 4 h before lysis. Upper panel; immunoprecipitates were probed with antibodies against β-catenin, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Lower panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub), β-catenin and Tollip, with actin as a loading control.
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pone.0130818.g003: Tollip requires an intact ubiquitin-binding domain to function in canonical Wnt signaling.(A) Schematic representation of deletion and point mutants of human Tollip. (B-D) myc-tagged Tollip wild-type (wt) and mutants were tested in the Super8xTOPFlash reporter assay (upper panels; B, deletion mutants; C, ubiquitin-binding deficient M240A/F241A mutant; D, phosphoinositide-binding deficient point mutants). Increasing amounts of mutant-encoding plasmids were transfected. Ctrl, cells transfected with an empty pcDNA plasmid instead of a Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 (C) or 4 (B, D) independent experiments; *P≤0.05, **P<0.01, ***P<0.001 (Mann-Whitney U test). Expression of mutated Tollip proteins was verified by Western blotting (WB) using anti-myc antibodies, with α-tubulin as a loading control (lower panels). (E) Expression of FGF9 and NRP1 genes upon overexpression of Tollip wt, K150E and M240A/F241A mutants in Wnt3a-stimulated HEK293 cells (Ctrl), measured by qPCR. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). Data are mean ± SEM from 3 independent experiments; *P≤0.05 (Mann-Whitney U test). (F) Immunoprecipitation of β-catenin from lysates of HEK293 cells transfected with an empty plasmid (Ctrl) or with constructs expressing wild-type Tollip (wt), M240A/F241A mutant or deletion 1 (del.1) mutant. Cells were either untreated or incubated with MG132 for 4 h before lysis. Upper panel; immunoprecipitates were probed with antibodies against β-catenin, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Lower panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub), β-catenin and Tollip, with actin as a loading control.

Mentions: Deletion mutagenesis was performed and the resulting five mutants (Fig 3A) were tested for their effects in the reporter assay (Fig 3B). The mutants devoid of the CUE domain (del.1 and del.2) lost the ability to inhibit the Wnt pathway activity. Instead, inhibition was retained in a mutant lacking the N-terminal TOM1-binding motif (del.3). Interestingly, the C2 domain alone (del.4) also inhibited the reporter activity, in contrast to a minor effect of the C-terminal fragment containing the CUE domain (del.5). We therefore concluded that the interaction with TOM1 via the N-terminus of Tollip is not important for the regulation of Wnt signaling. Instead, both the C2 and the CUE domains mediate this function of Tollip, suggesting that binding of phosphoinositides and/or ubiquitin may be involved. Consistent with this idea, both types of molecules are implicated in Wnt signaling. Phosphatidylinositol (4,5)-bisphosphate (PI4,5P2) is produced at the plasma membrane upon Wnt3a stimulation to drive formation of LRP6 signalosomes [52–54], while ubiquitin plays a number of roles, either proteolytic or regulatory, at various stages of the canonical Wnt cascade [55].


Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling.

Toruń A, Szymańska E, Castanon I, Wolińska-Nizioł L, Bartosik A, Jastrzębski K, Miętkowska M, González-Gaitán M, Miaczynska M - PLoS ONE (2015)

Tollip requires an intact ubiquitin-binding domain to function in canonical Wnt signaling.(A) Schematic representation of deletion and point mutants of human Tollip. (B-D) myc-tagged Tollip wild-type (wt) and mutants were tested in the Super8xTOPFlash reporter assay (upper panels; B, deletion mutants; C, ubiquitin-binding deficient M240A/F241A mutant; D, phosphoinositide-binding deficient point mutants). Increasing amounts of mutant-encoding plasmids were transfected. Ctrl, cells transfected with an empty pcDNA plasmid instead of a Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 (C) or 4 (B, D) independent experiments; *P≤0.05, **P<0.01, ***P<0.001 (Mann-Whitney U test). Expression of mutated Tollip proteins was verified by Western blotting (WB) using anti-myc antibodies, with α-tubulin as a loading control (lower panels). (E) Expression of FGF9 and NRP1 genes upon overexpression of Tollip wt, K150E and M240A/F241A mutants in Wnt3a-stimulated HEK293 cells (Ctrl), measured by qPCR. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). Data are mean ± SEM from 3 independent experiments; *P≤0.05 (Mann-Whitney U test). (F) Immunoprecipitation of β-catenin from lysates of HEK293 cells transfected with an empty plasmid (Ctrl) or with constructs expressing wild-type Tollip (wt), M240A/F241A mutant or deletion 1 (del.1) mutant. Cells were either untreated or incubated with MG132 for 4 h before lysis. Upper panel; immunoprecipitates were probed with antibodies against β-catenin, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Lower panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub), β-catenin and Tollip, with actin as a loading control.
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Related In: Results  -  Collection

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pone.0130818.g003: Tollip requires an intact ubiquitin-binding domain to function in canonical Wnt signaling.(A) Schematic representation of deletion and point mutants of human Tollip. (B-D) myc-tagged Tollip wild-type (wt) and mutants were tested in the Super8xTOPFlash reporter assay (upper panels; B, deletion mutants; C, ubiquitin-binding deficient M240A/F241A mutant; D, phosphoinositide-binding deficient point mutants). Increasing amounts of mutant-encoding plasmids were transfected. Ctrl, cells transfected with an empty pcDNA plasmid instead of a Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 (C) or 4 (B, D) independent experiments; *P≤0.05, **P<0.01, ***P<0.001 (Mann-Whitney U test). Expression of mutated Tollip proteins was verified by Western blotting (WB) using anti-myc antibodies, with α-tubulin as a loading control (lower panels). (E) Expression of FGF9 and NRP1 genes upon overexpression of Tollip wt, K150E and M240A/F241A mutants in Wnt3a-stimulated HEK293 cells (Ctrl), measured by qPCR. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). Data are mean ± SEM from 3 independent experiments; *P≤0.05 (Mann-Whitney U test). (F) Immunoprecipitation of β-catenin from lysates of HEK293 cells transfected with an empty plasmid (Ctrl) or with constructs expressing wild-type Tollip (wt), M240A/F241A mutant or deletion 1 (del.1) mutant. Cells were either untreated or incubated with MG132 for 4 h before lysis. Upper panel; immunoprecipitates were probed with antibodies against β-catenin, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Lower panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub), β-catenin and Tollip, with actin as a loading control.
Mentions: Deletion mutagenesis was performed and the resulting five mutants (Fig 3A) were tested for their effects in the reporter assay (Fig 3B). The mutants devoid of the CUE domain (del.1 and del.2) lost the ability to inhibit the Wnt pathway activity. Instead, inhibition was retained in a mutant lacking the N-terminal TOM1-binding motif (del.3). Interestingly, the C2 domain alone (del.4) also inhibited the reporter activity, in contrast to a minor effect of the C-terminal fragment containing the CUE domain (del.5). We therefore concluded that the interaction with TOM1 via the N-terminus of Tollip is not important for the regulation of Wnt signaling. Instead, both the C2 and the CUE domains mediate this function of Tollip, suggesting that binding of phosphoinositides and/or ubiquitin may be involved. Consistent with this idea, both types of molecules are implicated in Wnt signaling. Phosphatidylinositol (4,5)-bisphosphate (PI4,5P2) is produced at the plasma membrane upon Wnt3a stimulation to drive formation of LRP6 signalosomes [52–54], while ubiquitin plays a number of roles, either proteolytic or regulatory, at various stages of the canonical Wnt cascade [55].

Bottom Line: These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip.In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans.Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109, Warsaw, Poland.

ABSTRACT
Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

No MeSH data available.


Related in: MedlinePlus