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Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

View Article: PubMed Central - PubMed

ABSTRACT

Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with β-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.

No MeSH data available.


Parafibromin transactivates Hedgehog signalling in a tyrosine dephosphorylation-dependent manner.(a) Parafibromin knockdown results in reduced Hh signal activity. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (b) Parafibromin interacts with Gli1. TCLs prepared from SKES cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. (c) Phospho-resistant (PR)-parafibromin shows increased ability to activate Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (d) SHP2 expression promotes Hh signalling. Hh-responsive luciferase reporter assays were performed in A549 cells (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (e,f) PR-parafibromin shows increased binding to β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. (g,h) Non-phosphorylated parafibromin specifically interacts with β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting.
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f1: Parafibromin transactivates Hedgehog signalling in a tyrosine dephosphorylation-dependent manner.(a) Parafibromin knockdown results in reduced Hh signal activity. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (b) Parafibromin interacts with Gli1. TCLs prepared from SKES cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. (c) Phospho-resistant (PR)-parafibromin shows increased ability to activate Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (d) SHP2 expression promotes Hh signalling. Hh-responsive luciferase reporter assays were performed in A549 cells (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (e,f) PR-parafibromin shows increased binding to β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. (g,h) Non-phosphorylated parafibromin specifically interacts with β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting.

Mentions: In a previous study, Drosophila parafibromin Hyrax has been reported to activate Hh signalling23. To confirm the role of parafibromin in the regulation of the mammalian Hh signal, we knocked down endogenous parafibromin by specific shRNA in HEK293T human embryonic kidney cells and performed a Hh-dependent luciferase reporter assay. Inhibition of parafibromin resulted in significant reduction of Hh-reporter activity, which was restored by ectopic expression of RNAi-resistant parafibromin (Fig. 1a), suggesting that parafibromin plays a positive role in the nuclear Hh signalling. We next performed a co-immunoprecipitation study using SKES human Ewing sarcoma cells, in which Hh signalling is constitutively activated24, and found that endogenous Gli1 was co-precipitated with endogenous parafibromin (Fig. 1b). These results indicated that parafibromin potentiated Hh signalling by interacting with the Hh effector Gli1, although the data did not discriminate whether the observed interaction is direct or whether it is indirect, being mediated by another cellular component.


Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways
Parafibromin transactivates Hedgehog signalling in a tyrosine dephosphorylation-dependent manner.(a) Parafibromin knockdown results in reduced Hh signal activity. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (b) Parafibromin interacts with Gli1. TCLs prepared from SKES cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. (c) Phospho-resistant (PR)-parafibromin shows increased ability to activate Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (d) SHP2 expression promotes Hh signalling. Hh-responsive luciferase reporter assays were performed in A549 cells (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (e,f) PR-parafibromin shows increased binding to β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. (g,h) Non-phosphorylated parafibromin specifically interacts with β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting.
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f1: Parafibromin transactivates Hedgehog signalling in a tyrosine dephosphorylation-dependent manner.(a) Parafibromin knockdown results in reduced Hh signal activity. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (b) Parafibromin interacts with Gli1. TCLs prepared from SKES cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. (c) Phospho-resistant (PR)-parafibromin shows increased ability to activate Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (d) SHP2 expression promotes Hh signalling. Hh-responsive luciferase reporter assays were performed in A549 cells (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (e,f) PR-parafibromin shows increased binding to β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. (g,h) Non-phosphorylated parafibromin specifically interacts with β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting.
Mentions: In a previous study, Drosophila parafibromin Hyrax has been reported to activate Hh signalling23. To confirm the role of parafibromin in the regulation of the mammalian Hh signal, we knocked down endogenous parafibromin by specific shRNA in HEK293T human embryonic kidney cells and performed a Hh-dependent luciferase reporter assay. Inhibition of parafibromin resulted in significant reduction of Hh-reporter activity, which was restored by ectopic expression of RNAi-resistant parafibromin (Fig. 1a), suggesting that parafibromin plays a positive role in the nuclear Hh signalling. We next performed a co-immunoprecipitation study using SKES human Ewing sarcoma cells, in which Hh signalling is constitutively activated24, and found that endogenous Gli1 was co-precipitated with endogenous parafibromin (Fig. 1b). These results indicated that parafibromin potentiated Hh signalling by interacting with the Hh effector Gli1, although the data did not discriminate whether the observed interaction is direct or whether it is indirect, being mediated by another cellular component.

View Article: PubMed Central - PubMed

ABSTRACT

Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with &beta;-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.

No MeSH data available.