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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.


PTK6 phosphorylates parafibromin and impairs its scaffold function.(a) Immunohistochemistry of the mouse small intestine confirming the co-expression of PTK6 and parafibromin. The right panels show higher-magnification images. Scale bars, 20 μm. (b,c) PTK6 regulates parafibromin tyrosine phosphorylation. AGS cells were transfected with the indicated vectors (b) or si-PTK6 (c). Total cell lysates (TCLs) were sequentially immunoprecipitated (IP) with the indicated antibodies. (d) Y290/293/315F mutations abolish parafibromin phosphorylation by PTK6. AGS cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. (e) In vitro kinase assays indicating parafibromin is a direct substrate of PTK6. Purified recombinant parafibromin and PTK6 were incubated with 200 μM ATP. The reaction mixtures were subjected to immunoblotting. (f) PTK6 regulates parafibromin/β-catenin interaction. AGS cells were transfected with si-PTK6. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies. (g) PTK6 knockdown in AGS cells resulted in increased Wnt-reporter activity in the luciferase reporter assay (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (h–k) Ptk6−/− intestinal organoids show less-differentiated ‘cystic' growth and expanded Wnt signal activation. Immunoblot analysis (h), morphology (i), a percentage of cystic organoids (j) and CD44 immunostaining (k) of control and Ptk6−/−intestinal organoids (n=3, mean±s.d. *P<0.05; Wilcoxon rank sum test). Scale bar, 50 μm.
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f5: PTK6 phosphorylates parafibromin and impairs its scaffold function.(a) Immunohistochemistry of the mouse small intestine confirming the co-expression of PTK6 and parafibromin. The right panels show higher-magnification images. Scale bars, 20 μm. (b,c) PTK6 regulates parafibromin tyrosine phosphorylation. AGS cells were transfected with the indicated vectors (b) or si-PTK6 (c). Total cell lysates (TCLs) were sequentially immunoprecipitated (IP) with the indicated antibodies. (d) Y290/293/315F mutations abolish parafibromin phosphorylation by PTK6. AGS cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. (e) In vitro kinase assays indicating parafibromin is a direct substrate of PTK6. Purified recombinant parafibromin and PTK6 were incubated with 200 μM ATP. The reaction mixtures were subjected to immunoblotting. (f) PTK6 regulates parafibromin/β-catenin interaction. AGS cells were transfected with si-PTK6. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies. (g) PTK6 knockdown in AGS cells resulted in increased Wnt-reporter activity in the luciferase reporter assay (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (h–k) Ptk6−/− intestinal organoids show less-differentiated ‘cystic' growth and expanded Wnt signal activation. Immunoblot analysis (h), morphology (i), a percentage of cystic organoids (j) and CD44 immunostaining (k) of control and Ptk6−/−intestinal organoids (n=3, mean±s.d. *P<0.05; Wilcoxon rank sum test). Scale bar, 50 μm.

Mentions: Whereas SHP2 has been shown to mediate dephosphorylation of parafibromin21, little is known about a kinase(s) that inhibits the platform function of parafibromin through tyrosine phosphorylation. To address this issue, we focused on PTK6, also known as BRK, an intracellular tyrosine kinase distinctly related to Src family kinases27. Physiologically, PTK6 is primarily expressed in differentiated non-dividing epithelial cells of the gastrointestinal tract28. We observed that both PTK6 and parafibromin were expressed in the nucleus of differentiated epithelial cells above the crypt–villus border of the mouse small intestine (Fig. 5a). To test if PTK6 phosphorylates parafibromin, we co-expressed PTK6 and parafibromin in AGS human gastric cancer cells. We found that PTK6 expression markedly elevated the level of tyrosine phosphorylation of parafibromin (Fig. 5b). Conversely, knockdown of PTK6 in AGS cells resulted in reduced levels of parafibromin tyrosine phosphorylation (Fig. 5c and Supplementary Fig. 5a). Furthermore, PR-parafibromin (Y290/293/315F) was totally resistant to tyrosine phosphorylation by PTK6 (Fig. 5d). An in vitro kinase assay consolidated direct phosphorylation of parafibromin on Y290/293/315 (Fig. 5e). These observations collectively indicated that PTK6 is the kinase that mediates parafibromin phosphorylation at Y290/293/315, which are reciprocally dephosphorylated by SHP2 (ref. 21). To study the functional consequence of parafibromin phosphorylation by PTK6, we examined the effect of PTK6 inhibition on Wnt signal activation. Knockdown of PTK6 in AGS cells enhanced endogenous parafibromin/β-catenin complex formation (Fig. 5f) and potentiated Wnt-reporter activity (Fig. 5g). Thus, PTK6 actively suppressed the Wnt signalling pathway in AGS cells through tyrosine phosphorylation of parafibromin.


Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways
PTK6 phosphorylates parafibromin and impairs its scaffold function.(a) Immunohistochemistry of the mouse small intestine confirming the co-expression of PTK6 and parafibromin. The right panels show higher-magnification images. Scale bars, 20 μm. (b,c) PTK6 regulates parafibromin tyrosine phosphorylation. AGS cells were transfected with the indicated vectors (b) or si-PTK6 (c). Total cell lysates (TCLs) were sequentially immunoprecipitated (IP) with the indicated antibodies. (d) Y290/293/315F mutations abolish parafibromin phosphorylation by PTK6. AGS cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. (e) In vitro kinase assays indicating parafibromin is a direct substrate of PTK6. Purified recombinant parafibromin and PTK6 were incubated with 200 μM ATP. The reaction mixtures were subjected to immunoblotting. (f) PTK6 regulates parafibromin/β-catenin interaction. AGS cells were transfected with si-PTK6. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies. (g) PTK6 knockdown in AGS cells resulted in increased Wnt-reporter activity in the luciferase reporter assay (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (h–k) Ptk6−/− intestinal organoids show less-differentiated ‘cystic' growth and expanded Wnt signal activation. Immunoblot analysis (h), morphology (i), a percentage of cystic organoids (j) and CD44 immunostaining (k) of control and Ptk6−/−intestinal organoids (n=3, mean±s.d. *P<0.05; Wilcoxon rank sum test). Scale bar, 50 μm.
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f5: PTK6 phosphorylates parafibromin and impairs its scaffold function.(a) Immunohistochemistry of the mouse small intestine confirming the co-expression of PTK6 and parafibromin. The right panels show higher-magnification images. Scale bars, 20 μm. (b,c) PTK6 regulates parafibromin tyrosine phosphorylation. AGS cells were transfected with the indicated vectors (b) or si-PTK6 (c). Total cell lysates (TCLs) were sequentially immunoprecipitated (IP) with the indicated antibodies. (d) Y290/293/315F mutations abolish parafibromin phosphorylation by PTK6. AGS cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. (e) In vitro kinase assays indicating parafibromin is a direct substrate of PTK6. Purified recombinant parafibromin and PTK6 were incubated with 200 μM ATP. The reaction mixtures were subjected to immunoblotting. (f) PTK6 regulates parafibromin/β-catenin interaction. AGS cells were transfected with si-PTK6. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies. (g) PTK6 knockdown in AGS cells resulted in increased Wnt-reporter activity in the luciferase reporter assay (n=3, mean±s.d. **P<0.01; a two-tailed unpaired Student's t-test). (h–k) Ptk6−/− intestinal organoids show less-differentiated ‘cystic' growth and expanded Wnt signal activation. Immunoblot analysis (h), morphology (i), a percentage of cystic organoids (j) and CD44 immunostaining (k) of control and Ptk6−/−intestinal organoids (n=3, mean±s.d. *P<0.05; Wilcoxon rank sum test). Scale bar, 50 μm.
Mentions: Whereas SHP2 has been shown to mediate dephosphorylation of parafibromin21, little is known about a kinase(s) that inhibits the platform function of parafibromin through tyrosine phosphorylation. To address this issue, we focused on PTK6, also known as BRK, an intracellular tyrosine kinase distinctly related to Src family kinases27. Physiologically, PTK6 is primarily expressed in differentiated non-dividing epithelial cells of the gastrointestinal tract28. We observed that both PTK6 and parafibromin were expressed in the nucleus of differentiated epithelial cells above the crypt–villus border of the mouse small intestine (Fig. 5a). To test if PTK6 phosphorylates parafibromin, we co-expressed PTK6 and parafibromin in AGS human gastric cancer cells. We found that PTK6 expression markedly elevated the level of tyrosine phosphorylation of parafibromin (Fig. 5b). Conversely, knockdown of PTK6 in AGS cells resulted in reduced levels of parafibromin tyrosine phosphorylation (Fig. 5c and Supplementary Fig. 5a). Furthermore, PR-parafibromin (Y290/293/315F) was totally resistant to tyrosine phosphorylation by PTK6 (Fig. 5d). An in vitro kinase assay consolidated direct phosphorylation of parafibromin on Y290/293/315 (Fig. 5e). These observations collectively indicated that PTK6 is the kinase that mediates parafibromin phosphorylation at Y290/293/315, which are reciprocally dephosphorylated by SHP2 (ref. 21). To study the functional consequence of parafibromin phosphorylation by PTK6, we examined the effect of PTK6 inhibition on Wnt signal activation. Knockdown of PTK6 in AGS cells enhanced endogenous parafibromin/β-catenin complex formation (Fig. 5f) and potentiated Wnt-reporter activity (Fig. 5g). Thus, PTK6 actively suppressed the Wnt signalling pathway in AGS cells through tyrosine phosphorylation of parafibromin.

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.