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Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Mavrakis KJ, Andrew RL, Lee KL, Petropoulou C, Dixon JE, Navaratnam N, Norris DP, Episkopou V - PLoS Biol. (2007)

Bottom Line: Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance.Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo.As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Mammalian Neurogenesis, Medical Research Council, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

ABSTRACT
Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

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In the Absence of Arkadia, P-Smad2 Is More Stable and Correctly Localized in the Nucleus(A) Western blot of 8.5 dpc wild-type and Akd−/− embryo extracts. Each lane contains total protein extracts from three embryos. Note that P-Smad2 levels are at least 2-fold higher in mutants than in wild-type.(B and C) Western blot of extracts from Akd−/− and wild-type ES cells cultured with Activin for different intervals (in minutes (') and hours (h). Note that P-Smad2 is more stable in mutant than in wild-type cells. Loading control: PCNA, proliferation cell nuclear antigen.(D) Densitometry analysis of the bands on the blots in (C) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/−ES cells treated with Activin for 45 min is taken as 100%.(E) Western blot analysis showing P-Smad2 and Smad2 levels in ES cells stimulated with Activin for 1 h (1 h Act) and subsequently treated for different time points with H7 inhibitor as indicated.(F) Densitometry analysis of the blots in (E) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/− and wild-type ES cells treated with Activin for 1 h are represented as 100%.(G) Western blot showing P-Smad2 and Smad4 protein distribution in cytoplasmic and nuclear fractions of three different wild-type and Akd−/−ES cell lines as indicated. 30 μg of each protein sample was loaded for analysis. Histone H3, a nuclear protein, was used as a control for fractionation. C, cytoplasmic; N, nuclear.(H) Immunofluorescence with anti-P-Smad2 and anti-Smad2/3 (red) antibody on Akd −/− and wild-type ES cells treated with Activin A for 1.5 h prior to fixation, indicating no difference in the localization of P-Smad2 in the absence of Arkadia. Note the nuclear accumulation of P-Smad2, Smad2/3, and Smad4 in Akd−/− cells. White bar, 12 μm. WT, wild-type.
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pbio-0050067-g002: In the Absence of Arkadia, P-Smad2 Is More Stable and Correctly Localized in the Nucleus(A) Western blot of 8.5 dpc wild-type and Akd−/− embryo extracts. Each lane contains total protein extracts from three embryos. Note that P-Smad2 levels are at least 2-fold higher in mutants than in wild-type.(B and C) Western blot of extracts from Akd−/− and wild-type ES cells cultured with Activin for different intervals (in minutes (') and hours (h). Note that P-Smad2 is more stable in mutant than in wild-type cells. Loading control: PCNA, proliferation cell nuclear antigen.(D) Densitometry analysis of the bands on the blots in (C) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/−ES cells treated with Activin for 45 min is taken as 100%.(E) Western blot analysis showing P-Smad2 and Smad2 levels in ES cells stimulated with Activin for 1 h (1 h Act) and subsequently treated for different time points with H7 inhibitor as indicated.(F) Densitometry analysis of the blots in (E) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/− and wild-type ES cells treated with Activin for 1 h are represented as 100%.(G) Western blot showing P-Smad2 and Smad4 protein distribution in cytoplasmic and nuclear fractions of three different wild-type and Akd−/−ES cell lines as indicated. 30 μg of each protein sample was loaded for analysis. Histone H3, a nuclear protein, was used as a control for fractionation. C, cytoplasmic; N, nuclear.(H) Immunofluorescence with anti-P-Smad2 and anti-Smad2/3 (red) antibody on Akd −/− and wild-type ES cells treated with Activin A for 1.5 h prior to fixation, indicating no difference in the localization of P-Smad2 in the absence of Arkadia. Note the nuclear accumulation of P-Smad2, Smad2/3, and Smad4 in Akd−/− cells. White bar, 12 μm. WT, wild-type.

Mentions: To find at what position within the Nodal signaling cascade Arkadia functions, we compared the level of the receptor-activated (phosphorylated) signaling effector P-Smad2 in embryos and embryonic stem (ES) cells by Western blotting (Figure 2). We examined 20 wild-type and 20 Arkadia−/− embryos at 8.5 days post-coitum (dpc) (Figure 2A and unpublished data) and three wild-type and three Arkadia−/− blastocyst-derived ES cell lines (Figure 2G). We found that while the total levels of Smad2 protein remain the same, P-Smad2 was always at least two times higher in all Arkadia−/− embryos and ES cells compared to the wild-type samples. Similarly, the other Nodal signaling effector P-Smad3 was found to be elevated in Arkadia−/−ES cell lines (Figure S1). Therefore, in embryonic cells, P-Smad2/3 are more abundant in the absence of Arkadia than in its presence. As the phosphorylation of Smad2/3 depends on the kinase activity of the ligand-activated receptors, the data suggest that in the absence of Arkadia the receptors are more active or that P-Smad2/3 are more stable after their phosphorylation.


Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Mavrakis KJ, Andrew RL, Lee KL, Petropoulou C, Dixon JE, Navaratnam N, Norris DP, Episkopou V - PLoS Biol. (2007)

In the Absence of Arkadia, P-Smad2 Is More Stable and Correctly Localized in the Nucleus(A) Western blot of 8.5 dpc wild-type and Akd−/− embryo extracts. Each lane contains total protein extracts from three embryos. Note that P-Smad2 levels are at least 2-fold higher in mutants than in wild-type.(B and C) Western blot of extracts from Akd−/− and wild-type ES cells cultured with Activin for different intervals (in minutes (') and hours (h). Note that P-Smad2 is more stable in mutant than in wild-type cells. Loading control: PCNA, proliferation cell nuclear antigen.(D) Densitometry analysis of the bands on the blots in (C) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/−ES cells treated with Activin for 45 min is taken as 100%.(E) Western blot analysis showing P-Smad2 and Smad2 levels in ES cells stimulated with Activin for 1 h (1 h Act) and subsequently treated for different time points with H7 inhibitor as indicated.(F) Densitometry analysis of the blots in (E) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/− and wild-type ES cells treated with Activin for 1 h are represented as 100%.(G) Western blot showing P-Smad2 and Smad4 protein distribution in cytoplasmic and nuclear fractions of three different wild-type and Akd−/−ES cell lines as indicated. 30 μg of each protein sample was loaded for analysis. Histone H3, a nuclear protein, was used as a control for fractionation. C, cytoplasmic; N, nuclear.(H) Immunofluorescence with anti-P-Smad2 and anti-Smad2/3 (red) antibody on Akd −/− and wild-type ES cells treated with Activin A for 1.5 h prior to fixation, indicating no difference in the localization of P-Smad2 in the absence of Arkadia. Note the nuclear accumulation of P-Smad2, Smad2/3, and Smad4 in Akd−/− cells. White bar, 12 μm. WT, wild-type.
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Related In: Results  -  Collection

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pbio-0050067-g002: In the Absence of Arkadia, P-Smad2 Is More Stable and Correctly Localized in the Nucleus(A) Western blot of 8.5 dpc wild-type and Akd−/− embryo extracts. Each lane contains total protein extracts from three embryos. Note that P-Smad2 levels are at least 2-fold higher in mutants than in wild-type.(B and C) Western blot of extracts from Akd−/− and wild-type ES cells cultured with Activin for different intervals (in minutes (') and hours (h). Note that P-Smad2 is more stable in mutant than in wild-type cells. Loading control: PCNA, proliferation cell nuclear antigen.(D) Densitometry analysis of the bands on the blots in (C) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/−ES cells treated with Activin for 45 min is taken as 100%.(E) Western blot analysis showing P-Smad2 and Smad2 levels in ES cells stimulated with Activin for 1 h (1 h Act) and subsequently treated for different time points with H7 inhibitor as indicated.(F) Densitometry analysis of the blots in (E) showing the trend in P-Smad2 levels normalized against Smad2 where Akd−/− and wild-type ES cells treated with Activin for 1 h are represented as 100%.(G) Western blot showing P-Smad2 and Smad4 protein distribution in cytoplasmic and nuclear fractions of three different wild-type and Akd−/−ES cell lines as indicated. 30 μg of each protein sample was loaded for analysis. Histone H3, a nuclear protein, was used as a control for fractionation. C, cytoplasmic; N, nuclear.(H) Immunofluorescence with anti-P-Smad2 and anti-Smad2/3 (red) antibody on Akd −/− and wild-type ES cells treated with Activin A for 1.5 h prior to fixation, indicating no difference in the localization of P-Smad2 in the absence of Arkadia. Note the nuclear accumulation of P-Smad2, Smad2/3, and Smad4 in Akd−/− cells. White bar, 12 μm. WT, wild-type.
Mentions: To find at what position within the Nodal signaling cascade Arkadia functions, we compared the level of the receptor-activated (phosphorylated) signaling effector P-Smad2 in embryos and embryonic stem (ES) cells by Western blotting (Figure 2). We examined 20 wild-type and 20 Arkadia−/− embryos at 8.5 days post-coitum (dpc) (Figure 2A and unpublished data) and three wild-type and three Arkadia−/− blastocyst-derived ES cell lines (Figure 2G). We found that while the total levels of Smad2 protein remain the same, P-Smad2 was always at least two times higher in all Arkadia−/− embryos and ES cells compared to the wild-type samples. Similarly, the other Nodal signaling effector P-Smad3 was found to be elevated in Arkadia−/−ES cell lines (Figure S1). Therefore, in embryonic cells, P-Smad2/3 are more abundant in the absence of Arkadia than in its presence. As the phosphorylation of Smad2/3 depends on the kinase activity of the ligand-activated receptors, the data suggest that in the absence of Arkadia the receptors are more active or that P-Smad2/3 are more stable after their phosphorylation.

Bottom Line: Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance.Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo.As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Mammalian Neurogenesis, Medical Research Council, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

ABSTRACT
Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

Show MeSH
Related in: MedlinePlus