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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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Arkadia Is Required for Efficient P-Smad2/3 Target Gene Transcription and Super-Activates Signaling in Akd−/− ES Cells(A) Relative luciferase activity from three different wild-type and three different Akd−/− ES cell lines transfected with the Pitx2-luc reporter. All values are expressed as a percentage relative to wild-type cell line 3 (3) represented as 0%. Akd−/− ES cell lines have lower activity compared to wild-type, indicative of reduced target gene expression. Average differences (Av) between wild-type and Akd−/− ES cells are shown.(B) Nodal real-time PCR performed on two different wild-type and Akd−/− ES cell lines (1 and 2). The abundance of endogenous Nodal transcripts was quantified and normalized against two different housekeeping control genes, YWHAZ and GAPDH, in quadruplicate reactions (n = 4 for each primer pair). Both Akd−/− ES cell lines show reduced levels of endogenous Nodal, a known target gene of P-Smad2/FoxHI compared to wild-type.(C) Percentage change in Nodal/TGF-β signaling as quantified by Pitx2-luc assays upon transient transfection of GFP or GAkd plasmids in three wild-type and three Akd−/− ES cell lines. GAkd expression doubles Pitx2-luc reporter activity in Akd−/− ES cells and the enhancement is 50% higher than the average level (Av) of wild-type ES cells (horizontal line). The values are normalized against GFP-transfected Akd−/− cell line 3, which has the lowest signaling.(D) Percent (%) increase in luciferase activity generated by the transiently transfected Smad3 specific 9xCAGA-luc reporter in the presence of GFP- or GAkd-expressing plasmids in Akd−/− and wild-type ES cells showing that Arkadia super-activates Smad3-dependent transcription. The enhancement is 100% higher than that in wild-type ES cells (horizontal line). The luciferase values are expressed relative to that of Akd−/− cells transfected with GFP taken as 0%.(E) Super-activation is abolished when mutant Arkadias, GAkdR*, and GAkdNRG* are transfected, indicating that the enhancement requires Arkadia ubiquitin ligase activity and an interaction with P-Smad2/3.(F) Time course of super-activation shows that it is an early but transient phenomenon as quantified by the Pitx2-luc reporter. Relative luciferase activity of Akd−/− and wild-type ES cells are expressed as percent increase over the GFP control and compared at time points (h, hours) taken after transfection of GAkd or GFP. WT, wild-type.
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pbio-0050067-g005: Arkadia Is Required for Efficient P-Smad2/3 Target Gene Transcription and Super-Activates Signaling in Akd−/− ES Cells(A) Relative luciferase activity from three different wild-type and three different Akd−/− ES cell lines transfected with the Pitx2-luc reporter. All values are expressed as a percentage relative to wild-type cell line 3 (3) represented as 0%. Akd−/− ES cell lines have lower activity compared to wild-type, indicative of reduced target gene expression. Average differences (Av) between wild-type and Akd−/− ES cells are shown.(B) Nodal real-time PCR performed on two different wild-type and Akd−/− ES cell lines (1 and 2). The abundance of endogenous Nodal transcripts was quantified and normalized against two different housekeeping control genes, YWHAZ and GAPDH, in quadruplicate reactions (n = 4 for each primer pair). Both Akd−/− ES cell lines show reduced levels of endogenous Nodal, a known target gene of P-Smad2/FoxHI compared to wild-type.(C) Percentage change in Nodal/TGF-β signaling as quantified by Pitx2-luc assays upon transient transfection of GFP or GAkd plasmids in three wild-type and three Akd−/− ES cell lines. GAkd expression doubles Pitx2-luc reporter activity in Akd−/− ES cells and the enhancement is 50% higher than the average level (Av) of wild-type ES cells (horizontal line). The values are normalized against GFP-transfected Akd−/− cell line 3, which has the lowest signaling.(D) Percent (%) increase in luciferase activity generated by the transiently transfected Smad3 specific 9xCAGA-luc reporter in the presence of GFP- or GAkd-expressing plasmids in Akd−/− and wild-type ES cells showing that Arkadia super-activates Smad3-dependent transcription. The enhancement is 100% higher than that in wild-type ES cells (horizontal line). The luciferase values are expressed relative to that of Akd−/− cells transfected with GFP taken as 0%.(E) Super-activation is abolished when mutant Arkadias, GAkdR*, and GAkdNRG* are transfected, indicating that the enhancement requires Arkadia ubiquitin ligase activity and an interaction with P-Smad2/3.(F) Time course of super-activation shows that it is an early but transient phenomenon as quantified by the Pitx2-luc reporter. Relative luciferase activity of Akd−/− and wild-type ES cells are expressed as percent increase over the GFP control and compared at time points (h, hours) taken after transfection of GAkd or GFP. WT, wild-type.

Mentions: We showed above that loss of Arkadia leads to the stabilization and nuclear accumulation of P-Smad2/3. But do these higher levels correspond to an increase in target gene transcription? Analysis of Arkadia−/− and compound Arkadia−/−, Nodal+/− embryos showed that Nodal signaling is defective (Figure 1 and [39,40]) suggesting that Smad2/3 target gene transcription is compromised. We examined the transcriptional activity of P-Smad2/3 in three Arkadia−/− and three wild-type ES cell lines. To estimate the relative levels of P-Smad2/3 transcriptional activity, we used two different target gene luciferase reporters, 0.9-P1, (hereafter termed Pitx2-luc) regulated by P-Smad2/3 and its partner factor FoxH1, and 9xCAGA–luc, a Smad3 specific reporter [52,53]. Although the Arkadia−/− ES cell lines always have a higher amount of P-Smad2 protein compared to wild-type, they have on average 30% (Figure 5A) lower luciferase from the wild-type cell line with the lowest activity (WT 3 designated as reference = zero). Stimulation with Activin did not change significantly the luciferase reporter expression (unpublished data); indicating that under standard culture conditions ES cells exhibit ligand-saturated signaling (autocrine signaling). In addition, real-time PCR showed that the expression of the endogenous Nodal gene, which like the Pitx2-luc luciferase reporter is regulated by FoxH1/Smad2/3 binding sites (known as ASE) [12], is reduced by about 70% in Arkadia−/− ES cells (Figure 5B). Together, these observations suggest that Arkadia is necessary for efficient target gene expression and suggest that in its absence, the stable and high levels of P-Smad2/3 are hypoactive or prevented from activating their target genes. Therefore, although Arkadia degrades P-Smad2/3, it is necessary for efficient P-Smad2/3 transcriptional activity.


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)

Arkadia Is Required for Efficient P-Smad2/3 Target Gene Transcription and Super-Activates Signaling in Akd−/− ES Cells(A) Relative luciferase activity from three different wild-type and three different Akd−/− ES cell lines transfected with the Pitx2-luc reporter. All values are expressed as a percentage relative to wild-type cell line 3 (3) represented as 0%. Akd−/− ES cell lines have lower activity compared to wild-type, indicative of reduced target gene expression. Average differences (Av) between wild-type and Akd−/− ES cells are shown.(B) Nodal real-time PCR performed on two different wild-type and Akd−/− ES cell lines (1 and 2). The abundance of endogenous Nodal transcripts was quantified and normalized against two different housekeeping control genes, YWHAZ and GAPDH, in quadruplicate reactions (n = 4 for each primer pair). Both Akd−/− ES cell lines show reduced levels of endogenous Nodal, a known target gene of P-Smad2/FoxHI compared to wild-type.(C) Percentage change in Nodal/TGF-β signaling as quantified by Pitx2-luc assays upon transient transfection of GFP or GAkd plasmids in three wild-type and three Akd−/− ES cell lines. GAkd expression doubles Pitx2-luc reporter activity in Akd−/− ES cells and the enhancement is 50% higher than the average level (Av) of wild-type ES cells (horizontal line). The values are normalized against GFP-transfected Akd−/− cell line 3, which has the lowest signaling.(D) Percent (%) increase in luciferase activity generated by the transiently transfected Smad3 specific 9xCAGA-luc reporter in the presence of GFP- or GAkd-expressing plasmids in Akd−/− and wild-type ES cells showing that Arkadia super-activates Smad3-dependent transcription. The enhancement is 100% higher than that in wild-type ES cells (horizontal line). The luciferase values are expressed relative to that of Akd−/− cells transfected with GFP taken as 0%.(E) Super-activation is abolished when mutant Arkadias, GAkdR*, and GAkdNRG* are transfected, indicating that the enhancement requires Arkadia ubiquitin ligase activity and an interaction with P-Smad2/3.(F) Time course of super-activation shows that it is an early but transient phenomenon as quantified by the Pitx2-luc reporter. Relative luciferase activity of Akd−/− and wild-type ES cells are expressed as percent increase over the GFP control and compared at time points (h, hours) taken after transfection of GAkd or GFP. WT, wild-type.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1808117&req=5

pbio-0050067-g005: Arkadia Is Required for Efficient P-Smad2/3 Target Gene Transcription and Super-Activates Signaling in Akd−/− ES Cells(A) Relative luciferase activity from three different wild-type and three different Akd−/− ES cell lines transfected with the Pitx2-luc reporter. All values are expressed as a percentage relative to wild-type cell line 3 (3) represented as 0%. Akd−/− ES cell lines have lower activity compared to wild-type, indicative of reduced target gene expression. Average differences (Av) between wild-type and Akd−/− ES cells are shown.(B) Nodal real-time PCR performed on two different wild-type and Akd−/− ES cell lines (1 and 2). The abundance of endogenous Nodal transcripts was quantified and normalized against two different housekeeping control genes, YWHAZ and GAPDH, in quadruplicate reactions (n = 4 for each primer pair). Both Akd−/− ES cell lines show reduced levels of endogenous Nodal, a known target gene of P-Smad2/FoxHI compared to wild-type.(C) Percentage change in Nodal/TGF-β signaling as quantified by Pitx2-luc assays upon transient transfection of GFP or GAkd plasmids in three wild-type and three Akd−/− ES cell lines. GAkd expression doubles Pitx2-luc reporter activity in Akd−/− ES cells and the enhancement is 50% higher than the average level (Av) of wild-type ES cells (horizontal line). The values are normalized against GFP-transfected Akd−/− cell line 3, which has the lowest signaling.(D) Percent (%) increase in luciferase activity generated by the transiently transfected Smad3 specific 9xCAGA-luc reporter in the presence of GFP- or GAkd-expressing plasmids in Akd−/− and wild-type ES cells showing that Arkadia super-activates Smad3-dependent transcription. The enhancement is 100% higher than that in wild-type ES cells (horizontal line). The luciferase values are expressed relative to that of Akd−/− cells transfected with GFP taken as 0%.(E) Super-activation is abolished when mutant Arkadias, GAkdR*, and GAkdNRG* are transfected, indicating that the enhancement requires Arkadia ubiquitin ligase activity and an interaction with P-Smad2/3.(F) Time course of super-activation shows that it is an early but transient phenomenon as quantified by the Pitx2-luc reporter. Relative luciferase activity of Akd−/− and wild-type ES cells are expressed as percent increase over the GFP control and compared at time points (h, hours) taken after transfection of GAkd or GFP. WT, wild-type.
Mentions: We showed above that loss of Arkadia leads to the stabilization and nuclear accumulation of P-Smad2/3. But do these higher levels correspond to an increase in target gene transcription? Analysis of Arkadia−/− and compound Arkadia−/−, Nodal+/− embryos showed that Nodal signaling is defective (Figure 1 and [39,40]) suggesting that Smad2/3 target gene transcription is compromised. We examined the transcriptional activity of P-Smad2/3 in three Arkadia−/− and three wild-type ES cell lines. To estimate the relative levels of P-Smad2/3 transcriptional activity, we used two different target gene luciferase reporters, 0.9-P1, (hereafter termed Pitx2-luc) regulated by P-Smad2/3 and its partner factor FoxH1, and 9xCAGA–luc, a Smad3 specific reporter [52,53]. Although the Arkadia−/− ES cell lines always have a higher amount of P-Smad2 protein compared to wild-type, they have on average 30% (Figure 5A) lower luciferase from the wild-type cell line with the lowest activity (WT 3 designated as reference = zero). Stimulation with Activin did not change significantly the luciferase reporter expression (unpublished data); indicating that under standard culture conditions ES cells exhibit ligand-saturated signaling (autocrine signaling). In addition, real-time PCR showed that the expression of the endogenous Nodal gene, which like the Pitx2-luc luciferase reporter is regulated by FoxH1/Smad2/3 binding sites (known as ASE) [12], is reduced by about 70% in Arkadia−/− ES cells (Figure 5B). Together, these observations suggest that Arkadia is necessary for efficient target gene expression and suggest that in its absence, the stable and high levels of P-Smad2/3 are hypoactive or prevented from activating their target genes. Therefore, although Arkadia degrades P-Smad2/3, it is necessary for efficient P-Smad2/3 transcriptional activity.

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