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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 Ubiquitinates P-Smad2 and Mediates Its Degradation via the Proteasome(A and B) IP with anti-Flag antibody from Akd−/− MEFs transiently transfected with various plasmids as indicated, and Western blotted (WB) with anti-P-Smad2, anti-HA, or anti-ubiquitin antibodies. Note the presence of higher molecular weight P-Smad2 corresponding to protein modifications, including ubiquitin chains as shown by the presence of HA-ubiquitin tags (A) or ubiquitin (B). These are present only in cells transfected with full-length Arkadia and not with GFP (A and B) or mutant forms GAkdR* (A) and GAkdNRG* (B), which disrupt the ubiquitin ligase activity and the interaction with P-Smad2, respectively.(C) In vitro poly-ubiquitinated P-Smad2 (bracket) was detected by Western blotting with anti-P-Smad2 antibody. Note the presence of high molecular weight species only in the reactions containing in vitro transcribed/translated full-length (Akd) or the C-Akd (aa 510–989) Arkadia proteins (see WB with anti-ubiquitin in Figure S5A). Retic., Reticulocyte extract; E1 + E2, ubiquitination enzymes; GST-Ub, GST-tagged ubiquitin.(D and E) Nuclear extracts from Akd −/− MEFs transfected with either GAkd, GFP, or GAkdR* plasmids alone (E) or with Flag-Smad2 and Alk4* (D) and treated with either MG132 (+ ; 30 μM) or DMSO (−) for 4 h prior to lysis, and Western blotted with anti-P-Smad2 and either anti-PCNA (D) or anti-Tubulin (E) antibodies for loading controls. P-Smad2 protein levels decrease specifically in cells expressing Arkadia, and this does not occur when the proteasome is inhibited or when the RING domain is mutated (GAkdR*), indicating that the degradation is mediated via the proteasome and the ubiquitin ligase activity of Arkadia. (E) Graphical representation of relative endogenous P-Smad2 levels normalized against the housekeeping gene Tubulin shows approximately a 2.5-fold reduction of P-Smad2 in Arkadia-expressing MEFs in the presence of DMSO when compared to MG132. GFP- and GAkdR*-expressing MEF samples do not show any differences.(F) Western blots (WB) with anti-P-Smad2 and anti-Tubulin antibodies showing the rate of P-Smad2 degradation in wild-type and Akd−/− ES cells in the presence of MG132 or DMSO control. After initial stimulation with Activin (0*), P-Smad2 is rapidly degraded in wild-type ES cells but not in Akd−/−. This degradation in wild-type cells is reduced in the presence of MG132 compared to the DMSO control, indicating that it is mediated via the proteasome. SB, SB431542; WT, wild-type.
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pbio-0050067-g004: Arkadia Ubiquitinates P-Smad2 and Mediates Its Degradation via the Proteasome(A and B) IP with anti-Flag antibody from Akd−/− MEFs transiently transfected with various plasmids as indicated, and Western blotted (WB) with anti-P-Smad2, anti-HA, or anti-ubiquitin antibodies. Note the presence of higher molecular weight P-Smad2 corresponding to protein modifications, including ubiquitin chains as shown by the presence of HA-ubiquitin tags (A) or ubiquitin (B). These are present only in cells transfected with full-length Arkadia and not with GFP (A and B) or mutant forms GAkdR* (A) and GAkdNRG* (B), which disrupt the ubiquitin ligase activity and the interaction with P-Smad2, respectively.(C) In vitro poly-ubiquitinated P-Smad2 (bracket) was detected by Western blotting with anti-P-Smad2 antibody. Note the presence of high molecular weight species only in the reactions containing in vitro transcribed/translated full-length (Akd) or the C-Akd (aa 510–989) Arkadia proteins (see WB with anti-ubiquitin in Figure S5A). Retic., Reticulocyte extract; E1 + E2, ubiquitination enzymes; GST-Ub, GST-tagged ubiquitin.(D and E) Nuclear extracts from Akd −/− MEFs transfected with either GAkd, GFP, or GAkdR* plasmids alone (E) or with Flag-Smad2 and Alk4* (D) and treated with either MG132 (+ ; 30 μM) or DMSO (−) for 4 h prior to lysis, and Western blotted with anti-P-Smad2 and either anti-PCNA (D) or anti-Tubulin (E) antibodies for loading controls. P-Smad2 protein levels decrease specifically in cells expressing Arkadia, and this does not occur when the proteasome is inhibited or when the RING domain is mutated (GAkdR*), indicating that the degradation is mediated via the proteasome and the ubiquitin ligase activity of Arkadia. (E) Graphical representation of relative endogenous P-Smad2 levels normalized against the housekeeping gene Tubulin shows approximately a 2.5-fold reduction of P-Smad2 in Arkadia-expressing MEFs in the presence of DMSO when compared to MG132. GFP- and GAkdR*-expressing MEF samples do not show any differences.(F) Western blots (WB) with anti-P-Smad2 and anti-Tubulin antibodies showing the rate of P-Smad2 degradation in wild-type and Akd−/− ES cells in the presence of MG132 or DMSO control. After initial stimulation with Activin (0*), P-Smad2 is rapidly degraded in wild-type ES cells but not in Akd−/−. This degradation in wild-type cells is reduced in the presence of MG132 compared to the DMSO control, indicating that it is mediated via the proteasome. SB, SB431542; WT, wild-type.

Mentions: As P-Smad2/3 interact with Arkadia, it is possible that they are substrates of Arkadia ubiquitination. To address this, we examined the ubiquitination status of P-Smad2 in the presence or absence of Arkadia expression. We used Arkadia−/− mouse embryonic fibroblasts lines (MEFs) to exclude any endogenous Arkadia activity and introduced Flag-Smad2 and Alk4* to obtain phosphorylated Flag-Smad2, in the presence of full-length or mutant forms of Arkadia. Western blot analysis of the IPs with anti-P-Smad2 antibodies showed the existence of higher molecular weight forms of P-Smad2 associated specifically with the presence of full-length Arkadia (Figures 4A and 4B and S4) suggesting poly-ubiquitination. Probing with ubiquitin antibodies confirmed that these modifications contain ubiquitin chains (Figures 4A and 4B and S4). Furthermore, these blots show that P-Smad2 is not ubiquitinated in the presence of mutant Arkadia proteins lacking either ubiquitin ligase activity (GAkdR*) or the P-Smad2 interaction domain (GAkdNRG*; Figure 4A and 4B). Therefore, Arkadia ubiquitinates P-Smad2 in vivo, and this depends on both its ubiquitin ligase activity and the P-Smad2/3 interaction domain, suggesting that Arkadia ubiquitinates them directly.


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 Ubiquitinates P-Smad2 and Mediates Its Degradation via the Proteasome(A and B) IP with anti-Flag antibody from Akd−/− MEFs transiently transfected with various plasmids as indicated, and Western blotted (WB) with anti-P-Smad2, anti-HA, or anti-ubiquitin antibodies. Note the presence of higher molecular weight P-Smad2 corresponding to protein modifications, including ubiquitin chains as shown by the presence of HA-ubiquitin tags (A) or ubiquitin (B). These are present only in cells transfected with full-length Arkadia and not with GFP (A and B) or mutant forms GAkdR* (A) and GAkdNRG* (B), which disrupt the ubiquitin ligase activity and the interaction with P-Smad2, respectively.(C) In vitro poly-ubiquitinated P-Smad2 (bracket) was detected by Western blotting with anti-P-Smad2 antibody. Note the presence of high molecular weight species only in the reactions containing in vitro transcribed/translated full-length (Akd) or the C-Akd (aa 510–989) Arkadia proteins (see WB with anti-ubiquitin in Figure S5A). Retic., Reticulocyte extract; E1 + E2, ubiquitination enzymes; GST-Ub, GST-tagged ubiquitin.(D and E) Nuclear extracts from Akd −/− MEFs transfected with either GAkd, GFP, or GAkdR* plasmids alone (E) or with Flag-Smad2 and Alk4* (D) and treated with either MG132 (+ ; 30 μM) or DMSO (−) for 4 h prior to lysis, and Western blotted with anti-P-Smad2 and either anti-PCNA (D) or anti-Tubulin (E) antibodies for loading controls. P-Smad2 protein levels decrease specifically in cells expressing Arkadia, and this does not occur when the proteasome is inhibited or when the RING domain is mutated (GAkdR*), indicating that the degradation is mediated via the proteasome and the ubiquitin ligase activity of Arkadia. (E) Graphical representation of relative endogenous P-Smad2 levels normalized against the housekeeping gene Tubulin shows approximately a 2.5-fold reduction of P-Smad2 in Arkadia-expressing MEFs in the presence of DMSO when compared to MG132. GFP- and GAkdR*-expressing MEF samples do not show any differences.(F) Western blots (WB) with anti-P-Smad2 and anti-Tubulin antibodies showing the rate of P-Smad2 degradation in wild-type and Akd−/− ES cells in the presence of MG132 or DMSO control. After initial stimulation with Activin (0*), P-Smad2 is rapidly degraded in wild-type ES cells but not in Akd−/−. This degradation in wild-type cells is reduced in the presence of MG132 compared to the DMSO control, indicating that it is mediated via the proteasome. SB, SB431542; WT, wild-type.
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Related In: Results  -  Collection

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Show All Figures
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pbio-0050067-g004: Arkadia Ubiquitinates P-Smad2 and Mediates Its Degradation via the Proteasome(A and B) IP with anti-Flag antibody from Akd−/− MEFs transiently transfected with various plasmids as indicated, and Western blotted (WB) with anti-P-Smad2, anti-HA, or anti-ubiquitin antibodies. Note the presence of higher molecular weight P-Smad2 corresponding to protein modifications, including ubiquitin chains as shown by the presence of HA-ubiquitin tags (A) or ubiquitin (B). These are present only in cells transfected with full-length Arkadia and not with GFP (A and B) or mutant forms GAkdR* (A) and GAkdNRG* (B), which disrupt the ubiquitin ligase activity and the interaction with P-Smad2, respectively.(C) In vitro poly-ubiquitinated P-Smad2 (bracket) was detected by Western blotting with anti-P-Smad2 antibody. Note the presence of high molecular weight species only in the reactions containing in vitro transcribed/translated full-length (Akd) or the C-Akd (aa 510–989) Arkadia proteins (see WB with anti-ubiquitin in Figure S5A). Retic., Reticulocyte extract; E1 + E2, ubiquitination enzymes; GST-Ub, GST-tagged ubiquitin.(D and E) Nuclear extracts from Akd −/− MEFs transfected with either GAkd, GFP, or GAkdR* plasmids alone (E) or with Flag-Smad2 and Alk4* (D) and treated with either MG132 (+ ; 30 μM) or DMSO (−) for 4 h prior to lysis, and Western blotted with anti-P-Smad2 and either anti-PCNA (D) or anti-Tubulin (E) antibodies for loading controls. P-Smad2 protein levels decrease specifically in cells expressing Arkadia, and this does not occur when the proteasome is inhibited or when the RING domain is mutated (GAkdR*), indicating that the degradation is mediated via the proteasome and the ubiquitin ligase activity of Arkadia. (E) Graphical representation of relative endogenous P-Smad2 levels normalized against the housekeeping gene Tubulin shows approximately a 2.5-fold reduction of P-Smad2 in Arkadia-expressing MEFs in the presence of DMSO when compared to MG132. GFP- and GAkdR*-expressing MEF samples do not show any differences.(F) Western blots (WB) with anti-P-Smad2 and anti-Tubulin antibodies showing the rate of P-Smad2 degradation in wild-type and Akd−/− ES cells in the presence of MG132 or DMSO control. After initial stimulation with Activin (0*), P-Smad2 is rapidly degraded in wild-type ES cells but not in Akd−/−. This degradation in wild-type cells is reduced in the presence of MG132 compared to the DMSO control, indicating that it is mediated via the proteasome. SB, SB431542; WT, wild-type.
Mentions: As P-Smad2/3 interact with Arkadia, it is possible that they are substrates of Arkadia ubiquitination. To address this, we examined the ubiquitination status of P-Smad2 in the presence or absence of Arkadia expression. We used Arkadia−/− mouse embryonic fibroblasts lines (MEFs) to exclude any endogenous Arkadia activity and introduced Flag-Smad2 and Alk4* to obtain phosphorylated Flag-Smad2, in the presence of full-length or mutant forms of Arkadia. Western blot analysis of the IPs with anti-P-Smad2 antibodies showed the existence of higher molecular weight forms of P-Smad2 associated specifically with the presence of full-length Arkadia (Figures 4A and 4B and S4) suggesting poly-ubiquitination. Probing with ubiquitin antibodies confirmed that these modifications contain ubiquitin chains (Figures 4A and 4B and S4). Furthermore, these blots show that P-Smad2 is not ubiquitinated in the presence of mutant Arkadia proteins lacking either ubiquitin ligase activity (GAkdR*) or the P-Smad2 interaction domain (GAkdNRG*; Figure 4A and 4B). Therefore, Arkadia ubiquitinates P-Smad2 in vivo, and this depends on both its ubiquitin ligase activity and the P-Smad2/3 interaction domain, suggesting that Arkadia ubiquitinates them directly.

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