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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 Interacts Directly with P-Smad2/3 and This Requires the NRG Domain(A and B) IP with anti-Flag antibody from 293T cells stably expressing Flag-tagged Arkadia (Flag-Akd). Cells were cultured with Activin A (A) or SB and Western blotted with anti-P-Smad2 (A) or anti-P-Smad3 antibody (B). Note that Flag-Akd coIPs endogenous P-Smad2 (A) and P-Smad3 (B) in Activin and not in the SB-treated sample. Western blotting of IPs with anti-Smad2 antibody reveals this interaction to be specific to the phosphorylated form of Smad2 (A). The band in the total tissue cell lysates (TCL) and supernatants (SN) in SB-treated samples may correspond to P-Smad1 that does not coIP with Flag-Arkadia (B). Compare the intensity of the band in the TCL and SN to see the depletion of P-Smad3 after IP.(C) Autoradiograph showing in vitro transcribed/translated 35S-labeled full-length Arkadia (Akd), N-terminal portion (N-Akd), and luciferase (Luc) protein input. Flag-P-Smad2 (Figure S3B) attached to agarose beads was able to pull down specifically Akd and not N-Akd or Luc.(D) Map of Arkadia deletion constructs fused in frame with GFP on the N-terminus except for N-Akd. The numbers correspond to amino acid positions. NRG domain, dark gray; NLS, light gray; RING, black (see the amino acid sequence in Figure S3C). GAkdNRG* contains a partial internal deletion of the NRG domain (aa Δ 889–895).(E and F) IP with anti-GFP antibody from 293T cells transfected with GFP or GFP-tagged Arkadia constructs as indicated, along with Alk4* and either Flag-Smad2 (E) or myc-Smad3 (F) and Western blotted (WB) with antibodies as indicated. P-Smad2 protein is shown to coIP with the G-NRG-RING and not with other shorter forms missing the NRG domain. P-Smad3 protein is shown to coIP with full-length Arkadia (GAkd) and a RING domain deletion (GAkdR*). Note the presence of ladder and smear in GFP-tagged Arkadias including the G-NRG-RING, indicating that these proteins are unstable and heavily modified (including poly-ubiquitin chains; unpublished data).
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pbio-0050067-g003: Arkadia Interacts Directly with P-Smad2/3 and This Requires the NRG Domain(A and B) IP with anti-Flag antibody from 293T cells stably expressing Flag-tagged Arkadia (Flag-Akd). Cells were cultured with Activin A (A) or SB and Western blotted with anti-P-Smad2 (A) or anti-P-Smad3 antibody (B). Note that Flag-Akd coIPs endogenous P-Smad2 (A) and P-Smad3 (B) in Activin and not in the SB-treated sample. Western blotting of IPs with anti-Smad2 antibody reveals this interaction to be specific to the phosphorylated form of Smad2 (A). The band in the total tissue cell lysates (TCL) and supernatants (SN) in SB-treated samples may correspond to P-Smad1 that does not coIP with Flag-Arkadia (B). Compare the intensity of the band in the TCL and SN to see the depletion of P-Smad3 after IP.(C) Autoradiograph showing in vitro transcribed/translated 35S-labeled full-length Arkadia (Akd), N-terminal portion (N-Akd), and luciferase (Luc) protein input. Flag-P-Smad2 (Figure S3B) attached to agarose beads was able to pull down specifically Akd and not N-Akd or Luc.(D) Map of Arkadia deletion constructs fused in frame with GFP on the N-terminus except for N-Akd. The numbers correspond to amino acid positions. NRG domain, dark gray; NLS, light gray; RING, black (see the amino acid sequence in Figure S3C). GAkdNRG* contains a partial internal deletion of the NRG domain (aa Δ 889–895).(E and F) IP with anti-GFP antibody from 293T cells transfected with GFP or GFP-tagged Arkadia constructs as indicated, along with Alk4* and either Flag-Smad2 (E) or myc-Smad3 (F) and Western blotted (WB) with antibodies as indicated. P-Smad2 protein is shown to coIP with the G-NRG-RING and not with other shorter forms missing the NRG domain. P-Smad3 protein is shown to coIP with full-length Arkadia (GAkd) and a RING domain deletion (GAkdR*). Note the presence of ladder and smear in GFP-tagged Arkadias including the G-NRG-RING, indicating that these proteins are unstable and heavily modified (including poly-ubiquitin chains; unpublished data).

Mentions: Arkadia is an E3 ubiquitin ligase and could be destabilizing P-Smad2/3 directly by poly-ubiquitinating them, leading to their proteasome-dependent degradation. To test this hypothesis, we examined whether Arkadia interacts specifically with phosphorylated Smads. We used HEK293T (293T) cells stably expressing moderate levels of full-length Arkadia, tagged either with Flag on the N-terminus and Myc on the C-terminus, or with green fluorescent protein (GFP) fused to the N-terminus (GAkd). We performed immunoprecipitation (IP) with anti-Flag (Figure 3A and 3B), -Myc, or -GFP (unpublished data) antibodies and Western blotted with anti-P-Smad2, -Smad2 (Figure 3A), or -P-Smad3 (Figure 3B). The results show that with Activin stimulation, Arkadia coIPs with the phosphorylated endogenous Smad2 (Figure 3A) and Smad3 (Figure 3B). However, when the cells are treated with the SB receptor inhibitor, which eliminates Smad2/3 phosphorylation, Arkadia does not coIP unphosphorylated Smad2/3 (Figure 3A and unpublished data). Furthermore, we examined the interaction of Arkadia with other phosphorylated Smads (Smad1/5/8; Figure S3A) or with Smad4 (unpublished data) and found no evidence of interaction. We therefore conclude that Arkadia interacts specifically with the phosphorylated forms of Smad2/3.


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 Interacts Directly with P-Smad2/3 and This Requires the NRG Domain(A and B) IP with anti-Flag antibody from 293T cells stably expressing Flag-tagged Arkadia (Flag-Akd). Cells were cultured with Activin A (A) or SB and Western blotted with anti-P-Smad2 (A) or anti-P-Smad3 antibody (B). Note that Flag-Akd coIPs endogenous P-Smad2 (A) and P-Smad3 (B) in Activin and not in the SB-treated sample. Western blotting of IPs with anti-Smad2 antibody reveals this interaction to be specific to the phosphorylated form of Smad2 (A). The band in the total tissue cell lysates (TCL) and supernatants (SN) in SB-treated samples may correspond to P-Smad1 that does not coIP with Flag-Arkadia (B). Compare the intensity of the band in the TCL and SN to see the depletion of P-Smad3 after IP.(C) Autoradiograph showing in vitro transcribed/translated 35S-labeled full-length Arkadia (Akd), N-terminal portion (N-Akd), and luciferase (Luc) protein input. Flag-P-Smad2 (Figure S3B) attached to agarose beads was able to pull down specifically Akd and not N-Akd or Luc.(D) Map of Arkadia deletion constructs fused in frame with GFP on the N-terminus except for N-Akd. The numbers correspond to amino acid positions. NRG domain, dark gray; NLS, light gray; RING, black (see the amino acid sequence in Figure S3C). GAkdNRG* contains a partial internal deletion of the NRG domain (aa Δ 889–895).(E and F) IP with anti-GFP antibody from 293T cells transfected with GFP or GFP-tagged Arkadia constructs as indicated, along with Alk4* and either Flag-Smad2 (E) or myc-Smad3 (F) and Western blotted (WB) with antibodies as indicated. P-Smad2 protein is shown to coIP with the G-NRG-RING and not with other shorter forms missing the NRG domain. P-Smad3 protein is shown to coIP with full-length Arkadia (GAkd) and a RING domain deletion (GAkdR*). Note the presence of ladder and smear in GFP-tagged Arkadias including the G-NRG-RING, indicating that these proteins are unstable and heavily modified (including poly-ubiquitin chains; unpublished data).
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Related In: Results  -  Collection

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

pbio-0050067-g003: Arkadia Interacts Directly with P-Smad2/3 and This Requires the NRG Domain(A and B) IP with anti-Flag antibody from 293T cells stably expressing Flag-tagged Arkadia (Flag-Akd). Cells were cultured with Activin A (A) or SB and Western blotted with anti-P-Smad2 (A) or anti-P-Smad3 antibody (B). Note that Flag-Akd coIPs endogenous P-Smad2 (A) and P-Smad3 (B) in Activin and not in the SB-treated sample. Western blotting of IPs with anti-Smad2 antibody reveals this interaction to be specific to the phosphorylated form of Smad2 (A). The band in the total tissue cell lysates (TCL) and supernatants (SN) in SB-treated samples may correspond to P-Smad1 that does not coIP with Flag-Arkadia (B). Compare the intensity of the band in the TCL and SN to see the depletion of P-Smad3 after IP.(C) Autoradiograph showing in vitro transcribed/translated 35S-labeled full-length Arkadia (Akd), N-terminal portion (N-Akd), and luciferase (Luc) protein input. Flag-P-Smad2 (Figure S3B) attached to agarose beads was able to pull down specifically Akd and not N-Akd or Luc.(D) Map of Arkadia deletion constructs fused in frame with GFP on the N-terminus except for N-Akd. The numbers correspond to amino acid positions. NRG domain, dark gray; NLS, light gray; RING, black (see the amino acid sequence in Figure S3C). GAkdNRG* contains a partial internal deletion of the NRG domain (aa Δ 889–895).(E and F) IP with anti-GFP antibody from 293T cells transfected with GFP or GFP-tagged Arkadia constructs as indicated, along with Alk4* and either Flag-Smad2 (E) or myc-Smad3 (F) and Western blotted (WB) with antibodies as indicated. P-Smad2 protein is shown to coIP with the G-NRG-RING and not with other shorter forms missing the NRG domain. P-Smad3 protein is shown to coIP with full-length Arkadia (GAkd) and a RING domain deletion (GAkdR*). Note the presence of ladder and smear in GFP-tagged Arkadias including the G-NRG-RING, indicating that these proteins are unstable and heavily modified (including poly-ubiquitin chains; unpublished data).
Mentions: Arkadia is an E3 ubiquitin ligase and could be destabilizing P-Smad2/3 directly by poly-ubiquitinating them, leading to their proteasome-dependent degradation. To test this hypothesis, we examined whether Arkadia interacts specifically with phosphorylated Smads. We used HEK293T (293T) cells stably expressing moderate levels of full-length Arkadia, tagged either with Flag on the N-terminus and Myc on the C-terminus, or with green fluorescent protein (GFP) fused to the N-terminus (GAkd). We performed immunoprecipitation (IP) with anti-Flag (Figure 3A and 3B), -Myc, or -GFP (unpublished data) antibodies and Western blotted with anti-P-Smad2, -Smad2 (Figure 3A), or -P-Smad3 (Figure 3B). The results show that with Activin stimulation, Arkadia coIPs with the phosphorylated endogenous Smad2 (Figure 3A) and Smad3 (Figure 3B). However, when the cells are treated with the SB receptor inhibitor, which eliminates Smad2/3 phosphorylation, Arkadia does not coIP unphosphorylated Smad2/3 (Figure 3A and unpublished data). Furthermore, we examined the interaction of Arkadia with other phosphorylated Smads (Smad1/5/8; Figure S3A) or with Smad4 (unpublished data) and found no evidence of interaction. We therefore conclude that Arkadia interacts specifically with the phosphorylated forms of Smad2/3.

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