Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex
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Evaluation Summary:
The mechanisms of TGF-beta signaling have been intensively investigated. In this study, the authors reveal a novel mechanism of TGF-beta regulation, which suggests a higher order of signaling complexity. With some stronger experimental support, the paper will be of interest to those studying signal transduction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The hallmark event of the canonical transforming growth factor β (TGFβ) family signaling is the assembly of the Smad-complex, consisting of the common Smad, Smad4, and phosphorylated receptor-regulated Smads. How the Smad-complex is assembled and regulated is still unclear. Here, we report that active Arl15, an Arf-like small G protein, specifically binds to the MH2 domain of Smad4 and colocalizes with Smad4 at the endolysosome. The binding relieves the autoinhibition of Smad4, which is imposed by the intramolecular interaction between its MH1 and MH2 domains. Activated Smad4 subsequently interacts with phosphorylated receptor-regulated Smads, forming the Smad-complex. Our observations suggest that Smad4 functions as an effector and a GTPase activating protein (GAP) of Arl15. Assembly of the Smad-complex enhances the GAP activity of Smad4 toward Arl15, therefore dissociating Arl15 before the nuclear translocation of the Smad-complex. Our data further demonstrate that Arl15 positively regulates the TGFβ family signaling.
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Author Response
Reviewer #2 (Public Review):
The authors provide evidence that the small G protein Arl15 binds to the MH2 domain of Smad4, and propose, primarily on the basis of biochemical experiments that Arl15 controls the assembly of the heteromeric Smad (Smad4:R-Smad) complexes that form in response to TGF-b or BMP. They also propose that the Smad complex enhances the GAP activity of Smad4 toward Arl15. Finally, they propose that Arl15 acts as a global regulator of TGF-b family responses.
The initial observation that Arl15 interacts with the MH2 domain of Smad4 is intriguing and so are some of the biochemical interaction data. However, in the end, the proposed role of Arl15 in Smad complex formation in response to TGF-b or BMP and the proposed scenario are insufficiently supported by in vivo (in cells) data on the extent to …
Author Response
Reviewer #2 (Public Review):
The authors provide evidence that the small G protein Arl15 binds to the MH2 domain of Smad4, and propose, primarily on the basis of biochemical experiments that Arl15 controls the assembly of the heteromeric Smad (Smad4:R-Smad) complexes that form in response to TGF-b or BMP. They also propose that the Smad complex enhances the GAP activity of Smad4 toward Arl15. Finally, they propose that Arl15 acts as a global regulator of TGF-b family responses.
The initial observation that Arl15 interacts with the MH2 domain of Smad4 is intriguing and so are some of the biochemical interaction data. However, in the end, the proposed role of Arl15 in Smad complex formation in response to TGF-b or BMP and the proposed scenario are insufficiently supported by in vivo (in cells) data on the extent to which Arl15 controls the Smad complex formation and its activity. Indeed, experiments that I would intuitively see as the first and key questions to be addressed have not been done (or not been shown). More specifically: (1) Does Arl15 control/enhance the association of endogenous Smad4 with Smad2/3 or Smad1/5 in response to TGF-b or BMP, respectively? (2) Does Arl15 enhance the TGF-b- or BMP-induced nuclear localization of these endogenous complexes? (3) Since Arl15 enhances the direct target gene responses, does Arl15 enhance the TGF-b-induced binding of endogenous Smad complexes to regulatory sequences of target genes?
We would like to thank this reviewer for these insightful comments and constructive suggestions.
(1) Regarding whether "Arl15 controls/enhances the association of endogenous Smad4 with Smad2/3 or Smad1/5 in response to TGF-b or BMP, respectively", we found that, upon the depletion of Arl15, TGFβ1-induced interaction between Smad4 and phospho-Smad2/3 decreased substantially (see our response to point 8 of Reviewer #2's comments). Our finding supports our model that Arl15 promotes the assembly of the Smad-complex.
(2) Regarding whether "Arl15 enhances the TGF-b- or BMP-induced nuclear localization of these endogenous complexes", we demonstrated that depletion of Arl15 reduced the TGFβ1-induced nuclear localization of endogenous Smad-complex consisting of Smad4 and phospho-Smad2/3 (see our response to point 9 of Reviewer #2's comments).
(3) However, regarding whether "Arl15 enhances the TGF-b-induced binding of endogenous Smad-complexes to regulatory sequences of target genes", we did not produce experimental evidence since we think that the proposed experiment, e.g., CHIP of the Smad-complex, should not fall within the scope of our study. Please see our response to point 11 of Reviewer #2's comments.
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Evaluation Summary:
The mechanisms of TGF-beta signaling have been intensively investigated. In this study, the authors reveal a novel mechanism of TGF-beta regulation, which suggests a higher order of signaling complexity. With some stronger experimental support, the paper will be of interest to those studying signal transduction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This manuscript uncovers an apparently novel mechanism of TGF-β signaling. The authors identify Arl15 as an interacting partner of Smad4 using a two-hybrid screen. Arl15 binds directly to Smad4 through the MH2 domain on Smad4 when Arl15 is in the GTP-bound. Interestingly, their data suggest that when GTP bound, Arl15 displaces Smad4 from a closed to an open conformation. This leads to increased complex formation with R-Smads and appears to also facilitate Smad4 phosphorylation. In functional experiments the authors demonstrate that the GTPase activity of Arl15 promotes TGF-β-mediated activation of its transcriptional targets. Finally, the authors identify mutants of Arl15 in cancer patients that disrupted the interaction between Arl15 and Smad4. The authors put forth an intriguing model for how Arl15 through …
Reviewer #1 (Public Review):
This manuscript uncovers an apparently novel mechanism of TGF-β signaling. The authors identify Arl15 as an interacting partner of Smad4 using a two-hybrid screen. Arl15 binds directly to Smad4 through the MH2 domain on Smad4 when Arl15 is in the GTP-bound. Interestingly, their data suggest that when GTP bound, Arl15 displaces Smad4 from a closed to an open conformation. This leads to increased complex formation with R-Smads and appears to also facilitate Smad4 phosphorylation. In functional experiments the authors demonstrate that the GTPase activity of Arl15 promotes TGF-β-mediated activation of its transcriptional targets. Finally, the authors identify mutants of Arl15 in cancer patients that disrupted the interaction between Arl15 and Smad4. The authors put forth an intriguing model for how Arl15 through activation of its GTP-bound form opens up the closed Smad4 conformation to promote its activation and binding to R-Smads and subsequent stimulation of TGF-β target genes. Overall, this is a well-written manuscript and the data presented are of high quality and support the proposed model. The sub-cellular localization experiments, however, seem to be somewhat over-interpreted and the order in which these experiments are introduced into the paper is distracting. Nevertheless, the major aspects of this manuscript are strong and the results should provide new knowledge into the regulation of TGF-β signaling.
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Reviewer #2 (Public Review):
The authors provide evidence that the small G protein Arl15 binds to the MH2 domain of Smad4, and propose, primarily on the basis of biochemical experiments that Arl15 controls the assembly of the heteromeric Smad (Smad4:R-Smad) complexes that form in response to TGF-b or BMP. They also propose that the Smad complex enhances the GAP activity of Smad4 toward Arl15. Finally, they propose that Arl15 acts as a global regulator of TGF-b family responses.
The initial observation that Arl15 interacts with the MH2 domain of Smad4 is intriguing and so are some of the biochemical interaction data. However, in the end, the proposed role of Arl15 in Smad complex formation in response to TGF-b or BMP and the proposed scenario are insufficiently supported by in vivo (in cells) data on the extent to which Arl15 controls …
Reviewer #2 (Public Review):
The authors provide evidence that the small G protein Arl15 binds to the MH2 domain of Smad4, and propose, primarily on the basis of biochemical experiments that Arl15 controls the assembly of the heteromeric Smad (Smad4:R-Smad) complexes that form in response to TGF-b or BMP. They also propose that the Smad complex enhances the GAP activity of Smad4 toward Arl15. Finally, they propose that Arl15 acts as a global regulator of TGF-b family responses.
The initial observation that Arl15 interacts with the MH2 domain of Smad4 is intriguing and so are some of the biochemical interaction data. However, in the end, the proposed role of Arl15 in Smad complex formation in response to TGF-b or BMP and the proposed scenario are insufficiently supported by in vivo (in cells) data on the extent to which Arl15 controls the Smad complex formation and its activity. Indeed, experiments that I would intuitively see as the first and key questions to be addressed have not been done (or not been shown). More specifically: (1) Does Arl15 control/enhance the association of endogenous Smad4 with Smad2/3 or Smad1/5 in response to TGF-b or BMP, respectively? (2) Does Arl15 enhance the TGF-b- or BMP-induced nuclear localization of these endogenous complexes? (3) Since Arl15 enhances the direct target gene responses, does Arl15 enhance the TGF-b-induced binding of endogenous Smad complexes to regulatory sequences of target genes?
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