Abstract
As a central player in the canonical TGF-β signaling pathway, Smad2 transmits the activation of TGF-β receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-β to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-β receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-β receptor I that transduces TGF-β-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-β receptor complex, leading to its binding to the TGF-β receptor I. The PI(4,5)P2-binding activity of Smad2 is essential for the TGF-β-stimulated phosphorylation, nuclear transport, and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-β-mediated biological processes.
Highlights
This model is widely accepted, there is a major gap in our understanding of the spatiotemporal dynamics of the active transforming growth factor-β (TGF-β) receptor complex and the mechanism by which Smad2 and/or Smad3 are recruited to the complex
These findings suggest that clathrin-dependent internalization of the TGF-β receptors is a crucial step for Smad-mediated TGF-β signaling and that EEs are a main site for Smad-mediated TGF-β signaling, these notions have been challenged by other reports
It has been generally thought that Smad2 and Smad3 are targeted to the activated TGF-β signaling complex by direct interaction with TβRI [1, 2] or via Smad anchor for receptor activation (SARA) [7]
Summary
This model is widely accepted, there is a major gap in our understanding of the spatiotemporal dynamics of the active TGF-β receptor complex and the mechanism by which Smad2 and/or Smad3 are recruited to the complex. To quantitatively assess its membrane affinity, we determined the Kd values for selected vesicles by measuring RU/RUmax as a function of the Smad2 concentration (Fig. 1B).
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