Mechanism of Lipid Mediated Regulation of TGFβ Signaling

As a central player in the canonical TGF-beta signaling pathway, Smad2 transmits the activation of TGFβ receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been reported that activation of the TGFβ receptor complex leads to the recruitment of Smad2/3 to the activated signaling complex resulting in phosphorylation and activation of Smad2, the mechanism by which Smad2 is recruited to the activated TGFβ receptor complex 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 and partially overlaps with the reported TGFβ receptor I binding site. Multi-faceted imaging analyses show that PM recruitment of Smad2 is triggered by TGFβ-mediated local enrichment of PI(4,5)P2 near the TGF-beta receptor complex and mediated by coincident interaction of Smad2 with PI(4,5)P2 and TGFβ receptor I. The PI(4,5)P2 binding activity of Smad2 is essential for the TGFβ-stimulated nuclear transport and transcriptional activity of Smad2. Structural comparison of Smad MH2 domains and membrane binding analysis of Smad3 suggest that membrane binding activity may be common among Smad proteins and play an important regulatory role in their function in TGFβ signaling. The second project of mine is on evaluation of cholesterol concentration in the inner leaflet of the plasma membrane (IPM). Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the IPM is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.