Xenbase Image ID: 135811

Figure 4.  Smad6 is a phosphoprotein which binds to type I receptors nonspecifically and inhibits downstream of BMP receptors. (A) Smad6 nonspecifically interacts with type I receptors of the TGFβ superfamily. COS cells were transfected withFlag-tagged Smad6 and HA-tagged wild-type or constitutively active type I receptor (QD and TD) for BMPs (BMPR-IB), TGFβ (TβR-I), or activins (ActR–IB). Cell lysates were subjected to anti-Flag immunoprecipitation using a monoclonal antibody followed by immunoblotting using anti-HA polyclonal antibody (toppanel). Immunoprecipitates of cells transfected with receptor alone did not contain specific proteins (data not shown). Similar levels of Smad6 and receptor expression were confirmed by analyzing aliquots of total cell lysate by SDS-PAGE followed by immunoblotting (middle and bottom panels). (B) The level of phosphorylation of Smad6 is unchanged by BMP2 or TGFβ stimulation. R-1B/L17 cells were transiently transfected with an empty vector (pCMV5) or with theFlag-tagged Smads indicated at the bottom. Smad2- or Smad6-transfected cells (lanes 2–5) were cotransfected with TβR-I; Smad1- or Smad6-transfected cells (lanes 6–9) were cotransfected with BMPR-IB and BMPR-II. Cells were labeled with [32P]phosphate, stimulated with (+) or without (−) 100 pM TGFβ or 5 nM BMP2 for 20 min.Flag-tagged Smads were purified by immunoprecipitation with anti-Flag M2 antibody and analyzed by SDS-PAGE and autoradiography. (C) Smad6 inhibits receptor-independent Smad1 signaling. Expression of dominant-negative BMP type I receptor (tBMPR-IA, 1 ng of RNA) induces neural tissue (NRP-1 marker) in Xenopus animal caps. Smad1 (1 ng of RNA) prevents neuralization when coexpressed with tBMPR-IA, but its activity is inhibited by Smad6 and Smad6(C) (2 ng of RNA).EF1α is the loading control. Image published in: Hata A et al. (1998) Copyright © 1998. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article. Larger Image Printer Friendly View

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