Inhibition of TGF-β Signaling at the Nuclear Envelope: Characterization of Interactions Between MAN1, Smad2 and Smad3, and PPM1A

Signaling by transforming growth factor-β (TGF-β) is critical for various developmental processes and culminates in the activation of the transcription factors Smad2 and Smad3. MAN1, an integral protein of the inner nuclear membrane, inhibits TGF-β signaling by binding to Smad2 and Smad3. Depletion of the gene LEMD3 encoding MAN1 leads to developmental anomalies in mice, and heterozygous loss-of-function mutations in LEMD3 in humans cause sclerosing bone dysplasia. We modeled the three-dimensional structure of the MAN1-Smad2 complex from nuclear magnetic resonance and small-angle x-ray scattering data. As predicted by this model, we found that MAN1 competed in vitro and in cells with the transcription factor FAST1 (forkhead activin signal transducer 1) for binding to Smad2. The model further predicted that MAN1 bound to activated Smad2-Smad4 or Smad3-Smad4 complexes, which was confirmed by in vitro experiments; however, in cells, MAN1 bound only to Smad2 and Smad3 and not to the Smad4-containing complexes. Overexpression of MAN1 led to dephosphorylation of Smad2 and Smad3, thus hindering their recognition by Smad4, and MAN1 bound directly in vitro to the phosphatase PPM1A, which catalyzes the dephosphorylation of Smad2/3. These results demonstrate a nuclear envelope-localized mechanism of inactivating TGF-β signaling in which MAN1 competes with transcription factors for binding to Smad2 and Smad3 and facilitates their dephosphorylation by PPM1A.