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The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization

Nature Structural Biology volume 8, pages 248–253 (2001)Cite this article

Abstract

Smad proteins mediate the transforming growth factor β responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.

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Figure 1: Oligomerization states of S4AF, S3LC and S3LC(3E).
Figure 2: Ratio of Smad subunits within the heteromeric complex and the basis of subunit association.
Figure 3: The sulfate binding site within the L3 loop is critical for Smad heteromeric interaction.
Figure 4: Proposed model of phosphorylation induced Smad protein activation.

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Acknowledgements

We thank A. Roberts (NIH), M. Marinus (UMass), and K. Knight (UMass) for helpful suggestions of the manuscript, R. Derynck for the Smad4-Myc construct and the wild type GST fusion Smad3/Smad4 constructs, W. Kruijer for the SBE-Lux construct, S. Davis for technical assistance. This research is funded by the Sidney Kimmel Foundation Scholar Award and the center grant from the Diabetes and Endocrinology Research Center.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, 01655, Massachusetts, USA

    Benoy M. Chacko, Bin Qin, Suvana S. Lam & Kai Lin

  2. Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, 39216, Mississippi, USA

    John J. Correia

  3. Department of Nephrology, Vanderbilt University Medical Center, Nashville, 37232, Tennessee, USA

    Mark P. de Caestecker

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Correspondence to Kai Lin.

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Chacko, B., Qin, B., Correia, J. et al. The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization. Nat Struct Mol Biol 8, 248–253 (2001). https://doi.org/10.1038/84995

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