TβRI Phosphorylation of Smad2 on Ser465 and Ser467 Is Required for Smad2-Smad4 Complex Formation and Signaling*
- Shirin Abdollah‡,
- Marina Macı́as-Silva‡,
- Tomoo Tsukazaki‡,
- Hidetoshi Hayashi‡,
- Liliana Attisano§¶ and
- Jeffrey L. Wrana‡‖**
- From the ‡Program in Developmental Biology and Division of Gastroenterology, The Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1X8 and the ‖Department of Medical Genetics and Microbiology and §Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Abstract
Mothers against Dpp-related or Smad proteins are essential components of serine/threonine kinase receptor signaling pathways that are regulated by phosphorylation. Recently, it was demonstrated that Smad2 interacts transiently with and is a direct substrate of the transforming growth factor-β (TGF-β) type I receptor, TβRI. Phosphorylation sites on Smad2 were localized to a carboxyl-terminal fragment containing three serine residues at positions 464, 465, and 467. In this report, we show that TβRI specifically phosphorylates Smad2 on serines 465 and 467. Serine 464 is not a site of phosphorylation, but is important for efficient phosphorylation of Smad2. Phosphorylation at both sites is required to mediate association of Smad2 with Smad4 in mammalian cells, while in yeast, Smad2 interacts directly with Smad4 and does not require phosphorylation. Mutation of either serine residue 465 or 467 prevents dissociation of Smad2 from activated TβRI and blocks TGF-β-dependent signaling and Smad2 transcriptional activity. These results indicate that receptor-dependent phosphorylation of Smad2 on serines 465 and 467 is required in mammalian cells to permit association with Smad4 and to propagate TGF-β signals.
Footnotes
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↵* This work was supported by grants from the Medical Research Council of Canada (to J. L. W. and L. A.) and the National Cancer Institute of Canada (to J. L. W.) with funds from the Terry Fox Run.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Medical Research Council Scholar.
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↵** Medical Research Council Scholar. To whom correspondence should be addressed: Program in Developmental Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada, M5G 1X8. Tel.: 416-813-5173; Fax: 416-813-6531; E-mail:jwrana{at}sickkids.on.ca.
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↵1 The abbreviations used are: TGF-β, transforming growth factorβ; BMP, bone morphogenetic protein; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis.
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↵2 M. Macı́as-Silva and J. L. Wrana, unpublished data.
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- Received July 23, 1997.
