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Brown TA. Genomes. 2nd edition. Oxford: Wiley-Liss; 2002.

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Genomes. 2nd edition.

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Box 12.1Unraveling a signal transduction pathway

A typical set of experiments for studying the functions of proteins involved in a signal transduction pathway.

One of the most important extracellular signaling compounds is transforming growth factor-β (TGF-β), a family of some 30 related polypeptides that control processes such as cell division and differentiation in vertebrates. The cell surface receptors for TGF-β are serine-threonine kinases (see Table 12.2), which activate a variety of target proteins within the cell. Part of the signal transduction process initiated by TGF-β binding involves a set of proteins called the SMAD family, the name being an abbreviation of ‘SMA/MAD related’, referring to the proteins in Drosophila melanogaster and Caenorhabditis elegans, respectively, which were the original members of the family to be isolated.

SMAD signaling in vertebrates

Initially, five SMADs were discovered in vertebrate cells. Four of these - Smad1, Smad2, Smad3 and Smad5 - are called receptor-regulated SMADs because they associate directly with the cell surface receptor. Each of these SMADs is specific for a different type of serine-threonine receptor and hence responds to different members of the TGF-β family of signaling compounds. Binding of the extracellular signal induces a receptor to phosphorylate its SMAD, which then associates with Smad4, moves to the nucleus and, via interactions with DNA-binding proteins, activates a set of target genes. Smad4 is therefore a co-mediator that participates in the signaling pathway of each of the other four SMADs.

This interpretation of the SMAD pathway was complicated by the discovery of two additional SMADs - numbers 6 and 7 - that do not fit into the scheme. These SMADs lack the amino acid sequence motif serine-serine-X-serine (where X is valine or methionine), present in the C-terminal region of Smad1, 2, 3 and 5, that is phosphorylated by the receptor. Clearly, therefore, Smad6 and Smad7 do not respond directly to attachment of the extracellular signals to the receptor protein. Are they co-mediators similar to Smad4, or do they have some other role in TGF-β signal transduction?

The first step in understanding the functions of Smad6 and Smad7 was to determine the effect of overexpression of these proteins on TGF-β signal transduction. Overexpression was achieved by attaching the Smad6 or Smad7 gene to a strong promoter and then using cloning techniques to introduce the gene into cultured cells. The outcome was that nuclear genes normally switched on by TGF-β became non-responsive to the extracellular signal in cells overexpressing Smad6 or Smad7. This result gave the first indication that Smad6 and Smad7 have inhibitory effects on the TGF-β pathway.

Two models have been proposed to explain how the inhibitory SMADs, as Smad 6 and Smad7 are now called, repress the TGF-β pathway. The first model is based on the observation that in cell extracts the Smad6 and Smad7 proteins are associated with the intracellular parts of the cell surface receptors. The hypothesis is that Smad6 and Smad7 inhibit signal transduction by preventing the activated receptors from phosphorylating the other SMADs.

This model probably explains the inhibitory effects of overexpression of Smad6 and Smad7, but in normal cells there may not be enough copies of these proteins to block the cell surface receptors entirely. An alternative model has therefore been proposed, in which the inhibitory SMADs bind to one or more of the other SMADs, removing these from the pathway and hence stopping signal transduction. There is good evidence that this type of interaction is the explanation for the inhibitory effect that Smad6 has on Smad1. Yeast two-hybrid studies (Section 7.3.2) have shown that these two SMADs interact, and after binding to Smad6, Smad1 is unable to influence the transcriptional activators that it normally stimulates, even after it has been phosphorylated by the cell surface receptors.

Whatever the mechanism for Smad6 and Smad7 activity, the discovery of these inhibitory SMADs shows that TGF-β signaling via the SMAD pathway is more flexible than was originally envisaged. Rather than being an all-or-nothing response, the activity of the receptor-regulated SMADs can be modified by the inhibitory effects of Smad6 and Smad7, these proteins presumably responding to as-yet-unidentified intracellular signals in order to modulate the effects of TGF-β binding in an appropriate way.


  1. Imamura T, Takase M, Nishihara A. et al. Smad6 inhibits signalling by the TGF-β superfamily. Nature. (1998);389:622–626. [PubMed: 9335505]
  2. Whitman M. Feedback from inhibitory SMADs. Nature. (1998);389:549–551. [PubMed: 9335489]


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