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of Smad2 (activin/TGF ). Moreover, calmodulin prevents ERK2-mediated phosphorylation of Smads 1 and 2 and, conversely, phosphorylation by Erk2 inhibits the binding of calmodulin.88

Smurf-mediated Smad degradation

Interaction of receptor-regulated Smads with the E3-ubiquitin ligases Smurfs1 and 2 leads to their degradation by the proteasome (see page 472). Smurf1 binds Smads 1 and 5, whereas Smurf2 has broad specificity. Matters are

more complicated for Smad3 which first binds the transcription factor SnoN, priming it for ubiquitylation by Smurf2.89

Holding the TGF pathway in check

Inhibitory Smads

Smads 6 and 7 lack the C-terminal SxS phosphorylation site and have MH1 domains that are only distantly related to those of the other Smads (see Figure 20.8). In the absence of TGF , they are retained in the nucleus. Both their expression and then their translocation into the cytoplasm are induced by TGF , activin, and BMP. Here, they act as inhibitors (Figure 20.16a).90,91

In the cytoplasm, Smad6 forms complexes with phosphorylated Smad1, thereby preventing its interaction with Smad4 and in this way, inhibiting BMP signalling.92 For Smad7, the mechanism is much more elaborate. It is bound to Smurf2 and, for reasons not fully understood, is protected against ubiquitylation in the nucleus. On addition of TGF , the Smad–Smurf complex moves into the cytosol to associate with the membrane through the C2 domain in Smurf2. Here, Smad7 binds to the receptor T R-I. Ubiquitylation ensues and the complex is recognized by the proteasome for partial degradation (Figure 20.16a).93 The expression of Smad7 is also induced by inflammatory mediators (such as TNF- , IFN- , or IL-1 ) via the intermediate of NF- B/RelA and IRF3 transcription factors. This may be one mechanism by which these mediators lift the generally immunosuppressive influence of TGF .7,94

It seems that Smad7 originated as a transcription factor and then evolved to become a structural component of the Smurf ubiquitylation complex. Here it acts as a substrate receptor and also enables the association of the E2-ubiquitin conjugation protein (UbcH7) (Figure 20.16b). In view of this, it is possible that other Smad-interacting proteins are targets of ubiquitin-

mediated destruction. The finding that Smad7/Smurf2 also binds -catenin, causing its degradation, provides evidence for yet another mechanism of pathway cross-talk.95

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Fig 20.16  Induction of inhibitory Smad proteins.

(a) Transcripts induced by members of the TGF family, include those coding for Smads 6 and 7. The newly expressed Smad6 associates with phosphoSmad1, preventing the formation of functional Smad1/Smad4 complexes (1). BMP also causes the nuclear export of existing Smad6 (2) leading to the formation of the same non-functional complexes. The newly synthesized Smad7 protein (3) associates with Smurf2 and then with the type I receptor (4). It induces degradation of the Smad–receptor complex. TGF or activin also causes nuclear export of existing Smad7–Smurf complexes (5). Expression of Smad7 is also induced by inflammatory mediators such as TNF- , IFN- , or IL-1 and these may act to attenuate the TGF response (6). (b) Smad7 is an essential component of the Smurf2 ubiquitylation complex. It brings together Smurf2 (E3) and the E2-conjugating protein UbcH7, from which ubiquitin is transferred to the HECT domain of Smurf2 and then on to its substrate. Smad7 thus acts as a receptor, bringing Smurf2 and UbcH7 to the relevant substrates. This is not restricted to the type I TGF receptors, other proteins interacting with Smad7 are potential targets.

BAMBI, a signal inhibitory pseudo receptor

BMP, activin, and TGF induce the expression of BAMBI. Its expression correlates inversely with the metastatic potential of human melanoma cell lines. 96 It is a truncated type I receptor (closely resembling BMP-RI) that binds ligand but lacks GS and serine/threonine kinase domains. However, the cytosolic segment binds T RI receptors and, in so doing, prevents phosphorylation by type II receptors. The effect is reduced signalling output from TGF -receptors (see Figure 20.6e).30 It thus acts as a pseudo-receptor.

Smad phosphatases

A steady-state level of phosphorylation of Smads2 and 3 is achieved in cells within 15–30 min of adding TGF , and this can last for several hours. Addition of a receptor kinase inhibitor, to interrupt the T R-I signal, then allows rapid (30 min) dephosphorylation.49

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Fig 20.17  Signal attenuation by nuclear phosphatase PPM1A.

Nuclear Smad transcription complexes, dephosphorylated by PPM1A in the nucleus, disaggregate and transfer as monomers to the cytoplasm. Under conditions of low receptor activity, they remain here for some time and risk recognition by Smurf ubiquitin ligases. With high receptor activity, they are immediately rephosphorylated and return to the nucleus as hetero-oligomeric transcription factor complexes.

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Fig 20.18  Structure of PPM1A. R33 is a key residue for coordination of the phosphate. (1a6q97).

Finally, SCPs 1, 2, and 3 dephosphorylate the linker region of Smads 2 and 3 and the linker and the SxS motif of Smad1.100

TGF : tumour suppressor and metastatic promoter?

Given the importance of TGF in suppressing cell proliferation, terminal differentiation of haematopoietic cells, and the activation of cell death mechanisms, it is perhaps not surprising that mutations in the components of the signal transduction pathway can increase susceptibility to transformation. Together with other mutations that favour proliferation, these may ultimately result in the formation of tumours. The common mediator Smad4 was initially identified as one of the mutated or deleted genes linked to pancreatic, colorectal or other carcinomas.102 Most of these mutations are present in

the MH2 domain, which contains the SxS phosphorylation site. They act to prevent complex formation with other Smad proteins, decrease the stability of the protein, or prevent its interaction with non-Smad transcriptional partners.

The importance of Smad4 in colon cancer has been extensively studied in mice. Those that lack a single allele develop gastric/intestinal tumours within 1 year. These reveal a slow but continuous progression from initial

hyperplastic lesions to more advanced stages with clear dysplasia, similar to human colorectal cancer.103 In more advanced stages, the cells also lose the second (functional) Smad4 allele.104 Mice lacking one allele of both APC/C and Smad4 develop tumours earlier and they die earlier, suggesting that TGF - signalling is a component of APC/C-driven progression towards malignancy.

There are also hereditary and somatic forms of colorectal cancer in which the T R-II receptor is mutated, so that the cells lack their normal growthinhibitory signalling mechanism. Paradoxically, properly functional Smad4 is also required for TGF -induced epithelial–mesenchymal transition (EMT), a process generally considered to be tumorigenic. In breast cancer, this

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transition is linked with metastasis.105 Pancreatic tumour cells that are still sensitive to TGF -mediated growth inhibition become highly metastatic with over-expression of the T R-I receptor.106 The general view is that TGF acts as a tumour suppressor by holding proliferation in check, thus reducing the chance of transforming mutations (fewer replication cycles, fewer errors). On

the other hand, for cells already partly transformed (resistant to apoptosis and having reduced requirement of growth factors for proliferation), TGF may act as a metastasis promoter due to its capacity to induce the EMT.107

High levels of TGF also enable transformed cells to escape immune surveillance. There is much evidence for a role of the immune system in the elimination of cells in their early stages of transformation, and mice that lack essential components of the innate or adaptive immune system are prone to develop tumours.108 Transformed cells are recognized by cytotoxic T lymphocytes (CTL) (and other immune cells) and by tumour-specific

antibodies, leading to complement or NK-cell-dependent lysis. Activated CTLs recognize antigens on the tumour cell membrane so that they merge to form an immune synapse in which the CTLs release perforin and granzymes (serine

Fig 20.19  TGF suppresses immune surveillance of transformed cells.

Cytotoxic T lymphocytes (CTLs) recognize transformed cell antigens presented by MHC class I, through their T cell receptors and CD8 molecules. Immune synapses are formed into which granzymes (serine proteases)

and perforin are secreted. Perforin facilitates the uptake of granzymes into large endocytic vesicles in the target cell. Later the granzymes escape and enter the cell nucleus, where they destroy proteins. The CTLs also present FasL which binds to CD95/Fas (not shown; see page 489). Both the receptor engagement and the action of granzymes lead to target cell death. Released IFNalso has a cytolytic effect on transformed cells. TGF prevents expression of these cytolytic agents and therefore protects the transformed cell.

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Fig 20.20  Genetic programme underlying TGF -mediated EMT.

In the process of EMT, there are 728 genes regulated by TGF . They can be grouped on the basis of the different signalling pathways which operate them and their different phenotypic outcomes. Numerous genes require a contribution from the MEK–ERK pathway. A selection of genes, those that have been discussed in this book, are presented here.

Figure adapted from Zavadil et al.112

polarity and which is bound to the junctional ZO-1 (Figure 20.21, see also page 439).116 T R-II receptors are redistributed to the tight junctions on addition of TGF 1, causing phosphorylation and activation of the T R-I receptors and

phosphorylation of Par-6 which now recruits the E3-ubiquitin ligase Smurf1. This leads to destruction of RhoA and consequent dissolution of the tight junctions. The tissue dissociates, transforming into a mass of motile fibroblast-like cells. Loss of tight junctions also enables the expression of cyclin D, and by returning CDK4 to the nucleus, sets up a programme that initiates the cell cycle.

We conclude this chapter by pointing out that the Smads do not possess the exclusive rights to the relaying of TGF signals into the cell. In addition to the Par-6/RhoA pathway, TGF receptors also recruit PP2A ( -regulatory subunit), STRAP/PDK1, and eIF-2 , all of which affect protein synthesis. They also recruit FT , which affects Ras (and possibly ERK) signalling, and they recruit TAK1, which initiates the p38 and JNK pathway (see Figure 15.9, page 463).74

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Fig 20.21  TGF -mediated dissolution of the tight junction.

(a) T R-I is localized to the tight junction by association with occludin. Here it binds the cell polarity protein Par-6. Assembly of the tight junction relies on RhoA that dictates the configuration of the actin cytoskeleton. Addition of TGF leads to recruitment of T R-II into the tight junction to activate T RII/T RI and Par-6. (b) Phosphorylated Par-6 recruits Smurf1 (and its associated E2-ubiquitin conjugating protein) causing destruction of RhoA with consequent remodelling of the actin cytoskeleton and dissolution of the tight junction (c) Domain architecture of Par 6.

List of abbreviations

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628

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629

Signal Transduction

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630

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Continued

631