The Tumor Suppressor Smad4/DPC4 is Regulated by Phosphorylations that Integrate FGF, Wnt and TGF-beta Signaling

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Dorsal–ventral patterning: a morphogenetic gradient of BMP ligands

The earliest requirement for BMP signaling in an embryo is during the patterning of cell fates along its D–V axis. Formation of a D–V gradient of BMP signals has been evolutionary conserved and is utilized by both vertebrates and invertebrates (De Robertis, 2006; De Robertis, 2008; De Robertis and Sasai, 1996; O’Connor et al., 2006). In vertebrate embryos like Xenopus and zebrafish, BMPs pattern ventral cell fates, while BMP repression determines dorsal cell fate (Figure 2.1); this D–V polarity is reversed in invertebrate embryos such as Drosophila. The gradient of BMP signals subdivides the Xenopus ectoderm from ventral to dorsal into epidermis, neural crest, and central nervous system, while the mesoderm is subdivided into blood island, lateral plate mesoderm (kidney), somite, and notochord. Thus, a ventral gradient of extracellular BMPs regulates the initial tissue-type differentiations of the vertebrate embryo.
The main BMPs involved in D–V patterning in the Xenopus embryo are the ventrally expressed BMP4 and BMP7 and the dorsally expressed BMP2 and ADMP. Depletion of all four BMPs using injected antisense morpholino oligo nucleotides causes this robust morphogenetic field to collapse, resulting in complete neuralization of the developing embryo (Reversade and De Robertis, 2005). This is a spectacular transformation of the embryo, because the entire ectoderm becomes covered by central nervous system (CNS), in particular brain tissue. If any one of the four BMPs is not depleted, the embryo retains some D–V patterning. This indicates that both the dorsal and the ventral poles of the embryo serve as sources of BMP signals. In zebrafish embryos, mutation of bmp2b or bmp7 result in strong dorsalization or neuralization of the embryo (Kishimoto et al., 1997; Nguyen et al., 1997; Schmid et al., 2000), demonstrating that the requirement for BMPs in the specification of ventral fates has been evolutionary conserved (Little and Mullins, 2009).

Intracellular transduction of the BMP signal

BMPs transduce their intracellular signal via BMPR (BMP receptor) activation followed by transcription factor phosphorylation. BMPs first bind to and activate their transmembrane serine/threonine kinase receptors, which in turn phosphorylate the transcription factors Smad1/5/8 at its two C-terminal serines (SVS). Phosphorylated Smad1Cter binds to Smad4 (co-Smad) and translocates and accumulates in the nucleus, activating BMP-responsive genes (Figure 2.2) (Shi and Massague, 2003; Feng and Derynck, 2005), such as BMP4/7 and others. A dynamic D–V nuclear gradient of pSmad1Cter has been shown in a number of model organisms such as Drosophila (Dorfman and Shilo, 2001; Sutherland et al., 2003), zebrafish (Tucker et al., 2008) and Xenopus (Plouhinec and De Robertis, 2009). At very low levels of pSmad1Cter, caused by the extracellular inhibitory activity of Chordin and Noggin on BMP ligands, dorsally expressed genes are transcribed. Ventral genes are activated by BMP signals. The dorsal and ventral centers of the gastrula express molecules of similar biochemical activities but under reciprocal transcriptional control. This explains how a self-regulating field is maintained in the early embryo (Reversade and De Robertis, 2005). When the amount of one molecule is lowered in the dorsal side, the gradient can be restored by the expression of ventral counterparts (Reversade and De Robertis, 2005; Plouhinec and De Robertis, 2009). For example, the dorsal organizer expresses Chordin, while the ventral center expresses a Chordin-related BMP-binding molecule called CV2 (Crossveinless-2) (Coffinier et al., 2002; Conley et al., 2000). When Chordin and CV2 are depleted simultaneously, the embryo reaches very high BMP levels, indicating that CV2 in the ventral side can compensate for loss-of Chordin in the dorsal signaling center (Ambrosio et al., 2008).

Anterior–posterior patterning and Wnt signaling

The main determinant of the A–P axis in the early embryo is provided by Wnt signaling (Niehrs, 2004; Kiecker and Niehrs, 2001). A Wnt morphogen gradient is generated by a number of extracellular Wnt ligands, which are modulated by a diverse group of secreted Wnt antagonists such as Dkk-1 (Dickkopf-1) and sFRPs (secreted Frizzled-related proteins) (Logan and Nusse, 2004). In Xenopus and amphioxious embryos, the Wnt signaling gradient is maximal at the posterior blastopore (Niehrs, 2004; Christian and Moon, 1993; Yu et al., 2007), and its signal becomes lower in anterior regions (Figure 2.1). When neuralized Xenopus ectodermal explants are microinjected with varying doses of Wnt DNA, posterior markers are induced (McGrew et al., 1997). In planarians, A–P specification is also regulated by Wnt signaling, since inhibition of the canonical Wnt pathway by RNAi causes ectopic regeneration of head structures (Gurley et al., 2008; Reddien, 2008). A–P patterning by a Wnt gradient appears to be a universal property of animal development. At later stages, the A–P axis becomes subdivided into segments in many organisms. The A–P patterning within each segment also requires Wnt signals (De Robertis, 2008).


Regulation of Smad1 via linker phosphorylations downstream of BMP

The BMP transcription factor Smad1 is further regulated by inhibitory “linker” phosphorylations. The linker region of Smads lies between its MH1 (Mad homology domain, DNA binding) and MH2 (protein interaction) domains with a large number of potential phosphorylatable Serines and Threonines.

Inhibitory Smad1 linker phosphorylations by MAPK

Smad1 was first shown to be a target of growth factor signaling through the mitogen-activated protein kinase (MAPK) pathway in human cultured cell lines (Kretzschmar et al., 1997a). MAPK phosphorylations activated by epidermal growth factor receptor (EGFR) occur at four specific MAPK/Erk recognition consensus sites (PxS[PO3]P) within the linker region of Smad1. MAPK phosphorylation prevents nuclear accumulation of Smad1, and therefore inhibits its intracellular transcriptional activity (Kretzschmar et al., 1997a). Mutation of the Serines at the four MAPK sites into Alanines rendered Smad1 resistant to EGFR-induced phosphorylation and inhibition (Kretzschmar et al., 1997a). This discovery provided the first evidence of the antagonistic action of MAPK linker phosphorylation on the BMP signaling pathway.

Table of contents :

1.1 The Transforming Growth Factor-beta signaling pathway
1.1.1 Overview of the TGF-beta pathway
1.1.2 TGF-beta Ligands and Receptors
1.2 The Smad transcription factors
1.2.1 The Smad family of transcription factors
1.2.2 Structure of the Smad proteins
1.2.3 Activation of R-Smads
1.2.4 Dephosphorylation of Smad C-terminal motifs by phosphatases
1.2.5 Proteasomal degradation of Smad proteins
1.2.4 R-Smads oligomerization with Smad4
1.2.5 Smad nucleocytoplasmic shuttling
1.2.6 DNA recognition by Smad proteins
1.2.7 Negative regulation of R-Smads through linker phosphorylation
1.3 The canonical Wnt signaling pathway
1.3.1 Overview of the Wnt signaling pathway
1.3.2 The Wnt pathway regulates proteins stability
1.4 The FGF/EGF pathway
1.5 Conclusions
CHAPTER 2 Smad1/5/8 Linker Phosphorylations Integrate the BMP and Wnt Signaling Pathways
2.1 Introduction: embryonic axis formation and the double gradient model
2.2 Dorsal–ventral patterning: a morphogenetic gradient of BMP ligands
2.3 Intracellular transduction of the BMP signal
2.4 Anterior–posterior patterning and Wnt signaling
2.5 Regulation of Smad1 via linker phosphorylations downstream of BMP
2.5.1 Inhibitory Smad1 linker phosphorylations by MAPK
2.5.2 GSK3/Wnt regulates BMP/Smad1 signal termination
2.6 Asymmetric inheritance of Smad1
2.7 Smad1 signal duration: phenotypic similarities between BMP and Wnt antagonists in the developing embryo
2.8 Linker regulation of Drosophila Mad
2.8.1 Mad linker phosphorylations: BMP dependent or independent?
2.8.2 Phospho-resistant Mad mutants display Wg-like phenotypes
2.8.3 Mad and Smad1 are required for segment formation
2.8.4 The ancestry of segmentation
2.9 Conclusions
CHAPTER 3 Phosphorylation of Mad Controls Competition Between Wingless and BMP Signaling
3.1 Introduction
3.2 Results
3.2.1 GSK3 phosphorylation of Mad inhibits both BMP and Wg signaling
3.2.2 Mad activates Wg target genes independently of phosphorylation of its C terminus
3.2.3 Mad and Medea are required for Wg signal transduction
3.2.4 Mad binds to Pangolin in the absence of phosphorylation of its C terminus
3.2.5 The Pangolin-Mad-Armadillo complex binds to Tcf DNA binding sites
3.3 Discussion
3.4 Experimental Procedures
CHAPTER 4 The Tumor Suppressor Smad4/DPC4 is Regulated by Phosphorylations that Integrate FGF, Wnt and TGF-beta Signaling
4.1 Summary
4.2 Introduction
4.3 Results
4.3.1 Wnt and FGF regulate phosphorylation of Smad4 linker region
4.3.2 Wnt/GSK3 regulates the polyubiquitination and degradation of Smad4
4.3.3 Wnt/GSK3 regulates a Smad4 beta-TrCP phosphodegron
4.3.4 Wnt and TGF-beta signaling cross-talk via Smad4
4.3.5 The Smad4 linker contains a growth-factor regulated transcriptional activation domain
4.3.6 Phosphorylation by MAPK/Erk promotes Smad4 peak activity
4.3.7 Smad4 regulation by GSK3 determines germ layer specification
4.4 Discussion
4.4.1 Smad4 activity is regulated by growth factors
4.4.2 beta-TrCP binds to the Smad4 phosphodegron
4.4.3 Signalling insulation and crosstalk
4.4.4 Smad4 linker phosphorylation and tumor suppression
4.5 Experimental Procedures
5.1 One transcription factor, two signaling pathways: Mad as a transducer of Dpp and Wg.
5.2 One structure, two functions: Smad4 activity and stability are co-regulated.
5.3 Is Smad4 phosphorylated by GSK3 after TGF-beta stimulation?
5.4 Is Smad4 degraded in the Wnt destruction complex?
5.5 Smad4 and cancer: the loss-of-Smad4 and the progression of cancer
5.6 Smad4 degradation by beta-TrCP in pancreatic carcinoma.
5.7 Concluding remarks.


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