TGFβ and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1, is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGFβ pathways.

The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.

Due to its small size and compact fold, the WW domain became an attractive model for studies of protein stability and design [7^11]. Speci¢c residues have been identi¢ed that play a critical role in the structure and function of the domain and also in modulating its stability. In fact, the WW domain is the ¢rst protein module that has been successfully designed de novo, demonstrating the signi¢cant insight we already have regarding its fold [12]. Besides, the WW domain sequence is well conserved in length, even in its loops, which is a remarkable feature of this domain, compared with others, making protein modeling a useful tool for generating three-dimensional representations of their sequences. Nevertheless, attempts to predict binding targets for a speci¢c WW domain sequence or even for one of its subgroups or classes, with a good probability, have not been made so far.Based on the pattern of semi-conserved residues, WW domain sequences have been classi¢ed into three groups as described previously [12]. Group I contains the C-terminal tryptophan and the N-terminal proline, Group II sequences lack the N-terminal proline and ¢nally Group III with sequences without the second tryptophan. In another classi¢cation, based on the ligand predilection, WW domains were divided into two major and two minor groups [5]. One major group (Group I) binds polypeptides with the minimal core consensus PPxY, whereas the other binds ligands with the PPLP motif usually embedded in a long stretch of prolines (Group II). Group III WW domains select poly-P motifs £anked by R or K, whereas Group IV WW domains bind to short sequences with phospho-S or phospho-T followed by P, in a phosphorylation-dependent manner [5]. A sequence alignment of some selected WW sequences combining binding preferences and sequence conservation is shown in Fig. 1.In this contribution we will review the structural characteristics of WW domain^ligand complexes determined so far. On the basis of four WW domain structures in complex with di¡erent peptides and two structures of free WW domains [3,12^16], a three-dimensional structure has been modeled for the Npw38 WW domain that allows us to compare binding properties of WW and SH3 domains. WW domain as a phosphate-dependent SH3 domain?WW domains have the ability to bind proline-rich cores and/or phospho-SP/phospho-TP-containing motifs [5]. It is interesting that such a small and well-conserved module has a surprisingly large repertoire of potential ligands. The dissociation constants (K d ) for WW^ligand complexes lie in the high nM to low mM range for proline-rich ligands, and in the low mM range for phospho-SP-or phospho-TP-containing ligands [5].

Summary TGFβ induces phosphorylation of the transcription factors Smad2 and Smad3 at the C-terminus as well as at an interdomain linker region. TGFβ-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain Nedd4L specifically recognizes a TGFβ-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 poly-ubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGFβ activated Smads, restricts the amplitude and duration of TGFβ gene responses, and in mouse embryonic stem cells limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGFβ signal transduction.

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