ABSTRACT
Transforming growth factor (TGF)-ß and BMP signaling is mediated by Smads 1-5 (R-Smads and Co-Smads) and inhibited by Smad7, a major hub of regulation of TGF-ß and BMP receptors by negative feedback and antagonistic signals. The transcription coactivator YAP and the E3 ubiquitin ligases Smurf1/2 and Nedd4L target R-Smads for activation or degradation, respectively. Pairs of WW domain in these regulators bind PY motifs and adjacent CDK/MAPK and GSK3 phosphorylation sites in R-Smads in a selective and regulated manner. In contrast, here we show that Smad7 binds YAP, Smurf1, Smurf2, and Nedd4L constitutively, the binding involving a PY motif in Smad7 and no phosphorylation. We also provide a structural basis for how regulators that use WW domain pairs for selective interactions with R-Smads, resort to one single versatile WW domain for binding Smad7 to centralize regulation in the TGF-ß and BMP pathways.
Subject(s)
Smad7 Protein/chemistry , Transforming Growth Factor beta/physiology , Amino Acid Motifs , Calorimetry , Cell Cycle Proteins , Endosomal Sorting Complexes Required for Transport/chemistry , Humans , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Nedd4 Ubiquitin Protein Ligases , Nuclear Proteins/chemistry , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Protein Structure, Secondary , Signal Transduction , Surface Properties , Transcription Factors/chemistry , Ubiquitin-Protein Ligases/chemistryABSTRACT
When directed to the nucleus by TGF-ß or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-ß pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-ß signal delivery to turnover of the Smad signal transducers.
Subject(s)
Gene Expression Regulation , Phosphoserine/metabolism , Smad Proteins/metabolism , Amino Acid Sequence , Cell Cycle Proteins , Endosomal Sorting Complexes Required for Transport/metabolism , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , NIMA-Interacting Peptidylprolyl Isomerase , Nedd4 Ubiquitin Protein Ligases , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Peptidylprolyl Isomerase/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Smad Proteins/chemistry , Transcription Factors/chemistry , Transcription Factors/metabolism , Transforming Growth Factor beta1/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-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 TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it 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 TGF-beta signal transduction.
