Ye-Guang Chen

Crystal Structure of the Cytoplasmic Domain of the Type I TGF β Receptor in Complex with FKBP12

Activation of the type I TGFbeta receptor (TbetaR-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated TbetaR-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. TbetaR-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the TbetaR-II phosphorylation sites and further stabilizing the inactive conformation of TbetaR-I. Certain structural features at the catalytic center of TbetaR-I are characteristic of tyrosine kinases rather than Ser/Thr kinases.

PPM1A Functions as a Smad Phosphatase to Terminate TGFβ Signaling

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.

The TGF beta receptor activation process: an inhibitor- to substrate-binding switch.

The type I TGF beta receptor (T beta R-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain. We have studied the molecular mechanism of receptor activation using a homogeneously tetraphosphorylated form of T beta R-I, prepared using protein semisynthesis. Phosphorylation of the GS region dramatically enhances the specificity of T beta R-I for the critical C-terminal serines of Smad2. In addition, tetraphosphorylated T beta R-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12. Thus, phosphorylation activates T beta R-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate. Our observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the T beta R-I/Smad activation process.

Mechanism of TGFbeta receptor inhibition by FKBP12.

Transforming growth factor‐β (TGFβ) signaling requires phosphorylation of the type I receptor TβR‐I by TβR‐II. Although TGFβ promotes the association of TβR‐I with TβR‐II, these receptor components have affinity for each other which can lead to their ligand‐independent activation. The immunophilin FKBP12 binds to TβR‐I and inhibits its signaling function. We investigated the mechanism and functional significance of this effect. FKBP12 binding to TβR‐I involves the rapamycin/Leu–Pro binding pocket of FKBP12 and a Leu–Pro sequence located next to the activating phosphorylation sites in TβR‐I. Mutations in the binding sites of FKBP12 or TβR‐I abolish the interaction between these proteins, leading to receptor activation in the absence of added ligand. FKBP12 does not inhibit TβR‐I association with TβR‐II, but inhibits TβR‐I phosphorylation by TβR‐II. Rapamycin, which blocks FKBP12 binding to TβR‐I, reverses the inhibitory effect of FKBP12 on TβR‐I phosphorylation. By impeding the activation of TGFβ receptor complexes formed in the absence of ligand, FKBP12 may provide a safeguard against leaky signaling resulting from the innate tendency of TβR‐I and TβR‐II to interact with each other.

Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation

In eukaryotic cells, autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses. Autophagy is regulated by cell signalling such as the mTOR (mammalian target of rapamycin) pathway. However, the significance of autophagy in modulation of signal transduction is unclear. Here we show that autophagy negatively regulates Wnt signalling by promoting Dishevelled (Dvl) degradation. Von Hippel–Lindau protein-mediated ubiquitylation is critical for the binding of Dvl2 to p62, which in turn facilitates the aggregation and the LC3-mediated autophagosome recruitment of Dvl2 under starvation; the ubiquitylated Dvl2 aggregates are ultimately degraded through the autophagy–lysosome pathway. Moreover, a reverse correlation between Dvl expression and autophagy is observed in late stages of colon cancer development, indicating that autophagy may contribute to the aberrant activation of Wnt signalling in tumour formation.

The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF‐β receptors

Signal transduction specificity in the transforming growth factor‐β (TGF‐β) system is determined by ligand activation of a receptor complex which then recruits and phosphorylates a subset of SMAD proteins including Smads 1 and 2. These then associate with Smad4 and move into the nucleus where they regulate transcription. We have identified a discrete surface structure in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C‐terminal domain. The L3 loop sequence is invariant among TGF‐β‐ and bone morphogenetic protein (BMP)‐activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF‐β receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF‐β receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping both these two amino acids and four amino acids near the C‐terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD–receptor interactions, and indicate that the L3 loop, together with the C‐terminal tail, specifies SMAD activation.

Smad1 Recognition and Activation by the ALK1 Group of Transforming Growth Factor-β Family Receptors

Two structural elements, the L45 loop on the kinase domain of the transforming growth factor-β (TGF-β) family type I receptors and the L3 loop on the MH2 domain of Smad proteins, determine the specificity of the interactions between these receptors and Smad proteins. The L45 sequence of the TGF-β type I receptor (TβR-I) specifies Smad2 interaction, whereas the related L45 sequence of the bone morphogenetic protein (BMP) type I receptor (BMPR-I) specifies Smad1 interactions. Here we report that members of a third receptor group, which includes ALK1 and ALK2 from vertebrates and Saxophone from Drosophila, specifically phosphorylate and activate Smad1 even though the L45 sequence of this group is very divergent from that of BMPR-I. We investigated the structural elements that determine the specific recognition of Smad1 by ALK1 and ALK2. In addition to the receptor L45 loop and the Smad1 L3 loop, the specificity of this recognition requires the α-helix 1 of Smad1. The α-helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the Smad MH2 domain. Thus, Smad1 recognizes two distinct groups of receptors, the BMPR-I group and the ALK1 group, through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the Smad1 MH2 domain.

Smad7 Antagonizes Transforming Growth Factor β Signaling in the Nucleus by Interfering with Functional Smad-DNA Complex Formation

ABSTRACT Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor β (TGF-β) signaling by inhibiting TGF-β signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-β signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-β target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-β-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-β signaling in the nucleus by a novel mechanism.

The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation

The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation

Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis.

Activins, cytokine members of the transforming growth factor-β superfamily, have various effects on many physiological processes, including cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses endocrine function, etc. Activins interact with two structurally related serine/threonine kinase receptors, type I and type II, and initiate downstream signaling via Smads to regulate gene expression. Understanding how activin signaling is controlled extracellularly and intracellularly would not only lead to more complete understanding of cell growth and apoptosis, but would also provide the basis for therapeutic strategies to treat cancer and other related diseases. This review focuses on the recent progress on activin-receptor interactions, regulations of activin signaling by ligand-binding proteins, receptor-binding proteins, and nucleocytoplasmic shuttling of Smad proteins.