Maria J. Macias

Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-β Pathways

TGF-beta 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 TGF-beta pathways.

WW and SH3 domains, two different scaffolds to recognize proline-rich ligands.

WW domains are small protein modules composed of approximately 40 amino acids. These domains fold as a stable, triple stranded β‐sheet and recognize proline‐containing ligands. WW domains are found in many different signaling and structural proteins, often localized in the cytoplasm as well as in the cell nucleus. Based on analyses of seven structures of WW domains, we discuss their diverse binding preferences and sequence conservation patterns. While modeling WW domains for which structures have not been determined we uncovered a case of potential molecular and functional convergence between WW and SH3 domains. The binding surface of the modeled WW domain of Npw38 protein shows a remarkable similarity to the SH3 domain of Sem5 protein, confirming biochemical data on similar binding predilections of both domains.

Ubiquitin Ligase Nedd4L Targets Activated Smad2/3 to Limit TGF-β Signaling

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.

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β 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-β 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-β signal delivery to turnover of the Smad signal transducers.

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins.

BACKGROUND The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.

Structural determinants of SMAD function in TGF-β signaling

Smad transcription factors are central to the signal transduction pathway that mediates the numerous effects of the transforming growth factor β (TGF-β) superfamily of cytokines in metazoan embryo development as well as in adult tissue regeneration and homeostasis. Although Smad proteins are conserved, recent genome-sequencing projects have revealed their sequence variation in metazoan evolution, human polymorphisms, and cancer. Structural studies of Smads bound to partner proteins and target DNA provide a framework for understanding the significance of these evolutionary and pathologic sequence variations. We synthesize the extant mutational and structural data to suggest how genetic variation in Smads may affect the structure, regulation, and function of these proteins. We also present a web application that compares Smad sequences and displays Smad protein structures and their disease-associated variants.

Ultrafast folding of WW domains without structured aromatic clusters in the denatured state

Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a three-stranded antiparallel β-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38–44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 μs at 25°C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.

Solution Structure and Ligand Recognition of the WW Domain Pair of the Yeast Splicing Factor Prp40

The yeast splicing factor pre-mRNA processing protein 40 (Prp40) comprises two N-terminal WW domains, separated by a ten-residue linker, and six consecutive FF domains. In the spliceosome, the Prp40 WW domains participate in cross-intron bridging by interacting with proline-rich regions present in the branch-point binding protein (BBP) and the U5 small nuclear ribonucleoprotein component Prp8. Furthermore, binding of Prp40 to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to link splicing to transcription. To gain insight into this complex interaction network we have determined the solution structure of the tandem Prp40 WW domains by NMR spectroscopy and performed chemical shift mapping experiments with different proline-rich peptides. The WW domains each adopt the characteristic triple-stranded beta-sheet structure and are connected by a stable alpha-helical linker. On the basis of a detailed analysis of residual dipolar couplings (RDC) and 15N relaxation data we show that the tandem Prp40 WW domains behave in solution as a single folded unit with unique alignment and diffusion tensor, respectively. Using [1H-15N]-RDCs, we were able to accurately define the relative orientation of the WW domains revealing that the binding pockets of each domain face opposite sides of the structure. Furthermore, we found that both Prp40 WW domains interact with PPxY motifs (where x is any residue) present in peptides derived from the splicing factors BBP and Prp8. Moreover, the Prp40 WW domains are shown to bind proline-rich peptides devoid of aromatic residues, which are also recognised by the Abl-SH3 domain and the WW domain of the mammalian Prp40 orthologue formin binding protein 11. In contrast, no interaction was observed between the Prp40 WW domains and the CTD repeats used in this work.

Structural Basis for the Versatile Interactions of Smad7 with Regulator WW Domains in TGF-β Pathways

Transforming growth factor (TGF)-β 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-β 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-β and BMP pathways.

A small region in phosducin inhibits G-protein βγ-subunit function

G‐protein βγ‐subunits (Gβγ) are active transmembrane signalling components. Their function recently has been observed to be regulated by the cytosolic protein phosducin. We show here that a small fragment (amino acids 215–232) contained in the C‐terminus of phosducin is sufficient for high‐affinity interactions with Gβγ. Corresponding peptides not only disrupt Gβγ–Gα interactions, as defined by Gβγ‐stimulated GTPase activity of αo, but also other Gβγ‐mediated functions. The NMR structure of a peptide encompassing this region shows a loop exposing the side chains of Glu223 and Tyr224, and peptides with a substitution of either of these amino acids show a complete loss of activity towards Go. Mutation of this Tyr224 to Ala in full‐length phosducin reduced the functional activity of phosducin to that of phosducins isolated N‐terminus, indicating the importance of this residue within the short, structurally defined C‐terminal segment. This small peptide derived from phosducin may represent a model of a Gβγ inhibitor, and illustrates the potential of small compounds to affect Gβγ functions.