The postsynaptic density 95/disc-large/zona occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.     

Interaction prediction and classification of PDZ domains

Background  

PDZ domain is a well-conserved, structural protein domain found in hundreds of signaling proteins that are otherwise unrelated. PDZ domains can bind to the C-terminal peptides of different proteins and act as glue, clustering different protein complexes together, targeting specific proteins and routing these proteins in signaling pathways. These domains are classified into classes I, II and III, depending on their binding partners and the nature of bonds formed. Binding specificities of PDZ domains are very crucial in order to understand the complexity of signaling pathways. It is still an open question how these domains recognize and bind their partners.     

Identification of a specific inhibitor of the dishevelled PDZ domain

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.     

PDZ结构域介导的PIP脂质信号转导

PDZ结构域是调控蛋白质/蛋白质相互作用的一类重要结构域,能特异结合蛋白质C末端一段有规律的氨基酸序列。含有PDZ结构域的支架蛋白能够组装成超大的蛋白质复合体来调控细胞内的信号转导通路。最新研究表明,PDZ结构域还能与PIP脂质直接相互作用,从而参与调控PIP脂质信号通路。将综合最新研究进展,阐明PDZ结构域与PIP脂质的作用方式,以及对相关PIP脂质信号转导的调控过程。     

Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein

Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/β-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not β-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.     

PDZ domains - common players in the cell signaling

PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

PDZ domain from Dishevelled -- a specificity study

Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt β-catenin pathway at the expense of non-canonical Wnt signaling.     

PDZ domains – glue and guide

PDZ domains are small globular building blocks that are amongst the most abundant protein interaction domains in organisms. Over the past several years an avalanche of data has implicated these modules in the clustering, targeting and routing of associating proteins. An overview is given of the types of interactions displayed by PDZ domains and how this relates to the current knowledge on their spatial structure. Furthermore, the different levels on which PDZ – ligand binding can be regulated and the consequences of PDZ domain-mediated clustering for activity, routing and targeting of interacting proteins will be addressed. Finally, some cell and animal models that illustrate the impact of PDZ domain-containing proteins on (multi-) cellular processes will be discussed.     

Mutagenesis and selection of PDZ domains that bind new protein targets

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.     

The Tumor Suppressor SCRIB is a Negative Modulator of the Wnt/β‐Catenin Signaling Pathway

SCRIB is a scaffold protein containing leucine‐rich repeats (LRR) and PSD‐95/Dlg‐A/ZO‐1 domains (PDZ) that localizes at the basolateral membranes of polarized epithelial cells. Deregulation of its expression or localization leads to epithelial defects and tumorigenesis in part as a consequence of its repressive role on several signaling pathways including AKT, ERK, and HIPPO. In the present work, a proteomic approach is used to characterize the protein complexes associated to SCRIB and its paralogue LANO. Common and specific sets of proteins associated to SCRIB and LANO by MS are identified and an extensive landscape of their associated networks and the first comparative analysis of their respective interactomes are provided. Under proteasome inhibition, it is further found that SCRIB is associated to the β‐catenin destruction complex that is central in Wnt/β‐catenin signaling, a conserved pathway regulating embryonic development and cancer progression. It is shown that the SCRIB/β‐catenin interaction is potentiated upon Wnt3a stimulation and that SCRIB plays a repressing role on Wnt signaling. The data thus provide evidence for the importance of SCRIB in the regulation of the Wnt/β‐catenin pathway.     

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

PDZ结构域的结构特点及其识别特异性配体的机制

PDZ结构域作为介导蛋白质之间相互作用的重要结构域之一,参与到细胞内运输、离子通道、以及各种信号传导通路等多种生物学过程.PDZ结构域是由80~100个氨基酸组成的小的球状结构域,对某些多PDZ结构域蛋白来说,需要一前一后形成串联体才能正确折叠.另外,PDZ结构域相互之间也可以形成同源或异源二聚体.这些PDZ结构域的突出特点是能特异性地识别配体靶蛋白C末端短的氨基酸序列,但有些也能识别靶蛋白的内部β发夹结构.而一些支架蛋白的PDZ结构域与细胞膜上脂类的相互作用则增加了其与膜的亲和性.本文简要概括了PDZ结构域的结构特点及其对配体的各种特异性识别的机制,从而为研究各种PDZ蛋白的功能提供了结构基础.     

Protein modules as organizers of membrane structure.

Investigations conducted over the past 18 months have shed new light on how modular protein-binding domains, in particular PDZ domains, co-ordinate the assembly of functional plasma membrane domains. Members of the MAGUK (membrane-associated guanylate kinase) protein family, like PSD-95, use multiple domains to cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses, cellular junctions, and polarized membrane domains. Other PDZ proteins, like the Drosophila protein INAD and the epithelial Na(+)/H(+) regulatory factor (NHERF), organize cellular signaling by localizing transmembrane and cytosolic components to specific membrane domains and assembling these components into functional complexes. The organization of these proteins into discreet structures has functional consequences for downstream signaling.     

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.     

Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.     

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA

In Drosophila, phototransduction is mediated by G(q)-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.     

Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation.

PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na(+)/H(+) exchanger regulatory factor (NHERF-1; also called "EBP50") and its relative NHERF-2 (also called "E3KARP", "SIP-1", and "TKA-1") both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta(2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.     

MAGUKs: beyond ionic channel anchoring

A family of anchoring proteins named MAGUK (for membrane associated guanylate kinase) has emerged as a key element in the organization of protein complexes in specialized membrane regions. These proteins are characterized by the presence of multipe protein-protein interaction domains including PDZ and SH3 domains. The MAGUK family comprises the post-synaptic density 95 (PSD-95) protein and closely related molecules such as chapsyn-110, synapse-associated protein 102 (SAP-102), and SAP-97. These are located either on the pre- and/or post-synaptic sides of synapses or at cell-cell adhesion sites of epithelial cells. MAGUK proteins interact with glutamate receptors and various ionic channels. For instance, an interaction has been reported between the first two PDZ domains of MAGUK proteins and several channels via a consensus sequence Thr/Ser-X-Val/Leu usually located at their carboxy terminus. The role of these anchoring proteins in channel function is not fully understood. MAGUK proteins enhance the current density by increasing the number of functional channels to the sarcolemma. They can also facilitate signaling between channels and several enzymes or G protein-dependent signaling pathways. In the heart also, MAGUK proteins are abundantly expressed and they interact with various channels including Shaker Kv1.5 and connexins.