DIXDC1 Promotes Retinoic Acid-Induced Neuronal Differentiation and Inhibits Gliogenesis in P19 Cells

Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. XT Jing and HT Wu contributed equally to this work.     

Knockdown of DIXDC1 Inhibits the Proliferation and Migration of Human Glioma Cells

DIX domain containing 1 (DIXDC1), the human homolog of coiled-coil-DIX1 (Ccd1), is a positive regulator of Wnt signaling pathway. Recently, it was found to act as a candidate oncogene in colon cancer, non-small-cell lung cancer, and gastric cancer. In this study, we aimed to investigate the clinical significance of DIXDC1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that DIXDC1 was overexpressed in glioma tissues and glioma cell lines. The expression level of DIXDC1 was evidently linked to glioma pathological grade and Ki-67 expression. Kaplan–Meier curve showed that high expression of DIXDC1 may lead to poor outcome of glioma patients. Serum starvation and refeeding assay indicated that the expression of DIXDC1 was associated with cell cycle. To determine whether DIXDC1 could regulate the proliferation and migration of glioma cells, we transfected glioma cells with interfering RNA-targeting DIXDC1; investigated cell proliferation with Cell Counting Kit (CCK)-8, flow cytometry assays, and colony formation analyses; and investigated cell migration with wound healing assays and transwell assays. According to our data, knockdown of DIXDC1 significantly inhibited proliferation and migration of glioma cells. These data implied that DIXDC1 might participate in the development of glioma, suggesting that DIXDC1 can become a potential therapeutic strategy for glioma.     

DIXDC1 co-localizes and interacts with γ-tubulin in HEK293 cells

DIXDC1 is a Dishevelled-Axin (DIX) domain-containing protein involved in neural development and Wnt signaling pathway. Besides the DIX domain, DIXDC1 also contains a coiled-coil domain (MTH domain), which is a common feature of centrosomal proteins. We have demonstrated that exogenously expressed GFP-tag fused DIXDC1 co-localize with γ-tubulin both at interphase and mitotic phase in HEK293 cells. By immunostaining with anti-DIXDC1 and anti-γ-tubulin antibody, endogenous DIXDC1 was also co-localized with γ-tubulin at the centrosomes in HEK293 cells. We confirmed this interaction of DIXDC1 with γ-tubulin by co-immunoprecipitation. The findings suggest that DIXDC1 might play an important role in chromosome segregation and cell cycle regulation.     

Dvl2-DIX结构域的SiteⅢ相关突变体结晶及其与Ccd1-DIX的共结晶

Dvl(Dishevelled)是Wnt信号通路传递的核心分子,无论内源的还是过表达的Dvl在细胞体内都能因自聚而形成puncta．研究已报道,Dvl主要通过其DIX结构域上的三个作用区域来介导自聚：SiteⅠ、SiteⅡ和SiteⅢ,其中SiteⅠ和SiteⅡ还参与了Dvl-DIX与Ccd1-DIX的异聚．为了进一步得到Dvl2-DIX上SiteⅠ和SiteⅡ的直接三维结构,本研究设计了一系列的SiteⅢ突变体．通过体内和体外实验进一步证实了这些突变氨基酸确实参与了Dvl2-DIX的自聚,然后对这些SiteⅢ突变体蛋白成功地进行了纯化和结晶,最终得到3.1Å的Dvl2-DIX(G65A)晶体数据．分析表明该晶体存在片层位移现象,需对数据进行一定修正后才能进行后续的结构分析．体外实验又证实了这些突变氨基酸不影响Dvl2-DIX与Ccd1-DIX的异聚,为了进一步研究Dvl2-DIX与Ccd1-DIX相互作用,我们对这些SiteⅢ突变体蛋白与Ccd1-DIX进行共结晶．最终获得Dvl2-DIX(G65A)与Ccd1-DIX复合物的初晶,利于进一步的晶体优化及数据收集．     

Molecular basis of Wnt activation via the DIX domain protein Ccd1

The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into "head-to-tail" helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the "head" of Dvl-DIX and the "tail" of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s).     

Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.     

Desumoylation activity of Axam, a novel Axin-binding protein, is involved in downregulation of beta-catenin

Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of beta-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of beta-catenin. An Axam fragment which contains both domains was able to decrease the level of beta-catenin. On substitution of Ser for Cys(547) in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate beta-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, Axam(C547S) showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate beta-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.     

Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.     

Secondary structure, 1H, 13C and 15N resonance assignments and molecular interactions of the dishevelled DIX domain

Dishevelled (Dvl) is a positive regulator of the canonical Wnt signaling pathway, which regulates the levels of beta-catenin. The beta-catenin oncoprotein depends upon the association of Dvl and Axin proteins through their DIX domains, and its accumulation directs the expression of specific developmental-related genes at the nucleus. Here, the (1)H, (13)C and (15)N resonances of the human Dishevelled 2 DIX domain are assigned using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. In addition, helical and extended elements are identified based on the NMR data. The results establish a structural context for characterizing the actin and phospholipid interactions and binding sites of this novel domain, and provide insights into its role in protein localization to stress fibers and cytoplasmic vesicles during Wnt signaling.     

High-Affinity Dkk1 Receptor Kremen1 Is Internalized by Clathrin-Mediated Endocytosis

Kremens are high-affinity receptors for Dickkopf 1 (Dkk1) and regulate the Wnt/β-catenin signaling pathway by down-regulating the low-density lipoprotein receptor-related protein 6 (LRP6). Dkk1 competes with Wnt for binding to LRP6; binding of Dkk1 inhibits canonical signaling through formation of a ternary complex with Kremen. The majority of down-regulated clathrin-mediated endocytic receptors contain short conserved regions that recognize tyrosine or dileucine sorting motifs. In this study, we found that Kremen1 is internalized from the cell surface in a clathrin-dependent manner. Kremen1 contains an atypical dileucine motif with the sequence DXXXLV. Mutation of LV to AA in this motif blocked Kremen1 internalization; as reported previously for other proteins, the aspartic acid residue in Kremen1 is not critical. Inhibition of expression of the adaptor protein 2 (AP-2) or inhibition of clathrin by pitstop 2 also blocked Kremen1 internalization. The novel amino acid sequence identified in Kremen1 is similar to the motif previously identified in hydra, yeast, and other organisms known to signal from the trans-Golgi network to the endosomal compartment.     

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.     

Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons

Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.     

Lymphoid enhancer-binding factor-1 (LEF1) interacts with the DNA-binding domain of the vitamin D receptor

Ligand-independent actions of the vitamin D receptor (VDR) are required for normal post-morphogenic hair cycles; however, the molecular mechanisms by which the VDR exerts these actions are not clear. Previous studies demonstrated impaired regulation of the canonical Wnt signaling pathway in primary keratinocytes lacking the VDR. To identify the key effector of canonical Wnt signaling that interacts with the VDR, GST pulldown studies were performed. A novel interaction between the VDR and LEF1 (lymphoid enhancer-binding factor-1) that is independent of β-catenin was identified. This interaction is dependent upon sequences within the N-terminal region of the VDR, a domain required for VDR-DNA interactions and normal hair cycling in mice. Mutation of specific residues within the N-terminal region of the VDR not only abrogated interactions between the VDR and LEF1 but also impaired the ability of the VDR to enhance Wnt signaling in vdr(-/-) primary keratinocytes. Thus, this study demonstrates a novel interaction between the VDR and LEF1 that is mediated by the DNA-binding domain of the VDR and that is required for normal canonical Wnt signaling in keratinocytes.     

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.