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Mammalian genomes encode numerous antisense non-coding RNAs, which are assumed to be involved in the regulation of the sense gene expression. However, the mechanisms of their action and involvement in the development of diseases have not been well elucidated. The ANA/BTG3 protein is an antiproliferative protein whose expression is downregulated in prostate and lung cancers. Here we show that an antisense transcript of the ANA/BTG3 gene, termed ASBEL, negatively regulates the levels of ANA/BTG3 protein, but not of ANA/BTG3 mRNA and is required for proliferation and tumorigenicity of ovarian clear cell carcinoma. We further show that knockdown of ANA/BTG3 rescues growth inhibition caused by ASBEL knockdown. Moreover, we demonstrate that ASBEL forms duplexes with ANA/BTG3 mRNA in the nucleus and suppresses its cytoplasmic transportation. Our findings illustrate a novel function for an antisense transcript that critically promotes tumorigenesis by suppressing translation of the sense gene by inhibiting its cytoplasmic transportation.

The mammalian homolog of the Drosophila discs large tumor suppressor protein Dlg functions as a scaffolding protein that facilitates the transmission of diverse signals. In the present study, we attempted to identify the downstream target genes of Dlg, and found that Dlg up-regulates expression of the ELR+ CXC chemokine Scyb5, which has been implicated in the immune system. Our finding suggests that Scyb5 may play an important role in the tumor suppressor function of Dlg.

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the beta-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. TCF-binding elements have been found in the promoter regions of many TCF target genes and characterized by a highly conserved consensus sequence. Utilizing this consensus sequence, we performed an in silico screening for new TCF target genes. Through computational screening and subsequent experimental analysis, we identified a novel TCF target gene, DKK1, which has been shown to be a potent inhibitor of Wnt signaling. Our finding suggests the existence of a novel feedback loop in Wnt signaling.

Bone morphogenetic proteins (BMPs) play critical roles in cellular proliferation, differentiation, and programmed cell death in multiple tissues. An increasing body of recent evidence has suggested that classes of molecules collectively termed BMP antagonists play important roles for the local regulation of BMP actions by binding BMPs and neutralizing their activities. Uterine sensitization-associated gene-1 (USAG-1) was previously reported as a gene of unknown function, preferentially expressed in sensitized endometrium of the rat uterus. Here, we show that USAG-1 is abundantly expressed in the kidney and functions as a BMP antagonist. Recombinant USAG-1 binds directly to BMPs and antagonizes the BMP-mediated induction of alkaline phosphatase in C2C12 cells. USAG-1 also induces formation of secondary axis and/or hyperdorsalization when its mRNA is injected to Xenopus embryos. In the early stage of mouse embryogenesis, USAG-1 is expressed in the first and second branchial arches and in metanephros, while in later stages the expression is confined to renal tubules and ameloblasts of teeth. Postnatally, the expression is further restricted to distal tubules of kidney, in a pattern similar to the localization of BMP-7, which has been shown to be important in the development of kidney and preservation of adult renal functions under pathological stresses. Collectively, we suggest that USAG-1 is a BMP antagonist that interacts with BMP-7 in the developing and adult kidney.

The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.

Background: hDLG, the human homologue of the Drosophila tumour suppressor dlg, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. hDLG possesses multiple protein-binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain., Results: We studied the significance of the PDZ, SH3, HOOK and GK domains in the cytoplasmic localization of hDLG. We found that mutation of the SH3 or GK domain, but not the PDZ domain, resulted in a re-localization of hDLG to the nucleus. Furthermore, hDLG was found to possess a potential nuclear localization signal in the HOOK domain., Conclusion: These results suggest that the SH3, HOOK and GK domains of hDLG are important for its cytoplasmic localization.

DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.

Aberrant activation of Wnt signaling caused by mutations in adenomatous polyposis coli (APC) or beta-catenin is a critical event in the development of human colorectal tumors. Wnt signaling stabilizes beta-catenin, which in turn associates with TCF/LEF family transcription factors, ultimately altering the expression of Wnt target genes. We have recently identified ICAT, a beta-catenin-interacting protein that interferes with the interaction between beta-catenin and TCF-4, thereby negatively regulating Wnt signaling. In the present study, we generated a recombinant adenovirus encoding ICAT and examined its effect on the growth of tumor cells. We found that Icat inhibits proliferation of colorectal tumor cells mutated in APC or beta-catenin and hepatocellular carcinoma cells mutated in AXIN: By contrast, Icat did not inhibit growth of either normal or tumor cells containing the wild-type APC, beta-catenin, and Axin genes. Icat also inhibited the anchorage-independent growth of colorectal tumor cells and tumorigenic growth of colorectal tumor xenografts. Furthermore, we found that Icat inhibits both dephosphorylation of Cdc2 and nuclear translocation of cyclin B1 and induces G(2) arrest followed by cell death in colorectal tumor cells. These results suggest that Wnt signaling is critical for the growth of colorectal tumors and some hepatocellular carcinomas and that expression of ICAT or drugs which mimic its effects may be useful in the treatment of these tumors.

Recently, we have identified human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, as a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing eucine-rich repeats nd DZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), which is a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. Here, we investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localized with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC caused disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC. [ABSTRACT FROM AUTHOR]

The improved IGCR (In–Gel Competitive Reassociation) method was applied to the analysis of human gastric cancer genomic DNA to identify its alterations, and it appeared that the IGCR library contained a fragment of 3′-untranslated region (3′ UTR) of G-protein coupled receptor 30 (GPR30) mRNA. When we searched genomic DNA pairs of gastric cancer patients with this IGCR clone, we found the deletion polymorphism with or without 2 bp (Cytosine and Thymine; CT). We confirmed the existence of a novel mRNA in GPR30 3′UTR by northern blotting, cloned this novel mRNA and named it Leucine Rich Protein in GPR30 3′UTR (LERGU). The EST database search gave one alternative splicing form in this 3′ UTR, which was named as LERGU-1. A novel alternative splicing form of this mRNA was also identified from the stomach total RNA, which was named LERGU-2. The LERGU mRNA was also detected in eight gastric cancer cell lines, but GPR30 mRNA scarcely existed. Furthermore, we detected the 2 bp-deletion form in genomic DNAs and mRNAs derived from gastric cancers, but not in other type cancers. Since the 2 bp-deletion position on LERGU corresponds to its alternative splicing site, this deletion may produce a frame-shifted protein. Overall, our findings suggest that a mutation or disappearance of the normal LERGU protein may have a function in the development of gastric cancer. (Cancer Sci2005; 96: 191–196) [ABSTRACT FROM AUTHOR]

Abstract Background: The PSD-95 family of proteins possesses multiple protein binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain. The PSD-95 proteins function as scaffolding proteins that link ion channels such as the N-methyl-d-aspartate-receptors (NMDA-Rs) with cytoskeletal networks and signalling molecules, thereby controlling synaptic plasticity and learning. Results: We found that the PSD-95 family proteins interact via their GK domains with SPA-1-like protein (SPAL), a GTPase-activating protein (GAP) that is specific for Rap1. SPAL was contained within the NMDA-R-PSD-95 complex, and co-localized with PSD-95 and NMDA-R at the synapses in cultured hippocampal neurones. Furthermore, NMDA stimulation induced the dephosphorylation of SPAL in cultured hippocampal neurones. Conclusion: Our findings suggest that SPAL may be involved in the NMDA-mediated organization of cytoskeletal networks and signal transduction. [ABSTRACT FROM AUTHOR]

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of β-catenin, through its interaction with β-catenin, GSK-3β and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A?KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and β-catenin are transported along microtubules by KAP3?KIF3A?KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration. [ABSTRACT FROM AUTHOR]

Postsynaptic density (PSD)-95 interacts with and mediates clustering of the N-methyl-d-aspartate-receptors (NMDA-R). PSD-95 also interacts with the hDLG-associated protein DAP, which is also called Synapse-associated protein 90-associated protein (SAPAP), and Guanylate kinase-associated protein (GKAP). DAP interacted directly with the dynein light chain (DLC) family of proteins. DLC was contained in the NMDA-R-PSD-95-DAP-neuronal nitric oxide synthase (nNOS) complex. Furthermore, DAP interacted with nNOS and recruited it into the Triton X-100-insoluble fraction of transfected cells. DAP interacts directly with DLC and nNOS, and links these proteins to the NMDA-R-PSD-95 complex. [ABSTRACT FROM AUTHOR]

Background The synaptic protein PSD-95/SAP90 interacts with ion channels such as the N-methyl-D-aspartate-receptor (NMDA-R) via its PDZ domain, and is involved in their clustering. Moreover, it interacts with signalling molecules and plays an important role in coupling NMDA-R to pathways that control synaptic plasticity and learning. Results We report that PSD-95 interacts with the adenomatous polyposis coli (APC) tumour suppressor protein via its PDZ domain. Furthermore, we found that PSD-95, NMDA-R and APC are contained in the same complex in vivo. PSD-95-NMDA-R–APC association was found to require two cysteine residues conserved in the amino-terminus of PSD-95 that are known to be critical for its multimerization. Conclusion Our findings suggest that the PSD-95-NMDA-R-APC complex forms due to the multimerization of PSD-95 monomers, each of which can associate with either NMDA-R or APC. It is possible that APC is involved in the regulation of ion channel clustering and/or organization of signalling molecules. [ABSTRACT FROM AUTHOR]

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase α. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase α synergistically. B23 exists in two iso-forms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase α from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase α activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase α/retinoblastoma protei/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase α in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein [ABSTRACT FROM AUTHOR]

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10 – 100-fold the activity of DNA polymerase α from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase α more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase α. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase α antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase α. This result indicates that DNA polymerase α exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase α specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase α trapped to anti-DNA polymerase α antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication. [ABSTRACT FROM AUTHOR]

BCL-6: gene alterations have been observed in 27 – 45% of diffuse large B-cell lymphomas (DLBs) with chromosomal translocations at 3q27. The deregulated expression of normal BCL-6 protein caused by this chromosomal translocation is believed to be responsible for lymphomagenesis. Recently, we demonstrated that BCL-6 is expressed at high levels in germinal center B-cells as a 92-98 kDa nuclear protein in a constitutively phosphorylated form. In this study, we show that BCL-6 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro at the sites phosphorylated in vivo. These numerous phosphorylation sites were found to be located in its serine- and proline-clustered (SPC) region (amino acids-250-483). BCL-6 phosphorylation significantly increased in Ramos cells following stimulation with 12-o-tetradecanoylphorbol-13-acetate (TPA) or BCL-6- and erk1-transfected COS-7 cells stimulated with epidermal growth factor (EGF), and the increase of phosphorylation was inhibited by MEK1 inhibitor, PD98059. Furthermore, we observed that BCL-6 was associated with MAPK in vivo and its SPC region was important for this association. These results suggest that the functions of BCL-6 are regulated by phosphorylation mediated by the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract The human homologue of Drosophila tumor suppressor dlg, hDLG1, is one of the proteins known to interact with APC, a tumor suppressor for colorectal cancer. Alternative splicing of this gene generates transcripts either with [insertion 1 (I1)] or without 99 nucleotides in the 5' part of the dlg homology repeats (DHR) domain. We found almost equivalent expression of these two splicing variants in most human tissues; however, in skeletal muscle the transcript with the 99-bp insertion was predominant, and in the brain, that without the 99-bp insertion was expressed predominantly. We also examined alternative splicing in the region between the SH3 and GUK domains where two different sizes of insertions, 34 nucleotides (I2) or 100 nucleotides (I3), had been reported, and found various splicing patterns among the tissues examined. In brain we detected six different, alternatively spliced transcripts, two of which included a novel, 36-bp, brain-specific exon encoding a peptide bearing significant homology to a portion of rat synapse-associated protein, SAP97/PSD95. Subsequently, we investigated the splicing patterns of the hDLG1 gene in 24 neuroblastoma cell lines. In two-thirds of these lines, the splicing patterns were altered from those observed in normal brain tissue. As one-third retained the normal brain-splicing pattern, the loss of normal splicing of hDLG1 may not in itself cause formation of tumors, but it might reflect the biological character of individual neuroblastomas. [ABSTRACT FROM AUTHOR]

Abstract: DNA polymerase α (pol-α) is a heterotetrameric enzyme (p180–p68–p58–p48 in mouse) that is essential for the initiation of chain elongation during DNA replication. The catalytic (p180) and p68 subunits of pol-α are phosphorylated by Cdk–cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G2 phase of the cell cycle. The activity of Cdk2–cyclin A increases during late S phase and peaks in G2 phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-α and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-α by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-α activity by ppRb, suggesting that p68 might impede the association of ppRb with p180. Phosphorylation of p68 by Cdk2–cyclin A greatly reduced its inhibitory effect. Immunofluorescence analysis also revealed that ppRb localized at sites of DNA replication specifically in late S phase. These results suggest that Cdk–cyclin A can phosphorylate pol-α which may result in a conformational change in pol-α facilitating its interaction with and activation by ppRb. [Copyright &y& Elsevier]

Abstract: We examined the subcellular distribution of tumor suppressor adenomatous polyposis coli (APC) in developing cultured rat hippocampal neurons using both immunofluorescent microscopy and immunoelectron microscopy. APC initially localized at the distal tips of all the cell processes. Between 12 and 24h after plating, APC concentrated at the growth cone and in the distal portion of the longest process, which was growing very rapidly. The other processes, growing at a much slower rate, showed only weak immunoreactivity for APC. After maturation of the neurons, APC in the axons was restricted to the growth cones and distal portions. In contrast, APC formed a punctuated pattern along the dendritic processes. This subcellular distribution of APC was dependent on the organization of microtubules, but not actin filaments. Moreover, treatment of neurons with a PKCζ specific inhibitor caused defects in the staining pattern of APC. These results suggest that APC may be involved in neuronal process extension, and that APC may exert different functions on axons and dendritic processes. [Copyright &y& Elsevier]

The formation of the neuromuscular synapse requires muscle-specific receptor kinase (MUSK) to orchestrate postsynaptic differentiation, including the clustering of receptors for the neurotransmitter acetylcholine. Upon innervation, neural agrin activates MuSK to establish the postsynaptic apparatus, although agrin-independent formation of neuromuscular synapses can also occur experimentally in the absence of neurotransmission. Dok-7, a MuSK-interacting cytoplasmic protein, is essential for MuSK activation in cultured myotubes; in particular, the Dok-7 phosphotyrosine-binding domain and its target in MuSK are indispensable. Mice lacking Dok-7 formed neither acetylcholine receptor clusters nor neuromuscular synapses. Thus, Dok-7 is essential for neuromuscular synaptogenesis through its interaction with MUSK. [ABSTRACT FROM AUTHOR]

Cites the involvement of a novel protein RICS in the beta-catenin-N-cadherin and n-methyl-D-aspartate receptor signaling. Role of cadherin adhesion molecules in the synaptic plasticity; Regulation of synaptic structure; Dendritic spine morphology regulation.

Examines the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila. Role of Wnt/Wingless in cell development; Effect of Wnt/Wingless signaling; Interaction of the Drosophila protein D-Axin with Armadillo and D-APC; Impact of the mutation of D-Axin; Ectopic expression of D-Axin inhibiting Wingless signaling.

Cadherins mediate Ca2+-dependent cell--cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with β-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a β-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/β-catenin complex in the ER and ER exit site, resulting in a decrease in cell--cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with β-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner γ-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/β-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling. [ABSTRACT FROM AUTHOR]