Norio Masuko

Interaction of NE-dlg/SAP102, a Neuronal and Endocrine Tissue-specific Membrane-associated Guanylate Kinase Protein, with Calmodulin and PSD-95/SAP90 A POSSIBLE REGULATORY ROLE IN MOLECULAR CLUSTERING AT SYNAPTIC SITES

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends ofN-methyl-d-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K d value of 44 nm. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Cloning and characterization of NE-dlg: A novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dlg). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion: Possible involvement of down-regulation of β-catenin by NE-dlg expression

Membrane‐associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell‐cell contact. In Drosophila, mutations of the lethal (1)‐discs large (dlg) gene, which encodes a MAGUK protein, leads to post‐synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE‐dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE‐dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non‐neuronal cells. Overexpression of NE‐dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE‐dlg overexpression caused the down‐regulation of β‐catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE‐dlg were found to be essential for the growth suppression, loss of adhesive property and down‐regulation of β‐catenin. We propose that NE‐dlg regulates the cell growth and adhesive ability by controlling the level of β‐catenin through an APC‐independent pathway. Inactivation of NE‐dlg may therefore contribute to development and/or progression of human neoplasms. Int. J. Cancer 86:480–488, 2000.

A novel cinnamic acid derivative that inhibits Cdc25 dual-specificity phosphatase activity

The Cdc25 dual‐specificity phosphatases are key regulators of cell cycle progression through activation of cyclin‐dependent kinases (Cdk). Three homologs exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25A and Cdc25B have oncogenic properties and are overexpressed in some types of tumors. Compounds that inhibit Cdc25 dual‐specificity phosphatase activity might thus be potent anticancer agents. We screened several hundred compounds in a library using an in vitro phosphatase assay, with colorimetric measurement of the conversion of p‐nitrophenyl phosphate (pNPP) to p‐nitrophenol by the catalytic domain of recombinant human Cdc25, and discovered TPY‐835, which inhibits Cdc25A and Cdc25B activity (IC50 = 5.1 and 5.7 µM, respectively). TPY‐835 had mixed inhibition kinetics for Cdc25A and Cdc25B. TPY‐835 caused cell cycle arrest in the G1 phase in human lung cancer cells (A549 and SBC‐5) but not cell cycle arrest in the G2/M phase. After treatment with TPY‐835, the activation of Cdk2 was suppressed and phosphorylation of the retinoblastoma (Rb) protein was decreased in SBC‐5 cells. In addition, TPY‐835 induced an increase of the sub‐G1 phase cell population after 48–72 h treatment. The growth inhibitory effects of TPY‐835 against cisplatin (CDDP)‐, camptothecin‐ and 5‐FU‐resistant cell lines are comparable to the growth inhibitory effect on their parental lines, thus indicating that TPY‐835 did not show cross‐resistance to these cell lines. These results suggest that TPY‐835 is a promising candidate for constructing a novel class of antitumor agents that can control the cell cycle progression of cancer cells. (Cancer Sci 2005; 96: 614–619)

A Novel NE-dlg/SAP102-associated Protein, p51-nedasin, Related to the Amidohydrolase Superfamily, Interferes with the Association between NE-dlg/SAP102 and N-Methyl-d-aspartate Receptor

The membrane-associated guanylate kinase proteins have been known to interact various membrane receptors with their N-terminal segments designated the PDZ domains and to cluster these receptors at the target site of the cell membrane. NE-dlg/SAP102, a neuronal and endocrine tissue-specific MAGUK family protein, was found to be expressed in both dendrites and cell bodies in neuronal cells. Although NE-dlg/SAP102 localized at dendrites was shown to interact with N-methyl-d-aspartate receptor 2B via the PDZ domains to compose postsynaptic density, the binding proteins existing in the cell body of the neuron are still unknown. Here we report the isolation of a novel NE-dlg/SAP102-associated protein, p51-nedasin. Nedasin has a significant homology with amidohydrolase superfamily proteins and shows identical sequences to a recently identified protein that has guanine aminohydrolase activity. Nedasin has four alternative splice variants (S, V1, V2, and V3) that exhibited different C-terminal structures. NE-dlg/SAP102 is shown to interact with only the S form of nedasin which is predominantly expressed in brain. The expression of nedasin in neuronal cells increases in parallel with the progress of synaptogenesis and is mainly detected in cell bodies where it co-localizes with NE-dlg/SAP102. Furthermore, nedasin interferes with the association between NE-dlg/SAP102 and NMDA receptor 2B in vitro. These findings suggest that alternative splicing of nedasin may play a role in the formation and/or structural change in synapses during neuronal development by modifying clustering of neurotransmitter receptors at the synaptic sites.