Akihiko Matsumine

Binding of APC to the Human Homolog of the Drosophila Discs Large Tumor Suppressor Protein

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein β-catenin. Overexpression of APC blocks cell cycle progression. The APC-β-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum‐induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell‐cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down‐regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin‐CDK complexes.

Localization of the adenomatous polyposis coli tumour suppressor protein in the mouse central nervous system.

The adenomatous polyposis coli gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumours. The adenomatous polyposis coli gene product is a 300,000 mol. wt cytoplasmic protein that binds to at least three other proteins; beta-catenin, a cytoplasmic E-cadherin-associated protein; hDLG, a human homologue of the Drosophila discs large tumour suppressor protein and glycogen synthase kinase 3 beta, a mammalian homologue of the Drosophila ZESTE WHITE 3 protein. The adenomatous polyposis coli gene is highly expressed in the brain, suggesting that it may be involved in nerve function. Here we show that adenomatous polyposis coli is localized in the pericapillary astrocytic endfeet throughout the mouse central nervous system. Adenomatous polyposis coli is also localized in the astrocytic processes in the cerebellar granular layer, and displays concentrated expression in the terminal plexuses of the basket cell fibres around Purkinje cells. Adenomatous polyposis coli is further expressed in neuronal cell bodies and/or nerve fibres in the olfactory bulb, hippocampus, brain stem, spinal cord and dorsal root ganglia. Adenomatous polyposis coli is demonstrated to be co-localized with beta-catenin and/or hDLG in neurons and nerve fibres, but not in astrocytes. From these results, adenomatous polyposis coli is suggested to participate in a signal transduction pathway in astrocytes which is independent of beta-catenin and hDLG, and also in regulation of neuronal functions in association with beta-catenin and hDLG.

The colorectal tumour suppressor APC is present in the NMDA-receptor-PSD-95 complex in the brain

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.

Isolation and mapping of a human gene (RPD3L1) that is homologous to RPD3, a transcription factor in Saccharomyces cerevisiae

We have isolated a novel human gene RPD3L1, that is highly homologous to a transcription factor in Saccharomyces cerevisiae, RPD3 (reduced potassium dependency 3), from a human fetal lung cDNA library. The cDNA clone, hRPD3, consists of 2,100 nucleotides that contain an open reading frame of 1446 nucleotides encoding 482 amino acids. It shares 62% identity in nucleotide sequence and 52% identity in amino acid sequence to RPD3. This gene is expressed at various levels in all tissues examined. Furthermore, we were able to map it to chromosome band 1p34.1 by FISH.

SYT-SSX fusion proteins in synovial sarcomas : detection and characterization with new antibodies

To identify and characterize the SYT-SSX fusion proteins in synovial sarcomas, we developed two polyclonal antibodies against the N-terminal part and for the C-terminal part of the SYT-SSX2 protein. Specificity was demonstrated on COS-7 cells transfected with two subtypes of SYT-SSX fusion genes, SYT-SSX1 and SYT-SSX2. Both antibodies recognized a single protein of 61 kDa in an immunoprecipitation of the transfected COS-7 cell lysates. These antibodies also detected the native protein of 61 kDa in the lysate of a human synovial sarcoma cell line (HS-SY41) with immunoprecipitation, and in extracts of human synovial sarcomas with western blot analysis. An immunohistochemical study, using human synovial sarcoma tissues, demonstrated that the SYT-SSX fusion proteins localized in the nucleus of the tumor cells. These antibodies provide a useful method for studying the expression of the SYT-SSX fusion proteins.

Association of the MCC gene product with the plasma membrane and membrane organelles

The MCC (mutated in colorectal cancer) gene product was localized to the terminal web and microvilli and along the lateral plasma membranes in colon epithelial cells. In the cerebellum, the MCC protein was highly expressed in the molecular layer, and was associated with the plasma membrane and membrane organelles in neuronal components. In COS-7 cells overexpressing exogenous MCC, this protein was not only associated with the plasma membrane and membrane organelles but was also distributed throughout their cytoplasm. After permeabilization of the COS-7 cells, the MCC protein that remained in the cell was still associated with the plasma membrane and membrane organelles. These results indicate that the MCC protein binds to both plasma membrane and membrane organelles.