Human homologue of the Drosophila discs large tumor suppressor binds to p56lck tyrosine kinase and Shaker type Kv1.3 potassium channel in T lymphocytes.
Human homologue of the Drosophila discs large tumor suppressor protein (hDlg) belongs to a newly discovered family of proteins termed MAGUKs that appear to have structural as well as signaling functions. Consistent with the multi-domain organization of MAGUKs, hDlg consists of three copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate kinase-like domain. In addition, the hDlg contains an amino-terminal proline-rich domain that is absent in other MAGUKs. To explore the role of hDlg in cell signaling pathways, we used human T lymphocytes as a model system to investigate interaction of hDlg with known tyrosine kinases. In human T lymphocyte cell lines, binding properties of hDlg were studied by immunoprecipitation, immunoblotting, and immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg. Immunoblotting experiments revealed that the immunoprecipitates of hDlg contain p56lck, a member of the Src family of tyrosine kinases. The specificity of the interaction is demonstrated by the lack of p59fyn tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunoprecipitates. Direct interaction between hDlg and p56lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56lck expressed in the baculovirus-infected Sf9 cells. The p56lck binding site was localized within the amino-terminal segment of hDlg containing proline-rich domain. In addition, we show in vivo association of hDlg with Kv1.3 channel, which was expressed in T lymphocytes as an epitope-tagged protein using a vaccinia virus expression system. Taken together, these results provide the first evidence of a direct interaction between hDlg and p56lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.
Hanada
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Laboratory of Tumor Cell Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.
Lin
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Chandy
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D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D000074288:Discs Large Homolog 1 Protein; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase; D006801:Humans; D019169:Jurkat Cells; D051664:Kv1.3 Potassium Channel; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins; D015221:Potassium Channels / Q000302:isolation & purification / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; C496973:KCNA3 protein, human; D051664:Kv1.3 Potassium Channel; D008565:Membrane Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)