HUMAN HOMOLOG OF THE DROSOPHILA DISCS LARGE TUMOR-SUPPRESSOR BINDS TOP56(LCK) 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, Consis tent with the multi-domain organization of MAGUKs, hDlg consists of th ree copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate ki nase-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 prop erties of hDlg were studied by immunoprecipitation, immunoblotting, an d immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg, Im munoblotting experiments revealed that the immunoprecipitates of hDlg contain p56(lck), a member of the Src family of tyrosine kinases, The specificity of the interaction is demonstrated by the lack of p59(fyn) tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunop recipitates, Direct interaction between hDlg and p56(lck) is demonstra ted using glutathione S-transferase fusion proteins of hDlg and recomb inant p56(lck) expressed in the baculovirus infected Sf9 cells, The p5 6(lck) binding site was localized within the aminoterminal segment of hDlg containing proline-rich domain, In addition, we show in vivo asso ciation of hDlg with Kv1.3 channel, which was expressed in T lymphocyt es as an epitope-tagged protein using a vaccinia virus expression syst em, Taken together, these results provide the first evidence of a dire ct interaction between hDlg and p56(lck) tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.