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.