Volume 272, Number 43, Issue of October 24, 1997 pp. 26899-26904
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Homologue of the Drosophila Discs Large Tumor Suppressor Binds to p56^lck Tyrosine Kinase and Shaker Type Kv1.3 Potassium Channel in T Lymphocytes

(Received for publication, March 2, 1997, and in revised form, June 25, 1997)

Toshihiko Hanada , Lunhui Lin , K. George Chandy ^§ , S. Steven Oh and Athar H. Chishti

From the  Laboratory of Tumor Cell Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135 and the ^§ Departments of Microbiology and Molecular Genetics, University of California, Irvine, California 92697

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 (SD-95/iscs large/O-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 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 immunoprecipitates. Direct interaction between hDlg and p56^lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56^lck expressed in the baculovirus-infected Sf9 cells. The p56^lck 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 p56^lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.

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