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

doi:10.1074/jbc.272.43.26899

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*

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

Abstract

Human homologue of the Drosophiladiscs 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 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA66263 (to A. H. C.), GM-OD54872-01 (Shannon Award), and AI24783 (to K. G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: St. Elizabeth’s Medical Center, Bldg. ACH4, 736 Cambridge St., Boston, MA 02135. Tel.: 617-789-3118.
↵1 The abbreviations used are: GST, glutathioneS-transferase; PBS, phosphate-buffered saline; mAb, monoclonal antibody; MOPS, 4-morpholinepropanesulfonic acid; PI, phosphotidylinositol; PDZ, PSD-95/Discs large/zO-1.
↵2 T. Hanada and A. H. Chishti, unpublished data.
- Received March 2, 1997.
- Revision received June 25, 1997.