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
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
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DOI：
10.1074/jbc.272.43.26899
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发表时间：
1997-10-24
影响因子：
4.8
通讯作者：
Chishti, AH
中科院分区：
文献类型：
--
作者：
Hanada, T;Lin, LH;Chishti, AH
果蝇盘状大肿瘤抑制蛋白（Drosophila discs large tumor suppressor protein，hDlg）是一个新发现的具有结构和信号传导功能的MAGUKs蛋白家族。与MAGUKs的多结构域结构一致，hDlg由三个PDZ拷贝组成（PSD-95/Discs large/z 0 -1）结构域、SH 3基序和鸟苷酸激酶样结构域。此外，hDlg含有在其他MAGUK中不存在的氨基末端富含脯氨酸的结构域。为了探讨hDlg在细胞信号通路中的作用，我们以人T淋巴细胞为模型系统，研究了hDlg与已知酪氨酸激酶的相互作用。在人T淋巴细胞系中，通过免疫沉淀、免疫印迹和免疫复合物激酶测定研究了hDlg的结合特性。我们的研究结果表明蛋白酪氨酸激酶活性与hDlg免疫沉淀物有关，免疫印迹实验表明hDlg免疫沉淀物含有酪氨酸激酶Src家族成员p56（lck），hDlg免疫沉淀物中缺乏p59（fyn）酪氨酸激酶和磷脂酰肌醇3-激酶，利用杆状病毒感染的Sf 9细胞表达的hDlg和重组p56（lck）的谷胱甘肽S-转移酶融合蛋白，证明了hDlg和p56（lck）之间的直接相互作用。p56（lck）结合位点位于hDlg的氨基末端含有脯氨酸的结构域。我们显示了hDlg与Kv1.3通道的体内结合，所述Kv1.3通道使用牛痘病毒表达系统在T淋巴细胞中表达为表位标记的蛋白。总之，这些结果提供了hDlg和p56（lck）酪氨酸激酶之间直接相互作用的第一个证据，并提示了hDlg在偶联酪氨酸激酶和电压中的新功能。T淋巴细胞门控钾通道
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 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 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 aminoterminal 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.