Association of the T-cell protein tyrosine phosphatase with nuclear import factor p97
Association of the T-cell protein tyrosine phosphatase with nuclear import factor p97
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DOI：
10.1074/jbc.272.34.21548
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发表时间：
1997-08-22
影响因子：
4.8
通讯作者：
Tonks, NK
中科院分区：
文献类型：
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作者：
Tiganis, T;Flint, AJ;Tonks, NK
T细胞蛋白酪氨酸磷酸酶（TCPTP）转录物的选择性剪接产生两种形式的酶，它们在C末端不同：48-kDa内质网相关形式和45-kDa核形式。通过亲和层析，使用GST-TCPTP融合蛋白，我们分离了三种与TCPTP相互作用的120、116和97 kDa的胞质蛋白，p120蛋白与48-kDa形式的TCPTP的C末端的残基377-415相关，而p97和p116的识别位点被定位到包含TCPTP核定位序列（NLS）的残基350-381。TCPTP NLS显示为二分的，需要碱性残基350-358（基本群组一）和377-381（基本簇II），与p97和p116相互作用的位点，用于有效的核转位，p97，p116，TCPTP NLS的独特之处在于它们与SV 40大T抗原的经典NLS或核质蛋白的标准二分NLS不形成稳定的相互作用。p97基因序列分析表明其为核输入因子p97在透化细胞的体外测定中，p97对于TCPTP的最佳核输入是必需的，但不是充分的，我们发现，TCPTP共-与来自细胞裂解物的核输入因子p97免疫沉淀，纯化的重组p97与TCPTP在体外直接相互作用，这些结果表明p97和p116与TCPTP NLS结合的选择性，并表明p97可能介导不同于经典核输入过程的事件。这些结果表明，C-TCPTP的末端片段含有与蛋白质相互作用的对接位点，所述蛋白质可发挥作用以将酶靶向至限定的细胞内位置并在此过程中调节TCPTP功能。
Alternative splicing of the T-cell protein tyrosine phosphatase (TCPTP) transcript, generates two forms of the enzyme that differ at their extreme C termini: a 48-kDa endoplasmic reticulum-associated form and a 45-kDa nuclear form, By affinity chromatography, using GST-TCPTP fusion proteins, we have isolated three cytoplasmic proteins of 120, 116, and 97 kDa that interact with TCPTP, The p120 protein associated with residues 377-415 from the C terminus of the 48-kDa form of TCPTP, whereas the recognition site for p97 and p116 was mapped to residues 350-381 encompassing the TCPTP nuclear localization sequence (NLS). The TCPTP NLS was shown to be bipartite, requiring basic residues 350-358 (basic cluster I) and 377-381 (basic cluster II), the sites of interaction with p97 and p116, for efficient nuclear translocation, The interaction between p97, p116, and the TCPTP NLS appeared unique in that these proteins did not form a stable interaction with the classical NLS of SV40 large T antigen or the standard bipartite NLS of nucleoplasmin. Sequence analysis of p97 identified it as the nuclear import factor p97 (importin-beta), which is an essential component of the nuclear import machinery, In assays in vitro in permeabilized cells, p97 was necessary but not sufficient for optimal nuclear import of TCPTP, We found that TCPTP co-immunoprecipitated with the nuclear import factor p97 from cell lysates and that purified recombinant p97 and TCPTP interacted directly in vitro, These results indicate selectivity in the binding of p97 and p116 to the TCPTP NLS and suggest that p97 may mediate events that are distinct from the classical nuclear import process, Moreover, these results demonstrate that the C-terminal segment of TCPTP contains docking sites for interaction with proteins that may function to target the enzyme to defined intracellular locations and in the process regulate TCPTP function.