Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo.
Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo.
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DOI：
10.1042/bj3610525
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发表时间：
2002
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影响因子：
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通讯作者：
Wendy A. Kimber;L. Trinkle-Mulcahy;P. Cheung;M. Deák;Louisa J. Marsden;A. Kieloch;S. Watt;R. Javier;A. Gray;C. Downes;J. Lucocq;D. Alessi
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作者：
Wendy A. Kimber;L. Trinkle-Mulcahy;P. Cheung;M. Deák;Louisa J. Marsden;A. Kieloch;S. Watt;R. Javier;A. Gray;C. Downes;J. Lucocq;D. Alessi
PtdIns(3,4,5)P3 是生长因子和胰岛素诱导信号通路的既定第二信使。越来越多的证据表明，PtdIns(3,4,5)P3 的直接分解产物之一，即 PtdIns(3,4)P2，其水平可通过多种细胞外激动剂升高，也可能充当信号分子。最近，我们鉴定了两种相关的含普莱克斯特林同源 (PH) 结构域的蛋白，称为“串联 PH 结构域蛋白 1”(TAPP1) 和 TAPP2，它们在体外与 PtdIns(3,4)P2 具有高亲和力相互作用，但不结合 PtdIns(3,4,5)P3 或其他磷酸肌醇。在本研究中，我们证明用刺激 PtdIns(3,4)P2 产生的激动剂刺激 Swiss 3T3 或 293 细胞会导致 TAPP1 显着易位至质膜。这种募集依赖于功能性 PtdIns(3,4)P2 结合 PH 结构域，并受到渥曼青霉素（一种磷酸肌醇 3-激酶抑制剂，可防止 PtdIns(3,4)P2 生成）的抑制。对与 TAPP1 相互作用的蛋白质的研究发现了含有多 PDZ 的蛋白质，称为“MUPP1”，该蛋白质拥有 13 个 PDZ 结构域，并且没有其他已知的模块或催化结构域 [PDZ 是突触后密度蛋白 (PSD-95)/果蝇盘大肿瘤抑制蛋白 (dlg)/紧密连接蛋白 (ZO1)]。我们证明，对 293 细胞裂解物中内源表达的 TAPP1 进行免疫沉淀会导致内源 MUPP1 的免疫共沉淀，表明这些蛋白质可能在生理上彼此相互作用。我们发现 TAPP1 和 TAPP2 通过其 C 端氨基酸与 MUPP1 的第 10 个和第 13 个 PDZ 结构域相互作用。本研究的结果表明，TAPP1 和 TAPP2 可以在细胞中作为衔接蛋白发挥作用，以响应升高 PtdIns(3,4)P2 的信号，将 MUPP1 或它们可能与之相互作用的其他蛋白招募到质膜上。
PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed 'tandem-PH-domain-containing protein-1' (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed 'MUPP1', a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.