ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality.
ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality.
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DOI：
10.1016/j.jmb.2008.10.009
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发表时间：
2008-12-26
影响因子：
5.6
通讯作者：
Shisheva, Assia
中科院分区：
文献类型：
--
作者：
Sbrissa, Diego;Ikonomov, Ognian C.;Fenner, Homer;Shisheva, Assia
PtdIns(3,5)P2 对于多细胞生物的分化和发育至关重要，因为 PtdIns(3,5)P2 合成酶 PIKfyve 或其相关调节剂 ArPIKfyve 的敲除是致命的。在之前的内源蛋白研究中，我们发现 Sac3（一种转化 PtdIns(3,5)P2 的磷酸酶）与 PIKfyve-ArPIKfyve 生物合成复合物相关。然而，这三种蛋白质是否足以组织/维持该复合物（称为PAS复合物）、它们如何相互作用以及这种三元关联的功能相关性是什么，仍然悬而未决。在转染的哺乳动物细胞中使用免疫共沉淀分析，三种蛋白质的水平增加或减少，单独或双重或三重组合，我们在此报告，三联体足以形成和维持 PAS 复合物。 ArPIKfyve 是与 Sac3 和 PIKfyve 相互作用的主要组织者，而 Sac3 允许 PAS 复合体中最大的 PIKfyve-ArPIKfyve 关联。我们进一步发现 ArPIKfyve 通过同聚相互作用支撑 PAS 复合物，并通过其保守的 C 端结构域介导。 ArPIKfyve-ArPIKfyve 接触位点的 C 端肽片段的引入有效地分解了 PAS 复合物并降低了体外 PIKfyve 脂质激酶活性。通过探索 3T3L1 脂肪细胞中胰岛素调节的 GLUT4 易位作为功能读数，这一过程受到 PIKfyve 活性和 ArPIKfyve 水平的正向调节，我们确定 ArPIKfyve C 端肽的异位表达会抑制 GLUT4 表面积累。我们的数据表明，PAS 复合物的组织是为了提供最佳的 PIKfyve 功能，并通过 ArPIKfyve 同聚和异聚相互作用来维持。
PtdIns(3,5)P2 is vital in differentiation and development of multicellular organisms because the knockout of the PtdIns(3,5)P2-synthesizing enzyme PIKfyve or its associated regulator, ArPIKfyve, is lethal. In previous work with endogenous proteins we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P2, associates with the PIKfyve-ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex (referred to as PAS complex), how they interact with each other and what the functional relevance of this ternary association would be, remained unresolved. Using coimmunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double vs. triple combinations, we report here that the triad is sufficient to form and maintain the PAS complex. ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve, whereas Sac3 is permissive for maximal PIKfyve-ArPIKfyve association in the PAS complex. We further identified that ArPIKfyve scaffolds the PAS complex through homomeric interactions, mediated via its conserved C-terminal domain. Introduction of the C-terminal peptide fragment of the ArPIKfyve-ArPIKfyve contact sites effectively disassembled the PAS complex and reduced the in vitro PIKfyve lipid kinase activity. Exploring insulin-regulated GLUT4 translocation in 3T3L1 adipocytes as a functional readout, a process that is positively regulated by PIKfyve activity and ArPIKfyve levels, we determined that ectopic expression of the ArPIKfyve C-terminal peptide inhibits GLUT4 surface accumulation. Our data indicate that the PAS complex is organized to provide optimal PIKfyve functionality and is maintained via ArPIKfyve homomeric and heteromeric interactions.
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影响因子：
4.8
作者：
Sbrissa, D;Ikonomov, OC;Shisheva, A
通讯作者：
Shisheva, A
影响因子：
4.8
作者：
Ikonomov, OC;Sbrissa, D;Shisheva, A
通讯作者：
Shisheva, A
影响因子：
3.3
作者：
Ikonomov, OC;Sbrissa, D;Shisheva, A
通讯作者：
Shisheva, A
影响因子：
4.8
作者：
Ishiki, M;Randhawa, VK;Klip, A
通讯作者：
Klip, A
影响因子：
4
作者：
Rutherford AC;Traer C;Wassmer T;Pattni K;Bujny MV;Carlton JG;Stenmark H;Cullen PJ
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Cullen PJ