Type Iγ661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis
Type Iγ661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis
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DOI：
10.1074/jbc.m601465200
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发表时间：
2006-07-21
影响因子：
4.8
通讯作者：
Anderson, Richard A.
中科院分区：
文献类型：
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作者：
Bairstow, Shawn F.;Ling, Kun;Anderson, Richard A.
网格蛋白包被的囊泡介导膜结合蛋白的分选和细胞内转运。这些外壳的形成是由接合蛋白（adaptor proteins， AP）的组装启动的，接合蛋白通过识别内吞分选基序特异性地与膜货物蛋白结合。脂质信号分子磷脂酰肌醇4,5-二磷酸（PI(4,5)P-2）在这一过程中至关重要，因为它既是一个靶向因子，也是一个调节因子。PI(4,5) P2是由I型磷脂酰肌醇磷酸激酶（PIPKI）合成的。我们通过酵母双杂交筛选发现了AP2复合物的mu 2亚基与PIPKI gamma 661之间的直接相互作用。这种相互作用通过谷胱甘肽s转移酶拉降中的mu 2亚基和HEK293细胞中内源性PIPKI γ 661与AP2复合物的共免疫沉淀得到证实。这种相互作用在体内是由PIPKI γ 661的26个氨基酸尾部的酪氨酸基序介导的。由于AP2调节质膜上转铁蛋白受体的内吞作用，我们还使用流式细胞术内吞试验检测了PIPKI γ 661的作用。我们观察到野生型PIPKI γ 661在Madin-Darby犬肾细胞中的稳定表达增强了转铁蛋白的摄取，而激酶死亡的PIPKI γ 661的稳定表达具有抑制作用。两种情况都不影响PI(4,5) P2的整体细胞水平。使用相同的检测系统，基于RNA干扰的HeLa细胞中PIPKI γ 661的敲低也对转铁蛋白内吞有抑制作用。总的来说，这一证据表明PIPKI γ 661在ap2介导的转铁蛋白内吞作用中起重要作用。
Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is critical for this process, as it serves as both a targeting and regulatory factor. PI( 4,5) P2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu 2-subunit of the AP2 complex and PIPKI gamma 661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu 2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKI gamma 661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKI gamma 661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKI gamma 661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKI gamma 661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKI gamma 661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5) P2. RNA interference-based knockdown of PIPKI gamma 661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKI gamma 661 in the AP2-mediated endocytosis of transferrin.