Identification of Focal Adhesion Kinase (FAK) and Phosphatidylinositol 3-kinase (PI3-kinase) as Par3 Partners by Proteomic Analysis
Identification of Focal Adhesion Kinase (FAK) and Phosphatidylinositol 3-kinase (PI3-kinase) as Par3 Partners by Proteomic Analysis
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DOI：
10.1002/cm.20444
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发表时间：
2010-05-01
期刊：
影响因子：
2.9
通讯作者：
Kaibuchi, Kozo
中科院分区：
文献类型：
--
作者：
Itoh, Norimichi;Nakayama, Masanori;Kaibuchi, Kozo
Partition defective 3 （Par3）参与多种极性事件，包括上皮细胞顶基极性的建立、神经元轴突/树突的特化以及前后极性细胞的定向迁移。Par3被认为作为一种支架蛋白，通过与各种伙伴蛋白如Par6、aPKC、Tiam1/2和Numb相互作用来调节细胞极性。然而，Par3在极化迁移中的作用模式在很大程度上仍然未知。为了探索Par3的功能，我们将Par3亲和柱层析和液相色谱-串联质谱（LC-MS/MS）的霰弹枪分析相结合，筛选Par3相互作用蛋白。我们从大鼠脑细胞质中获得了大约200个par3相互作用蛋白。其中，我们重点研究了FAK和PI3-kinase，因为它们都参与了细胞的定向迁移。FAK与PDZ结构域和Par3的卷曲卷曲区域相关，pi3激酶的p110与Par3的卷曲卷曲区域相关。Par3在扩散细胞中与FAK部分共定位。RNA干扰耗尽Par3可抑制粘附诱导的FAK和pi3激酶的激活，RNA干扰抵抗Par3可恢复其抑制作用。此外，黏附诱导的细胞扩散以及细胞向胶原的定向迁移都需要Par3。这些结果表明Par3直接与FAK和pi3激酶相互作用，增强了它们极化细胞迁移的活性。(C) 2010 Wiley-Liss, Inc
Partition defective 3 (Par3) is involved in a variety of polarity events including establishment of apico-basal polarity of epithelial cell, axon/dendrite specification of neurons and directional migration of cells with front-rear polarity. Par3 is thought to regulate cell polarity as a scaffold protein by interacting with various partner proteins such as Par6, aPKC, Tiam1/2 and Numb. However, the mode of actions of Par3 in polarized migration remains largely unknown. To explore Par3 functions, we screened Par3-interacting proteins by combining Par3 affinity column chromatography and shotgun analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We obtained about two hundred Par3-interacting proteins from the rat brain cytosol fraction. Among them, we focused on FAK and PI3-kinase, as both of them participate in directional cell migration. FAK associated with the PDZ domain and the coiled-coil region of Par3 and p110 of PI3-kinase associated with the coiled-coil region of Par3. Par3 was partially colocalized with FAK in spreading cells. Depletion of Par3 by RNA interference inhibited adhesion-induced activation of FAK and PI3-kinase, and RNA interference-resistant Par3 restored the inhibitory effects. In addition, Par3 was required for the adhesion-induced cell spreading as well as for directional cell migration toward collagen. These results suggest that Par3 directly interacts with FAK and PI3-kinase, enhancing their activities for polarized cell migration. (C) 2010 Wiley-Liss, Inc