Fujikawa Akihiro, Matsumoto Masahito, Kuboyama Kazuya, Suzuki Ryoko, Noda Masaharu
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan; School of Life Science, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan.
PLoS One. 2015 Mar 5;10(3):e0119361. doi: 10.1371/journal.pone.0119361. eCollection 2015.
G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.
G蛋白偶联受体激酶相互作用蛋白1（Git1）作为一种衔接蛋白，将Pix和PAK等信号蛋白与粘着斑蛋白连接起来，从而参与细胞运动的控制。我们之前证明Git1是一种多酪氨酸磷酸化蛋白，其主要磷酸化位点是粘着斑靶向同源（FAH）结构域附近的Tyr-554，并且该位点可被Z型蛋白酪氨酸磷酸酶受体（Ptprz）选择性去磷酸化。在本研究中，基于使用Git1的Tyr-554突变体进行的免疫沉淀实验，我们发现Tyr-554磷酸化降低了Git1与FAH结构域结合蛋白桩蛋白和Hic-5的结合。与野生型小鼠相比，Ptprz缺陷小鼠大脑中Git1的Tyr-554磷酸化水平更高，且其与桩蛋白的结合始终更低。然后，我们使用三种不同方法研究了Tyr-554磷酸化在细胞运动控制中的作用：随机细胞运动、伤口愈合和博伊登小室试验。内源性Git1的短发夹RNA介导的敲低损害了A7r5平滑肌细胞的运动。野生型Git1和一个Git1突变体（在多个潜在酪氨酸磷酸化位点中仅保留Tyr-554）的外源性表达挽救了运动缺陷，但Tyr-554磷酸化缺陷或磷酸化状态模拟的Git1突变体则不能。我们的结果表明，Git1的Tyr-554位点的循环磷酸化 - 去磷酸化对于Git1与桩蛋白/Hic-5之间的动态相互作用至关重要，以确保细胞运动的协调。
