An aPKC-exocyst complex controls paxillin phosphorylation and migration through localised JNK1 activation.
An aPKC-exocyst complex controls paxillin phosphorylation and migration through localised JNK1 activation.
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DOI：
10.1371/journal.pbio.1000235
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发表时间：
2009-11
期刊：
影响因子：
9.8
通讯作者：
Parker PJ
中科院分区：
文献类型：
--
作者：
Rosse C;Formstecher E;Boeckeler K;Zhao Y;Kremerskothen J;White MD;Camonis JH;Parker PJ
外囊/aPKC复合物控制迁移细胞前沿的激酶JNK和ERK的时空激活，从而控制细胞迁移过程中粘附蛋白桩蛋白的动态行为。非典型蛋白激酶C（aPKC）亚型通过与Cdc 42和Par 6结合参与细胞极化和迁移。在不同的迁移模型中，外囊复合物已被证明参与分泌事件和迁移。通过RNA干扰（RNAi），我们表明外囊到迁移NRK细胞的前沿的极化递送依赖于aPKC。反过来，我们证明了aPKC定位在前沿是依赖于外囊。这种相互依赖性的基础来自双杂交、质谱和免疫共沉淀研究，这些研究表明存在由Kibra介导的aPKC-外囊相互作用。使用RNAi和小分子抑制剂，显示aPKC、Kibra和外囊对于NRK细胞迁移是必需的，并且进一步证明它们对于JNK在前沿的局部活化是必需的。通过aPKC对JNK的迁移相关控制决定了质膜底物桩蛋白（Paxillin）的JNK磷酸化，但不决定核JNK底物c-jun的磷酸化。该研究将aPKC的极化行为与外囊复合物的促迁移特性相结合，定义了与迁移细胞前沿JNK局部激活相关的高阶复合物，该复合物决定了迁移速率。细胞迁移是多细胞生物在胚胎发育、免疫应答和伤口愈合等事件中的一个重要过程。细胞迁移也有助于病理学的发展，例如癌细胞侵入健康组织。为了使细胞移动，关键分子必须以协调的方式参与;了解哪些分子以及它们如何以及何时工作（例如，在生理与病理条件下）将影响旨在抑制异常迁移的新疗法。迁移细胞必须协调两个关键过程：细胞的前边缘或“前导”边缘的延伸和后边缘的收缩。这两个过程都需要被称为粘着斑复合物的蛋白质组装的周转。在本文中，我们表明，两个不同的群体的调节剂的迁移- aPKC，蛋白激酶，和胞吐，一个复杂的蛋白质也被称为所需的胞吐-物理相互作用通过支架蛋白kibra。所有这些组分都是有效细胞迁移所需的，并且都以相互依赖的方式在移动细胞的前缘富集。在前沿，这些组分控制两种额外的蛋白激酶ERK和JNK的局部活化。在迁移细胞前部的ERK和JNK的激活反过来控制桩蛋白的磷酸化，桩蛋白是粘着斑的一个组成部分。桩蛋白的磷酸化与更动态的局灶性粘连的存在相关。因此，我们的数据表明，aPKC-kibra-外囊复合物在向迁移细胞的前沿传递局部刺激信号中起着至关重要的作用。
The exocyst/aPKC complex controls the spatiotemporal activation of the kinases JNK and ERK at the leading edge of migrating cells and thereby controls the dynamic behaviour of the adhesion protein paxillin during cell migration. Atypical protein kinase C (aPKC) isoforms have been implicated in cell polarisation and migration through association with Cdc42 and Par6. In distinct migratory models, the Exocyst complex has been shown to be involved in secretory events and migration. By RNA interference (RNAi) we show that the polarised delivery of the Exocyst to the leading edge of migrating NRK cells is dependent upon aPKCs. Reciprocally we demonstrate that aPKC localisation at the leading edge is dependent upon the Exocyst. The basis of this inter-dependence derives from two-hybrid, mass spectrometry, and co-immunoprecipitation studies, which demonstrate the existence of an aPKC–Exocyst interaction mediated by Kibra. Using RNAi and small molecule inhibitors, the aPKCs, Kibra, and the Exocyst are shown to be required for NRK cell migration and it is further demonstrated that they are necessary for the localized activation of JNK at the leading edge. The migration associated control of JNK by aPKCs determines JNK phosphorylation of the plasma membrane substrate Paxillin, but not the phosphorylation of the nuclear JNK substrate, c-jun. This plasma membrane localized JNK cascade serves to control the stability of focal adhesion complexes, regulating migration. The study integrates the polarising behaviour of aPKCs with the pro-migratory properties of the Exocyst complex, defining a higher order complex associated with the localised activation of JNK at the leading edge of migrating cells that determines migration rate. Cell migration is an essential process in multicellular organisms during such events as embryonic development, the immune response, and wound healing. Cell migration is also instrumental in the development of pathologies such as cancer cell invasion of healthy tissues. To make cells move, key molecules must be engaged in a coordinated manner; understanding which molecules, and how and when they work (for example, under physiological versus pathological conditions) will impact on new therapies designed to suppress abnormal migration. Migrating cells must coordinate two key processes: extension of the front or ‘leading’ edge of the cell and retraction of the back edge. Both processes require the turnover of protein assemblies known as focal adhesion complexes. In this paper we show that two different groups of regulators of migration – aPKC, a protein kinase, and exocyst, a complex of proteins also known to be required for exocytosis – interact physically via the scaffold protein kibra. All these components are required for efficient cell migration and all are enriched at the leading edge of moving cells, in a mutually dependent manner. At the leading edge, these components control the local activation of two additional protein kinases, ERK and JNK. The activation of ERK and JNK at the front of migrating cells in turn controls the phosphorylation of paxillin, a component of focal adhesions. Phosphorylation of paxillin is associated with the presence of more dynamic focal adhesions. Our data thus indicate that an aPKC-kibra-exocyst complex plays a crucial role in delivering local stimulatory signals to the leading edge of migrating cells.
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