Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation
Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation
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DOI：
10.1016/j.bbamcr.2020.118739
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发表时间：
2020-09-01
影响因子：
5.1
通讯作者：
McCulloch, Christopher A.
中科院分区：
文献类型：
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作者：
Ding, Isabel;Ostrowska-Podhorodecka, Zofia;McCulloch, Christopher A.
在细胞迁移的延伸的形成需要紧密协调的重组的所有三种细胞骨架聚合物，但中间丝网络与肌动蛋白相互作用，以产生扩展的机制还没有得到很好的定义。我们研究了肌动蛋白结合蛋白细丝蛋白A（FLNA）与波形蛋白在成纤维细胞延伸形成中的相互作用。成纤维细胞中波形蛋白的敲低（KD）使细胞延伸的长度减少50%（p < 0.001）。细胞与纤连蛋白结合后，波形蛋白中丝氨酸39、56和72的磷酸化程度随时间增加，这与波形蛋白丝组装有关。在能够磷酸化波形蛋白的FLNA相互作用激酶中，我们将重点放在PAK1上，我们通过与FLNA相关的相互免疫沉淀发现了PAK1。酶抑制剂研究和siRNA KD表明，PAK1是波形蛋白磷酸化和细胞延伸形成所必需的。在沉降测定中，波形蛋白仅在表达FLNA的细胞的不溶性沉淀部分中检测到，而在FLNA KD细胞中，FLN KD细胞的上清液中的波形蛋白增加。与野生型相比，FLNA KD细胞显示波形蛋白中丝氨酸56和72的磷酸化损失，并且细胞延伸的数量和长度减少> 4倍。我们认为PAK1与FLNA的关联使得波形蛋白磷酸化和细丝组装成为可能，这在细胞迁移过程中细胞延伸的发展和稳定中非常重要。
The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by > 4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.