de Graauw Marjo, Tijdens Ine, Smeets Mirjam B, Hensbergen Paul J, Deelder André M, van de Water Bob
Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratoria, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Mol Cell Biol. 2008 Feb;28(3):1029-40. doi: 10.1128/MCB.01247-07. Epub 2007 Dec 10.
Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.
肌动蛋白细胞骨架的动态重塑是细胞铺展、运动和迁移所必需的，并且可以由酪氨酸激酶活性调节。磷酸化酪氨酸蛋白质组学筛选显示，脂质、钙和肌动蛋白结合蛋白膜联蛋白A2（AnxA2）在酪氨酸23位点的磷酸化是ts-v-Src激酶诱导的细胞散射之前的一个主要事件。磷酸化模拟突变体Y23E-AnxA2本身的表达足以诱导MDCK细胞中的肌动蛋白重组和细胞散射。虽然Y23E-AnxA2而非Y23A-AnxA2增强了Src或肝细胞生长因子（HGF）诱导的细胞散射，但短发夹RNA介导的AnxA2敲低抑制了v-Src和HGF诱导的细胞散射。仅在存在HGF的情况下，在表达野生型AnxA2的细胞中诱导三维分支形态发生，而Y23E-AnxA2诱导不依赖HGF的分支形态发生。AnxA2的敲低阻止了囊肿形成过程中的管腔形成。Y23E-AnxA2诱导的散射与肌动蛋白切割蛋白丝切蛋白的去磷酸化/激活有关。同样，无活性的S3E-丝切蛋白和组成型活性的LIM激酶（丝切蛋白的直接上游激酶）抑制Y23E-AnxA2诱导的散射。总之，我们的研究表明AnxA2磷酸化在细胞散射和分支形态发生的背景下调节丝切蛋白依赖性肌动蛋白细胞骨架动力学中起着至关重要的作用。
