Cell-matrix adhesions differentially regulate fascin phosphorylation
Cell-matrix adhesions differentially regulate fascin phosphorylation
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DOI：
10.1091/mbc.10.12.4177
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发表时间：
1999-12-01
影响因子：
3.3
通讯作者：
Zhang, LL
中科院分区：
文献类型：
--
作者：
Adams, JC;Clelland, JD;Zhang, LL
细胞粘附到细胞外基质的单个大分子上对肌动蛋白捆绑蛋白：肌成束蛋白的亚细胞定位和细胞形成稳定肌成束蛋白微刺的能力具有显著影响。肌成束蛋白的肌动蛋白结合活性被磷酸化下调，我们使用两种分化的细胞类型，C2 C12骨骼肌成肌细胞和LLC-PK 1肾上皮细胞，以检查细胞粘附到基质成分纤连蛋白，层粘连蛋白-l和血小板反应蛋白-l差异调节的假设：肌成束蛋白磷酸化。在这两种细胞类型中，用PKC激活剂12-tetradecanoyl phorbol 13-acetate（TPA）或粘附于纤连蛋白的处理导致1小时后肌成束蛋白的弥漫性分布。C2 C12细胞含有PKC家族成员α、γ和λ，并且PKC α定位在细胞粘附至纤连蛋白后改变。二维等电聚焦/SDS-聚丙烯酰胺凝胶被用来确定成束蛋白成为磷酸化的细胞粘附：纤连蛋白和PKC抑制剂calphostin C和chelerythrine氯化物抑制。在粘附于血小板反应蛋白-1或层粘连蛋白-1的细胞中未检测到肌成束蛋白磷酸化。表达绿色荧光蛋白（GFP）-肌成束蛋白的LLC-PK 1细胞也显示出类似的肌成束蛋白磷酸化调节。LLC-PK 1细胞表达GFP-肌成束蛋白S39 A，一个nonphosphorylatable突变体，没有经历传播和局部接触组织纤连蛋白，而细胞表达GFP-肌成束蛋白S39 D突变体与组成性负电荷传播更广泛比：野生型细胞。相比之下，共表达S39 A肌成束蛋白与内源性肌成束蛋白的C2 C12细胞仍然能够在血小板反应蛋白-1上形成微尖峰，并且表达肌成束蛋白S39 D的细胞附着于血小板反应蛋白-1但不形成微尖峰。通过TPA诱导的下调阻断PKC α活性导致纤维连接蛋白粘附的C2 C12和LLC-PK 1细胞中野生型肌成束蛋白的肌动蛋白结合，但不改变S39 A或S39 D肌成束蛋白的分布。肌成束蛋白与肌动蛋白在纤维连接蛋白粘附细胞中的关联在整合素α 5亚基的抑制性抗体的存在下也是明显的，这些新的结果建立了基质启动的PKC依赖性调节肌成束蛋白在丝氨酸39处的磷酸化作为基质粘附与细胞骨架结构的组织相耦合的机制。
Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein:fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-l, and thrombospondin-l differentially regulates:fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1h. C2C12 cells contain the PKC family members alpha, gamma, and lambda, and PKC alpha localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent:to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-l or to laminin-l. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively-than :wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-l, and cells that expressed fascin S39D attached to thrombospondin-l but did not form microspikes. Blockade of PKC alpha activity-by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin alpha 5 subunit, These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.