Bradbury Peta M, Turner Kylie, Mitchell Camilla, Griffin Kaitlyn R, Middlemiss Shiloh, Lau Loretta, Dagg Rebecca, Taran Elena, Cooper-White Justin, Fabry Ben, O'Neill Geraldine M
Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales 2000, Australia.
J Cell Sci. 2017 Apr 1;130(7):1263-1273. doi: 10.1242/jcs.192930. Epub 2017 Feb 21.
Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.
粘着斑蛋白的Cas家族成员包含一个高度保守的C端粘着斑靶向（FAT）结构域。为了确定FAT结构域在这些蛋白中的作用，我们将野生型外源性NEDD9与一种杂交构建体进行了比较，在该杂交构建体中，NEDD9的FAT结构域已被p130Cas（也称为BCAR1）的FAT结构域所取代。光漂白后荧光恢复（FRAP）显示，融合蛋白在粘着斑处的交换显著减慢，二维迁移也显著变慢。使用原子力显微镜（AFM）测量的细胞硬度以及用磁镊装置测量的细胞粘附力均未检测到差异。因此，迁移减慢并非由于细胞硬度或粘附强度的变化。对在刚性增加的表面上细胞迁移的分析显示，表达p130Cas FAT结构域的细胞中细胞运动性显著降低。p130Cas FAT结构域诱导了NEDD9内酪氨酸残基的刚性依赖性磷酸化。这反过来又减少了NEDD9的翻译后切割，我们的研究表明这会抑制NEDD9诱导的迁移。因此，我们的数据共同表明，p130Cas FAT结构域独特地赋予了一种机械传感功能。
