A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin
A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin
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DOI：
10.1242/jcs.003806
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发表时间：
2007-11-15
影响因子：
4
通讯作者：
Burridge, Keith
中科院分区：
文献类型：
--
作者：
Dubash, Adi D.;Wennerberg, Krister;Burridge, Keith
细胞与细胞外基质蛋白如纤连蛋白的粘附启动影响细胞形态、迁移和存活的信号级联。这些信号通路中的一些涉及GTP酶的Rho家族，例如Cdc 42、Rac 1和RhoA，它们在调节细胞骨架的组织中起关键作用。虽然在理解Rho蛋白如何控制细胞骨架结构方面取得了重大进展，但对控制GTP酶本身激活的信号知之甚少。本研究的重点是确定哪些鸟嘌呤核苷酸交换因子（S）负责激活RhoA下游的粘附到纤连蛋白。使用亲和下拉测定激活的交换因子，我们表明，RhoA特异性交换因子LSC/p115 RhoGEF和LARG被激活时，细胞被铺板到纤连蛋白，但不是其他交换因子，如Ect 2或Db 1。Lsc和LARG一起敲低显著降低RhoA活化和应力纤维的形成以及纤连蛋白粘附下游的粘着斑。类似地，Lsc/p115 RhoGEF的催化失活突变体的过表达抑制RhoA活性和应力纤维的形成以及纤连蛋白上的粘着斑。这些数据建立了一个以前未表征的作用，交换因子LSC/p115 RhoGEF和LARG连接纤连蛋白信号下游RhoA激活。
Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.