Bryan Brad, Cai Yi, Wrighton Katharine, Wu Gangyi, Feng Xin-Hua, Liu Mingyao
Alkek Institute of Biosciences and Technology, Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, Houston, TX 77030, USA.
FEBS Lett. 2005 Feb 14;579(5):1015-9. doi: 10.1016/j.febslet.2004.12.074. Epub 2005 Jan 13.
The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in non-neuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCzeta-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance.
小GTP酶的Rho家族由神经突生长、轴突导向和树突分支的重要调节因子组成。先前的研究在非神经元细胞类型中表明，E3泛素连接酶Smurf1通过PKCζ依赖性募集到细胞突出部位，以及随后RhoA的泛素化和蛋白酶体降解来调节细胞极性和突出活性。在本研究中，我们表明Smurf1增强了Neuro2a神经母细胞瘤细胞中的神经突生长。我们证明RhoA被泛素化，并且Smurf1和RhoA在体内存在物理相互作用。有趣的是，在Neuro2a细胞中过表达Smurf1在二丁酰环磷酸腺苷诱导神经突生长期间显著降低RhoA蛋白水平，但在视黄酸诱导神经突生长时则不然。使用通用蛋白酶体抑制剂MG132可消除这种Smurf1依赖性的RhoA蛋白水平降低，这表明RhoA通过Smurf1被靶向进行泛素化和降解。总之，我们的数据表明，特定导向信号对Rho GTP酶不同亚群的局部调节导致RhoA活性的细胞内不对称，这可能调节神经突生长和导向。
