Nusser Nóra, Gosmanova Elvira, Makarova Natalia, Fujiwara Yuko, Yang Linda, Guo Fukun, Luo Yongneng, Zheng Yi, Tigyi Gábor
Department of Physiology, University of Tennessee Health Science Center, Memphis, 894 Union Ave, Memphis, TN 38163, USA.
Cell Signal. 2006 May;18(5):704-14. doi: 10.1016/j.cellsig.2005.06.010. Epub 2005 Aug 16.
Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.
RhoA的激活会阻止神经生长因子（NGF）诱导的神经突生长，并导致包括PC12细胞在内的神经元细胞中的神经突回缩。尽管NGF对神经突生长有抑制作用，但它会激活RhoA的GTP负载以及效应物与之结合，这就产生了一个明显的矛盾。根据GTP酶功能的分子开关假说，RhoA的GTP负载应该足以统一激活其效应物。然而，当监测NGF诱导的GTP-RhoA与多个靶点的结合时，我们注意到它与效应物的相互作用存在差异。我们发现，NGF会引发蛋白激酶A介导的RhoA丝氨酸（188）位点的磷酸化，这使其无法与Rho相关激酶（ROK）结合，而它仍保留与其他RhoA靶点相互作用的能力，包括rhotekin、mDia-1和PKN。我们在体外和体内均表明，丝氨酸（188）的磷酸化代表了另一种开关，能够在效应物途径之间传导信号。在PC12细胞分化的背景下，NGF诱导的RhoA丝氨酸（188）位点的磷酸化使其无法与ROK相互作用，否则ROK会阻断神经突生长。将RhoA（S188A）突变体转染到PC12细胞中会阻止NGF诱导的神经突生长，就像组成型激活的RhoA（14V）一样，这表明该磷酸化位点的必要性。用模拟磷酸化的谷氨酸残基替换RhoA（V14/S188E）中的丝氨酸（188）会选择性地损害其与ROK的相互作用，并且当转染到PC12细胞中时会恢复NGF诱导的神经突生长。因此，丝氨酸（188）的磷酸化可能作为RhoA的一种新型二级开关，能够将GTP结合引发的效应物激活作用覆盖到与RhoA C末端相互作用的一部分靶点（如ROK）上。
