Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof Dr E De Robertis-CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.
PLoS One. 2012;7(2):e32087. doi: 10.1371/journal.pone.0032087. Epub 2012 Feb 23.
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.
芽蛋白（Spry）家族蛋白是受受体酪氨酸激酶（RTKs）诱导的下游信号通路的内源性调节剂。通过实时 PCR，我们检测到 Spry4 mRNA 的表达在 NGF 刺激下显著增加，表明 Spry4 可以调节 NGF 及其受体 TrkA 诱导的细胞内信号通路和生物学过程。在这项工作中，我们证明 Spry4 的野生型过表达导致 NGF 诱导的 MAPK 和 Rac1 激活以及神经突生长显著减少。在分子水平上，我们的发现表明，一种突变形式的 Spry4（Y53A）的异位表达，其中一个保守的酪氨酸残基被取代，不能阻断 TrkA 介导的 Erk/MAPK 激活和 NGF 诱导的神经突生长，表明 Spry4 对 NGF 信号的抑制作用需要完整的酪氨酸 53 位。使用小干扰 RNA 敲低实验下调 Spry4 增强了 PC12 细胞对 NGF 的分化和 MAPK 激活。总之，这些发现建立了一个新的生理机制，通过该机制 Spry4 调节神经突生长，不仅减少了 MAPK 通路，而且限制了 Rac1 激活对 NGF 的反应。
