Konishi Hiroyuki, Namikawa Kazuhiko, Shikata Keiji, Kobatake Yuji, Tachibana Taro, Kiyama Hiroshi
Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, Osaka 545-8585, Japan.
J Biol Chem. 2007 Aug 10;282(32):23491-9. doi: 10.1074/jbc.M611703200. Epub 2007 Jun 14.
Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.
Akt介导的信号通路激活对于受损神经元的存活和再生至关重要。在本研究中，我们试图通过使用一种识别由Akt磷酸化的共有基序的抗体来鉴定新的Akt底物。使用过表达组成型活性Akt的PC12细胞。通过二维聚丙烯酰胺凝胶电泳，我们鉴定出抗体免疫染色增加的蛋白质斑点。质谱分析显示几个主要斑点为神经元中间丝蛋白外周蛋白。使用几个外周蛋白片段，在体外确定其磷酸化位点为其头部结构域中的Ser(66)。此外，免疫共沉淀实验表明，在HEK 293T细胞中Akt与外周蛋白的头部结构域相互作用。制备了一种针对磷酸化外周蛋白的抗体，不仅在Akt激活的培养细胞中，而且在神经损伤的舌下运动神经元中都观察到了磷酸化外周蛋白的诱导。这些结果表明，外周蛋白是体内Akt的一种新底物，其磷酸化可能在运动神经再生中起作用。
