Department of Biochemistry and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, South Korea.
Proteomics. 2013 Jan;13(2):355-67. doi: 10.1002/pmic.201200251.
Tropomyosin-related kinase A (TrkA) is a receptor-type protein tyrosine kinase and exploits pleiotypic roles via nerve growth factor (NGF)-dependent or NGF-independent mechanisms in various cell types. Here, we showed that the inhibition of TrkA activity by GW441756 resulted in the suppression of tyrosine phosphorylation of cellular proteins including extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). To find novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways, we investigated GW441756 effects on TrkA-dependent targets in SK-N-MC neuroblastoma cells by proteomic analysis. The major TrkA-dependent protein spots controlled by GW441756 were determined by PDQuest image analysis, identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS, and verified by 2DE/Western blot analysis. Thus, we found that most of the identified protein spots were modified forms in a normal condition, and their modifications were regulated by TrkA activity. Especially, our results demonstrated that the modifications of α-tubulin and heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) were significantly upregulated by TrkA, whereas α-enolase modification was downregulated by TrkA, and it was suppressed by GW441756, indicating that TrkA activity is required for their modifications. Taken together, we suggest here that the major novel TrkA-dependent targets such as α-tubulin, hnRNP C1/C2, and α-enolase could play an essential role in TrkA-mediated tyrosine phosphorylation signaling pathways via regulation of their posttranslational modifications.
原肌球蛋白相关激酶 A（TrkA）是一种受体型蛋白酪氨酸激酶，通过神经生长因子（NGF）依赖或非依赖机制在各种细胞类型中发挥多效性作用。在这里，我们表明 GW441756 抑制 TrkA 活性导致细胞蛋白酪氨酸磷酸化的抑制，包括细胞外信号调节蛋白激酶（ERK）和 c-Jun N-末端激酶（JNK）。为了寻找与 TrkA 介导的酪氨酸磷酸化信号通路相关的新靶标，我们通过蛋白质组学分析研究了 GW441756 对 SK-N-MC 神经母细胞瘤细胞中 TrkA 依赖性靶标的影响。通过 PDQuest 图像分析确定了由 GW441756 控制的主要 TrkA 依赖性蛋白点，通过 MALDI-TOF MS 和 MALDI-TOF/TOF MS/MS 鉴定，并通过 2DE/Western blot 分析验证。因此，我们发现大多数鉴定的蛋白点在正常条件下是修饰形式，其修饰受 TrkA 活性调节。特别是，我们的结果表明，α-微管蛋白和异质性核核糖核蛋白 C1/C2（hnRNP C1/C2）的修饰形式由 TrkA 显著上调，而α-烯醇化酶的修饰形式由 TrkA 下调，由 GW441756 抑制，表明 TrkA 活性是其修饰所必需的。总之，我们在这里提出，主要的新型 TrkA 依赖性靶标，如α-微管蛋白、hnRNP C1/C2 和α-烯醇化酶，可能通过调节其翻译后修饰在 TrkA 介导的酪氨酸磷酸化信号通路中发挥重要作用。
