Proteomic analysis of novel targets associated with TrkA‐mediated tyrosine phosphorylation signaling pathways in SK ‐ N ‐ MC neuroblastoma cells

Tropomyosin‐related kinase A ( T rk A ) 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 T rk A activity by GW 441756 resulted in the suppression of tyrosine phosphorylation of cellular proteins including extracellular signal‐regulated protein kinase (ERK) and c‐ J un N ‐terminal kinase (JNK). To find novel targets associated with T rk A ‐mediated tyrosine phosphorylation signaling pathways, we investigated GW 441756 effects on T rk A ‐dependent targets in SK ‐ N ‐ MC neuroblastoma cells by proteomic analysis. The major T rk A ‐dependent protein spots controlled by GW 441756 were determined by PDQ uest image analysis, identified by MALDI ‐ TOF MS and MALDI ‐ TOF / TOF MS / MS , and verified by 2 DE / W estern 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 T rk A activity. Especially, our results demonstrated that the modifications of α‐tubulin and heterogeneous nuclear ribonucleoproteins C 1/ C 2 (hnRNP C1/C2) were significantly upregulated by T rk A , whereas α‐enolase modification was downregulated by T rk A , and it was suppressed by GW 441756, indicating that T rk A activity is required for their modifications. Taken together, we suggest here that the major novel T rk A ‐dependent targets such as α‐tubulin, hnRNP C1/C2, and α‐enolase could play an essential role in T rk A ‐mediated tyrosine phosphorylation signaling pathways via regulation of their posttranslational modifications.