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

Abstract

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.

Figure 1

Morphological change of SK-N-MC neuroblastoma cells by TrkA overexpression. SK-N-MC-TrkA cells (stable clones #9 and #21) were cultured in the absence (–Tet) or presence (+Tet) of tetracycline for 20 h. The cells were fixed and then stained with TRITC-labeled red against anti-TrkA antibody. Differential interference contrast (DIC) image and the intracellular localization of ectopic TrkA were analyzed by confocal microscopy. Merge panel shows combined image of DIC and TrkA.

Figure 2

GW441756 effects on the cellular processes by TrkA overexpression. (A) SK-N-MC-TrkA cells (#9) were untreated or treated with 2 μM of GW441756 for 12 h in the absence or presence of tetracycline. (B) SK-N-MC-TrkA cells (#9) were cultured in the absence or presence of tetracycline for 12 h and then untreated or treated with 2 μM of GW441756 for 24 h, leading to 36 h Tet-On induction. Whole cell extracts were analyzed by Western blot using antibodies against TrkA, phospho-Tyr, phospho-Y204-ERK, phospho-T183/Y185-JNK, and β-actin.

Figure 3

Representative 2DE images of GW441756 effects on TrkA-dependent targets. SK-N-MC-TrkA cells (#9) were cultured in the absence or presence of tetracycline for 12 h and then untreated or treated with 2 μM of GW441756 for 24 h. The cells were analyzed by 2DE and Commassie Blue stain.

Figure 4

Quantitative analysis of GW441756 inhibitory effects on TrkA-dependent protein spots. PDQuest image analysis of GW441756 effects on TrkA-dependent protein spots was performed with two sets of Commassie Blue stained 2DE images. Seven protein spots, which had same identification results in MALDI-TOF MS and MALDI-TOF/TOF MS/MS analysis, were more than twofold upregulated (A) or downregulated (B) by TrkA overexpression, and it was suppressed by GW441756 (compare protein spots indicated by arrows in left four panels). Relative intensities of the protein spots determined by PDQuest software have shown in right panels. Each bar represents the mean ± SD for two independent experiments, and data significance was evaluated with a Student's t-test, *p < 0.05. (C) 2DE profile of GW441756-controlled TrkA-dependent protein spots that had matched identification results in MALDI-TOF MS and MALDI-TOF/TOF MS/MS analysis.

Figure 5

Quantitative analysis of GW441756 inhibitory effects on a potential TrkA-dependent protein spots. Three protein spots of SSP 1122, 8105, and 8106 were more than twofold upregulated (A) or downregulated (B) by TrkA, and it was significantly suppressed by GW441756.

Figure 6

Verification of protein spots identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS analysis. Each protein spot and a piece of β-actin spot were excised from Commassie Blue stained gels and performed by 10% SDS-PAGE. The proteins were analyzed by Western blot using antibodies against α-tubulin (A), β-tubulin (B), hnRNP C1/C2 (C), and α-enolase (D). The NC membranes were reprobed by β-actin.

Figure 7

Investigation of TrkA and GW441756 effects on the identified proteins by 1DE/Western blot analysis. Protein samples (5 μg each) prepared for 2DE were separated by 10% SDS-PAGE and analyzed by Western blot using antibodies against phospho-TrkA, α-tubulin, acetylated α-tubulin, β-tubulin, hnRNP C1/C2, α-enolase, and β-actin.

Figure 8

Characterization of TrkA effects on the identified proteins by 2DE/Western blot analysis. Protein samples (10 μg each) prepared for 2DE were performed by 1D isoelectric focusing using 7 cm IPG strip (pH 4–7), separated by 10% SDS-PAGE and transferred to NC membrane. Panels a and b were detected by Western blot analysis using α-tubulin antibody, and the blots were reprobed with α-tubulin and β-actin antibodies (panels a’ and b’). Panels c and d were detected with hnRNP C1/C2 antibody, and panels e and f were detected with α-enolase antibody. Each arrow indicates major area modified by TrkA.

Figure 9

Confirmation of GW441756 effects on the identified protein spots by 2DE/Western blot analysis. Protein samples (50 μg each) were performed by 1D isoelectric focusing using 17 cm IPG strip (pH 4–7) and separated on 8.5–14% sucrose gradient polyacrylamide gels. The areas of α-tubulin and hnRNP C1/C2 protein spots were excised and transferred to NC membrane. Panels a and b were performed by Western blot analysis using α-tubulin antibody, and the blots were reprobed with α-tubulin and β-actin antibodies (panels a’ and b’). Panels c and d were detected with hnRNP C1/C2 antibody, and the blots were reprobed with hnRNP C1/C2 and β-actin antibodies (panels c’ and d’).