Abstract
The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.
Highlights
The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), respectively
As the identification of specific effector proteins is of major interest for the understanding of the biological role of Trk receptors in particular in solid tumors, we here aimed brain-derived neurotrophic factor; DIGE, difference gel electrophoresis; PMF, peptide mass fingerprint; 2D, two-dimensional; 2-DE, twodimensional gel electrophoresis
To identify specific protein expression changes following TrkA or TrkB activation in neuroblastoma cells by their respective ligands, NGF or BDNF, we analyzed the proteome of SY5Y-TrkA, SY5Y-TrkB, or untreated control cells using the 2DE-based DIGE system
Summary
The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. We used the difference gel electrophoresis (DIGE) system together with MALDI-peptide mass fingerprint (PMF) MS analysis to detect reproducible proteome changes caused by ligand activation of SY5Y-TrkA or -TrkB cells in a time course from 0 to 24 h in five biologically independent experiments. Protein spots differentially expressed (p Ͻ 0.05) between either SY5Y-TrkA or SY5Y-TrkB cells and untreated controls in the time course following neurotrophin treatment were identified using MS.
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