Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

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. 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. Additions & CorrectionsMolecular & Cellular ProteomicsVol. 4Issue 8PreviewVol. 4 (2005) 291–299 Full-Text PDF Open Access Neurotrophins mediate growth, survival, and differentiation of normal sympathetic neurons. The tyrosine kinases TrkA, TrkB, and TrkC have been identified as the biologically active receptors for the neurotrophins nerve growth factor (NGF), 1The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; DIGE, difference gel electrophoresis; PMF, peptide mass fingerprint; 2D, two-dimensional; 2-DE, two-dimensional gel electrophoresis. brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3), respectively (1.Miller F.D. Kaplan D.R. On Trk for retrograde signaling.Neuron. 2001; 32: 767-770Google Scholar). Ligand binding induces Trk receptor dimerization and autophosphorylation of cytoplasmic tyrosines leading to the activation of various signaling pathways, including the Ras/MAPK pathway, the PLCγ pathway, and the PI3K pathway (2.Patapoutian