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

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 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 A. Reichardt L.F. Trk receptors: mediators of neurotrophin action.Curr. Opin. Neurobiol. 2001; 11: 272-280Google Scholar). The physiological functions of Trk receptors in the nervous system vary markedly ranging from induction of proliferation to synaptic modulation and axonal path-finding (reviewed in Refs. 3.Segal R.A. Selectivity in neurotrophin signaling: theme and variations.Annu. Rev. Neurosci. 2003; 26: 299-330Google Scholar and 4.Roux P.P. Barker P.A. Neurotrophin signaling through the p75 neurotrophin receptor.Prog. Neurobiol. 2002; 67: 203-233Google Scholar). There is considerable interest in aberrant Trk receptor expression or signaling as a hallmark of various diseases, including Alzheimer′s disease (5.Olivieri G. Otten U. Meier F. Baysang G. Dimitriades-Schmutz B. Muller-Spahn F. Savaskan E. Beta-amyloid modulates tyrosine kinase B receptor expression in SHSY5Y neuroblastoma cells: Influence of the antioxidant melatonin.Neuroscience. 2003; 120: 659-665Google Scholar) and solid tumors such as carcinomas (6.Koizumi H. Morita M. Mikami S. Shibayama E. Uchikoshi T. Immunohistochemical analysis of TrkA neurotrophin receptor expression in human non-neuronal carcinomas.Pathol. Int. 1998; 48: 93-101Google Scholar) and neuroblastoma (7.Nakagawara A. Azar C.G. Scavarda N.J. Brodeur G.M. Expression and function of TRK-B and BDNF in human neuroblastomas.Mol. Cell. Biol. 1994; 14: 759-767Google Scholar). Despite of the high degree of sequence similarity and broadly overlapping signaling pathways, activation of TrkA or TrkB causes divergent biological responses and phenotypes when expressed in the same cell type. For example, high expression of TrkA in neuroblastoma is associated with a favorable biology and a good patient outcome, whereas TrkB is mainly expressed on aggressive neuroblastomas with a poor patient outcome (7.Nakagawara A. Azar C.G. Scavarda N.J. Brodeur G.M. Expression and function of TRK-B and BDNF in human neuroblastomas.Mol. Cell. Biol. 1994; 14: 759-767Google Scholar, 8.Nakagawara A. Arima-Nakagawara M. Scavarda N.J. Azar C.G. Cantor A.B. Brodeur G.M. Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma.N. Engl. J. Med. 1993; 328: 847-854Google Scholar). The biological effect of Trk receptor signaling also varies between cell types. Activation of TrkA by NGF causes differentiation in neuronal precursors and neuroblastoma cells, whereas it induces proliferation in fibroblasts and apoptosis in medulloblastoma cells (9.Muragaki Y. Chou T.T. Kaplan D.R. Trojanowski J.Q. Lee V.M.-Y. Nerve growth factor induces apoptosis in human medulloblastoma cell lines that express TrkA receptors.J. Neurosci. 1997; 17: 530-542Google Scholar, 10.Micera A. Vigneti E. Pickholtz D. Reich R. Pappo O. Bonini S. Maquart F.X. Aloe L. Levi-Schaffer F. Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair.Proc. Natl. Acad. Sci. U S A. 2001; 98: 6162-6167Google Scholar). In medullary thyroid carcinoma, TrkB expression is associated with favorable biology and good prognosis (11.Sakamoto Y. Kitajima Y. Edakuni G. Sasatomi E. Mori M. Kitahara K. Miyazaki K. Expression of Trk tyrosine kinase receptor is a biologic marker for cell proliferation and perineural invasion of human pancreatic ductal adenocarcinoma.Oncol. Rep. 2001; 8: 477-484Google Scholar), which is in contrast to its role in neuroblastoma. The high level of sequence homology and overlapping signaling pathways of Trk receptors suggest the existence of specific effector molecules critical for receptor and cell-type-specific responses. Although some target molecules for tyrosine kinase signaling elements have been reported (12.Graves L.M. Guy H.I. Kozlowski P. Huang M. Lazarowski E. Pope R.M. Collins M.A. Dahlstrand E.N. Earp 3rd, H.S. Evans D.R. Regulation of carbamoyl phosphate synthetase by MAP kinase.Nature. 2000; 403: 328-332Google Scholar, 13.Pyronnet S. Imataka H. Gingras A.C. Fukunaga R. Hunter T. Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.EMBO J. 1999; 18: 270-279Google Scholar), there is still no comprehensive view of neurotrophin signaling at the proteome level. 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 to gain deeper insights into TrkA and TrkB signaling pathways of the childhood tumor neuroblastoma by a proteomic approach. The human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA was used a model system. The resulting biological phenotypes of these cell lines have been extensively analyzed (14.Eggert A. Ikegaki N. Liu X.-G. Chou T.T. Lee V.M. Trojanowski J.Q. Brodeur G.M. Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.Oncogene. 2000; 19: 2043-2051Google Scholar, 15.Eggert A. Grotzer M.A. Ikegaki N. Liu X.G. Evans A.E. Brodeur G.M. Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells.Cancer Res. 2002; 62: 1802-1808Google Scholar). Briefly, activation of SY5Y-TrkA cells by NGF resulted in neuronal differentiation, growth inhibition, and inhibition of angiogenesis (14.Eggert A. Ikegaki N. Liu X.-G. Chou T.T. Lee V.M. Trojanowski J.Q. Brodeur G.M. Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.Oncogene. 2000; 19: 2043-2051Google Scholar, 15.Eggert A. Grotzer M.A. Ikegaki N. Liu X.G. Evans A.E. Brodeur G.M. Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells.Cancer Res. 2002; 62: 1802-1808Google Scholar), whereas activation of SY5Y-TrkB cells by BDNF resulted in enhanced proliferation and resistance to chemotherapy (16.Ho R. Eggert A. Hishiki T. Minturn J.E. Ikegaki N. Foster P. Camoratto A.M. Evans A.E. Brodeur G.M. Resistance to chemotherapy mediated by TrkB in neuroblastomas.Cancer Res. 2002; 62: 6462-6466Google Scholar). Thus, the biological phenotypes of TrkA/B-transfected SY5Y cells corresponded well with the excellent outcome of TrkA-expressing neuroblastomas and the poor outcome of TrkB-expressing neuroblastomas. 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. SH-SY5Y is a subclone from the human SK-N-SH neuroblastoma cell line and lacks endogenous Trk receptor expression (17.Biedler J.L. Helson L. Spengler B.A. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture.Cancer Res. 1973; 33: 2643-2649Google Scholar). Full-length TrkA or TrkB cDNA were cloned into the retroviral expression vectors pLNCX or pLNCX2, respectively (Clontech, Palo Alto, CA). The resulting SY5Y-TrkA and SY5Y-TrkB cell lines have been characterized previously (14.Eggert A. Ikegaki N. Liu X.-G. Chou T.T. Lee V.M. Trojanowski J.Q. Brodeur G.M. Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.Oncogene. 2000; 19: 2043-2051Google Scholar). In contrast to SY5Y, the SY5Y-TrkA and SY5YTrkB cell lines demonstrated high and comparable expression of the inserted receptor on mRNA and protein levels (14.Eggert A. Ikegaki N. Liu X.-G. Chou T.T. Lee V.M. Trojanowski J.Q. Brodeur G.M. Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.Oncogene. 2000; 19: 2043-2051Google Scholar). Cells were grown in RPMI 1640 containing 10% FBS, l-glutamine, and 500 μg/ml geneticin (Life Technologies, Inc., Eggenstein, Germany). Neither BDNF nor NGF was detectable by ELISA in the FCS used (data not shown). Ligand-induced activation of Trk receptors was accomplished by supplementing the culture medium with either 100 ng/ml NGF or 50 ng/ml BDNF. Cells were harvested at five consecutive time points (0, 0.5, 1, 6, and 24 h) after addition of the ligand. These time points were chosen to monitor immediate responses to neurotrophins as well as late effector proteins. To allow for statistically robust analyses, five independent experiments were conducted. Cells were washed with 3 ml of ice-cold PBS, scraped from the dish, and centrifuged 5 min at 4 °C and 1,000 × g. The cell pellet was resuspended in 1 ml of ice-cold PBS and centrifuged for another 5 min at 4 °C and 2,000 × g. After determining the wet weight of the cell pellet, it was frozen in liquid nitrogen and stored at −80 °C. Proteins in each sample were fluorescently tagged with one of a set of matched fluorescent dyes designed to minimally interfere with protein mobility during two-dimensional electrophoresis (2-DE). Cell pellets (100 mg) were lysed by sonication (6 × 10-s pulses on ice) in 148 μl of sample buffer (30 mm TrisHCl; 2 m thiourea; 7 m urea; 4% CHAPS, pH 8.5). After centrifugation at 1,500 × g for 5 min, protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Stock cyanine dyes (1 nmol/μl) were diluted in anhydrous DMF p.a. to 400 pmol/μl (Sigma, St. Louis, MO), and 8 pmol dye was added per μg protein in the cell lysate. The sample was vortexed, centrifuged briefly, and left on ice for 0.5 h in the dark. The labeling reaction was stopped by adding 1 μl per 400 pmol dye of 10 mm l-lysine (Sigma). After vortexing and centrifuging, the sample was left on ice for 10 min in the dark. Proteins extracted from NGF-treated and untreated SY5Y-TrkA cells were labeled with Cy3 and Cy5, respectively, mixed with Cy2-labeled internal standards (see and in one gel. The internal of proteins including samples from time points of one which to for internal S. R.M. analysis of human by two-dimensional difference gel electrophoresis and mass Scholar). Proteins of SY5Y-TrkB cells were labeled with internal proteins of SY5Y-TrkA or SY5Y-TrkB cells and untreated were and per 100 μl cell 10 μl and 10 μl (Amersham were was performed using × containing and a in in J. U. electrophoresis of and for a analysis of the Scholar). After the were in buffer mm mm pH for 10 min and stored on a gel at −80 °C. The was performed on using a system. were the × × and using containing dye Germany). For identification of proteins by 400 μg protein was to × and to × × was performed using a with MS M. of for of proteins in 1994; Scholar). were using a (Amersham and were chosen for each of the dyes to of the were using the (Amersham and were performed using the and of (Amersham respectively. were to the internal (see and proteins were identified by Protein differentially expressed between either SY5Y-TrkA or SY5Y-TrkB cells and untreated in the time course following neurotrophin were identified using of interest were of the with and extracted from the gel as previously K. D. J. of proteins by two-dimensional gel electrophoresis and analyzed by mass and of 2000; Scholar). were used for MALDI target to the The were on the MALDI target with μl of in and For analysis of MS was using the with a MALDI target to The were in the with a target of and a of The was set to and to The internal of was performed using the of with the and The were using the or and the were with the proteome in to identify the proteins. were from using the system for protein identification using mass peptide 2000; Scholar). was used as for protein The following were with to in the of and no protein mass and The were in the human protein of The gene for the identified proteins was using S. P. P. A. M.A. the of the and Res. 2003; Scholar). To identify specific protein expression changes following TrkA or TrkB activation in neuroblastoma cells by their ligands, NGF or BDNF, we analyzed the proteome of or untreated cells using the DIGE system. To detect immediate proteome changes as well as late we five time points between 0 and 24 h following ligand proteins were together with untreated proteins and the Cy2-labeled protein of of samples at a time in one gel We the and the DIGE by the buffer system for proteome of of the of protein in the of In of protein were by the and The analysis of the expression of SY5Y-TrkB cells using resulted in 24 and by BDNF and by NGF in SY5Y-TrkA cells of specific for SY5Y-TrkB cells and were specific for SY5Y-TrkA cells were in cell demonstrated the same one was between SY5Y-TrkA and SY5Y-TrkB of proteins following neurotrophin receptor activation in SY5Y-TrkA or SY5Y-TrkB identified by of protein following neurotrophin of SY5Y-TrkA or Neurotrophin resulted in in SY5Y-TrkA cells, in SY5Y-TrkB cells, and in cell The proteins identified in this study are in For identification of were and analyzed by were to the proteome using system for protein identification using mass peptide 2000; Scholar). We identified 22 of 24 proteins and 9 of proteins. of these proteins in in and the and points and to the proteins to differentially were at late time points Proteins identified during this study were using S. P. P. A. M.A. the of the and Res. 2003; Scholar). of the proteins were to organization and These and were identified, signal and and are members of the and and are in the The majority of the proteins identified in this study have not been in TrkA/TrkB signaling or the biology of neuroblastoma. The identified proteins into (1.Miller F.D. Kaplan D.R. On Trk for retrograde signaling.Neuron. 2001; 32: 767-770Google Scholar, A. Reichardt L.F. Trk receptors: mediators of neurotrophin action.Curr. Opin. Neurobiol. 2001; 11: 272-280Google Scholar, 3.Segal R.A. Selectivity in neurotrophin signaling: theme and variations.Annu. Rev. Neurosci. 2003; 26: 299-330Google Scholar) to their expression during cell stimulation with their specific proteins time points proteins between 0.5 and 1 and proteins that were differentially expressed in the late of stimulation The expression of one protein from each of these is in The majority of the proteins differentially expressed upon neurotrophin receptor activation were in the late stimulation in SY5Y-TrkA and in The of differentially expressed in the are in and B of proteins using the DIGE system is in a system are by is to differentially expressed proteins from To between the DIGE system and we performed of a of a of time points of SY5Y-TrkA and SY5Y-TrkB cell on the gel allowed the identification of Cy3 resulted in using the The protein in the were comparable it that some proteins are by not by DIGE as in as in between and for proteins in the mass not were to in the proteins as in is most by the mass of the dye to the which is detectable for proteins. high of proteins on gel allowed the of proteins to differentially expressed in and their identification by We here the of the DIGE for identification of proteins differentially expressed following Trk receptor activation in SY5Y neuroblastoma cells stably transfected with TrkA or TrkB cDNA. These cell lines are excellent for the response to Although signal transduction of Trk receptors have been analyzed A. B. S. H. S. in and late responses between and SH-SY5Y neuroblastoma 2001; Scholar, M. S. N.J. R. is for sympathetic neuronal by NGF not by p75 Cell Biol. 2002; Scholar), late effector proteins have not been for the for proteins associated with and to the biological changes by Trk receptor activation in neuroblastoma. The DIGE identification of protein changes between samples on the same and DIGE a of of changes A. L. E. S. for two-dimensional gel difference gel electrophoresis a internal 2003; Scholar, S. K. G. G. proteomic analysis by two-dimensional 2003; Scholar, S. P. A. R. of two-dimensional gel electrophoresis for proteomic expression analysis of a model cell Cell. 2002; Scholar, R. J. B. R. S. J. F. E. M. and of two-dimensional gel electrophoresis 2001; Scholar, M. gel gel for changes in protein 1997; 18: Scholar). We that DIGE and are the identification of differentially expressed proteins by MS The of the system was by the time course in five independent cell culture the of cellular and of for identification of proteins by labeling is of the protein in the mass when using a Thus, the mass of the to into when the for identification by MS these we here a reproducible for identification of differentially expressed proteins. The of identified in this to the phenotypes of cells The gene for was to in to TrkA H. P.A. of the human gene to by in Cell Scholar). this protein we identified the protein following activation of TrkA or TrkB Thus, the of to the phenotypes of SY5Y-TrkA and SY5Y-TrkB cells of to in cells been Chou in and changes in Cell Biol. Scholar). of into a neuronal cell line was with cell mobility and of G. F. P. of function and by Biol. Cell. 2003; 14: Scholar). Thus, of by also to the aggressive of TrkB-expressing SY5Y cells. In of by to the and favorable of TrkA-expressing neuroblastoma cells. of a protein were also differentially between SY5Y-TrkA and SY5Y-TrkB cells. expression is involved in differentiation of cells and in response to NGF 3rd, factor of cytoplasmic expression Neurosci. Res. 2001; Scholar). Thus, it also important for differentiation signaling in SY5Y-TrkA cells. a protein responses E. A. J. expression and effect of on of and 2001; Scholar) as well as the induction of cell apoptosis and cell J. of by cell Biol. Scholar), is in SY5Y-TrkB cells and to the of aggressive neuroblastoma cells. In expression was to to tumor cells S. H. G. J. H. D. J. K. Association of not with proliferation in human neuroblastomas and cell lung Res. 2002; Scholar). As proliferation of SY5Y-TrkB cells is enhanced following BDNF stimulation in also a to proliferation in these cells. was in SY5Y-TrkB cells protein as a which functions as a in cells to translation of proteins in cells. In neuroblastoma, a role for to and are following TrkB activation and are associated with aggressive tumor cell and of differentiation in line with the of neuroblastomas. that the of Trk responses on receptors are on cell on cell or the of the cell Neurotrophins Trk receptors at nerve the and receptor and retrograde of The is a the (reviewed in cellular for Neurobiol. Scholar). the expression of in model system. suggest that the protein expression of is differentially between SY5Y-TrkA and SY5Y-TrkB cells in response to their specific are to there are differences in retrograde and the role of protein is to proteins are involved in the of differentiation of SY5Y-TrkA cells and proliferation of SY5Y-TrkB cells. the proteins identified here are not to the for the of Trk receptors in neuroblastoma cells to to the high levels of leading to and the biological of the well with the favorable such as differentiation and of neuroblastomas high levels of TrkA, and the including and of neuroblastomas TrkB, effects In resistance to chemotherapy was to the of TrkB expressed in this model system in proteome associated with the at time points following neurotrophin receptor activation. the of monitor a of the some of the changes in protein the phenotypes of SY5Y-TrkA and SY5Y-TrkB are the in the or of proteins in and cells. from study that of differentiation on level 24 h following NGF of SY5Y-TrkA detectable on protein level at time to the that and gel are M.A. D. G. of gene expression by pH in human and proteomic 2003; Scholar) and direct of and protein is by differences in mRNA and protein level of which at that the phenotypes of SY5Y-TrkA and SY5Y-TrkB cells by a of factors proteomic in the of to a protein were The of proteins as well as differences in protein or to In of the of analysis of the to neurotrophin receptor of the signaling pathways and analysis of effector proteins identified in this study insights into the for the phenotypes of SY5Y-TrkA and SY5Y-TrkB cells.