Characterization of the B-Raf interactome in mouse hippocampal neuronal cells

B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hi...

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Autores principales:	Bonfiglio, J.J., Maccarrone, G., Rewerts, C., Holsboer, F., Arzt, E., Turck, C.W., Silberstein, S.
Formato:	JOUR
Materias:	B-Raf Interactome Mass spectrometry Neurons Proteomics B Raf kinase cell surface receptor chaperone mitogen activated protein kinase amino acid sequence animal cell article enzyme activation enzyme regulation gel electrophoresis hippocampus matrix assisted laser desorption ionization time of flight mass spectrometry mouse nerve cell nonhuman priority journal protein analysis protein expression protein interaction proteomics RNA metabolism RNA translation signal transduction Animals Blotting, Western Cells, Cultured Chromatography, Liquid Cytoskeleton Electrophoresis, Gel, Two-Dimensional Hippocampus Mice Molecular Chaperones Proto-Oncogene Proteins B-raf RNA, Messenger Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_18743919_v74_n2_p186_Bonfiglio
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_18743919_v74_n2_p186_Bonfiglio
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spelling	todo:paper_18743919_v74_n2_p186_Bonfiglio2023-10-03T16:34:07Z Characterization of the B-Raf interactome in mouse hippocampal neuronal cells Bonfiglio, J.J. Maccarrone, G. Rewerts, C. Holsboer, F. Arzt, E. Turck, C.W. Silberstein, S. B-Raf Interactome Mass spectrometry Neurons Proteomics B Raf kinase cell surface receptor chaperone mitogen activated protein kinase amino acid sequence animal cell article enzyme activation enzyme regulation gel electrophoresis hippocampus matrix assisted laser desorption ionization time of flight mass spectrometry mouse nerve cell nonhuman priority journal protein analysis protein expression protein interaction proteomics RNA metabolism RNA translation signal transduction Animals Blotting, Western Cells, Cultured Chromatography, Liquid Cytoskeleton Electrophoresis, Gel, Two-Dimensional Hippocampus Mice Molecular Chaperones Neurons Proteomics Proto-Oncogene Proteins B-raf RNA, Messenger Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons. © 2010 Elsevier B.V. Fil:Bonfiglio, J.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Silberstein, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_18743919_v74_n2_p186_Bonfiglio
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	B-Raf Interactome Mass spectrometry Neurons Proteomics B Raf kinase cell surface receptor chaperone mitogen activated protein kinase amino acid sequence animal cell article enzyme activation enzyme regulation gel electrophoresis hippocampus matrix assisted laser desorption ionization time of flight mass spectrometry mouse nerve cell nonhuman priority journal protein analysis protein expression protein interaction proteomics RNA metabolism RNA translation signal transduction Animals Blotting, Western Cells, Cultured Chromatography, Liquid Cytoskeleton Electrophoresis, Gel, Two-Dimensional Hippocampus Mice Molecular Chaperones Neurons Proteomics Proto-Oncogene Proteins B-raf RNA, Messenger Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
spellingShingle	B-Raf Interactome Mass spectrometry Neurons Proteomics B Raf kinase cell surface receptor chaperone mitogen activated protein kinase amino acid sequence animal cell article enzyme activation enzyme regulation gel electrophoresis hippocampus matrix assisted laser desorption ionization time of flight mass spectrometry mouse nerve cell nonhuman priority journal protein analysis protein expression protein interaction proteomics RNA metabolism RNA translation signal transduction Animals Blotting, Western Cells, Cultured Chromatography, Liquid Cytoskeleton Electrophoresis, Gel, Two-Dimensional Hippocampus Mice Molecular Chaperones Neurons Proteomics Proto-Oncogene Proteins B-raf RNA, Messenger Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Bonfiglio, J.J. Maccarrone, G. Rewerts, C. Holsboer, F. Arzt, E. Turck, C.W. Silberstein, S. Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
topic_facet	B-Raf Interactome Mass spectrometry Neurons Proteomics B Raf kinase cell surface receptor chaperone mitogen activated protein kinase amino acid sequence animal cell article enzyme activation enzyme regulation gel electrophoresis hippocampus matrix assisted laser desorption ionization time of flight mass spectrometry mouse nerve cell nonhuman priority journal protein analysis protein expression protein interaction proteomics RNA metabolism RNA translation signal transduction Animals Blotting, Western Cells, Cultured Chromatography, Liquid Cytoskeleton Electrophoresis, Gel, Two-Dimensional Hippocampus Mice Molecular Chaperones Neurons Proteomics Proto-Oncogene Proteins B-raf RNA, Messenger Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
description	B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons. © 2010 Elsevier B.V.
format	JOUR
author	Bonfiglio, J.J. Maccarrone, G. Rewerts, C. Holsboer, F. Arzt, E. Turck, C.W. Silberstein, S.
author_facet	Bonfiglio, J.J. Maccarrone, G. Rewerts, C. Holsboer, F. Arzt, E. Turck, C.W. Silberstein, S.
author_sort	Bonfiglio, J.J.
title	Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
title_short	Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
title_full	Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
title_fullStr	Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
title_full_unstemmed	Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
title_sort	characterization of the b-raf interactome in mouse hippocampal neuronal cells
url	http://hdl.handle.net/20.500.12110/paper_18743919_v74_n2_p186_Bonfiglio
work_keys_str_mv	AT bonfigliojj characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT maccarroneg characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT rewertsc characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT holsboerf characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT arzte characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT turckcw characterizationofthebrafinteractomeinmousehippocampalneuronalcells AT silbersteins characterizationofthebrafinteractomeinmousehippocampalneuronalcells
_version_	1807314795190288384

Characterization of the B-Raf interactome in mouse hippocampal neuronal cells

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