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 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.
Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires, IFIBYNE-CONICET, Buenos Aires, Argentina.
Juan J
JJ
Bonfiglio
Giuseppina
G
Maccarrone
Christiane
C
Rewerts
Florian
F
Holsboer
Eduardo
E
Arzt
Christoph W
CW
Turck
Susana
S
Silberstein
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D012333:RNA, Messenger; D048493:Proto-Oncogene Proteins B-raf