title: Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics.
abstract: The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein-protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it's N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.
authors.affiliation: Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, I-80131 Naples, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Lamberti
authors.forename: Carmen
authors.initials: C
authors.lastname: Sanges
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Nunzia
authors.initials: N
authors.lastname: Migliaccio
authors.forename: Francesco
authors.initials: F
authors.lastname: Rosso
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Papale
authors.forename: Monica
authors.initials: M
authors.lastname: Marra
authors.forename: Augusto
authors.initials: A
authors.lastname: Parente
authors.forename: Michele
authors.initials: M
authors.lastname: Caraglia
authors.forename: Alberto
authors.initials: A
authors.lastname: Abbruzzese
authors.forename: Paolo
authors.initials: P
authors.lastname: Arcari
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015977:Eukaryotic Initiation Factor-1; D018398:Homeodomain Proteins; D016601:RNA-Binding Proteins; C490755:SORBS2 protein, human