title: Identification of ubiquitin-modified lysine residues and novel phosphorylation sites on eukaryotic initiation factor 2B epsilon.
abstract: Eukaryotic initiation factor 2Bε (eIF2Bε) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Bε followed by ubiquitin. Tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Bε. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC-MS/MS analysis, and an interaction between endogenous eIF2Bε with NEDD4 was confirmed by co-immunoprecipitation.
authors.affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Tuckow
authors.forename: Abid A
authors.initials: AA
authors.lastname: Kazi
authors.forename: Scot R
authors.initials: SR
authors.lastname: Kimball
authors.forename: Leonard S
authors.initials: LS
authors.lastname: Jefferson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes; D020717:Eukaryotic Initiation Factor-2B; D025801:Ubiquitin; D013912:Threonine; D012694:Serine; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine