title: The N-terminal domain of the human eIF2beta subunit and the CK2 phosphorylation sites are required for its function.
abstract: CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2alpha evidenced the direct involvement of this protein kinase in eIF2beta phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2beta or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2beta phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2beta also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2beta S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Delta2-138 N-terminal-truncated form of eIF2beta (eIF2beta-CT). Mutation at Ser2 and Ser67 did not affect eIF2beta integrating into the eIF2 trimer or being able to complex with eIF5 and CK2alpha. The eIF2beta-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2beta slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2beta S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2beta-CT form, being detectable at doses where eIF2beta and eIF2beta S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2beta for its function in mammals.
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Ciències, Universitat Autònoma de Barcelona, Edifici Cs, Campus de Bellaterra, 08193 Bellaterra, Barcelona, Spain. Franc.llorens@uab.es
authors.forename: Franc
authors.initials: F
authors.lastname: Llorens
authors.forename: Anna
authors.initials: A
authors.lastname: Duarri
authors.forename: Eduard
authors.initials: E
authors.lastname: Sarró
authors.forename: Nerea
authors.initials: N
authors.lastname: Roher
authors.forename: Maria
authors.initials: M
authors.lastname: Plana
authors.forename: Emilio
authors.initials: E
authors.lastname: Itarte
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry* / Q000378:metabolism*; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary
chemical_list: D015852:Eukaryotic Initiation Factor-2; D047390:Casein Kinase II