title: Interaction of recombinant human eIF2 subunits with eIF2B and eIF2alpha kinases.
abstract: The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2alpha kinase, is found to interact with wt and mutants of eIF2alpha in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2alpha. The dissociation constant of alpha-subunit-PERK complex (Kd alpha-subunit) is 0.74 microM and the interaction is stoichiometric.
authors.affiliation: Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Rajasekhar N V S
authors.initials: RN
authors.lastname: Suragani
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kamindla
authors.forename: N Z
authors.initials: NZ
authors.lastname: Ehtesham
authors.forename: Kolluru V A
authors.initials: KV
authors.lastname: Ramaiah
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B / Q000378:metabolism*; D006801:Humans; D021122:Protein Subunits / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D021122:Protein Subunits; D011994:Recombinant Proteins; D019892:eIF-2 Kinase