Structural Characterization of the Human Eukaryotic Initiation Factor 3 Protein Complex by Mass Spectrometry

Abstract

Protein synthesis in mammalian cells requires initiation factor eIF3, an ϳ800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a-m) with a sequence coverage of ϳ79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex. Molecular & Cellular Proteomics 6:1135-1146, 2007.The initiation phase of eukaryotic protein synthesis involves formation of an 80 S ribosomal complex containing the initiator methionyl-tRNA i bound to the initiation codon in the mRNA. This is a multistep process promoted by proteins called eukaryotic initiation factors (eIFs).1 Currently at least 12 eIFs, composed of at least 29 distinct subunits, have been identified (1). Mammalian eIF3, the largest initiation factor, is a multisubunit complex with an apparent molecular mass of ϳ800 kDa. This protein complex plays an essential role in translation by binding directly to the 40 S ribosomal subunit and promoting formation of the 43 S preinitiation complex consisting of the Met-tRNA i ⅐eIF2⅐GTP ternary complex, eIF1, eIF1A, and the 40 S ribosomal subunit (2, 3). The ability of eIF3 to bind to 40 S subunits in the absence of other initiation factors is enhanced by the presence of its loosely associated eIF3j subunit (4). The eIF3 complex also promotes binding of 5Ј-m 7 G-capped mRNA through its interaction with eIF4G, the largest member of the eIF4F cap-binding complex (5, 6). The 43 S preinitiation complex then scans the mRNA (together forming the 48 S complex) in a 5Ј ...

An Oncogenic Role for the Phosphorylated h-Subunit of Human Translation Initiation Factor eIF3

“…It will be interesting to determine whether eIF3h functions similarly in regulating apoptosis. Phosphorylated eIF3h Is the Activated Isoform of eIF3h-eIF3h is phosphorylated in vivo at residue Ser 183 in HeLa cells (19). Many initiation factors are phosphoproteins, and phosphorylation has been shown to function as a general mechanism for the regulation of translation initiation (20,21).…”

Phosphorylation of Plant Translation Initiation Factors by CK2 Enhances the in Vitro Interaction of Multifactor Complex Components

“…It is likely that the phosphorylation of the eIF3c-NTD by CK2 enhances its interaction with positively charged binding domains in its MFC partners, as basic domains containing high levels of lysine and arginine residues in yeast eIF1 (4) and eIF5 (9) play a critical role in their association with yeast eIF3c. A hexaphosphorylated tryptic peptide has been identified in vivo from the N-ter-minal region of the mammalian eIF3c (37). The potential kinases responsible for their phosphorylation were predicted by Scansite to be the acidophilic serine kinases, CK1 or CK2.…”

“…Conservation of similar CK2 sites are highlighted. In vivo wheat factor phosphorylation sites observed in this study are underlined, as well as the in vivo phosphorylation of the eIF3c N terminus at a CK2 consensus site reported by large scale phosphoproteomic analysis for A. thaliana (28) or mammals (37). CK2 phosphorylation sites in the C terminus of both S. cerevisiae and human eIF5 (18,40) have also been observed both in vitro and in vivo and are also underlined in their respective sequences.…”

Regulation of protein synthesis and the role of eIF3 in cancer

“…Yet there has been little evidence to indicate that the factor's activity is regulated. Many of the 13 eIF3 subunits are phosphoproteins, and an increase in their phosphorylation correlates with activation of protein synthesis (27), but regulation of eIF3 activity by phosphorylation has been documented only in a few cases (see below). Alternatively, specific subunits may associate with other proteins that regulate protein synthesis or other processes.…”

“…eIF5B and PABP can be phosphorylated, but possible functional effects are not known. Finally, numerous phosphorylation sites have been mapped to various subunits of eIF3 (27). Phosphorylation at two sites in eIF3f by CDK11p46 during apoptosis enhances the protein's ability to inhibit protein synthesis (28), whereas phosphorylation at a single site in eIF3h promotes cell malignancy, as described below.…”