Mouse p56 Blocks a Distinct Function of Eukaryotic Initiation Factor 3 in Translation Initiation

Daniel J. Hui, Fulvia Terenzi, William C. Merrick, Ganes C. Sen
2004 Journal of Biological Chemistry  
Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2⅐GTP⅐Met-tRNA i (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However,
more » ... anslation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3⅐ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit⅐eIF3⅐ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits. One of the key features of the innate immune response is the induction of numerous cellular genes in response to viral stress. Viral stress conditions in cells are triggered by mechanisms commonly associated with a cell undergoing viral infection, such as the production of double-stranded RNA, the production of interferons, as well as other virus-mediated pathways that have yet to be elucidated. Previous studies from our laboratory have characterized the human viral stress-inducible protein p56, a 56-kDa protein (1). Human p56 (Hup56) has been shown to act as an inhibitor of protein synthesis through its association with the "e" subunit of eukaryotic initiation factor 3 (eIF3 1 ; the e subunit is also known as p48 or Int6) (2, 3). We have shown recently (4) that the inhibitory activity of human p56 occurs at the step of ternary complex stabilization by eIF3, a key step in the initiation pathway for protein synthesis in eukaryotes. Eukaryotic initiation factor 3 is the largest of the 11 or more factors required for the initiation of protein synthesis in eukaryotes. eIF3 is composed of 12 subunits named eIF3a to eIF3l, although the exact stoichiometry and arrangement of the subunits are poorly understood (5). eIF3 has many functions in translation initiation, one of which is to serve as a ribosome dissociation factor by binding to the 40 S ribosomal subunit and preventing its re-association with 60 S subunits (6, 7). eIF3 also plays a role in stabilizing interactions with other components of the initiation pathway such as the ternary complex that consists of eIF2⅐GTP⅐Met-tRNA i as well as stabilizing the formation of the 43 S complex that is formed when the ternary complex joins the 40 S ribosome (8, 9) . Finally, eIF3 is also involved in binding to eIF4G of the heterotrimeric eIF4F complex, stabilizing its association with the 43 S complex (10, 11). The p56 family of proteins includes several similar sized proteins in humans (p54, p56, p58, and p60) (12-15) as well as other species including hamster (16), mouse (17), and fish (18) . The p56 family members share a structural homology consisting of a series of loosely conserved, 34-amino acid tetratricopeptide (TPR) tandem repeats (19) . TPR motifs are known to mediate protein-protein interactions (20), and these motifs have been shown to be required for the interaction between human p56 and eIF3e (21). Conversely, the region of eIF3e that Hup56 interacts with is another loosely conserved structural motif known as the PCI motif. Named for Proteasome, COP9 signalosome and Initiation Factor, the three families of multisubunit complexes that feature this motif, PCI motif-containing proteins, are potential target proteins for interaction with p56 family members based on structural homology (22). Three subunits of eIF3, "a," "c," and "e," contain PCI motifs (23, 24). By extending the in vitro protein synthesis studies that showed that Hup56 can inhibit initiation of translation by interfering with eIF3 function, we followed a systematic investigation to reveal that all functions of eIF3 are not blocked by Hup56; it blocks only one specific step, namely the stabilization of the ternary complex of eIF2⅐GTP⅐Met-tRNA i . Hup56 does not interfere with the interactions of eIF3 with 40 S ribosomal subunits or eIF4F. Because we are interested in studying the functions of the p56 family of proteins in interferon-infected animal models, we have extended our investigation to mouse (Mu)p56. Here we report that, like Hup56, Mup56 inhibited translation by binding to eIF3. However, unlike Hup56, it bound to the eIF3c (p110) subunit and not eIF3e. Consequently, Mup56 inhibited a function of eIF3 that is different from the one inhibited by Hup56. MATERIALS AND METHODS Constructs- The plasmid encoding full-length Hup56 was constructed by excising the full-length p56 cDNA from pBluescript KS(II) and inserting into pcDNA3 (Invitrogen). A full-length Mup56 plasmid for expression in mammalian cells was generated by PCR using an existing clone and then subcloning the cDNA sequence into
doi:10.1074/jbc.m406700200 pmid:15561726 fatcat:abfsmydgv5hlbpdmw7hpqe3iyi