The

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m 7 G(5)ppp(5)N, where N is any nucleotide] present at the 5 termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.

Interferon-induced protein kinase (PKR) is a member of a family of kinases that regulate translation initiation through phosphorylation of eukaryotic initiation factor 2␣. In addition to the conserved catalytic subdomains that are present in all serine/threonine kinases, the eukaryotic initiation factor 2␣ kinases possess an insert region between catalytic subdomains IV and V that has been termed the kinase insert domain. To investigate the importance of the kinase insert domain of PKR, several deletions and point mutations were introduced within this domain and analyzed for kinase activity both in vitro and in vivo. Here we show that deletion of the kinase insert sequence or mutation of serine 355, which lies within this region, abrogates kinase activity. In addition, the kinase insert domain of PKR and adjacent amino acids (LFIQME) in catalytic subdomain V are not required for binding of the pseudosubstrate inhibitor K3L from vaccinia virus. A portion of the catalytic domain of PKR between amino acids 366 and 415 confers K3L binding in vivo, suggesting a possible role for this region of PKR in substrate interaction.The double-stranded RNA (dsRNA) 1 -activated protein kinase, PKR (also called p68, DAI, and P1 kinase) is an interferon-inducible protein that plays a key role in the antiviral and antiproliferative interferon response (for a review see Ref.