REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

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The regulation of protein synthesis in mammalian cells under stress conditions as well as the regulation of GCN4-specific translation in the yeast Saccharomyces cerevisiae under amino acid starvation conditions is mediated by phosphorylation of the eukaryotic translation initiation factor (eIF)-2. We have been studying how the mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically recognize and phosphorylate serine-51 on the a subunit of eIF2 to regulate translation. We have established a system to suppress the toxicity of PKR expression in yeast by co-expressing viral inhibitors of PKR. The vaccinia virus K3L protein is a pseudosubstrate inhibitor of PKR, and can suppress PKR toxicity in yeast. A conserved amino acid sequence motif found near the carboxyl-terminus of K3L and between residues 73-83 in eIF2alpha is critical for K3L inhibition of PKR. Biochemical analyses have revealed that this motif, located over 30 residues from the site of phosphorylation in eIF2alpha is important for K3L binding to PKR. The corresponding residues in eIF2a are required for proper regulation of GCN4 expression and mutations in this motif in eIF2alpha impair phosphorylation of Ser-51 in vivo. These results suggest that contacts over 30 residues from site of phosphorylation are important for kinase recognition of eIF2alpha. In another approach to study kinase-substrate recognition we have identified 14 PKR alleles that are resistant to K3L inhibition. The PKR mutations cluster in the carboxyl-terminal half of the kinase domain and, based on the x-ray structure of the cAMP-dependent protein kinase, are predicted to alter contacts between the kinase and substrate. The pk2 protein from the baculovirus Autographa californica resembles a truncated eIF2alpha kinase domain and we have found that pk2 is an eIF2alpha kinase inhibitor. The pk2 protein directly interacted with PKR, and pk2 was found to protect baculovirus from the anti-viral effects of PKR. Analysis of substrate specificity of the eIF2alpha kinases revealed that PKR is a dual-specificity protein kinase capable of phosphorylating eIF2alpha on Ser, Thr, or Tyr at residue-51. A second major area of investigation involves the yeast protein FUN12 which resembles bacterial IF2, a translation factor that like eIF2 delivers Met-tRNAiMet to the ribosome. In model assays of protein synthesis the FUN12 protein could substitute for eIF2, and polyribosome profiles from fun12-deleted strains showed defects in translation initiation. These results, combined with a number of genetic observations in fun12-deletion strains, suggest that FUN12 functions in general or alternate mechanisms of translation initiation. Finally, the FUN12 protein has also been identified in archaea and we have identified a human homolog of FUN12 suggesting that this factor plays an important, and conserved, role in cellular protein synthesis.