PHOSPHORYLATION SITES IN ISOFORMS OF INITIATION FACTOR EIF4E IN CELEGANS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of the mRNA cap-binding protein eIF4E, an initiation factor involved in the recruitment of mRNA to the ribosome. This process determines the rate of protein synthesis, the spectrum of mRNAs translated, and the rate of mRNA turnover. This specific project is centered on the physiological role of eIF4E phosphorylation. Although the rate of protein synthesis and the phosphorylation of eIF4E are highly correlated, and although eIF4E phosphorylation has been evolutionarily conserved in all animals examined, the biochemical consequences of eIF4E phosphorylation remain elusive and controversial. The most definitive data are likely to come from genetically tractable animal models like C. elegans. We have discovered and extensively characterized five isoforms of eIF4E in C. elegans, termed IFE-1, IFE-2, etc., including the production of knockout strains for all five isoforms. In one case, we have been able to rescue the wild-type phenotype by transgenic expression of the missing IFE. In mammals, eIF4E is phosphorylated at a single site in the C-terminus and by two kinases termed Mnk1 and Mnk2. These are tethered to eIF4G, a component of translational initiation complexes. We propose to determine the phosphorylation sites in each of the five IFEs by mass spectrometry. Then we will alter the site in one particular IFE to prevent phosphorylation. We will determine the result of expressing the modified form in knockout worms with regard to overall rate of protein synthesis, tissue and developmental expression of the IFE, half-life of IFE, spectrum of mRNAs translated, and overall phenotype of the organism. Knowledge of the phosphorylation sites will also assist us in finding the kinase(s) responsible for IFE phosphorylation and specifically determine whether a C. elegans homologue of Mnk is responsible. Since we have discovered the kinase(s) responsible, we can study their upstream signaling pathways using biochemical and genetic approaches.