STRUCTURE/FUNCTION OF EFF-2 KINASE

We have recently identified a new class of protein kinases, which are structurally and evolutionarily unrelated to members of the eukaryotic protein kinase superfamily. The prototype member of this class is Ca2+/calmodulin-dependent elongation factor-2 kinase (eEF-2 kinase), a ubiquitous protein kinase present in various eukaryotic organisms. The major function of eEF-2 kinase is to phosphorylate and inactivate eEF-2, and thus, regulate the elongation phase of protein synthesis. Preliminary evidence suggests that eEF-2 kinase has a novel type of catalytic domain, and utilizes a novel mechanism of substrate recognition. The aim of this proposal is to identify and characterize the functional domains of eEF-2 kinase, to study its mechanism of substrate recognition, and to reveal its specific physiological role(s). In vitro mutagenesis of human eEF-2 kinase will be performed to identify and characterize the catalytic and calmodulin-binding domains. We will also study the mechanism of substrate recognition by eEF-2 kinase. An oriented peptide library will be screened to identify the consensus sequence recognized by eEF- 2 kinase. Next, we will test the hypothesis that an alpha- helical conformation of the peptide at the phosphorylation site of the substrate is required for recognition by eEF-2 kinase. Peptides mimicking phosphorylation sites with varying degrees of alpha-helicity will be synthesized and tested for their ability to undergo phosphorylation. To identify other potential substrates for eEF-2 kinase, two approaches will be used: a solid-phase phosphorylation expression screening method and the yeast two-hybrid system. To reveal the exact physiological function of eEF-2 kinase, Caenorhabditis elegans will be used. We will analyze the effect of eEF-2 kinase gene inactivation on phenotype and overall developmental pattern using three different approaches: Tc1 mutagenesis, deletion mutagenesis with rescue by plasmid transgenics, and an antisense approach. This work will reveal the structural organization and function of a new class of protein kinases, which will provide new and important information about the mechanism of protein phosphorylation.