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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057300-05
Application #
6627220
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Ikeda, Richard A
Project Start
1999-01-01
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2005-12-31
Support Year
5
Fiscal Year
2003
Total Cost
$262,113
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
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Chu, Hsueh-Ping; Liao, Yi; Novak, James S et al. (2014) Germline quality control: eEF2K stands guard to eliminate defective oocytes. Dev Cell 28:561-572
Perraud, Anne-Laure; Zhao, Xiaoyun; Ryazanov, Alexey G et al. (2011) The channel-kinase TRPM7 regulates phosphorylation of the translational factor eEF2 via eEF2-k. Cell Signal 23:586-93
Park, Sungjin; Park, Joo Min; Kim, Sangmok et al. (2008) Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD. Neuron 59:70-83
Hermosura, Meredith C; Nayakanti, Hannah; Dorovkov, Maxim V et al. (2005) A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc Natl Acad Sci U S A 102:11510-5
Heine, Martin; Cramm-Behrens, Catharina I; Ansari, Athar et al. (2005) Alpha-kinase 1, a new component in apical protein transport. J Biol Chem 280:25637-43
Dorovkov, Maxim V; Ryazanov, Alexey G (2004) Phosphorylation of annexin I by TRPM7 channel-kinase. J Biol Chem 279:50643-6
Dorovkov, Maxim V; Pavur, Karen S; Petrov, Alexey N et al. (2002) Regulation of elongation factor-2 kinase by pH. Biochemistry 41:13444-50
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Pavur, K S; Petrov, A N; Ryazanov, A G (2000) Mapping the functional domains of elongation factor-2 kinase. Biochemistry 39:12216-24

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