ID OF IN VITRO PHOSPHORYLATION SITES WITHIN ELONGATION FACTOR 2 KINASE

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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. Elongation factor 2 (EF2) is a key component involved in protein synthesis. Upon phosphorylation by the specific Ca2+/calmodulin-dependent protein kinase EF2 kinase (EF2K), EF2 is completely inactivated. The physiological role of phosphorylation of EF2 is unknown but may involve regulation of cell growth in dividing cells and of local protein synthesis in non-dividing cells. Turnover of EF2K appears to be regulated by its phosphorylation state, possibly by targeting of the enzyme to a ubiquitination/proteosome pathway by phosphorylation of the DSGYPS motif (residues 439-444) by unknown kinases. These kinases may act in concert with PKA to regulate the enzyme. The goal of ongoing studies is to identify protein kinases that are able to phosphorylate EF2K, either within the DSGYPS motif or elsewhere. In the current work, AMP kinase was shown to phosphorylate EF2K. After incubation with the kinase in vitro, EF2K was digested and analyzed by mass spectrometry to discover site(s) of phosphorylation, if any. Our data indicates that EF2K is phosphorylated by AMP kinase at one site within the region 394-400. Work to identify the exact site of phosphorylation is ongoing.