Mapping the functional domains of elongation factor-2 kinase

A new class of eukaryotic protein kinases that are not homologous to member s of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., ct al. (1995) J. Biol. Chem. 270, 523-529; Ryazan ov, A. G., et al. (1997) Proc. Natl. Acad. Sci, U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein ki nases that art: not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R35]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2 , and thus can modulate the rate of polypeptide chain elongation during tra nslation, eEF-2 was the only known substrate for eEF-3 kinase. We demonstra te here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid pep tide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylatio n site in Dictyostelium myosin heavy chains. This enabled us to develop a r apid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST -tagged fusion protein, and then performed systematic in vitro deletion mut agenesis. We analyzed eEF-2 kinase deletion mutants for the ability to auto phosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, M H-1. Mutants with deletions between amino acids 51 and 335 were unable to a utophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mut ants with deletions between amino acids 521 and 725 were unable to phosphor ylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-I. The kinases with deletions between amino acids 2 and 50 and 336 and 5 20 were able to catalyze all three reactions. In addition, the C-terminal d omain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitatio n assay. These results suggest that eEF-2 kinase consists of two domains co nnected by a linker region. The aminoterminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-3, targeting d omain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".