title: Mapping the functional domains of elongation factor-2 kinase.
abstract: A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, 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 kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. 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 translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid 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 mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. 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".
authors.affiliation: Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Pavur
authors.forename: A N
authors.initials: AN
authors.lastname: Petrov
authors.forename: A G
authors.initials: AG
authors.lastname: Ryazanov
mesh_terms: D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D054736:Elongation Factor 2 Kinase; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D002147:Calmodulin; D020652:Peptide Elongation Factor 2; D010455:Peptides; D011994:Recombinant Proteins; C517689:EEF2K protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase