Expression of recombinant eEF-2 kinase in vitro. Plasmid DNA from clones Cefk-1, Cefk-2, as well as mouse and human eEF-2 kinase cDNA were used in the TnT wheat germ extract coupled transcription/translation system (Promega). [³⁵S]Methionine-labeled products were then analyzed by SDS/PAGE.

Activity of recombinant eEF-2 kinase in vitro. A large-scale (0.5 ml) reaction using a mixture of Cefk-1 and Cefk-2 plasmids was run as in Fig. 1, with the omission of labeled methionine. In the control experiment, the reaction was run with a plasmid containing a luciferase gene. (A) The reaction mixtures were separated by chromatography on a Mono Q column as described. (B) eEF-2 kinase activity in fractions was measured as the ability to phosphorylate purified rabbit eEF-2 in the presence of [γ-³²P]ATP. Purified rabbit reticulocyte eEF-2 kinase was used in the (+) control experiments. (C) Ca²⁺/calmodulin-dependency of recombinant C. elegans eEF-2 kinase. Mono Q fraction 25 was assayed in a standard eEF-2 kinase assay in the presence and absence of Ca²⁺ and calmodulin and 20 μM trifluoperazine (TFP) or N-(6-aminohexyl)-5-chloro-1-napthalene-sulfonamide (W7). (D) Ca²⁺/calmodulin-dependency of recombinant human eEF-2 kinase. Human eEF-2 kinase cDNA was expressed in a coupled transcription/translation system as described above and eEF-2 kinase activity was assayed without further purification.

Northern blot analysis of tissue distribution of mouse eEF-2 kinase mRNA. Northern blots of mouse tissues containing 2 μg of polyadenylylated RNA per lane were probed with the random-primed ³²P-labeled mouse eEF-2 kinase cDNA (31). The major transcript appeared at 3.1 kb and minor transcripts at 6.1 and 2.5 kb were also apparent (exposure time, 5 days). The same blots were stripped and rehybridized with a human eEF-2 cDNA (exposure time, 4 days).

Sequence alignment of C. elegans, mouse, human eEF-2 kinase, and the catalytic domain of Dictyostelium discoideum MHCK A. Identical amino acids are indicated by dark blue boxed regions and chemically conserved amino acids are indicated by light blue shaded regions. Amino acids in the human sequence that are identical to the mouse sequence are represented by dots. Amino acids underlined in black correspond to the six regions that match peptides obtained from the sequencing of purified rabbit reticulocyte eEF-2 kinase. The GXGXXG nucleotide-binding motif is underlined in red. The blue dashed line over residues 625–632 in C. elegans eEF-2 kinase designates the amino acids corresponding to exon 4, which is missing in Cefk-2.

Substrate specificity of eEF-2 kinase and MHCK A. Phosphorylation assays containing eEF-2 kinase (≈50 ng) or MHCK A (0.2 μg) and either 0.5 μg rabbit reticulocyte eEF-2 or 0.1 μg Dictyostelium myosin were performed under standard conditions except that incubation time was extended to 10 min.

Schematic representation of the structure of mammalian and C. elegans eEF-2 kinase and MHCK A. The homologous regions are represented by blue shading. The regions of weak similarity are represented by green shading. The position of the GXGXXG motif is indicated by vertical arrows.