Analysis of the domain structure of elongation factor-2 kinase by mutagenesis.
A number of elongation factor-2 kinase (eEF-2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF-2K had no effect on activity, showing that such a lysine residue is not important in eEF-2K activity. Mutation of a conserved pair of cysteine residues C-terminal to the GXGXXG sequence, however, completely inactivated eEF-2K. The eEF-2K CaM binding domain was localised to residues 77-99 which reside N-terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF-2K activity. Removal of approximately 130 residues from the C-terminus of eEF-2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF-2 kinase activity but not autokinase activity, suggesting that a short region at the C-terminal end may be important in interacting with eEF-2. Likewise, removal of between 75 and 100 residues from the N-terminal end completely abolished eEF-2K activity.
Diggle
T A
TA
Department of Biochemistry, University of Leicester, University Road, Leicester, UK.
Seehra
C K
CK
Hase
S
S
Redpath
N T
NT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D054736:Elongation Factor 2 Kinase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000378:metabolism
D014364:Tryptophan; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase