Purification and phosphorylation of elongation factor-2 kinase from rabbit reticulocytes.
Eukaryotic elongation-factor-2 kinase has been purified to homogeneity from rabbit reticulocytes through a seven-step procedure and has been identified as a protein with a molecular mass of approximately 103 kDa as judged by SDS/PAGE. A degradation product of about 95 kDa was also evident in some preparations. The activity of the purified kinase was completely dependent on calcium and calmodulin. The kinase rapidly underwent extensive autophosphorylation, incorporating 1 mol phosphate/mol within 1 min; 5 mol phosphate/mol were incorporated within 1 h. The autophosphorylation was Ca2+/calmodulin-dependent; phosphopeptide mapping revealed multiple phosphopeptides even after just 0.5 min of autophosphorylation, suggesting that a number of sites became rapidly phosphorylated. Autophosphorylation occurred on serine and threonine residues. Preincubation in the presence of Ca2+, Mg2+ and ATP produced a rapid 2-3-fold activation of the kinase and also induced partial Ca(2+)-independent activity. Preincubation in the absence of the ligands showed that all three were required for full activation and induction of Ca(2+)-independent activity.
Redpath
N T
NT
Department of Biochemistry, School of Medical Sciences, University of Bristol, England.
Proud
C G
CG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D006360:Heat-Shock Proteins / Q000302:isolation & purification; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D008274:Magnesium; D002118:Calcium