Kinetics of calcium and calmodulin‐dependent protein kinase III from embryonic chicken leg muscle cells

Abstract

Embryonic chicken muscle cells (CELM) contain the calmodulin-dependent protein kinase that specifically phosphorylates eukaryotic elongation factor 2. The kinase requires Ca 2+and maximum activity in CELM was observed at 10 uM Ca 2+ . The ATP concentration required for half the maximum activity of CaM PKIII in CELM was calculated to be 0.15 mM. In CELM, dephosphorylation of eEF-2 was catalyzed by phosphoprotein phosphatase PP2A alone. The activity of PP2A was relatively low and the half-life of added phosphorylated eEF-2 was more than 15 min. Due to the low phosphoprotein phosphatase activity, inhibition of the PP2A activity by addition of okadaic acid had little effect on the eEF-2 phosphorylation kinetics.

Mechanisms for Increased Levels of Phosphorylation of Elongation Factor-2 during Hibernation in Ground Squirrels

“…Activation of eEF-2 kinase occurs in response to a variety of hormones, neurotransmitters, and growth factors that influence cellular Ca 2+ levels ( , ). As a consequence, the phosphorylation of eEF-2 and the resulting inhibition of protein synthesis are often transient in nature.…”

“…Phosphorylation of eIF2R contributes to a substantial decrease in the rate of protein synthesis initation in hibernators (10), but the mechanism-(s) involved in the regulation of elongation in hibernators is not known. mRNA translocation on the ribosome involves elongation factor-2 (eEF-2), 1 and eEF-2 is phosphorylated and inhibited by a specific kinase (eEF-2 kinase), with phosphorylation reducing the ability of the factor to promote translocation possibly by decreasing its affinity for pretranslocation ribosomes (1,24,32,36,37). Increased levels of phosphorylation of eEF-2 occur in response to cellular stimulation by mitogens, growth factors, and neurotransmitters that influence intracellular Ca 2+ levels (31,35,37).…”

Mutation of Tyr307 and Leu309 in the Protein Phosphatase 2A Catalytic Subunit Favors Association with the α4 Subunit Which Promotes Dephosphorylation of Elongation Factor-2

“…Possible candidates are the R subunit of eukaryotic initiation factor 2 (eIF-2R) that is phosphorylated and inhibited by several eIF-2R protein kinases, and eukaryotic elongation factor 2 (eEF2) that is phosphorylated and inhibited by eEF2 kinase (previously called CaM kinase III) (32)(33)(34). There is good evidence that eIF-2R is dephosphorylated by PP1 (20)(21)(22)(26)(27)(28), while various studies show that eEF2 is dephosphorylated with considerable specificity by PP2A (23)(24)(25)(32)(33)(34). We found here that transient overexpression of FLAG-R4 produced a significant and selective decrease in the level of phospho-eEF2.…”

“…Possible candidates are the α subunit of eukaryotic initiation factor-2 (eIF-2α) or eukaryotic elongation factor-2 (eEF2). There is good evidence that eIF-2α is dephosphorylated by PP1 ( − ), while eEF2 is dephosphorylated by PP2A ( − ). In the present study, we found that overexpression of FLAG-α4 resulted in a selective decrease in phospho-eEF2.…”

Heterogeneity of cardiac rat and human elongation factor 2

“…The other is phosphorylation of the threonine residues 53, 56 and 58 by a specific Ca ++ /calmodulin-dependent EF-2 kinase (CaM kinase III) [3]. ADP-ribosylated as well as phosphorylated EF-2 have reduced affinity for the pretranslocation type of ribosome and are thus unable to promote translocation, which leads to a reduced rate of protein synthesis [4,5]. While ADP-ribosylation of EF-2 in vivo is irreversible, phosphorylated EF-2 (P-EF-2) is physiologically dephosphorylated by phosphoprotein phosphatases 2A and 2C.…”