Autophosphorylation Of CaM Kinase Research Articles

Studies on the site of phosphorylation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase.

The phosphorylation site(s) involved in the activation of CaM-kinase IV by CaM-kinase kinase alpha was studied using a mutant CaM-kinase IV (K71R) in which Lys71 (ATP-binding site) was replaced with Arg, because the autophosphorylation of CaM-kinase IV occurring at multiple sites made it difficult to study phosphorylation of the enzyme by CaM-kinase kinase. Sequence analysis of the phosphopeptide from the trypsin digest of CaM-kinase IV (K71R) phosphorylated by CaM-kinase kinase alpha suggested that the phosphorylation of CaM-kinase IV by CaM-kinase kinase only occurred at Thr196. The recombinant mutant CaM-kinase IV in which Thr196 or Thr200 was replaced with nonphosphorylatable alanine showed little activity in the presence and absence of the kinase kinase. The mutant enzyme in which Thr196 was replaced with negatively charged aspartic acid showed almost 25 times as high activity as the wild-type enzyme in the absence of the kinase kinase, and no more activation was observed in its presence. In contrast, the enzyme in which Thr200 was replaced with aspartic acid showed little enzyme activity. Thus, it may be concluded that the phosphorylation of Thr196 in CaM-kinase IV by CaM-kinase kinase is necessary for the subsequent autophosphorylation and activation of CaM-kinase IV.

Inactivation and Self-association of Ca/Calmodulin-dependent Protein Kinase II during Autophosphorylation

The time-dependent loss in enzyme activity associated with the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase) was altered by both pH and ATP concentration. These parameters also influenced the extent to which soluble CaM-kinase undergoes self-association to form large aggregates of sedimentable enzyme. Specifically, autophosphorylation of CaM-kinase in 0.01 mM ATP at pH 6.5 resulted in the formation of sedimentable enzyme and a 70% loss of enzyme activity. Under similar conditions at pH 7.5, the enzyme lost only 30% of its activity, and no sedimentable enzyme was detected. In contrast to 0.01 mM ATP, autophosphorylation of CaM-kinase at pH 6.5 in 1 mM ATP did not result in a loss of activity or the production of sedimentable enzyme, even though the stoichiometry of autophosphorylation was comparable. Electron microscopy studies of CaM-kinase autophosphorylated at pH 6.5 in 0.01 mM ATP revealed particles 100-300 nm in diameter that clustered into branched complexes. Inactivation and self-association of CaM-kinase were influenced by the conditions of autophosphorylation in vitro, suggesting that both the catalytic and physical properties of the enzyme may be sensitive to fluctuations in ATP concentration and pH in vivo.

Regulation of Ca2+/calmodulin-dependent protein kinase II in the cell systems in response to cellular stimuli

Calcium ion (Ca2+) is considered to be involved in the regulation of numerous cellular processes. CaM kinase II is present at the highest concentration in the brain and is considered to be involved in the regulation and coordination of numerous cellular processes. CaM kinase II is activated by Ca2+/calmodulin and simultaneously undergoes autophosphorylation. It has not been determined whether the enzyme is activated in the cell systems in response to the increase in cytoplasmic Ca2+ concentration. We have studied CaM kinase II in several kinds of cells including the primary cultures of cerebellar granule cells and the cell lines of rat embryo fibroblast 3Y1 cells, neuroblastoma cells, PC12 cells and C6 glioma cells. The immunohistochemical analysis demonstrated the presence of CaM kinase II in all of the cells examined. Furthermore, the kinase in cerebellar granule cells was activated by the stimulation of the glutamic acid receptor. Autophosphorylation of CaM kinase II in 3Y1 cells was stimulated by the addition of growth factors. These results suggest that CaM kinase II undergoes activation and autophosphorylation in response to various stimuli to the cells and is regulated in the dynamic state.