Differentiation increases type II calmodulin-dependent protein kinase in the neuroblastoma/glioma cell line 108CC15 (NG108-15).
A type II calcium/calmodulin-dependent protein kinase (CaM kinase II) was purified approximately 300-fold from cultured neuroblastoma/glioma (NG108) cell homogenate. The purification of the kinase, which used a combination of differential centrifugation and chromatography on cation-exchange, calmodulin-affinity, and gel-filtration resins, was monitored by the ability of the kinase to phosphorylate the high-molecular-weight microtubule-associated protein 2 (MAP-2). The kinase was compared with authentic CaM kinase II purified from rat brain cytosol. Based upon holoenzyme molecular weight, subunit composition and molecular weight, calcium-dependent calmodulin-binding to subunits, calcium/calmodulin-dependent autophosphorylation of subunits, substrate specificity, apparent km's for ATP and calmodulin, phosphopeptide maps of subunits, time course, and heat lability, the kinase was identified as a type II calcium/calmodulin-dependent protein kinase. When cellular differentiation was induced under specific conditions of cell culture, a significant increase in the apparent activity and amount of the kinase per mg protein was observed relative to control cells. These studies suggest that there is an increase in CaM kinase II expression during cellular differentiation, which may relate to the concurrent development of electrical excitability, synaptogenesis, and elaboration of cytoskeletal elements. Thus, the NG108 cell should provide a useful model to study the physiological functions of CaM kinase II.
Vallano
M L
ML
Department of Pharmacology, State University of New York, Syracuse 13210.
Beaman-Hall
C M
CM
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002454:Cell Differentiation; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D010449:Peptide Mapping; D010748:Phosphopeptides; D011494:Protein Kinases / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D010748:Phosphopeptides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases