Characterization Of Calmodulin-dependent Protein Kinase Research Articles

Neurite outgrowth of neuroblastoma cells overexpressing α and β isoforms of Ca 2+/calmodulin-dependent protein kinase II—effects of protein kinase inhibitors

Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735–12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol.Chem. 258 (1983) 12632–12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293–1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141–144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15–21 [21]]. The α and β isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca 2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559–601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II α- or β-isoform, and of mutant α-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant α-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca 2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759–16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca 2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59–70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca 2+-independent activity of calmodulin-dependent protein kinase II α subunit, J. Biochem. 109 (1991) 137–143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca 2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559–567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of α and β isoforms of Ca 2+/calmodulin-dependent protein kinase II in neuroblastoma cells—H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129–141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the β than α cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. Themes: Development and regeneration Topics: Process outgrowth, growth cones, and sprouting

Purification and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas

Calmodulin-dependent protein kinase III (CaM kinase III) phosphorylates and thereby inactivates eukaryotic elongation factor-2 (EF-2). This enzyme, purified to homogeneity from either rabbit reticulocytes or rat pancreas, had similar properties: it migrated with an apparent M(r) of 140,000 by gel filtration, was comprised of a major polypeptide of M(r) 95,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a high affinity for CaM (half-maximal activation < 1 nM). The M(r) 95,000 polypeptide was autophosphorylated by an intramolecular mechanism on seryl residues in the presence of Ca2+, CaM, and ATP, and phosphopeptide mapping indicated that several sites were phosphorylated. Autophosphorylation of CaM kinase III resulted in the generation of a partially Ca2+/calmodulin-independent activity. The enzyme could also be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Amino acid sequencing of CaM kinase III indicated that it is distinct from other known proteins including the heat-shock protein hsp90, which was recently suggested to be identical to CaM kinase III (Nygärd, O., Nilsson, A., Carlberg, U., Nilsson, L., and Amons, R. (1991) J. Biol. Chem. 266, 16425-16430). Furthermore, hsp90 did not copurify with CaM kinase III, and the M(r) 95,000 protein did not cross-react with antibodies to hsp90. CaM kinase III exhibited Michaelis-Menten kinetics toward its substrates ATP and EF-2, with Km values of 15 and 2 microM, respectively. CaM kinase III was able to phosphorylate yeast EF-2 with an Km of 2 microM, but the enzyme did not significantly phosphorylate a variety of other substrates, confirming its identification as a novel protein kinase.

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.