Investigation of the Ca(2+)-independent form of Ca(2+)/calmodulin-dependent protein kinase II in neurite outgrowth.
Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays important roles in the control of nerve functions in response to intracellular Ca(2+) (for reviews [Annu. Rev. Physiol. 57 (1995) 417-445; Trends Neurosci. 17 (1994) 406-412]). Brief Ca(2+) signals activate CaM kinase II, and stimulate an autophosphorylation of Thr-286 which allows the kinase to maintain its activated state even after the Ca(2+) concentration has returned to basal levels [J. Biol. Chem. 264 (1989) 16759-16763; Neuron 3 (1989) 59-70; J. Biochem. 109 (1991) 137-143]. Autophosphorylation of CaM kinase II occurs in situ, but it occurs relatively quickly, within just a few minutes [Endocrinology 134 (1994) 2245-2250; J. Biol. Chem. 268 (1993) 7863-7867; J. Biol. Chem. 265 (1990) 18055-18058]. In the present study, we investigated the involvement of the autophosphorylated/Ca(2+)-independent form of CaM kinase II in neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they formed neurites. The autophosphorylation of Thr-286 and appearance of Ca(2+)-independent activity preceded the neurite formation. The effect of mutating of the kinase autophosphorylation site replacing Thr-286 with Ala (alpha T286A kinase) or Asp (alpha T286D kinase) was examined. alpha T286A kinase was not converted to a Ca(2+)-independent form, and alpha T286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alpha T286D kinase had much longer neurites than Nb2a/alpha cells, whereas cells with alpha T286A kinase did not form neurites. These results indicated that the Ca(2+)-independent form of CaM kinase II autophosphorylated at Thr-286 is involved in neurite outgrowth.
Sogawa
Y
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1, Tokushima 770-8505, Japan.
Yoshimura
Y
Y
Yamauchi
T
T
D000818:Animals; D002118:Calcium / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D016501:Neurites / Q000201:enzymology* / Q000648:ultrastructure; D009447:Neuroblastoma / Q000378:metabolism; D010766:Phosphorylation; D014162:Transfection
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium