Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility.
To elucidate the functional role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal cells, we studied the phenotypic effects of overexpression of the CaM kinase II wild-type alpha subunit and a mutant enzyme alpha isoform (Ala-286), in which formation of the Ca(2+)-independent form by autophosphorylation is markedly suppressed by replacement of Thr-286 with Ala, using Neuro2a (Nb2a) and NG108-15 neuroblastoma cell lines. The cDNAs inserted into the EcoRI site of pEF321 expression vector were introduced into Nb2a and NG108-15 cells with pEF321-neo (neo). Stable clones were obtained by G418 selection. The specific activities of CaM kinase II in alpha and Ala-286 transfectants were two to four times higher than those in non-transfectants and in cells transfected with neo alone. Indirect immunofluorescence using a monoclonal antibody specific to the CaM kinase II alpha isoform revealed that CaM kinase II was mainly localized in the perikaryal and dendritic cytoplasm of the alpha and Ala-286 transfectants. Immediately after plating, Nb2a and NG108-15 cells transfected with neo, alpha and Ala-286 cDNAs appeared round. Several hours after plating, alpha transfectants showed cell flattening and initiation of neurite outgrowth, and thereafter extended numerous long and branching neurites. Numerous filopodia protruded from flat growth cones, some of which were accompanied by extensive veil formation. Non- and neo transfectants remained round. In Ala-286 transfectants, however, the phenotypic changes were remarkably less than in alpha transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Y
Y
Goshima
S
S
Ohsako
T
T
Yamauchi
D000818:Animals; D015152:Blotting, Northern; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005910:Glioma / Q000378:metabolism* / Q000648:ultrastructure; D006822:Hybrid Cells; D051379:Mice; D016296:Mutagenesis; D016501:Neurites / Q000502:physiology / Q000648:ultrastructure; D009447:Neuroblastoma / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases