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Journal of Neuroscience, Vol 13, 559-567, Copyright © 1993 by Society for Neuroscience
Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility
Y Goshima, S Ohsako and T Yamauchi
Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Japan.
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)
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