Brain Research 881 2000 165–175 www.elsevier.com locate bres
Research report
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Ca -independent activity of Ca
calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in
neuroblastoma cells
a a
b , a
Yoshimi Sogawa , Yoshiyuki Yoshimura , Akira Otaka , Takashi Yamauchi
a
Department of Biochemistry , Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1, Tokushima 770-8505, Japan
b
Graduate School of Pharmaceutical Sciences , Kyoto University, Kyoto 606-8501, Japan
Accepted 8 August 2000
Abstract
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We investigated the involvement of Ca -independent activity of Ca
calmodulin-dependent protein kinase II CaM kinase II in stimulation of neurite outgrowth. When neuroblastoma Neruo2a Nb2a cells expressing the a isoform of CaM kinase II Nb2a a cells
were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation.
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Ca -independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually
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decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca -independent activity. The
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autophosphorylation and appearance of Ca -independent activity preceded the formation of neurites. The effect of mutation of the
autophosphorylation site in the kinase whose Thr-286 was replaced with Ala aT286A kinase or Asp aT286D kinase was examined.
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aT286A kinase was not converted to a Ca -independent form, and aT286D kinase had Ca
-independent activity significantly as an autophosphorylated kinase. Cells expressing aT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells.
Cells expressing aT286D kinase had much longer neurites than Nb2a a cells expressing the wild type kinase, although the initiation of
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neurite outgrowth was very late. These results indicated that Ca -independent activity of the kinase autophosphorylated at Thr-286
involves for neurite outgrowth.
2000 Elsevier Science B.V. All rights reserved.
Theme : Development and regulation
Topic : Process outgrowth, growth cones, and sprouting
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Keywords : Ca
calmodulin-dependent protein kinase II; Autophosphorylation; Neurite outgrowth; Ca -independent activity; Neuroblastoma
1. Introduction brain, and in particular in cortical and hippocampal
neurons. Many of its substrates are involved in neuronal
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Ca calmodulin-dependent protein kinase II CaM
signaling. Neuronal CaM kinase II plays important roles in kinase II is a multifunctional mediator of the activity
the control of nerve functions in response to intracellular
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dependent calcium
increase in
excitable cells
Ca , including the synthesis and secretion of neuro-
[3,13,21,43,44]. It is expressed at very high levels in the transmitters, a receptor function, structural modification of
the cytoskeleton, axonal transport, long term potentiation
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LTP and learning and memory [6,25]. It has been
Abbreviations: CaM kinase II, Ca calmodulin-dependent protein
reported that transgenic mice lacking the a isoform of this
kinase II; DMEM, Dulbecco’s modified Eagle’s medium; EGTA, ethyl- ene glycol bis b-aminoethylether-N,N,N9,N9-tetra acetic acid; FBS, fetal
kinase are defective in long-term potentiation and spatial
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bovine serum; GFP, green fluorescence protein; Nb2a, Neuro 2a; PAGE,
learning [2,36]. Brief Ca signals activate CaM kinase II,
polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluo-
and stimulate an autophosphorylation of Thr-286 which
ride; SDS, sodium dodecyl sulfate
allows the kinase to maintain its activated state after the
Corresponding author. Tel.: 181-88-633-7250; fax: 181-88-633-
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Ca concentration has returned to basal levels [11,18,32].
9514. E-mail address
: yamauchiph.tokushima-u.ac.jp T. Yamauchi.
Autophosphorylation of CaM kinase II occurs in situ. For
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 3 8 - 9
166 Y
example, depolarization or electrical stimulation increased cence reagent were purchased from DuPont New England
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Ca -independent activity in PC 12 cells [28], pituitary
Nuclear Research, G418 geneticin from Sigma, horserad- cells [10] and hippocampal neurons [12]. However, since
ish peroxidase-coupled anti-mouse goat IgG and anti-rabbit
TM6
the autophosphorylation of the kinase occurs relatively goat IgG from Bio-Rad, FuGENE
transfection reagent quickly, in just a few minutes, little is known about how
from Bohringer Mannheim, pEGFP-C1 vectors from
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the Ca -independent activity of the kinase influences
CLONTECH. The CaM kinase II substrate peptide au- physiological events.
tocamtide 2 KKALRRQETVDAL [18] was synthesized CaM kinase II is a multigene family, in which each of
by Sawady Tech, Tokyo. Anti-phospho-CaM kinase II four isoforms a, b, g, and d is encoded by a separate
antibody was provided by Dr. Yoko Yamagata, National gene [4,24,39,40]. The a and b isoforms are expressed
Institute for Physiological Sciences, Okazaki, Japan, and almost exclusively in the nervous system [6]. Brain CaM
was also prepared by our hand as described [42]. Phospho- kinase II consists of a distinct homo-oligomer of either a
peptide containing an autophosphorylation site was syn- or b polypeptides [20]. The concentration of a and b
thesized with a ABI peptide synthesizer. CaM kinase II isoforms varies markedly in different brain regions with
was purified from rat forebrain as described previously postnatal age [38]. The role of each isoform in the
[44]. Monoclonal antibody specific to the a isoform of regulation of nerve function has been investigated in
CaM kinase II was as described [19]. neuroblastoma cells expressing these isoforms [15,31].
CaM kinase II promotes neurite outgrowth, and the b isoform induces a greater change in morphology than the a
2.2. Cell culture isoform [31].
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Neurite outgrowth is triggered by the influx of Ca in
Mouse neuroblastoma Neuro 2a cells Nb2a and human response to various stimuli [1]. The neuronal growth cone,
embryonal kidney cells HEK 293T were cultured in a specialized structure at the distal end of a developing
Dulbecco’s modified Eagle’s medium DMEM sup- neurite, is responsible for neurite outgrowth and path
plemented with 10 fetal bovine serum FBS, penicillin, finding in the developing nervous system [29]. The motili-
and streptomycin at 378C in a 7 CO humidified
2
ty of neurites and growth cones is important to synaptic incubator, and the media changed every 2 to 3 days as
plasticity. Alterations in cell shape result from directed needed. Cells transfected with neo alone, a CaM kinase II
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changes in cytoskeletal dynamics regulated by Ca and its mutant cDNAs were maintained in the presence of
signaling. CaM kinase II is one of the major mediators of 0.7 mg ml G418. Assessments of neurite outgrowth were
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Ca signaling in the central nervous system [6]. It
carried out as described previously [15]. Cells with one regulates polymerization-depolymerization of microtubules
process greater than one cell diameter were considered and microtubule-microfilament interaction through phos-
neurite bearing. Neurite length for a given cells was phorylation of microtubule-associated proteins [14,45,46].
quantified as the average radial distance from the neurite Furthermore, it plays important roles in synaptic plasticity
tips to the soma. [6,25]. We have shown that CaM kinase II is involved in
controlling neuronal morphogenesis using neuroblastoma cells expressing the kinase [15,31], suggesting that these
2.3. Isolation of Nb2a cells stably expressing a isoform cells could be used as a model system for studying the
of CaM kinase II and its mutants motility of neurites. On the other hand, calcineurin is a
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Ca calmodulin-dependent protein phosphatase that has
The wild type and mutant cDNAs of the a isoform of been reported to regulate neurite outgrowth [9,23,27].
CaM kinase II were inserted into the EcoRI site of the Moreover, growth cones retain the ability to modulate in
pEF321 and pcDNA3 expression vectors in the sense
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response to Ca elevation [34], suggesting that Ca
direction under the control of human elongation factor 1 signaling plays an important role in the regulation of
and CMV promoters, respectively. pEF321- or pcDNA3-a, neurite outgrowth.
or a-mutant DNAs aT286A and aT286D DNAs and
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To elucidate the mechanism of Ca signaling through
pEF321-neo DNA pEF321-neo were introduced into the action of CaM kinase II on the control of neurite
Nb2a cells by the calcium phosphate gel method [15], or
TM6
outgrowth, we investigated the relation between neurite by treatment with FuGENE
transfection reagent ac-
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outgrowth and Ca -independent activity induced by
cording to the manufacturer’s instructions. Mutant cDNAs autophosphorylation of the kinase in neuroblastoma cells
of aT286A and aT286D were prepared from the a isoform expressing the a isoform and its mutants.
of CaM kinase II cDNA by site directed mutagenesis as described previously [32]. After 2 to 4 weeks of growth in
selection medium containing G-418, several individual
2. Materials and methods
