166 Y
example, depolarization or electrical stimulation increased cence reagent were purchased from DuPont New England
21
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
21
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].
21
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
21
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
21
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
21
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
21 21
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
21
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-
21
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
G-418-resistant clones were isolated. The cells were further cloned from a single cell by a limited dilution
2.1. Materials method. Expression of the wild and mutant kinases was
32
[g- P]ATP and immunoblot enhanced chemilumines- confirmed by immunoblotting Fig. 1A.
Y . Sogawa et al. Brain Research 881 2000 165 –175
167
Fig. 1. Detection of CaM kinase II and its mutants in Nb2a cells stably expressing the enzymes and GFP-tagged kinases by immunoblotting. A Cell extracts 50 mg of protein of Nb2a cells stably expressing the a isoform of CaM kinase II and its mutants were subjected to immunoblotting. Lane 1,
purified CaM kinase II 30 ng; lanes 2–5, Nb2a control cells, Nb2a a cells, Nb2a aT286A cells, and Nb2a aT286D cells, respectively. Molecular markers are shown on the left. B pEGFP-C1 a and pEGFP-C1 aT286D DNAs were transfected to HEK 293T cells, and cultured for 24 h, and then cells
were extracted as described under Materials and methods. Cell extracts 50 mg of protein were subjected to immunoblotting. Lane 1, transfected with pEGFP-C1 a DNA; lane 2, transfected with pEGFP-C1 aT286D DNA; lane 3, purified CaM kinase II 30 ng. An arrow indicates the GFP-tagged kinase.
Molecular markers are shown on the left.
2.4. Expression of GFP-CaM kinase II 2.5. Preparation of cell extracts
The sequence corresponding to the a isoform of rat Cells were cultured in tissue culture dishes, washed once
21 21
brain CaM kinase II was ligated with EcoRI and HindIII with Ca
-Mg -free DMEM, harvested, and then soni-
sites at the 59 and 39 noncoding regions, respectively, and cated in 200 ml of extraction buffer 10 cm dish. The
cloned into pEGFP-C1 expression vector to yield plasmid extraction buffer consisted of 40 mM Tris–HCl, pH 7.6, 1
pEGFP-C1 a in frame. GFP was tagged to the N-terminus mM dithiothreitol, 1 mM EGTA, 10 glycerol, 1 mM
5
of CaM kinase II. Nb2a cells were seeded at 2310 35 phenylmethylsulfonyl fluoride PMSF, 0.1 mM okadaic
mm dish and cultured for 24 h. The plasmid DNA 2 mg acid, 0.1 mM calyculin A, and 10 mg ml each of the
TM6
was introduced to the cells with 3 ml of FuGENE antibiotic protease inhibitors, antipain, leupeptin, and
transfection reagent. The number of cells, and amount of pepstatin A. After centrifugation at 18,5003g for 30 min,
TM6
FuGENE transfection reagent and plasmid DNA were
the soluble and particulate fractions were separated. changed in proportion to the size of the culture dish. After
an appropriate period of incubation, the medium was 2.6. Immunoblot analysis
changed to DMEM without phenol red, and the expression of GFP-fusion protein was monitored by fluorescent
Immunoblot analysis of transfected cells was carried out microscopy.
as described previously [19]. The soluble fraction of To confirm that GFP-fusion protein was expressed and
transfected cells was separated by 10 polyacrylaminde– had kinase activity, pEGFP-C1 a plasmid DNA was
SDS gel electrophoresis and electrophoretically transferred introduced to HEK 293T cells as described above. HEK
to a nitrocellulose membrane. The transblot was preincu- 293T cells had less endogenous kinase activity and greater
bated with 5 non-fat dry skim milk in phosphate-buf- efficiency of transfection than Nb2a cells under the ex-
fered saline, pH 7.4 PBS, and then incubated for 1 h with perimental conditions. After 24 h of transfection, cells
monoclonal antibodies specific to the a isoform of CaM were extracted, and the kinase activity was determined.
kinase II or phospho-CaM kinase II. The membrane was The expression of GFP-tagged kinases was confirmed by
washed with PBS or Tris-buffered saline TBS containing immunoblotting as shown in Fig. 1B.
0.05 Tween 20 or 0.1 Triton X-100, and then incu-
168 Y
Table 1
bated with horseradish peroxidase-coupled antibody di-
a
Neurite length of Nb2a a cells
luted 1:2000. Immunoreactive bands were detected using
Time after stimulation Ratio of neurite length to size of cell body
enhanced chemiluminescence reagents. For reprobing, membranes were washed with PBS2
h No neurites
0–1 1–2
2–3 3
0.05 Tween 20, and then incubated with 62.5 mM
Cells 100 cells
Tris–HCl, pH 6.8, containing 2 SDS and 100 mM
0.25 86
14
2-mercaptoethanol for 30 min at 508C. After the mem-
0.5 72
25 3
1.0 49
46 4
1
branes had been washed with PBS-0.05 Tween 20 and
2.0 28
44 25
2 1
reused, the detection reaction was started from the block-
4.0 22
51 21
5 1
ing step as described above.
8.0 17
56 23
3 1
Protein was measured with bovine serum albumin as a
24 26
35 32
4 3
standard as described [5]. SDS–PAGE was performed by
a 5
Nb2a a cells were seeded at 2310 well of 24-well plates, and
the method of Laemmli [22].
photographed at 15 min, 30 min, 60 min, 2 h, 4 h, 8 h, and 24 h after stimulation. Neurite length for a given cell was quantified as the average
2.7. Assay of CaM kinase II
radial distance from the neurite tips to the soma. The values are representative of data obtained from four independent experiments.
CaM kinase II activity was assayed by phosphorylation of autocamtide 2 at 308C for 1 min as described previously
[38]. The standard reaction mixture contained 50 mM increased in number and composed about 27 of the total
32 5
Hepes buffer, pH 8.0, 50 mM [g- P]ATP 4–6310 cpm, at 2 h Fig. 2 and Table 1. Neurite-bearing cells were not
8 mM MgCH COO , 10 mM autocamtide 2, 2 mM increased significantly after 2 h. Neurite length increased
3 2
calmodulin, 0.2 mM CaCl , 0.1 mM EGTA, and a suitable markedly from 30 min to 2 h after stimulation, indicating
2
amount of enzyme in a total volume of 50 ml. The that the formation of neurites was initiated soon after
21
Ca -independent activity was measured under the same
stimulation. Control Nb2a cells did not form neurites, conditions except that 1 mM EGTA was added in place of
consistent with previous results [31]. Neurite outgrowth of
21
Ca .
Nb2a a cells was observed when cells were stimulated by
21
ionomycin, a Ca -ionophore data not shown. But, the
2.8. Autophosphorylation of CaM kinase II morphological change could only be investigated for a
short time, since this reagent had strong cell toxicity. The Autophosphorylation of CaM kinase II was carried out
initiation of neurites was faster than that of differentiation at 08C. The reaction mixture contained 50 mM Hepes
of neuroblastoma cells by cAMP [30] or that of pheochro- buffer, pH 8.0, 100 mM ATP, 8 mM Mg CH COO , 0.2
mocytoma PC12 cells by nerve growth factor [16], sug-
3 2
mM CaCl , 0.1 mM EGTA, 2 mM calmodulin, l mM gesting that the action of CaM kinase II was related to the
2
dithiothreitol, and suitable amounts of CaM kinase II in a morphological change of the growth cone.
total volume of 30 ml. Control reactions were carried out in
21
the absence of Ca and calmodulin. After incubation for
3.2. Expression of GFP-CaM kinase II 10 min, aliquots of the reactions were removed and
assayed for enzymatic activity. To investigate the involvement of CaM kinase II in
neurite outgrowth directly, the distribution of CaM kinase II in neurites was examined using GFP-fusion protein.
3. Results When pEGFP-C1 a plasmid DNA was introduced in HEK
