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
293T cells, expression of enzyme protein was detected by 3.1. Neurite outgrowth
immunoblotting Fig. 1B. The molecular mass of the a isoform of CaM kinase II and GFP is 50 and 27 kDa,
We previously demonstrated that CaM kinase II pro- respectively. GFP-CaM kinase II was found to be 77 kDa,
moted neurite outgrowth in neuroblastoma cells, and these consistent with the mass of respective proteins. Specific
cells formed neurites within 2 h of stimulation [31]. To enzyme activity was 9.40 and 5.71 nmol min mg protein
clarify the involvement of CaM kinase II in neurite in soluble and particulate fractions, respectively, indicating
outgrowth, morphological change was investigated in the that the activity of the GFP-tagged kinase was comparable
21
period just after stimulation. Cells were stimulated by to that of the native kinase. The activity was Ca
and
21
plating. When Nb2a cells expressing the a isoform of CaM calmodulin dependent, and Ca
-independent activity was kinase II Nb2a a cells were stimulated, they showed
induced by autophosphorylation of the kinase data not flattening and formed neurites as long as about one-third of
shown. These results indicated that GFP-CaM kinase II the cell diameter at 15 min Fig. 2. Neurites were short
had similar enzymatic properties to the wild type enzyme. early on, being less than 1 cell diameter in about half of
Then, pEGFP-C1 a DNA was transfected to Nb2a or the cells bearing neurites at 60 min. Cells bearing neurites
Nb2a a cells. After 24 h, GFP-CaM kinase II was
Y . Sogawa et al. Brain Research 881 2000 165 –175
169
5
Fig. 2. Morphological change of Nb2a a cells. Nb2a a cells were seeded at 2310 well of 24-well plates, and photographed at 15 min, 30 min, 60 min, 2 h, 4 h, and 8 h after stimulation by plating A–F, respectively. The center of each well was chosen as the site for analysis. A phase contrast
photomicrograph is shown. Bar, 40 mm.
transiently expressed in Nb2a cells Fig. 3. These cells 30 min, and then gradually decreased. The enzyme activity
had neurites and the GFP-tagged kinase was found to be was significantly high at 4 to 8 h as compared with the
21
distributed in the neurites as well as in the cell body. Cells basal level. However, after 24 h, the Ca
-independent
21
transfected with control pEGFP-C1 plasmid DNA did not activity was only slightly increased. The Ca
-independent change cell morphology and did not have neurites. The
activity accounted for about 4 of total activity at 30 min.
21
efficiency of the transfection was about 30. These results This may be physiologically significant, since the Ca
- indicated that CaM kinase II localized in neurites and
independent activity of the extract composed at maximum promoted neurite outgrowth even in cells transiently
about 50 of total activity when the kinase was auto- expressing the kinase. The kinase was clearly detectable in
phosphorylated in vitro, and the kinase in neurites may be neurites of Nb2a a cells when pEGFP-C1 a DNA was
present at relatively low concentrations as judged from the introduced into Nb2a a cells Fig. 3E and F. The kinase
distribution of GFP-CaM kinase II. was detected throughout the neurite. These results suggest
Immunoblotting using phospho-kinase specific antibody that the kinase may phosphorylate some proteins involved
demonstrated phosphorylation of Thr-286 at 15 min, and in neurite outgrowth.
with a maximum at 30 min and then a decrease Fig. 4B. Phospho-kinase was detected at 2 and 4 h after stimulation.
21
3.3. Detection of Ca -independent activity and
After 24 h, phospho-kinase activity significantly increased autophosphorylation of CaM kinase II
again. The a isoform of CaM kinase II was expressed at almost the same level in these cell extracts Fig. 4B, lower
21
To clarify the relation of Ca calmodulin-independent
panel. The change of autophosphorylated kinase was
21
activity with neurite outgrowth, autophosphorylated kinase compatible with that of Ca
-independent activity. These
21
was investigated in Nb2a a cells by immunoblotting and results indicated that autophosphorylation and Ca
-in- activity assay Fig. 4. When cells were seeded on new
dependent activity preceded the initiation of neurite out-
21 21
dishes in DMEM supplemented with 10 FBS, Ca -
growth, suggesting that Ca -independent activity plays an
independent activity was increased at 15 min and peaked at important role in neurite outgrowth.
170 Y
Fig. 3. Expression of GFP-CaM kinase II in Nb2a or Nb2a a cells. Nb2a or Nb2a a cells were transfected with pEGFP-C1 a DNA as described under Materials and methods. Cells were cultured for 24 h and then photographed. A and B control pEGFP-C1 DNA transfected to Nb2a cells; C and D
pEGFP-C1 a DNA transfected to Nb2a cells; E and F, pEGFP-C1 a DNA transfected to Nb2a a cells; A and C, phase contrast photomicrographs; B, D, E and F fluorescent photomicrographs. E and F show typical samples bearing short and long neurites, respectively.
3.4. Effect of mutation of Thr-286 in CaM kinase II on growth was investigated in cells expressing aT286A and
neurite outgrowth aT286D kinases Nb2a aT286A and Nb2a aT286D cells,
21
respectively. aT286A kinase did not generate Ca -in-
21
The effect of a mutation at Thr-286 on neurite out- dependent activity. aT286D kinase had Ca
-independent
Y . Sogawa et al. Brain Research 881 2000 165 –175
171
significantly high as compared with that of wild type less
21
than 1 and aT286A kinase about 1. The Ca -
independent activity of aT286D and aT286A kinases did not increase significantly on autophosphorylation, whereas
that of the wild type increased about 50. These experi- ments were done in three independent clones, and similar
results were obtained in each clone data not shown.
The morphological change caused by the stimulation was investigated as described above. Nb2a aT286A cells
did not form neurites and were indistinguishable from control Nb2a cells at all time points data not shown,
consistent with a previous report [15]. On the other hand, Nb2a aT286D cells had neurites, although they formed
very late. The adhesion of Nb2a aT286D cells to culture dishes was weak as compared with that of Nb2a a cells.
The cells did not proliferate for 24 h, but then increased with a doubling time of about 24–30 h, which was almost
the same as that of Nb2a a and Nb2a aT286A cells data not shown. Nb2a aT286D cells did not change shape and
were round for a period of 2 days. After 48 h, the cells were flattened and neurite outgrowth initiated, with a rapid
increase in the number of processes. About 20 of cells bore short neurites at that time. From 48 to 96 h, the
neurites lengthened considerably, and relatively large differences in length were observed between the cells
Figs. 5 and 6, suggesting that the initiation of neurite outgrowth was rate limiting step of these process. The
expression of the kinase in the growth cone was clearly demonstrated using the GFP-tagged kinase Fig. 6E and
F. On the other hand, Nb2a a cells had neurites at an early period. Neurites gradually increased in number from
24 to 72 h, and did not change after 72 h. These results indicated that initiation of neurite outgrowth might require
21
Fig. 4. Appearance of Ca -independent activity and autophosphorylated
a threshold level of CaM kinase II activity, and that
6
21
kinase. Nb2a a cells 5310 were stimulating by plating on 10-cm
Ca -independent activity of the kinase induced by auto-
dishes and extracted at the times indicated. A Time course of appear-
21
phosphorylation of Thr-286 involved in neurite outgrowth
ance of Ca -independent activity. An aliquot was withdrawn, and total
21
of neuroblastoma cells.
and Ca -independent activities of the kinase were determined. The
enzyme activity was normalized to that of control cells without stimula-
21
tion, and expressed as a percent age of Ca -independent activity to total
activity. Data are representative of at least 5 experiments done in duplicate. B Time course of the change of phospho-kinase detected by
4. Discussion
