Departments of 1 Molecular and Cellular Physiology, 2 Molecular Genetics, and 3 Genome Sciences, University of Cincinnati Medical School, Cincinnati, Ohio 45267
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ca2+/calmodulin-regulated protein kinase II (CaMKII) mediates many cellular events. The four CaMKII isoforms have numerous splice variants, three of which contain nuclear localization signals. Little is known about the role of nuclear localized CaMKII in neuronal development. To study this process, PC12 cells were transfected to produce CaMKII targeted to either the cytoplasm or the nucleus and then treated with nerve growth factor (NGF). NGF triggers a signaling cascade (MAPK) that results in the differentiation of PC12 cells into a neuronal phenotype, marked by neurite outgrowth. The present study found that cells expressing nuclear targeted CaMKII failed to grow neurites, whereas cells expressing cytoplasmic CaMKII readily produced neurites. Inhibition of neuronal differentiation by nuclear CaMKII was independent of MAPK signaling, as sustained Erk phosphorylation was not affected. Phosphorylation of CREB was also unaffected. Thus nuclear CaMKII modifies neuronal differentiation by a mechanism independent of MAPK and CREB activation.
calcium/calmodulin-dependent protein kinase II; mitogen-activated protein kinase; calmodulin; neurite outgrowth; CaMKII isoforms; extracellular signal-regulated protein kinase; cAMP-response element-binding protein
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WHEN THE LEVEL OF CA2+ INCREASES inside a cell, several Ca2+-regulated signal transduction cascades are initiated. One such pathway involves calmodulin (CaM), which activates several downstream targets, including calcium/calmodulin-regulated protein kinase II (CaMKII) (for reviews see Refs. 5 and 39). CaMKII regulates a wide variety of cellular events, from controlling cell cycle progression to regulating cellular differentiation and influencing apoptosis (1).
There are four isoforms of CaMKII, ,
,
, and
, each encoded
by a separate gene but having very high sequence homology (1, 8,
46, 49). Each CaMKII isoform is composed of a catalytic domain,
a regulatory domain, and an association domain. The association
domain is responsible for 8-12 individual CaMKII molecules
forming a holoenzyme complex that can be composed of one or several
different isoforms (1, 24, 40). At the carboxyl end of the
regulatory domain, there is a short sequence known as the variable
region (1). Outside this variable region, the amino acid
sequence homology is 80-90% (46, 49). The functional differences between isoforms provide unique developmental, regional, or
subcellular expression patterns. The substrate specificity, kinetics,
and calmodulin affinities are extremely similar (1, 3, 4, 25,
46).
Alternative splicing of these four genes gives rise to a large number
of variants (for reviews see Refs. 1, 22,
24, 40). The - and
-isoforms are found
in neuronal tissue, whereas the
- and
-isoforms are more widely
distributed (49); all cells express at least one gene
type. The details of tissue distribution and developmental regulation
of these splice variants are under investigation. Three of the
isoforms,
B,
A, and
B,
contain nuclear localization signals (NLS) within the variable region (3, 10, 22). The nuclear localized
B-isoform was first identified in rat hearts
(10) and has subsequently been found in the rat cerebellum
and, to a lesser extent, in the cerebrum (47). The nuclear
targeted
B-isoform is found in astrocytes (46), whereas mRNA for it has been detected in the
diencephalon and midbrain of rats but not in the hippocampus or cortex
(3). For the
B- and
B-isoforms, nuclear localization can be blocked by
phosphorylation of a serine located immediately adjacent to the NLS
(23), which decreases binding of CaMKII to a nuclear import receptor. Nuclear targeting may therefore be a dynamic event,
changing as a result of several influences, potentially including
kinases and phosphatases.
Studies with several cytoplasmic isoforms of CaMKII have shown them to
be involved in neurite outgrowth, their exact role depending on both
the cell type and the isoform studied. When the
E-isoform (an embryonic mouse type) is expressed in
NIH/3T3 cells, it locates in the perinuclear region and induces the
spontaneous formation of processes coming from the cell (4,
25). This localization is in contrast to the
C-isoform, which remains in the cytoplasm when expressed
in NIH/3T3 cells and does not induce these extensions (4,
25). A constitutively active mutant of the
C-isoform is capable of inducing neurite outgrowth in P19 cells (8). The role of the
-isoform in neurite
outgrowth has been studied extensively. When overexpressed in CAD cells (9), Nb2a cells (15, 41), and
NG108-15 cells (15),
-CaMKII causes
spontaneous neurite outgrowth. However, when
-CaMKII is overexpressed in pheochromocytoma (PC12) cells, it has been shown to inhibit neurite outgrowth that is normally induced by either nerve
growth factor (NGF) (31) or cAMP (48). These
latter two studies are of particular interest, because they both employ PC12 cells, and they will be considered in more detail in
DISCUSSION.
A further influence on the role of various CaMKII isoforms is the fact
that their expression in a cell can change as a result of cellular
development. For instance, a central nervous system (CNS) cell line,
CAD cells, shows increased CaMKII activity on differentiation, as well
as a shift in the predominant isoform expressed, from the cytoplasmic
C (
2)-isoform in undifferentiated cells to another
cytoplasmic isoform,
D (
4), after differentiation (8). In P19 cells undergoing differentiation, expression
of the
C-isoform is decreased (8). However,
this does not happen when PC12 cells are differentiated; rather, the
D- and
A (
1)-isoforms are increased,
whereas the
C remains constant (8). This
has led Donai et al. (8) to suggest that the lineage and
maturity of the cell may determine which CaMKII isoforms are expressed.
Because of the importance of the role of selected CaMKII isoforms in
neurite outgrowth, the current study was undertaken to explore how the
subcellular location of CaMKII influences NGF-induced neuronal
differentiation. The similarity in kinetics, substrate specificity, and
CaM-binding properties between the different CaMKII isoforms has
allowed the -isoform to be used in many studies of CaMKII function
(9, 15, 31, 41, 48). Thus transgenes were constructed to
target expression of
-CaMKII to either the nucleus or the cytoplasm,
and these transgenes were transfected into rat PC12 cells. The effects
of targeted CaMKII proteins on NGF-induced neurite outgrowth were then studied.
PC12 cells respond to NGF by elaborating neurites within a few days (18). This process requires both new RNA transcription and ongoing protein synthesis (17). The binding of NGF to its high-affinity receptor Trk A initiates several intracellular responses. Among these are an early, transient increase in intracellular Ca2+ concentration (33, 34) and the initiation of a signaling cascade leading to phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases Erk1 and Erk2 (26, 36, 54). Several factors converge to maintain Erk phosphorylation and, hence, its activation over a sustained time period. The initial phosphorylation of Erk depends on CaM (11) and Ras (54), whereas at later time points it is mediated by PKA (53) and Rap1 (54). This sustained activation is necessary for neurite outgrowth in PC12 cells, since epidermal growth factor (EGF), which phosphorylates Erk for only a short time period, does not initiate neurites (26).
The experiments described here use the PC12 cell neuronal model,
transfected with targeted -CaMKII transgenes, to point to a role for
nuclear localized CaMKII in controlling neuronal differentiation. Cell
lines expressing CaMKII in the nucleus failed to grow neurites in
response to NGF, whereas lines with cytoplasmic CaMKII responded to NGF
similar to control cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. Rat PC12 cells (18) were cultured in a 1:1 mix of DMEM-F12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (all from GIBCO-BRL). For transfected cell lines, medium was supplemented with 500 µg/ml G418 antibiotic (BioWhittaker). Cells were grown on tissue culture plastic at 37°C in 5% CO2 and subcultured before reaching confluence. PC12 cells were a gift of Dr. Maria Czyzyk-Krzeska (University of Cincinnati) and were used between passages 7 and 20 of receipt. Transfected cell lines were used between passages 3 and 15 from initial selection. Stocks of all cell lines were kept on liquid N2 and thawed as needed. To assess the overall viability of transfected cell lines, the MTT assay for cell growth was used. Following the protocol of Loo and Rillema (30), cells were plated in one-half of a 96-well plate at 1.5 × 103 cells/well and grown under standard conditions in the presence of G418 antibiotic (if appropriate). On a given assay date, medium was removed from the wells and replaced with medium containing 1.25 mg/ml MTT reagent [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and incubated at 37°C for 2 h. An equal volume of extraction buffer (10% SDS dissolved in a 1:1 mix of H2O and N,N-dimethylformamide) was then added to each well, and the plates were incubated overnight. The optical density was measured at 570 nm and averaged over the 30 center wells of the 48 treated wells (omitting the outside edge wells). The experiment was repeated three to five times for each growth curve. These experiments showed that cell viability and proliferation rate were not changed in cell lines expressing either nuclear or cytoplasmic CaMKII (data not shown).
Neuronal differentiation with NGF.
Lines of PC12 cells were plated at 5 × 103
cells/cm2 on glass coverslips coated with
poly-L-lysine. A thin film of poly-L-lysine was
applied to the coverslips in tissue culture dishes and allowed to dry
for 2-4 h. Dishes were then washed four times with sterile water
and once with complete medium. One day after cells were plated, medium
was changed to serum-free medium with 50 ng/ml NGF (Alamone
Laboratories) and 500 µg/ml G418 antibiotic for stable cell lines.
After the given incubation time, the coverslips were fixed in 10%
formalin and mounted on glass slides. To measure neurite outgrowth, six
adjacent microscope fields were photographed from each coverslip and
scanned (Nikon Coolscan II) as JPEG files along with an image of a
stage micrometer. The JPEG file names were encoded so that images could
be analyzed blinded as to treatment. Neurites were measured on enlarged
photomicrographs with ImagePro software (Media Cybernetics), with
spatial calibration taken from the stage micrometer. The criteria used
to define a neurite-bearing cell required one process to extend 15
µm from the cell body. The length of the longest neurite coming from
any given cell was also recorded. For branched neurites, the portion
giving the greatest total length was used. In cases where the beginning
point of a neurite could come from one of two cells, the neurite was
arbitrarily assigned to one of the cells, and the other cell was scored
as nonneurite bearing. Likewise, if two neurites overlapped and the end
points could not be established, only one neurite and cell were
included in the analysis. Because assigning individual neurites to
their appropriate cell body was difficult in large clumps, groups of
more than six cells were excluded from analysis. After all measurements
for one experiment had been completed, the coding on the JPEG image
files was broken, and the data were analyzed using an Excel (Microsoft)
spreadsheet. For fluorescent photomicrographs, coverslips were fixed in
formalin and then permeabilized in ice-cold acetone for 6-8 min
and blocked with preimmune serum for a minimum of 30 min at 37°C.
Coverslips were incubated with appropriate primary antibody either
overnight at 4°C or for 1 h at room temperature. Coverslips were
then washed four times with PBS and incubated with an FITC-conjugated
secondary antibody at room temperature for 60 min. Coverslips were
again washed four times with PBS and mounted in 90% glycerol on glass
slides. Images were taken with Kodak film on a Nikon Optiphot inverted microscope.
Producing stable PC12 cell lines expressing targeted -CaMKII.
Constructs for targeted CaMKII are shown in Fig.
1. An epitope-specific sequence (the
"FLAG tag," amino acids DYKDDDDK), used to identify the transgenic
protein, and a Kozak sequence (nucleotides ACCACC) were added to the
amino terminus of
-CaMKII DNA. Nuclear targeting was achieved by
including an SV40 NLS (amino acids PKKKRKVE); this construct is
designated NLS-CaMKII. The cytoplasmic targeting was achieved by
leaving the NLS out; that construct is designated Cyto-CaMKII.
Constructs were cloned into the HindIII/EcoR1
sites of the pcDNA3 mammalian expression vector. Transfast (Qiagen) reagents were used to cotransfect wild-type PC12 cells with a plasmid
coding for neomycin resistance and either the cytoplasmic or nuclear
localized, FLAG-tagged CaMKII. Cell lines were selected for antibiotic
resistance with the neomycin analog G418 and then screened for properly
targeted CaMKII expression by immunocytochemistry by use of a
monoclonal anti-FLAG antibody (Eastman Kodak), as described. A cell
line transfected to express neomycin resistance alone was also produced
to be used as a mock transfected control. Three independent clones of
the Cyto-CaMKII transfected cells (Cyto-A2, Cyto-I2, and Cyto-B4), and
three clones of NLS-CaMKII transfected cells (NLS-TC2, NLS-KA1, and
NLS-KB5) were chosen for further analysis.
|
Measuring CaMKII activity.
The CaMKII activity assay followed the protocol of Hanson and Schulman
(21). This assay measures CaMKII activity on the basis of
the incorporation of 32P into an artificial CaMKII
substrate, autocamtide III. Control or transfected PC12 cell
lines were plated at 1-2 × 106 cells in a 60-mm
dish on the day before assay. Cells were rinsed with PBS and lysed 2 min before the beginning of the autocamtide phosphorylation reaction by
use of an extraction buffer containing PIPES (20 mM), EGTA (0.5 mM),
leupeptin (10 µg/µl), Na-pyrophosphate (10 mM), ammonium molybdate
(0.4 mM), Triton X-100 (0.1%), and dithiothreitol (20 µM).
Cell lysate (20 µl) was then added to a reaction tube containing ATP
(0.05 mM), PIPES (50 mM), BSA (0.2 mg/ml), MgCl2 (20 mM),
and ~0.2-0.25 µCi of [-32P]ATP. To measure
CaMKII activity in the lysate, CaCl2 (1 mM), calmodulin (20 µg/ml), and autocamtide III (15 µM) were included; for
Ca2+-independent activity, the CaCl2 and CaM
were replaced with EGTA (1 mM). The reaction mix was incubated at
30°C for 2 min and then quenched by adding ice-cold trichloroacetic
acid (3% final) and storing on ice. Proteins were pelleted, and 20 µl of supernatant were spotted onto phosphocellulose paper.
Radioactivity, corresponding to incorporation of 32P into
autocamtide III by CaMKII, was measured in a scintillation counter. All
assays were performed in duplicate and averaged for one experiment.
Three to fourteen experiments were performed on each cell line. For
CaMKII assays in subcellular fractions, the nuclear and cytoplasmic
fractions were separated (see next section), and the CaMKII activity
assay was run on samples of each, as above.
Separation of nuclear and cytoplasmic fractions. The protocol of Kroll et al. (29) was used to isolate nuclear and cytoplasmic fractions from PC12 cells. Control or transfected PC12 cell lines were plated at ~1 × 106 cells in 60-mm tissue culture dishes 1-2 days before fractions were separated. Cells were scraped off three to four dishes, combined, and pelleted. The cells were resuspended in ice-cold PBS and repelleted. The plasma membrane was disrupted by resuspending cells in a 10 mM Tris buffer containing NaCl (10 mM), MgCl2 (3 mM), and NP-40 (0.5%) and incubating for 1 min on ice before centrifuging. Nuclei were pelleted, and the supernatant (cytoplasmic fraction) was removed. Nuclei were washed once in ice-cold PBS with EGTA (1 mM) and then resuspended in the NP-40-containing buffer and sonicated to disrupt nuclear membrane. Debris was pelleted and the supernatant (nuclear fraction) removed.
Immunoblots.
Transfected or wild-type cell lines were plated at 8 × 105 cells in 35-mm dishes the day before protein
collection. Plates were rinsed once in PBS and then scraped in the
presence of warm lysis buffer. Lysates were heated to 95-100°C
for 5 min and triturated through a 22-gauge × 0.5-in. needle five to
six times to shear the DNA before it was aliquoted and stored at
80°C. Total protein content of lysates was measured using a Lowry
protocol modified to accommodate a 96-well format (see Ref.
43). Equal protein loads (usually 20 µg of total
protein) were subjected to SDS-PAGE separation in a 12% gel followed
by transfer to nitrocellulose (Schleicher & Schuell). The transfer was
confirmed with Ponceau S staining and the position of molecular weight
markers identified. Membranes were blocked in TBS containing 5% nonfat
dry milk and 0.1% Tween 20 (Bio-Rad Laboratories) for 1-2 h and
then incubated with primary antibody overnight at 4°C. Membranes were
rinsed three times in blocking solution for 5-15 min each,
followed by incubation in the appropriate horseradish
peroxidase-conjugated secondary antibody for 1-2 h at room
temperature. Blots were developed with a chemiluminescence reagent kit
(Amersham) and exposed to film. The density of protein bands was
quantified by backlighting the film and using ImagePro software (Media
Cybernetics) to measure the integrated optical density (IOD) of a fixed
rectangular area corresponding to the largest band measured on a given
film. The IOD of an identically sized adjacent background area was
subtracted from the band IOD, and data were expressed as either total
or ratioed IOD, as noted.
Reagents and antibodies.
All chemicals were from Sigma unless noted otherwise. Media and cell
culture supplies were from GIBCO-BRL unless noted otherwise. Antibodies
to Erk, phospho-Erk, cAMP-response element-binding protein (CREB),
and phospho-CREB Ser133 were from Cell Signaling
Technologies. The anti-FLAG antibody was from Sigma, and the -CaMKII
antibody was from Zymed. The antibody to phospho-CREB
Ser142 was the kind gift of Drs. Michael Greenberg and Jon
Kornhauser (Harvard Medical School).
Statistics. Data were averaged using an Excel spreadsheet (Microsoft). Unless noted otherwise, the two-tailed Student's paired t-test was performed. A significant difference is defined as P < 0.05; a highly significant difference is defined as P < 0.01. Error bars in graphs are ±1 SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subcellular targeting of CaMKII in PC12 cells.
To investigate the role that subcellular localization of CaMKII plays
in neuronal differentiation, PC12 cells were transfected with
transgenes (Fig. 1A) to produce an epitope-tagged -CaMKII targeted to concentrate in either the cytoplasm (Cyto-CaMKII) or the
nucleus (NLS-CaMKII). Cytoplasmic localization is conferred by the
native
-CaMKII sequence, whereas nuclear localization was achieved
by adding an SV40 NLS. To confirm proper targeting of the protein,
transfected cells were fixed on glass coverslips and analyzed by
immunofluorescence microscopy with an anti-FLAG primary antibody. This
revealed that the protein produced by the Cyto-CaMKII construct retains
its cytoplasmic localization, whereas the protein produced by the
NLS-CaMKII construct accumulates exclusively in the nucleus (Fig.
1B). To generate stable cell lines expressing these
proteins, the appropriate CaMKII plasmid was cotransfected into PC12
cells along with a neomycin resistance plasmid. Colonies were selected
with G418 antibiotic and screened for appropriately targeted
FLAG-tagged protein production by immunocytochemistry (as in Fig.
1B). Several colonies from each construct were identified for further analysis based on the presence of correctly targeted anti-FLAG staining. The identity of this protein as
-CaMKII was confirmed by immunoblot analysis using an anti-
-CaMKII antibody (Fig. 1C). PC12 cells lack the
-isoform of CaMKII,
expressing primarily the
- and
-isoforms (48). The
absence of
-CaMKII in control PC12 cells is shown in lanes
1 (untransfected) and 2 (mock transfected) of Fig.
1C. Lanes 3-5 show cell lines with nuclear
targeted
-CaMKII, which migrates slightly higher than cytoplasmic
CaMKII, consistent with its extra eight amino acids coded for by the
NLS. Lanes 6-8 show
-CaMKII in cell lines with cytoplasmic targeting. These data confirm that the transfected PC12
cells express a properly localized, FLAG-tagged
-CaMKII protein.
Three independent clonal cell lines expressing cytoplasmic CaMKII
(Cyto-A2, Cyto-I2, and Cyto-B4) and three lines expressing nuclear
CaMKII (NLS-TC2, NLS-KB5, and NLS-KA1) were selected for further analysis.
|
Effect of targeted CaMKII on neuronal differentiation.
NGF initiates differentiation of PC12 cells into a neuron-like
phenotype characterized by a decrease in proliferation, an increase in
catecholamine synthesis, and the appearance of long, branching neurites
(18). To assess the role of targeted CaMKII in this
differentiation process, cells expressing CaMKII in either the nucleus
or the cytoplasm were treated with 50 ng/ml NGF for 6-7 days, and
the extent of neurite formation was measured (see representative
photomicrographs in Fig. 3A).
The concentration of NGF chosen (50 ng/ml) causes ~50% of control
cells to respond with neurite outgrowth; thus either an increase or
decrease in neuronal differentiation could be detected. Untransfected
and mock transfected PC12 cells were used as controls. To quantify neurite outgrowth, computer-aided image analysis (ImagePro) was used on
enlarged photomicrographs to measure the single longest identifiable
process coming from a given cell. By tracing along the neurite path,
the computer-aided system allowed measurement of actual neurite length,
which was taken from the cell body to the tip of the growth cone.
Processes of 15 µm or longer were scored as neurites (see
MATERIALS AND METHODS for full criteria of inclusion). Thus
not only was the percentage of neurite-bearing cells measured but the
lengths of neurites were also compared. After 6-7 days of NGF
treatment, 52% of untransfected control cells and 63% of mock
transfected cells had at least one defined neurite (Fig.
3B). All of the cell lines expressing Cyto-CaMKII had a
slight, but not statistically significant, increase in the percentage
of neurite-bearing cells (62-66%) compared with untransfected controls (52%). In contrast, the cells expressing NLS-CaMKII had very
few neurites. Of the three independent cell lines studied, from 4 to
25% of the cells had any processes of 15 µm or longer (Fig.
3B), which was significantly fewer than controls.
Analyzing the length of neurites, when formed, gives a similar picture: Cyto-CaMKII-expressing cells have longer neurites than controls, whereas NLS-CaMKII-expressing cells have significantly shorter neurites. Figure 3C shows histograms of combined neurite
length data. Figure 3C, top, shows neurite length
of control cell lines (untransfected and mock transfected combined). A
total of 2,528 cells were analyzed, of which 1,502 cells (59%) had
neurites. The largest percentage of cells (mode) had neurites of
22 ± 1 µm in length, whereas the average length was 41.2 ± 3.5 µm. Many cells had neurites up to, and occasionally longer
than, 100 µm. Figure 3C, middle, shows neurites
from all cell lines expressing Cyto-CaMKII. A total of 1,025 cells were
analyzed, with 682 (66%) being neurite bearing. The mode of neurite
length for these lines is slightly longer than controls, at 26-27
µm, whereas the average neurite length, at 48.6 ± 3.7 µm, was
statistically increased over control cells (P < 0.001, Fig. 3C). Figure 3C, bottom, shows the
combined data for cell lines expressing NLS-CaMKII. A total of 1,982 cells were analyzed, with 298 (15%) having neurites. When cells did
grow neurites, these neurites generally just made the cutoff, with a
mode of 15 µm and an average length of 30.7 ± 3.7 µm,
significantly (P < 0.001) shorter than controls; very few neurites grew longer than 50 µm.
|
Modulation of the MAPK pathway.
Decreased neurite outgrowth in the NLS-CaMKII cells could be caused by
a disruption in the integrity of the NGF signal transduction pathways,
which are characterized by several parallel signaling cascades
(36, 42, 53, 54). One effect of NGF binding to its
high-affinity receptor (Trk A) on PC12 cells is the initiation of
signaling cascades leading to the sustained (>2 h) phosphorylation of
the MAPKs Erk1 and Erk2 (Ref. 36 and Fig. 5). The
sustained activation of this pathway has been shown to be necessary for neuronal differentiation and neurite outgrowth in PC12 cells (26, 36). To address the integrity of this MAPK pathway, PC12 cells were cultured overnight in low-serum medium (to reduce background Erk
phosphorylation) and then stimulated with NGF for 10-120 min before proteins were extracted. Immunoblots were run, probing for both
total Erk level and the presence of Erk phosphorylated at
Thr183 and Tyr185. Untransfected control cells
display rapid initial phosphorylation of Erk (10 min), followed by a
sustained phosphorylation that does not fully decay over the 2-h time
period studied (Fig. 4). This same
phosphorylation pattern is seen in cell lines expressing either nuclear
or cytoplasmic CaMKII (Fig. 4), indicating that this MAPK-signaling
pathway remains intact and unmodified. Thus the lack of NGF-induced
neuronal differentiation in cells expressing NLS-CaMKII cannot be
accounted for by changes in the phosphorylation of Erk1 and Erk2.
|
Expression and phosphorylation of CREB.
The transcription factor CREB is rapidly phosphorylated at
Ser133 after a number of different extracellular stimuli.
This phosphorylation is accomplished by several intracellular signaling
pathways (see reviews in Refs. 12 and 38)
including two MAPK cascades in PC12 cells (52) and by PKA
after increases in intracellular Ca2+ concentration
(19). Phosphorylation at Ser133 is a necessary
step for CREB activation, and activated CREB is important in the
maintenance of neuronal cell populations (12). Because NGF
can activate CREB through several different signaling pathways, the
hypothesis was tested that either the total amount of CREB present or
its phosphorylation at Ser133 in response to NGF was
modified by targeted expression of -CaMKII. Control and
CaMKII-transfected cell lines were exposed to medium with or without
NGF (50 ng/ml) for 15 min before cells were lysed and proteins
extracted. Immunoblots using antibodies to total CREB or phospho-CREB
Ser133 were run on these proteins (Fig.
5A). Quantification of band density from the blots shows that the basal level of CREB is unchanged in the cell lines studied (Fig. 5B). There is a uniform,
slight decrease in band density of CREB after NGF treatment (Fig.
5B, left), but because of the rapid time frame
involved (15 min) this may indicate a slight decrease in antibody
affinity for the phosphorylated form of CREB rather than an actual
decrease in CREB content. The degree of Ser133
phosphorylation was evaluated in parallel blots by use of a
phosphospecific antibody to Ser133. Treating untransfected
control cells with NGF for 15 min led to a robust increase in
phospho-CREB Ser133 (Fig. 5, A and
B), as others have reported (14). In the Cyto-CaMKII and NLS-CaMKII cell lines, treatment with NGF also led to rapid phosphorylation of CREB Ser133 (Fig. 5, A
and B), which was not statistically different from controls. These data show that the inhibition of neuronal
differentiation in cells expressing NLS-CaMKII did not arise from
changes in either total CREB or phospho-CREB Ser133.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CaMKII mediates many signal transduction systems and has been found in virtually every cell type examined (for reviews see Refs. 1 and 24). It is particularly abundant in neuronal populations, where it has been implicated in such diverse functions as promoting neuronal survival and regulating long-term potentiation. Although it appears necessary for proper neuronal development, the exact mechanisms of CaMKII's actions are still being defined.
In the area of neurite outgrowth, results of several studies have been inconclusive. A CNS cell line, CAD cells, which normally differentiate on serum withdrawal, underwent spontaneous neuronal differentiation after being transfected to produce CaMKII (9). In two neuroblastoma cell lines, overexpressing CaMKII led to an increase in both the percentage of cells with neurites and the neurite length (15). However, when CaMKII was overexpressed in PC12 cells, which were then stimulated with either NGF (31) or cAMP (48), neurite formation was inhibited. These latter two studies are similar to the experiments performed here and will be discussed separately.
Perhaps one reason for the incomplete picture arises from differences
in the subcellular locations of various CaMKII splice variants. At
least three CaMKII splice variants, B,
A,
and
B, contain NLSs (3, 10, 46). Some
progress has been made in determining tissue distribution of these
isoforms (10, 47). Two of the variants,
B
and
B, have been shown to concentrate in the nucleus of
astrocytes (46), whereas the
B-isoform has also been seen in cerebellar granule cells (47). Little is
known about the role that these isoforms play in normal cellular
function. It is known that expression levels of various isoforms can
change for a given cell type, depending on the stimuli to which the
cells are exposed or the stage in their development (3,
8). Therefore, this study has set about specifically to
investigate the role of nuclear targeted vs. cytoplasmic targeted
CaMKII in neuronal differentiation with the PC12 cell line as a test system.
PC12 cells were transfected with an -CaMKII gene, either containing
or omitting an NLS. The CaMKII protein produced by these constructs is
efficiently expressed in one of two subcellular locations, either the
cytoplasm (Cyto-CaMKII) or the nucleus (NLS-CaMKII). Furthermore, the
activity of CaMKII was increased in the appropriate compartment
relative to either untransfected controls or cell lines with the
opposite CaMKII targeting. These experiments demonstrate that
functional CaMKII can be targeted to mimic various endogenous isoforms.
Control or transfected cell lines expressing Cyto-CaMKII or NLS-CaMKII were treated with a submaximal concentration of NGF (one that leads to ~50% of control cells differentiating and growing neurites within 6-7 days), and the percentage of neurite-bearing cells and neurite length were measured as an index of neuronal differentiation. In three independent clonal cell lines expressing Cyto-CaMKII, the percentage of neurite-bearing cells was similar to the percentage of neurite-bearing cells in untransfected or mock transfected PC12 cells. In addition, the length of neurites formed was increased compared with the control cell lines. These data indicate that an increase in cytoplasmic CaMKII above the endogenous levels found in PC12 cells does not inhibit neurite outgrowth.
This result is consistent with studies done on several neuroblastoma
cell lines. When Nb2A (15, 41), NG108-15
(15), or CAD cells (9) are transfected with
an -CaMKII gene, the extent of neurite outgrowth after appropriate
stimulation is markedly increased over untransfected control cells.
Masse and Kelly (31), however, produced PC12 cell lines
expressing
-CaMKII and showed a decrease in the percentage of
neurite-bearing cells after NGF treatment. Several differences were
observed in cellular growth properties, and one important difference in
technique was noted between that study and this one. Their cell lines
transfected with
-CaMKII showed a decreased initial replication rate
compared with controls. The CaMKII-transfected cells studied here all
proliferated similarly to untransfected controls (data not shown). In
addition, Masse and Kelly reported increased cell-to-substrate adhesion in transfected cells compared with controls, something not seen in the
cell lines used for the present study (data not shown). Experimentally,
the culture medium used by Masse and Kelly to maintain their PC12 cells
contained 10% horse serum and 5% fetal calf serum. In the present
study, 10% FBS was used and no horse serum. Because serum is an
undefined component known to contain numerous cytokines and growth
factors, it seems reasonable that this difference in sera may have
influenced the basal state of the cells. Therefore, the interaction of
NGF signaling and CaMKII could be occurring in distinct intracellular
contexts. These differences seem to include changes in
cell-to-substrate adhesion, an important parameter for neurite growth.
Another group that has overexpressed -CaMKII in the cytoplasm of
PC12 cells, Tashima et al. (48), used cAMP to stimulate neurite outgrowth. They also saw a decrease in neurite-bearing cells in
lines expressing
-CaMKII (48). However, when a
different pathway from NGF is used, cAMP induces neurite outgrowth; the neurites appear much more rapidly and exhibit a different morphology (20, 35, 48). A similar effect to that of Tashima et al. was seen in the Cyto-CaMKII cells in our study. Neurite outgrowth was
greatly reduced in Cyto-CaMKII cells following cAMP treatment compared
with untransfected control cells (Kutcher LW, unpublished observation).
A novel feature of this study is employing an NLS to target CaMKII to
the nucleus. When -CaMKII is targeted to the nucleus of PC12 cells,
a different picture emerges. In contrast to the Cyto-CaMKII-expressing
cells, three independent clonal cell lines expressing NLS-CaMKII all
showed significantly fewer neurite-bearing cells after NGF stimulation.
When neurites did form in the NLS-CaMKII-transfected cells, they were
shorter than the neurites formed in either control or
Cyto-CaMKII-expressing cells. Thus, on the basis of the percentage of
neurite-bearing cells and the length of neurites formed, nuclear targeted CaMKII inhibits NGF-induced neuronal differentiation in PC12 cells.
Several steps in the NGF-signaling cascade were evaluated to investigate the mechanisms leading to this phenomenon. NGF initiates neurite outgrowth in PC12 cells by a complex signaling pathway, with many sites of potential regulation (reviewed in Refs. 6, 27, and 45; see also Ref. 54). Binding of NGF to its high-affinity receptor, Trk A, initiates several intracellular events, including a transient increase in intracellular Ca2+ concentration (33, 34) and the sustained phosphorylation of the MAPKs Erk1 and Erk2 (36, 53). This Erk activation is mediated via Ras (36, 54) and Rap1 (54) and is necessary for neurite outgrowth (26). Stimuli other than NGF, such as EGF and activation of CaM (11), can transiently phosphorylate Erk without initiating neurite outgrowth, suggesting that the sustained phosphorylation of Erk is critical to neurite induction. To evaluate the integrity of this MAPK-signaling cascade, both the total amount of Erk and its phosphorylation in response to NGF were evaluated in Cyto-CaMKII and NLS-CaMKII cell lines. These cell lines show robust phosphorylation of Erk after 10 min of NGF, which is maintained at levels similar to controls for 120 min. Therefore, neither immediate nor longer-term Erk phosphorylation is affected by the presence of either NLS-CaMKII or Cyto-CaMKII. Thus the observed differences in neuronal differentiation are not related to this NGF-stimulated pathway, at least to the point of Erk phosphorylation.
NGF-signaling cascades ultimately activate the transcription
factor CREB. Several kinases, such as PKA, PKC, RSK2, and CaM kinases, phosphorylate CREB at Ser133. This
phosphorylation recruits coactivators into a signaling complex capable
of driving transcription of genes that have the cAMP response element
(CRE) in their promoters (see reviews in Refs. 7,
32, and 38). Xing et al. (52)
have determined that NGF leads to the phosphorylation of CREB at
Ser133 by two distinct mechanisms. The first is via the
sustained activation of Erk (discussed above), which activates the
kinases RSK1, -2, and -3; blocking this pathway decreases,
but does not eliminate, CREB Ser133 phosphorylation. A
second MAPK pathway, involving p38 MAPK activation of MAPK-activated
protein kinase 2, is also initiated by NGF and leads to
Ser133 phosphorylation (52). In hippocampal
cells, the upstream activator of CaMKII, CaM, can translocate to the
nucleus, where it correlates with increased CREB phosphorylation
(6). It has been suggested that a nuclear targeted isoform
of CaMKII, 3, may be involved in CREB regulation (47).
Therefore, the hypothesis was tested that either total CREB or
phospho-CREB Ser133 was altered in NLS-CaMKII cell lines.
Immunoblots probing for these proteins in NLS-CaMKII, Cyto-CaMKII, and
control cell lines all show equivalent amounts of CREB, and the cell
lines respond to NGF with equivalent levels of Ser133
phosphorylation. Thus the inhibited neuronal differentiation in
NLS-CaMKII cells does not arise from differences in either total CREB
or its ability to be phosphorylated at a site critical for activation,
Ser133.
CREB can be phosphorylated at sites in addition to Ser133. Phosphopeptide mapping shows that Ser142 (28, 44) and Ser143 (28) can both be phosphorylated by CaMKII. In vitro assays indicate that phosphorylation of Ser142 inhibits dimerization of CREB and subsequent binding of the CREB-binding protein (CBP) (51). Because CBP is critical to the activated CREB complex (32, 38), phosphorylation at Ser142 by CaMKII would be expected to inhibit CREB activity, which seems to happen in vitro. In cortical neurons stimulated with KCl (28) and in GH3 cells (44), full activity of a CREB/Gal4 reporter gene requires that the Ser142 of CREB be mutated to an alanine.
Recent in vivo work, however, indicates that the picture may be more complex; CREB Ser142 phosphorylation may actually increase gene transcription in certain contexts (13, 28). Gau et al. (13) produced transgenic mice with a Ser142-to-Ala mutation in CREB. These animals had alterations in their circadian clock that correlated with decreased expression of c-Fos (13), a transcription factor controlled by CREB (14).
Because of the emerging role of Ser142 phosphorylation in CREB activity, an antibody to phospho-CREB Ser142 (28) was used to probe for this phosphorylation in rat cortical neurons, control PC12 cells, and NLS-CaMKII- and Cyto-CaMKII-expressing PC12 cells. Immunoblots of primary cortical neurons treated for 15 min with depolarizing concentrations of KCl show robust phosphorylation of CREB Ser142 similar to that seen by Kornhauser et al. (28). In control or transfected PC12 cells, the basal level of CREB Ser142 phosphorylation was very low, equivalent to untreated cortical neurons. There was no increase in this phosphorylation when the cells were treated with NGF and only a slight increase when they were depolarized with KCl. The data shown here indicate that, in PC12 cells, CREB is not phosphorylated at Ser142 in response to NGF. Furthermore, the presence of a nuclear localized CaMKII is not sufficient to drive that phosphorylation either with or without treatment. In addition, NGF-stimulated c-Fos expression was not decreased by either NLS-CaMKII or Cyto-CaMKII (data not shown), indicating that CREB remains fully competent to drive gene expression in these cell lines. Therefore, the decreased neuronal differentiation in NLS-CaMKII-expressing PC12 cells does not seem to be due to an inhibitory phosphorylation of CREB at Ser142.
In conclusion, this study has used transgenes encoding targeted
-CaMKII to elevate CaMKII kinase activity in specific subcellular compartments and has shown that nuclear CaMKII inhibits NGF-induced neuronal differentiation of PC12 cells, whereas cytoplasmically localized CaMKII does not. The inhibition of differentiation is independent of MAPK activation, which is required for neurite outgrowth. The inhibited neurite outgrowth is also independent of the
transcription factor CREB, as total CREB remained constant in cells
with NLS-CaMKII compared with controls, nor was phosphorylation at
Ser133 or Ser142 changed. Thus it would appear
that the role of CaMKII in promoting or inhibiting neuronal
differentiation depends on the subcellular location of the expressed
CaMKII isoform. As more is learned about which neuronal populations
express nuclear localized CaMKII isoforms and at what developmental
stages these expressions occur, a more complete picture of the role for
nuclear CaMKII in neurons will emerge.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Maria Czyzyk-Krzeska (University of Cincinnati) for the gift of PC12 cells and Drs. Michael Greenberg and Jon Kornhauser (Harvard Medical School) for the anti-phospho-CREB Ser142 antibody. We also thank Dr. Linda Levin (University of Cincinnati) for expert advice with the statistical analysis.
![]() |
FOOTNOTES |
---|
This work was partially funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46433 to J. R. Dedman.
Address for reprint requests and other correspondence: J. Dedman, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Wy, Cincinnati, OH 45267 (E-mail: John.Dedman{at}UC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 5, 2003;10.1152/ajpcell.00510.2002
Received 5 November 2002; accepted in final form 21 January 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Braun, AP,
and
Schulman H.
The multifunctional calcium/calmodulin-dependent protein kinase: from form to function.
Annu Rev Physiol
57:
417-445,
1995[ISI][Medline].
2.
Brickey, DA,
Bann JG,
Fong YL,
Perrino L,
Brennan RG,
and
Soderling TR.
Mutational analysis of the autoinhibitory domain of calmodulin kinase II.
J Biol Chem
269:
29047-29054,
1994
3.
Brocke, L,
Srinivasan M,
and
Schulman H.
Developmental and regional expression of multifunctional Ca2+/calmodulin-dependent protein kinase isoforms in rat brain.
J Neurosci
15:
6797-6808,
1995[ISI][Medline].
4.
Caran, N,
Johnson LD,
Jenkins KJ,
and
Tombes RM.
Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II.
J Biol Chem
276:
42514-42519,
2001
5.
Dedman, JR,
and
Kaetzel MA.
Calcium as an intracellular second messenger: mediation by calcium binding proteins.
In: Cell Physiology Source Book. New York: Academic, 1995, p. 128-136.
6.
Deisseroth, K,
Heist EK,
and
Tsien RW.
Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons.
Nature
392:
198-202,
1998[ISI][Medline].
7.
Deisseroth, K,
and
Tsien RW.
Dynamic multiphosphorylation passwords for activity-dependent gene expression.
Neuron
34:
179-182,
2002[ISI][Medline].
8.
Donai, H,
Murakami T,
Amano T,
Sogawa Y,
and
Yamauchi T.
Induction and alternative splicing of delta isoform of Ca(2+)/calmodulin-dependent protein kinase II during neural differentiation of P19 embryonal carcinoma cells and during brain development.
Brain Res Mol Brain Res
85:
189-199,
2000[ISI][Medline].
9.
Donai, H,
Nakamura M,
Sogawa Y,
Wang JK,
Urushihara M,
and
Yamauchi T.
Involvement of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth induced by cAMP treatment and serum deprivation in a central nervous system cell line, CAD derived from rat brain.
Neurosci Lett
293:
111-114,
2000[ISI][Medline].
10.
Edman, CF,
and
Schulman H.
Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms.
Biochim Biophys Acta
1221:
89-101,
1994[ISI][Medline].
11.
Egea, J,
Espinet C,
Soler RM,
Peiro S,
Rocamora N,
and
Comella JX.
Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca(2+) and calmodulin.
Mol Cell Biol
20:
1931-1946,
2000
12.
Finkbeiner, S.
CREB couples neurotrophin signals to survival messages.
Neuron
25:
11-14,
2000[ISI][Medline].
13.
Gau, D,
Lemberger T,
von Gall C,
Kretz O,
Le Minh N,
Gass P,
Schmid W,
Schibler U,
Korf HW,
and
Schutz G.
Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock.
Neuron
34:
245-253,
2002[ISI][Medline].
14.
Ginty, DD,
Bonni A,
and
Greenberg ME.
Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB.
Cell
77:
713-725,
1994[ISI][Medline].
15.
Goshima, Y,
Ohsako S,
and
Yamauchi T.
Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility.
J Neurosci
13:
559-567,
1993[Abstract].
16.
Green, SH.
The use of PC12 cells for the study of the mechanism of action of neurotrophic factors: signal transduction and programmed cell death.
In: Methods: A Companion to Methods in Enzymology. New York: Academic, 1995, p. 222-237.
17.
Greene LA. The importance of both early and delayed responses in
the biological actions of nerve growth factor. Trends
Neurosci: 91-94, 1984.
18.
Greene, LA,
and
Tischler AS.
Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.
Proc Natl Acad Sci USA
73:
2424-2428,
1976[Abstract].
19.
Grewal, SS,
Fass DM,
Yao H,
Ellig CL,
Goodman RH,
and
Stork PJ.
Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway.
J Biol Chem
275:
34433-34441,
2000
20.
Gunning, PW,
Landreth GE,
Bothwell MA,
and
Shooter EM.
Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells.
J Cell Biol
89:
240-245,
1981[Abstract].
21.
Hanson, PI,
and
Schulman H.
Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis.
J Biol Chem
267:
17216-17224,
1992
22.
Heist, EK,
and
Schulman H.
The role of Ca2+/calmodulin-dependent protein kinases within the nucleus.
Cell Calcium
23:
103-114,
1998[ISI][Medline].
23.
Heist, EK,
Srinivasan M,
and
Schulman H.
Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear targeting.
J Biol Chem
273:
19763-19771,
1998
24.
Hook, SS,
and
Means AR.
Ca(2+)/CaM-dependent kinases: from activation to function.
Annu Rev Pharmacol Toxicol
41:
471-505,
2001[ISI][Medline].
25.
Johnson, LD,
Willoughby CA,
Burke SH,
Paik DS,
Jenkins KJ,
and
Tombes RM.
Delta Ca(2+)/calmodulin-dependent protein kinase II isozyme-specific induction of neurite outgrowth in P19 embryonal carcinoma cells.
J Neurochem
75:
2380-2391,
2000[ISI][Medline].
26.
Klesse, LJ,
Meyers KA,
Marshall CJ,
and
Parada LF.
Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells.
Oncogene
18:
2055-2068,
1999[ISI][Medline].
27.
Klesse, LJ,
and
Parada LF.
Trks: signal transduction and intracellular pathways.
Microsc Res Tech
45:
210-216,
1999[ISI][Medline].
28.
Kornhauser, JM,
Cowan CW,
Shaywitz AJ,
Dolmetsch RE,
Griffith EC,
Hu LS,
Haddad C,
Xia Z,
and
Greenberg ME.
CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events.
Neuron
34:
221-233,
2002[ISI][Medline].
29.
Kroll, SL,
Paulding WR,
Schnell PO,
Barton MC,
Conaway JW,
Conaway RC,
and
Czyzyk-Krzeska MF.
Von Hippel-Lindau protein induces hypoxia-regulated arrest of tyrosine hydroxylase transcript elongation in pheochromocytoma cells.
J Biol Chem
274:
30109-30114,
1999
30.
Loo, DT,
and
Rillema JR.
Measurement of cell death.
Methods Cell Biol
57:
251-264,
1998[ISI][Medline].
31.
Masse, T,
and
Kelly PT.
Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation.
J Neurosci
17:
924-931,
1997
32.
Mayr, B,
and
Montminy M.
Transcriptional regulation by the phosphorylation-dependent factor CREB.
Nat Rev Mol Cell Biol
2:
599-609,
2001[ISI][Medline].
33.
Nikodijevic, B,
and
Guroff G.
Nerve growth factor-induced increase in calcium uptake by PC12 cells.
J Neurosci Res
28:
192-199,
1991[ISI][Medline].
34.
Pandiella-Alonso, A,
Malgaroli A,
Vicentini LM,
and
Meldolesi J.
Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells.
FEBS Lett
208:
48-51,
1986[ISI][Medline].
35.
Pollock, JD,
Krempin M,
and
Rudy B.
Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells.
J Neurosci
10:
2626-2637,
1990[Abstract].
36.
Qiu, MS,
and
Green SH.
PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.
Neuron
9:
705-717,
1992[ISI][Medline].
37.
Rich, RC,
and
Schulman H.
Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.
J Biol Chem
273:
28424-28429,
1998
38.
Shaywitz, AJ,
and
Greenberg ME.
CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.
Annu Rev Biochem
68:
821-861,
1999[ISI][Medline].
39.
Soderling, TR.
The Ca-calmodulin-dependent protein kinase cascade.
Trends Biochem Sci
24:
232-236,
1999[ISI][Medline].
40.
Soderling, TR,
Chang B,
and
Brickey D.
Cellular signaling through multifunctional Ca2+/calmodulin-dependent protein kinase II.
J Biol Chem
276:
3719-3722,
2001
41.
Sogawa, Y,
Yoshimura Y,
Otaka A,
and
Yamauchi T.
Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells.
Brain Res
881:
165-175,
2000[ISI][Medline].
42.
Stephens, RM,
Loeb DM,
Copeland TD,
Pawson T,
Greene LA,
and
Kaplan DR.
Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses.
Neuron
12:
691-705,
1994[ISI][Medline].
43.
Stoscheck, CM.
Quantitation of protein.
Methods Enzymol
182:
50-68,
1990[ISI][Medline].
44.
Sun, P,
Enslen H,
Myung PS,
and
Maurer RA.
Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.
Genes Dev
8:
2527-2539,
1994[Abstract].
45.
Szeberenyi, J.
Gene activation pathways of nerve growth factor signaling: a minireview.
Neurobiology (Bp)
4:
1-11,
1996[Medline].
46.
Takeuchi, Y,
Yamamoto H,
Fukunaga K,
Miyakawa T,
and
Miyamoto E.
Identification of the isoforms of Ca(2+)/calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization.
J Neurochem
74:
2557-2567,
2000[ISI][Medline].
47.
Takeuchi, Y,
Yamamoto H,
Matsumoto K,
Kimura T,
Katsuragi S,
Miyakawa T,
and
Miyamoto E.
Nuclear localization of the delta subunit of Ca2+/calmodulin-dependent protein kinase II in rat cerebellar granule cells.
J Neurochem
72:
815-825,
1999[ISI][Medline].
48.
Tashima, K,
Yamamoto H,
Setoyama C,
Ono T,
and
Miyamoto E.
Overexpression of Ca2+/calmodulin-dependent protein kinase II inhibits neurite outgrowth of PC12 cells.
J Neurochem
66:
57-64,
1996[ISI][Medline].
49.
Tobimatsu, T,
and
Fujisawa H.
Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs.
J Biol Chem
264:
17907-17912,
1989
50.
Tombes, RM,
Mikkelsen RB,
Jarvis WD,
and
Grant S.
Downregulation of delta CaM kinase II in human tumor cells.
Biochim Biophys Acta
1452:
1-11,
1999[ISI][Medline].
51.
Wu, X,
and
McMurray CT.
Calmodulin kinase II attenuation of gene transcription by preventing cAMP response element-binding protein (CREB) dimerization and binding of the CREB-binding protein.
J Biol Chem
276:
1735-1741,
2001
52.
Xing, J,
Kornhauser JM,
Xia Z,
Thiele EA,
and
Greenberg ME.
Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.
Mol Cell Biol
18:
1946-1955,
1998
53.
Yao, H,
York RD,
Misra-Press A,
Carr DW,
and
Stork PJ.
The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor.
J Biol Chem
273:
8240-8247,
1998
54.
York, RD,
Yao H,
Dillon T,
Ellig CL,
Eckert SP,
McCleskey EW,
and
Stork PJ.
Rap1 mediates sustained MAP kinase activation induced by nerve growth factor.
Nature
392:
622-626,
1998[ISI][Medline].