From the Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100 Israel
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Gene expression is necessary for the formation
and consolidation of long term memory in both invertebrates and
vertebrates. Here, we describe the expression and characterization of
candidate plasticity gene 16 (cpg16), a protein
serine/threonine kinase that was previously isolated from rat
hippocampus as a plasticity-related gene. CPG16, when expressed in and
purified from bacteria and COS7 cells, was only capable of
autophosphorylation and phosphorylation of myelin basic protein but
failed to phosphorylate many other peptides and proteins in in
vitro phosphorylation assays. Recombinant CPG16, when
overexpressed and purified from COS7 cells, had a relatively low level
of autophosphorylation activity. This activity was significantly
stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were
added to the cells but not by any other extracellular stimuli tested,
e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a
direct substrate for this kinase. This suggests that CPG16 may be
activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon
stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems
to partially inhibit cAMP-stimulated activity of the transcription
factor CREB (cAMP response element-binding protein), suggesting its
involvement in the down-regulation of cAMP-induced transcription. Thus,
CPG16 is a protein serine/threonine kinase that may be involved in a
novel signaling pathway downstream of cAMP-dependent protein kinase.
Learning and memory processes in the brain are characterized by
plasticity changes in central nervous system neurons. In a comprehensive search for candidate plasticity-related genes
(CPGs1 (1, 2)), a novel
cDNA encoding for a putative protein kinase, termed
cpg16, has been isolated from kainate-treated rat
hippocampus. Here we show that CPG16 is a protein serine/threonine
kinase that, when expressed in COS7 cells, is activated by cAMP via a
cAMP-dependent protein kinase (PKA)-induced mechanism.
Studies on the effect of CPG16 on transcription have revealed that it
may be involved in the down-regulation of cAMP response element-binding
protein (CREB) activity. Thus, it is possible that CPG16 participates in the regulation of neuronal plasticity by down-regulating
PKA-stimulated transcription.
Northern Blot Analysis--
Northern blotting was performed as
described previously (3) with 5 µg of RNA/lane. The probe used for
detection was the 1600-base pair cpg16 cDNA (2), which
was labeled by the random priming technique (U. S. Biochemical Corp.)
using [ Construction of Expression Vectors for CPG16--
The polymerase
chain reaction method was used to clone the cDNA encoding the open
reading frame for cpg16 (2) in-frame with glutathione
S-transferase (GST) into the pGEX2T expression vector and
purified as described (4). To construct a hemagglutinin (HA)-cpg16 plasmid, polymerase chain reaction was used to
clone the cpg16 in-frame with the HA epitope tag in a pCGN
vector (5). The HA-CPG16 open reading frame was sequenced to ensure its composition.
Transfection of COS7 and NIH-3T3 Cells--
COS-7 or NIH-3T3
cells were grown in 6-cm tissue culture plates containing Dulbecco's
modified Eagle's medium and 10% fetal calf serum (FCS) to
approximately 60% confluency. The cells were then transfected with
plasmid DNA, prepared using Qiagen columns (Qiagen). Subsequently, the
COS7 cells were transfected by the DEAE-dextran-mediated method as
described previously (6) using 2.5 µg/ml DNA. After 10%
Me2SO shock, cells were cultured in normal growth media and
maintained in a humidified incubator (5% CO2) at 37 °C.
NIH-3T3 cells were transfected by the calcium phosphate method using 5 µg of DNA (7). After the transfection, the cells were cultured in
normal growth media and maintained in a humidified incubator (5%
CO2) at 37 °C.
Activation of Transfected COS7 and Immunoprecipitation of
HA-CPG16--
Control and HA-cpg16-transfected COS-7 cells
in 6-cm tissue culture plates were starved for 16 h in 0.1% FCS
in Dulbecco's modified Eagle's medium. The cells were then treated
with either 10 µM forskolin (Sigma), 10 µM
8-bromo-cAMP (8-Br-cAMP, Sigma), 10% FCS, or other stimuli for various
times. If isobutylmethylxanthine (Sigma, 10 µM) was
included in an experiment, it was added 5 min before other treatments.
After stimulation, the cells were washed once with ice-cold
phosphate-buffered saline (PBS) and lysed (10 min on ice) in Buffer H
(50 mM Autophosphorylation and in Vitro Kinase Assays--
GST-CPG16 or
HA-CPG16 autophosphorylation was performed at 30 °C in a buffer
containing 50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 20 µM ATP (20 cpm/fmol), 10 µg/ml leupeptin,
10 µg/ml aprotinin, 50 mM Phosphoamino Acid Analysis--
32P-Labeled proteins
were separated by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride (Immobilon-P; Millipore
Corp.). Membrane slices containing the proteins were hydrolyzed in
constant boiling 5.7 N HCl and analyzed by two-dimensional electrophoresis as described (11).
Immunostaining--
NIH-3T3 cells were transfected using the
calcium phosphate method using 5 µg of DNA. After transfection, the
cells were replated on glass coverslips contained in a 6-well tissue
culture dish and allowed to grow for 36 h after which the cells
were starved in Dulbecco's modified Eagle's medium and 0.1% calf
serum for 16 h. Cells were activated with the appropriate
compounds and then fixed with 3% paraformaldehyde for 20 min. After
one wash with PBS, cells were permeabilized with 0.2% Triton X-100 in
PBS for 5 min and blocked in 3% bovine serum albumin in PBS for 30 min. The primary monoclonal antibody, anti-HA antibody (antibody unit,
WIS) was applied to the cells at a final concentration of 1 µg/ml in
PBS for 45 min, washed with PBS, and incubated again with lissamine
rhodamine-conjugated goat anti-mouse IgG and IgM (Jackson
ImmunoResearch Laboratories) 1:200 in PBS for 45 min. After washing
with PBS, coverslips were mounted, analyzed, and photographed using a
Zeiss Axiophot camera.
CREB-Luciferase Assay--
The assay was performed as described
by Spengler et al. (12). Briefly, Madin-Darby canine kidney
cells expressing a stable luciferase reporter construct containing five
copies of the CRE element were transfected by electroporation with an
expression vector (1 µg) containing HA-cpg16. Six h after
transfection, serum was removed from the cells, and after a total of
24 h, stimulants were added. Four h after stimulation, the medium
was discarded, and the cells were washed with PBS. The cells were then
lysed by adding luciferase lysis buffer (Labsystems, Inc.). Cell
extracts were transferred to the luminometer (Dynatech Laboratories,
Inc.), and luciferase activity was recorded. The resulting data were analyzed using Excel software (Microsoft Corp.).
Northern Blot Analysis of cpg16--
cpg16 was recently
identified (2) in a differential cDNA screen from
kainate-stimulated rat hippocampus dentate gyrus. We performed Northern
blot analysis of poly(A)-selected mRNA from total rat brain, rat
dentate gyrus, kainate-activated rat dentate gyrus, heart, liver,
spleen, and kidney with a 32P-labeled probe of the
1600-base pair cDNA of cpg16 as described above.
Hybridization was found in the rat brain and rat dentate gyrus, with
strong up-regulation of the cpg16 message after treatment with kainic acid. In most of the other tissues examined, no expression of cpg16 was detected, although a weak signal could be seen
in the kidney (Fig. 1). Because a similar
(but not identical) sequence was recently detected in a mouse skin
library, the expression of this gene may not be limited to the brain,
and it could play a role in additional tissues.
Expression of cpg16 in Bacteria and in Mammalian Cells--
The
predicted open reading frame of cpg16 encodes a putative
protein of approximately 50 kDa. Based on sequence homology, the
primary structure of CPG16 predicted a protein serine/threonine kinase
containing all 15 characteristic amino acids of these kinases (13). To
determine whether CPG16 is indeed a 50-kDa functional protein kinase
and to characterize its enzymatic properties, cpg16 was
fused in-frame with cDNA of GST, expressed in Escherichia coli and purified using glutathione beads. Coomassie Blue staining of the purified preparation revealed one main protein at 75 kDa (Fig.
2), which corresponded to the expected
molecular mass for the GST-CPG16 fusion protein. Another band that
could be detected in the purified fraction at the molecular mass of 27 kDa is probably the GST protein alone. In the presence of
Mg2+ and ATP, the purified GST fusion underwent
autophosphorylation in a time-dependent manner, indicating
that the cpg16 cDNA indeed encodes for a functional
kinase (Fig. 2). A lower molecular weight band (35 kDa) that was
detected upon autophosphorylation most likely represents a degradation
product of the GST-CPG16 itself, as judged by its increase when
protease inhibitors were omitted from the bacterial extraction buffer
in the course of purification (data not shown).
To allow for studies of CPG16 in mammalian cells, the cDNA of
cpg16 was inserted into a mammalian expression vector
containing an HA tag. The HA-cpg16 was transfected into COS7
cells followed by starvation of the cells, lysis, and
immunoprecipitation of the HA-containing proteins. One band was
detected when the purified preparation was subjected to Western blot
analysis with the anti-HA antibody (52 kDa, Fig.
3A), which corresponded to the
expected molecular mass of CPG16 fused to HA. Because a band with an
identical molecular mass was autophosphorylated upon incubation of the
purified proteins with ATP, the identified band is most likely the
autophosphorylated HA-CPG16.
Substrate Specificity of CPG16--
CPG16 with both HA and GST
tags showed low (8 nmol/min/mg) autophosphorylation activity. For a
clue regarding a possible physiological substrate(s) for CPG16, we
performed an in vitro substrate specificity assay in which
we tested a group of peptides, proteins, and cytosolic extracts for
their ability to be phosphorylated by CPG16. Using Mg2+ and
ATP as cofactors, CPG16 appeared to have a restricted specificity, as
it showed activity only toward MBP at a low rate of 12 nmol/min/mg (Table I and Fig. 3). This specificity of
CPG16 was most apparent when denatured brain extract, which contains
thousands of proteins, was used as a substrate. In most of the
experiments performed, CPG16 failed to phosphorylate any of the
proteins in the tested preparation (Fig. 3), although in some of the
experiments a phosphorylated 45-kDa band could be detected upon
phosphorylation with CPG16 (data not shown). Phosphoamino analysis of
the autophosphorylated GST-CPG16 and of CPG16-phosphorylated MBP
revealed that the phosphorylation occurred only on serine and threonine
(Fig. 3B). This is consistent with the predicted protein
serine/threonine kinase sequence of cpg16 (2, 13).
When compared with other protein kinases, CPG16 shows the highest
homology to members of the
Ca2+/calmodulin-dependent protein kinases and
in particular to Ca2+/calmodulin-dependent
protein kinase II (CaMKII). Interestingly, CaMKII was shown to be a key
player in hippocampal plasticity (14, 15), mainly by affecting the
transcription factor CREB (16). We therefore examined whether
Ca2+ and calmodulin are capable of modulating the
autophosphorylation activity of CPG16. We found that neither
Ca2+ and calmodulin nor the CaM kinase inhibitors EGTA,
calmidazolium, and W7 had any effect on the autophosphorylation of the
bacterially expressed GST-CPG16 (Fig. 4
and data not shown). Similarly, there was no effect of Ca2+
or calmodulin on GST-CPG16 activity toward any of the substrates examined in in vitro phosphorylation assays (data not
shown). Moreover, when the sequence of cpg16 was aligned
with the sequence of CaMKII, the calmodulin binding region of CaMKII
differed from the corresponding region in CPG16 and predicted a cryptic
calmodulin-binding site. Indeed, GST-CPG16 did not bind to a
calmodulin-agarose column under any condition tested (data not shown),
strongly suggesting that CPG16 is not dependent on Ca2+ or
calmodulin for its direct enzymatic activity.
CPG16 Is Stimulated by cAMP in COS7 Cells--
Because the
activity of many protein serine/threonine kinases can be increased in
response to extracellular agents, we undertook to determine whether the
low basal activity of CPG16 can be modulated by any extracellular
stimuli. Thus, COS7 cells were transiently transfected with HA-CPG16,
serum-starved, and treated with agents that activate intracellular
signaling pathways. HA-CPG16 was then immunoprecipitated using anti-HA
antibodies and subjected to in vitro kinase assays.
Interestingly, both forskolin and 8-Br-cAMP agents, which increase the
intracellular levels of cAMP, caused a substantial transient activation
of CPG16. Specifically, HA-CPG16 purified from forskolin-treated cells
exhibited a 6-8-fold increase in its autophosphorylation activity,
which peaked at 30 min after treatment and decreased again to basal
levels within 60-90 min after stimulation (Fig.
5). 8-Br-cAMP and dibutyryl cAMP yielded similar results (Fig. 5 and data not shown), although the time course
of activation in response to 8-Br-cAMP was somewhat slower and peaked
at around 45 min after treatment. The addition of
isobutylmethylxanthine to inhibit phosphodiesterases and further
increase cAMP had only a small effect on the kinetics of the
phosphorylation of HA-CPG16.
Other extracellular agents that were used to identify stimulators or
activators of HA-CPG16 included the Ca2+ ionophore, A23187,
the protein kinase C activator phorbol-12-myristate-13-acetate, the
protein-tyrosine phosphatase inhibitor vanadate, epidermal growth
factor, and FCS. All these compounds are known to activate several
downstream signaling pathways including MAPK cascades and others but
had no detectable effect on the autophosphorylation activity of CPG16
(Fig. 5 and data not shown). Moreover, even after purification from
stimulated cells, there was no increase of CPG16 activity toward any of
the in vitro substrates examined besides MBP (data not shown).
CPG16 Is Activated Downstream of PKA--
From the above
experiments performed on COS7 cells, it appears that CPG16 is
exclusively activated by cAMP-elevating agents, which transmit their
signals primarily through the activation of the protein kinase PKA.
Therefore, we used the specific PKA inhibitor H89 to examine whether
PKA is indeed involved in CPG16 activation. Thus, H89 was added to the
examined cells before stimulation by forskolin or 8-Br-cAMP (17), and
the autophosphorylation activity of CPG16 was determined as described
above. Our results show that H89 significantly inhibited the forskolin
stimulation of CPG16 when added to the transfected COS7 cells 15 min
before stimulation (Fig. 6). Therefore,
these results clearly indicate the involvement of PKA in the activation
process of CPG16.
We then examined whether CPG16 can be a direct substrate of either PKA
or ERK/MAPK that was suspected to phosphorylate a PXSP consensus sequence in the N terminus of CPG16 (18, 19). Although both
the catalytic subunit of PKA and activated ERK2 were very active in
phosphorylating vitronectin and MBP, respectively, no phosphorylation
of purified HA-CPG16 by these kinases could be detected even after a
prolonged phosphorylation reaction (Fig. 7). Recombinant active MAPK/ERK kinase
(7) and casein kinase II also failed to phosphorylate HA-CPG16 (data
not shown), and CPG16 had no apparent effect on all four tested
kinases. Therefore, the activation of CPG16 may occur by a
cAMP-dependent PKA-induced mechanism (probably a kinase
cascade) similar to the activation of phosphorylase kinase downstream
of PKA (20).
Studies on a Possible Role of CPG16 in the Nucleus--
To obtain
information on the subcellular localization of CPG16, we performed
immunolocalization studies in COS7 and in NIH-3T3 cells transiently
transfected with HA-cpg16. In both cell types, most of the
HA staining was detected in the cytosol, although some enrichment in
nuclear staining could be observed after 8-Br-cAMP and forskolin
stimulation of the cells (Fig. 8 and data
not shown). This nuclear enrichment could not be detected when the
cells were stimulated with FCS under conditions in which ERKs are
translocated to the nucleus (7). Although mostly cytosolic, the limited translocation indicated that CPG16 may have a nuclear role upon cAMP
elevation.
The translocation of CPG16 to the nucleus prompted the notion that it
might participate in the regulation of transcription. Because the
phosphorylation of CPG16 is affected by cAMP, we tested the possibility
that overexpression of CPG16 would influence gene expression through
the transcription factor CREB. Madin-Darby canine kidney cells
expressing a stable luciferase reporter construct containing five
copies of the CRE element were stimulated with various cAMP-elevating
agents, and their luciferase reporter activity was determined (12). As
expected, activation of these cells with forskolin, 8-Br-cAMP,
epidermal growth factor, and isoproterenol but not calcium ionophore,
peroxovanadate, and phorbol ester (phorbol-12-myristate-13-acetate) increased the activity of the luciferase reporter gene (Fig.
9). However, when these cells were
transfected with a large amount of expression vector containing the
cDNA for CPG16 (5 µg/plate), a moderate inhibition compared with
the control vector was detected in the luciferase activity (Fig. 9).
Lesser amounts of the vector containing the CPG16 did not have a
significant effect on this activity. Thus, it is possible that CPG16
participates in the down-regulation of cAMP-induced transcription,
although the small amount of inhibition may suggest that this
inhibition might be an indirect process.
Glutamate receptors produce long term plasticity changes in
central nervous system neurons, at least in part by induction of
transcription cascades (21, 22). Glutamate-activated transcription factors induce a first wave of gene expression, the immediate-early gene, many of which encode transcription factors, for example, c-fos (23). The immediate-early gene products in turn induce hundreds of downstream genes, many of which are thought to produce plastic changes in brain neurons and therefore are known as candidate plasticity-related genes (CPGs (1)). Recently, Hevroni et
al. (2) identified about 400 genes that are either induced or
down-regulated upon kainate (glutamate analog) induction. One of the
most intriguing genes identified was CPG16, which demonstrated sequence
homology with the plasticity-related CaMKII.
In this study we tried first to check whether or not CPG16 is indeed a
Ca2+/calmodulin-dependent kinase. A careful
analysis of the C-terminal region of CPG16 revealed that six amino
acids essential for calmodulin binding are missing. Indeed, we found
that CPG16 failed to bind to a calmodulin-agarose column, indicating
that the cryptic site was not sufficient to promote calmodulin binding.
Moreover, no effect of Ca2+ or calmodulin on the in
vitro or in vivo activity of CPG16 could be detected
(Fig. 4). Therefore, our results clearly indicate that CPG16 is
regulated by a different mechanism than that involved in the activation
of Ca2+/calmodulin-regulated kinases.
Although not influenced by calmodulin and Ca2+, a different
regulator was identified when COS7 cells were transiently transfected with CPG16. Of several stimuli that were tested for their ability to
modulate CPG16 activity immunoprecipitation, cAMP-elevating agents were
shown to have a stimulatory effect. Thus, forskolin or 8-Br-cAMP
increased CPG16 autophosphorylation activity by about 6-8-fold over
its basal activity, whereas other stimuli did not have any effect. The
H89 inhibitor, which selectively inhibits PKA activity, abolished the
cAMP-dependent stimulation, indicating that CPG16 acts
downstream of PKA. However, CPG16 could not be phosphorylated or
directly activated by PKA in vitro. Moreover, the activation
observed was much smaller than the activation of PKA itself, which
under similar conditions may usually reach more than 50-fold. Taken
together, these data suggest that CPG16 may be activated by a
PKA-induced mechanism that could be a PKA-induced kinase cascade.
Activation because of phosphorylation by an intermediate kinase is
likely also because of the existence of threonine residue in the
activation loop of CPG16 that is analogous to the activation sites of
many activated protein kinases such as ERK and MAPK/ERK kinase (8).
However, at this stage, the direct mechanism of activation is not
clear. The relatively small activation of 6-8-fold could be explained
by higher susceptibility to modulating enzymes such as phosphatases. An
alternative explanation could be a possible dependence on cofactors or
intermediate molecules that may be essential to secure full activation
but are not expressed in high quantities in the COS7 cells. It is also
possible that the activation toward the real substrate of CPG16 is
higher than the activation of its autophosphorylation or activity
toward MBP.
The involvement of cAMP in neuronal plasticity is well established, and
many studies have documented the involvement of this second messenger
in the synaptic plasticity of memory and behavior (25). cAMP is known
to transmit its signals primarily through several PKA isoforms that may
function either in the cytosol or translocate to the nucleus and
regulate processes there (26). In the cytosol, the PKAs may regulate
metabolic as well as protein synthesis processes (26). However, the
main function of PKA in the nucleus is to regulate the transcription of
cAMP-dependent genes, which is mediated primarily by
phosphorylation of the transcription factor CREB (16). In this regard,
the induction of CPG16 by kainate and its activation by cAMP may
indicate that this kinase is one of the components that participates in
plasticity determination. However, unlike overexpression of the
catalytic subunit of PKA, the overexpression of CPG16 seems to slightly
inhibit the cAMP-stimulated CREB activity. Therefore, unlike the
stimulatory effect of PKA, CPG16 is likely to participate in the
down-regulation of cAMP signals in the nucleus. Inhibitory processes
have been shown in all levels of the PKA cascades, including the
down-regulation of receptors by phosphorylation, hydrolysis of cAMP by
phosphodiesterases, or phosphorylation/dephosphorylation inhibition of
PKA targets. Interestingly, cAMP-regulated protein phosphatase activity
has been shown recently to down-regulate CaMKII activity during
long-term potentiation (27). Therefore, CPG16 may participate in the
regulation of CREB either by phosphorylating its inhibitory residues or
alternatively, via the activation of phosphatases that are involved in
the inactivation of CREB.
Although the role of CPG16 in the nucleus is intriguing, it should be
noted that its localization is primarily cytosolic, and only a small
amount of nuclear translocation was observed upon 8-Br-cAMP treatment
of cells. Therefore, CPG16 probably phosphorylates and modulates the
activity of other downstream targets mainly in the cytosol; however,
these targets have yet to be identified. An intriguing clue regarding a
possible substrate for CPG16 came from the recent identification of
KIAA0369 (28). This cDNA was found to have a high degree of
identity to both CPG16 and doublecortin (9, 24), the later is linked to
the genetic disease x-linked lissencephaly and double cortex syndrome.
It is therefore possible that CPG16 is involved in the regulation of
doublecortin itself or to one of its related proteins; however, more
work is needed to confirm this hypothesis.
In summary, we have characterized a neuronal plasticity-related protein
kinase, CPG16. Although it shows high sequence homology to
Ca2+/calmodulin protein kinases, we have found that it is
not influenced by Ca2+ or calmodulin but is activated by
cAMP-elevating agents. However, CPG16 is not an in vitro
substrate for PKA, suggesting that it may be activated by a
PKA-dependent cascade. CPG16 was also shown to translocate
into the nucleus upon stimulation and to have a weak inhibitory effect
on cAMP-stimulated transcriptional activity of CREB. Therefore, CPG16
may participate in the down-regulation of cAMP signals toward
plasticity-related genes.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]dCTP according to the manufacturer's instructions.
-glycerophosphate, pH 7.3, 1.5 mM
EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 µg/ml pepstatin-A
(8)). The cells were then scraped from the plate and centrifuged
(14,000 × g, 20 min, 4 °C). The supernatant containing HA-CPG16 was collected and incubated with 2 µg of anti-HA antibody (4 °C) for 1 h, then precipitated with protein
A-agarose (Sigma) for 1 h. The complexes were washed (4 °C)
three times with PBS + 0.1% Nonidet P-40, once with 0.5 M
LiCl in PBS, and twice with buffer A ((50 mM
-glycerophosphate, pH 7.3, 1.5 mM EGTA, 1 mM
EDTA, 1 mM dithiothreitol, and 0.1 mM
Na3VO4 (8)).
-glycerophosphate, 100 µM orthovanadate in a final volume of 30 µl. The
reactions were terminated by adding SDS-polyacrylamide gel
electrophoresis sample buffer and boiled for 5 min. In vitro
kinase assays were performed using the same reaction mixture with the
following substrates or protein kinases. Recombinant Jun N-terminal
kinase, recombinant external-signal responsive kinase (ERK),
recombinant p38 mitogen-activated protein kinase (p38MAPK), recombinant
MAPK/ERK kinase, and recombinant
N-EE-MAPK/ERK kinase were prepared
as described (7). Activated ERK was prepared by phosphorylation with
active MAPK/ERK kinase (7). Nuclear K-7 was the kind gift from Dr. K. Bomsztyk, University of Washington, Seattle. Purified vitronectin and
PKA (catalytic subunit) were gifts from Dr. S. Shaltiel, The Weizmann
Institute of Science, Israel (WIS). GST-Jun 1-74 and GST-ATF2 16-96
as well as GST-Rho and GST-ARF were expressed in bacteria and purified as described (10). MAP2 was a gift from Dr. Z. Eleazar (WIS). Peptides
based on the following sequences were synthesized by the biological
services at the WIS: epidermal growth factor receptor 661-675,
glycogen synthase kinase 6-20, Jun 60-78, and Far1 134-150. Myosin
light chain, histone IIIs, myelin basic protein (MBP),
-casein, and
protamine were purchased from Sigma.
RESULTS
View larger version (64K):
[in a new window]
Fig. 1.
Tissue distribution Northern blot of
cpg16. A Northern blot containing 5 µg of poly(A+) RNA from
kainate-treated dentate gyrus (Den +), untreated dentate
(Den ), total brain, kidney, liver, testis, heart, and
lung was probed with the cpg16 cDNA clone (upper panel)
and with a glyceraldehyde 3-phosphate dehydrogenase (G3PD)
probe as a control (lower panel).
View larger version (53K):
[in a new window]
Fig. 2.
Expression and autophosphorylation of
GST-CPG16 and HA-CPG16. A, purification of GST-CPG16
from BL21(DE3) bacteria was as described under "Experimental
Procedures." The following samples were loaded on a 10%
SDS-polyacrylamide gel electrophoresis, which was stained with
Coomassie Blue: lane 1, purified GST-CPG16;
lane 2, purified GST. B, time course of GST-CPG16
autophosphorylation. Autophosphorylation reaction was carried out as
described under "Experimental Procedures," and aliquots from the
reaction were sampled at the following time intervals and examined by
SDS-polyacrylamide gel electrophoresis and autoradiography: lane
3, time 0; lane 4, 5 min; lane 5,
15 min; and lane 6, 40 min. C,
immunoprecipitation and Western blot detection of either HA-cpg16 or
vector control-transfected COS-7 cells. Transfection,
immunoprecipitation with anti-HA monoclonal antibody, and
immunodetection with anti-HA polyclonal antibody were carried out as
described under "Experimental Procedures." Samples from the
following treatments of COS7 cells were loaded: lane 7,
nontransfected cells; lane 8, vector control-transfected
cells; lane 9, HA-cpg16-transfected cells. D,
autophosphorylation of HA-cpg16 immunoprecipitated from transfected
COS-7 cells. HA-CPG16 was immunoprecipitated using anti-HA monoclonal
antibody from the following COS7 cells and subjected to
autophosphorylation reaction as described under "Experimental
Procedures." Lane 10, nontransfected cells; lane
11, vector control-transfected cells; and lane 12,
HA-cpg16-transfected cells. The location of GST-CPG16, GST alone
(GST), HA-CPG16, and the HA antibodies (Ab) are
indicated.
View larger version (49K):
[in a new window]
Fig. 3.
In vitro phosphorylation by CPG16
and phosphoamino acid analysis. A, autophosphorylation,
phosphorylation of MBP, and of total denatured brain extract. In
vitro kinase assay is shown with buffer control (lanes
1 and 3), bead-conjugated GST-CPG16 (lanes 2 and 4), bead-conjugated HA-CPG16 (lanes 6 and
7), and control immunoprecipitation (no HA antibodies,
lane 5) from HA-cpg16-transfected COS7 cells. The following
substrates were incorporated in the assays: lanes 4 and
6, MBP (10 µg); lane 7, denatured (65 °C, 10 min) brain extract. The reaction was performed as described under
"Experimental Procedures." B, 1, phosphoamino
acid analysis of autophosphorylated GST-CPG16; 2,
phosphoamino acid analysis of MBP phosphorylated by HA-CPG16.
PT, phosphothreonine; PS, phosphoserine;
PY, phosphotyrosine).
Substrate specificity for CPG-16
View larger version (28K):
[in a new window]
Fig. 4.
Effect of Ca2+ and calmodulin on
the autophosphorylation of CPG16. Autophosphorylation of GST-CPG16
was carried out as described under "Experimental Procedures" with
no addition (upper left), with 10 µM
CaCl2 and 30 µM Calmodulin (upper
right), with 10 µM CaCl2, 5 µM calmodulin, and 100 µM EGTA (lower
left), and with 10 µM CaCl2, 30 µM calmodulin, and 125 µM calmidazolium
(CMZ, lower right).
View larger version (32K):
[in a new window]
Fig. 5.
Effect of cAMP on CPG16. COS7 cells were
transfected with either HA-cpg16 (panels A,
B, and C, HA-CPG16) or vector control
(A and C, Control), allowed to grow
for 36 h, and then serum-starved for an additional 16 h. Then
the cells were treated for the indicated times with Me2SO
(panels A and B, time 0), with
forskolin (10 µM, panel A, both in
Control and HA-CPG16), with 8-Br-cAMP
(panel B), with FCS (10%, panel C, 10, 30, and
60 min, both in Control and HA-CPG16), or left
untreated (panel C, time 0). The cells were then harvested,
and the autophosphorylation activity of CPG16 was determined as
described under "Experimental Procedures." The amount of HA-CPG16
precipitated was determined in some of the experiments by Western blot
with anti-HA polyclonal antibody and was found to be equal among the
different points (data not shown). The graphs (panel
D) represent the average of the densitometric readings of the
autophosphorylated HA-CPG16 from three similar experiments. MW,
molecular mass.
View larger version (38K):
[in a new window]
Fig. 6.
Inhibition of forskolin activation of CPG16
by H89. A, COS7 cells were transfected with HA-cpg16,
then allowed to grow for 36 h and serum-starved for an additional
16 h. Then, the cells were treated with 10 µM
forskolin for 30 min (+Forsk.) or pretreated with 4 µM H-89 (CalBiochem) for 15 min and then treated with
forskolin (Forsk.+H89) or left untreated (+H89).
Autophosphorylation activity of HA-CPG16 was determined as described
under "Experimental Procedures." The results of the
autophosphorylation reaction (Phos.) are shown in the
upper panel. The amount of immunoprecipitated HA-CPG16 was
determined by Western blotting (Blot) using anti-HA
polyclonal antibody (Ab). These results are shown in the
lower panel, two lanes for each treatment.
B, quantitation of the experiment. The results are the mean
of two distinct experiments that were done in duplicate.
View larger version (42K):
[in a new window]
Fig. 7.
Phosphorylation of CPG16 by PKA and
ERK2. Phosphorylation reactions were performed as described under
"Experimental Procedures" with the following components:
1, immunoprecipitated HA-CPG16 (~0.2 µg); 2,
PKA (0.1 µg); 3, PKA + vitronectin (1 µg); 4,
HA-CPG16 + PKA; 5, buffer alone; 6, active ERK
(0.05 µg); 7, active ERK + MBP (5 µg); 8,
HA-CPG16 + active ERK. Reactions proceeded for 30 min before
inactivation by boiling in a sample buffer.
View larger version (74K):
[in a new window]
Fig. 8.
Localization of HA-CPG16 in NIH-3T3.
NIH-3T3 cells transfected with HA-cpg16 were grown on coverslips for
36 h after transfection and serum-starved for an additional
16 h. Then the cells were stimulated with 8-Br-cAMP (10 µM, 10 and 30 min), with FCS (10%, 60 min), or left
untreated (control). The cells were fixed and stained with anti-HA
monoclonal antibody as described under "Experimental Procedures."
Staining of HA-CPG16-transfected cells with secondary antibody alone or
staining of untransfected cells did not result in any significant
fluorescence (data not shown).
View larger version (39K):
[in a new window]
Fig. 9.
Effect of CPG16 on cAMP-induced activation of
CREB. Madin-Darby canine kidney cells expressing a stable
luciferase reporter construct containing five copies of the CREB
element were transfected by electroporation with an expression vector
(5 µg) containing HA-cpg16. Six h after transfection, the
cells were serum-starved, and after a total of 24 h, the following
stimulants were added: forskolin (10 µM), 8-Br-cAMP (10 µM), epidermal growth factor (EGF, 50 ng/ml),
isoproterenol (10 µM), A23187 (Ionophore, 20 µM), peroxovanadate (VOOH, 100 µM), and 12-O-tetradecanoylphorbol-13-acetate
(TPA) (100 µM). Luciferase activity was
determined as described under "Experimental Procedures."
DISCUSSION
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Muriel Zohar, Zeev Gechtman, and Elly Nedivi for valuable discussions and comments on the manuscript as well as Tami Hanoch for her skillful technical assistance and Drs. Uri Gat and David De graaf for their assistance with the CREB-luciferase assays.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from MINERVA and from the Kekst Family Foundation for Molecular Biology.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.
Supported by the WIS post-doctoral fellowship program. Current
address: C.R.O.E.T., L606, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201. E-mail:
silvermm{at}ohsu.edu.
This paper is dedicated to the memory of Dr. Yoav Citri, who was
tragically killed in a car accident in December, 1995.
§ An incumbent of the Samuel and Isabela Friedman Career Development Chair and to whom correspondence should be addressed. Tel.: 972-8-9343602; Fax: 972-8-9344116; E-mail: bmseger{at}weizmann.weizmann.ac.il.
The abbreviations used are: CPG, candidate plasticity-related gene; 8-Br-cAMP, 8-bromo-cAMP; PKA, cAMP-dependent protein kinase; MAPK, mitogen-activated protein kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CREB, cAMP response element-binding protein; ERK, external signal-responsive kinase; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline; WIS, Weizmann Institute of Science, Israel; FCS, fetal calf serum; MBP, myelin basic protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|