The Type and the Localization of cAMP-dependent
Protein Kinase Regulate Transmission of cAMP Signals to the Nucleus in
Cortical and Cerebellar Granule Cells*
Mayra
Paolillo
,
Antonio
Feliciello§,
Antonio
Porcellini§§§,
Corrado
Garbi§,
Maurizio
Bifulco¶,
Sergio
Schinelli
,
Carmelo
Ventra
¶¶,
Eugenio
Stabile§,
Gaetano
Ricciardelli
,
Gennaro
Schettini**, and
Enrico V.
Avvedimento§¶
From the
Istituto di Farmacologia, Facoltà di
Farmacia, Università di Pavia, § Dipartimento di
Biologia e Patologia Cellulare e Molecolare, CEOS, CNR,
Dipartimento di Biochimica e Biotecnologie Mediche,
Facoltà di Medicina, Università Federico II, Napoli,
** Istituto di Farmacologia, Facoltà di Medicina and IST, Servizio
di Farmacologia e Neuroscienze-IST-CBA, Genova, and ¶ Dipartimento
di Medicina Sperimentale, Facoltà di Medicina di Catanzaro,
Università di Catanzaro, Italy
 |
ABSTRACT |
cAMP signals are received and transmitted by
multiple isoforms of cAMP-dependent protein kinases,
typically determined by their specific regulatory subunits.
In the brain the major regulatory isoform RII
and the RII-anchor
protein, AKAP150 (rat) or 75 (bovine), are differentially expressed.
Cortical neurons express RII
and AKAP75; conversely, granule
cerebellar cells express predominantly RI
and RII
. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon
cAMP induction, whereas granule cerebellar cells fail to do so.
Down-regulation of RII
synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RII
and AKAP75 genes by
microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a
cAMP-responsive element promoter.
These data indicate that the composition of PKA in cortical and granule
cells underlies the differential ability of these cells to transmit
cAMP signals to the nucleus.
 |
INTRODUCTION |
cAMP formed by adenylyl cyclases after stimulation of
G-protein-coupled receptors binds the regulatory subunits (R) of the tetrameric PKA1 holoenzyme
and promotes dissociation of the catalytic subunits (C-PKA). A fraction
of C-PKA translocates to the nucleus and stimulates cAMP-dependent gene expression (1-3). Multiple isoforms of
PKA are determined by their specific regulatory subunits. Four
regulatory subunits (RI
, RI
, RII
, and RII
) have been
cloned. PKA containing RII
is the predominant PKA isoform in the
brain and is expressed in the cortex, whereas in the brainstem and
cerebellum (except Purkinje cells) RII
has not been found (4-6). In
mammalian brain, signals triggered by cAMP are targeted to specific
effector sites by the tethering of cAMP-dependent protein
kinases to intracellular compartments (4, 7, 8). PKAII is bound to
membranes via specific anchor proteins (AKAPs), which bind R
subunits. Bovine brain AKAP75 has been studied as prototype of kinase A
anchor protein and shares high homology with human AKAP79 and rat
AKAP150 (9, 10). These proteins have similar properties, related sequences, and are recognized by the same antibodies (9-11).
AKAP150/75 and RII
are co-localized in the dendritic cytoskeleton
and perikarya of forebrain neurons; both proteins have not been found
in cerebellar granule cells (6).
Although the structure and expression pattern of the PKA regulatory
subunits and AKAPs are well documented, the functional role of these
proteins in the transduction of cAMP signals is still poorly
understood. It is not known how the different PKA isoforms in different
districts of the central nervous system receive and transmit cAMP signals.
We have chosen primary cortical and granule cerebellar neurons as
prototype cells with different PKA composition and localization. PKA in
cortical neurons is mainly of II
type and is membrane-anchored by
AKAP150/75. Conversely, granule cerebellar cells do not express AKAP75/150 and RII
. The R subunits expressed by these cells are RI
and RII
(6, 12).
We have studied the activation by cAMP of these enzymes and the
transmission of the signals to the nucleus by measuring the accumulation of C-PKA in the nucleus, CREB phosphorylation, and the
transcription of a cAMP-induced promoter following cAMP stimulation. Also, we have manipulated the composition of PKA in granule and cortical cells by down-regulating RII
in cortical cells or by expressing AKAP75 and RII
in granule cells, respectively.
The results presented here indicate that RII
and the PKA-anchor
protein, AKAP75, amplify the transmission of cAMP signals to the
nucleus and suggest that the composition of PKA might influence the
ability of the cell to receive and transmit cAMP signals to the nucleus.
 |
MATERIALS AND METHODS |
Primary Cultures of Cortical and Cerebellar Granule
Cells--
Primary cultures were obtained as described previously for
striatal neurons with some modifications (13). Briefly, cortices of
16-day-old rat embryos were dissected and incubated with papain. Tissue
fragments were mechanically dissociated and the cells plated in
polylysine-coated dishes in 1:1 minimum Eagle's medium/F12 medium
containing 2 mM glutamine and 10% fetal calf serum.
Cerebellar granule cells were obtained from 7-day-old rat pups. Tissue
fragments were digested with trypsin and mechanically dissociated. The
cells were plated in polylysine-coated dishes in 25 mM
K+ BME containing 10% fetal calf serum. 24 h after
plating, 10 µM Cytosine C Arabinoside was added to the
cultures to prevent the growth of non-neuronal cells. Under these
conditions the contamination of glial cells, measured by staining with
glial fibrillary protein, was less than 10%. The cells were grown for
7 days and stimulated with 10 µM forskolin and 0.5 mM IBMX (Sigma) or increasing concentrations of
dibutyryl-cAMP (Calbiochem). PKA inhibition was achieved by treating
the cells with 10 µM of the PKA inhibitor H89 (Biomol, Plymouth Meeting, PA) (14). We have also found that a 2-h treatment with PKI-amide (50 µg/ml) (Life Technologies, Inc.) inhibits 50% of
PKA activity in vivo.
PKA Assay--
PKA was measured by phosphorylation of the
synthetic peptide, kemptide, in the presence of [32P]ATP
(DuPont) (3,000 Ci/mmol, final specific activity 125-150 cpm/pmol)
with 5 µg of cytoplasmic extracts in the presence or absence of 0.5 mM cAMP for 10 min of incubation at 30 °C. PKA activity
was expressed in picomoles of phosphorylated kemptide/min/µg of
cytoplasmic or nuclear proteins (15, 16). C-PKA/total PKA ratio is a
measure of PKA activation. The activation of cytoplasmic PKA is shown
as the ratio between C-PKA (
cAMP) and total PKA (+0.5 mM
cAMP). Nuclear PKA catalytic activity was measured in sucrose
gradient-fractionated nuclei in the presence or absence of 10 µM of the PKA-specific inhibitor pseudosubstrate (PKI)
(16).
Treatment of the Cells with Specific Antisense
Oligonucleotides--
Antisense oligonucleotides specific to the
NH2 terminus of rat RII
protein
(5'-CCGCGGGATCTCGATGCTCA-3') (GenTech, France) or mismatched
phosphorotioate oligonucleotides were directly added to the culture
medium for 72 h (addition every 24 h, 6 µM
final concentration). Each experimental condition was performed in
triplicate. Cell viability was determined by trypan blue staining. The
cells showed 90-95% viability after 72 h of treatment with oligonucleotides.
Immunoblot Analysis--
50 µg of cytoplasmic or 30 µg of
nuclear proteins were resolved in 10% SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose filters. The filters
were rinsed in Tris-buffered saline with Tween 20 (10 mM
Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20) and with
10% nonfat dry milk in the same buffer with Tween 20. The filters were
incubated with specific antibodies in 5% nonfat dry milk for 1 h
(RII
, RII
) or 6 h (CREB, PCREB). After washing, the filters
were incubated with peroxidase-conjugated monoclonal anti-rabbit IgG
(Sigma) in 5% nonfat dry milk in Tris-buffered saline with Tween 20 for 1 h and developed using the Renaissance chemiluminescence kit
(NEN Life Science Products). Anti-rat PCREB antibodies (17) were
purchased from Upstate Biotechnology, Lake Placid, NY. Specific
anti-RII
or anti-RII
antibodies were generated by immunizing
rabbits with a synthetic RII
peptide (peptide 31-57 from the AUG of
the rat sequence) or RII
(peptide containing the residues 53-73
from the start codon of the rat protein) cross-linked to soybean
trypsin inhibitor. The total IgGs were purified, and the specificity of
each preparation was tested by immunoprecipitation, immunofluorescence,
and immunoblot by preadsorbing the antibodies to the specific peptides
or control peptides (12, 18).
Microinjection of DNA Expression Vectors--
Cerebellar granule
cells were grown for 7 days and injected with the DNA solutions in
phosphate buffer (25 ng/ml of each plasmid). The plasmid vectors used
were: CMV-GFP (CLONTECH); RSV-lacZ;
CRE-lacZ containing five CRE elements and the vasoactive
intestinal peptide promoter driving lacZ gene (19, 20). In
some experiments plasmid vectors expressing C-PKA were used (21, 22).
AKAP plasmids contained the sequence of AKAP75 or AKAP45 driven by the
cytomegalovirus promoter (23). All plasmids preparations were tested in
the rat thyroid cell line, FRTL-5, and in the PC12 cell line by stable transfection. The expression of the specific proteins was measured by
immunoanalyses (immunoblot, immunofluorescence, and
immunoprecipitation) and Northern blot (18, 24).
The injection apparatus consisted of a phase contrast microscope
connected to a computer-aided image analyzer (AIS automatic injection
system, Zeiss, Germany) and a computer-operated microinjector (Eppendorf, Germany). In each experiment 150 cells/dish and 2 dishes/DNA were injected. 18 h after the injection the cells were stimulated with 0.1 mM forskolin in the presence of 100 mM IBMX for 4 h, washed in phosphate-buffered saline
solution and fixed with 4% paraformaldehyde for 30 min. Cells were
permeabilized with 0.1% Triton X-100 and after extensive washes were
incubated first with the monoclonal anti-
-galactosidase antibody
(Sigma) and then with fluorescein-tagged goat anti-mouse IgG antibody (Sigma) in phosphate-buffered saline containing 0.2% skin porcine gelatin for 30 min at room temperature. In all experiments, the fluorescein-tagged anti-mouse IgG antibodies were injected alone as
control of the microinjection procedures. The specificity of anti-
-galactosidase antibodies was tested by omitting the first antibody. Under our conditions the efficiency of injection was about
20%.
 |
RESULTS |
Differential Response to cAMP of PKA in Granule Cells and Cortical
Neurons--
The binding of cAMP to the inactive PKA tetrameric
holoenzyme induces its dissociation, thereby releasing active catalytic subunits in the cytoplasm. We have studied cAMP signaling in primary cultures of cortical neurons and cerebellar granule cells by measuring the cytoplasmic and nuclear PKA catalytic activity following cAMP stimulation for various periods of time. We have used forskolin, a
stimulator of adenylyl cyclase, and IBMX, the inhibitor of cAMP phosphodiesterases, to maintain constant cAMP levels. Fig.
1A shows the time course of
cytoplasmic PKA activity in cortical and granule cells stimulated by 10 µM forskolin in the presence of 0.5 mM IBMX.
In granule cells, cAMP-induced PKA activity was marked and persistent.
In cortical cells PKA activation was lower, peaking at 15 min of cAMP
stimulation and returning to the basal value within 20 min. The
activation of PKA in granule cerebellar cells reached the plateau
between 1 and 5 min and remained constant up to 30 min after the
initial cAMP stimulation. The cAMP dose-response curve indicated that
the cerebellar granule cells PKA was activated at lower cAMP
concentration relative to PKA in cortical neurons (Fig.
1B).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
Different PKA responses to cAMP in cortical
and cerebellar granule cells. A, cortical neurons and
cerebellar granule cells were stimulated with 10 µM
forskolin for the indicated time, and free PKA catalytic activity in
the cytoplasm was measured as described under "Materials and
Methods." The activation of cytoplasmic PKA is shown by the ratio
between C-PKA ( cAMP) and total PKA (+0.5 mM cAMP). The
basal C-PKA activity in the cytoplasm was 6.3 ± 0.5 pmol/min/µg
protein in granule cells and 4.6 ± 0.4 pmol/min/µg protein in
cortical cells. The undissociated PKA holoenzyme (+cAMP) was 42.2 ± 4.1 in cortical cells and 28.6 ± 3.0 pmol/min/µg in granule
cells, respectively. B, cortical and granule cerebellar
granule cells were stimulated for 15 min with different cAMP
concentrations. C-PKA and total PKA were determined in total cell
extracts, and the data are shown as increase of C-PKA over the basal.
C, nuclear PKA catalytic activity in granule and cortical
cells stimulated with 10 µM forskolin or 1 µM dibutyryl cAMP for 10 min. Nuclei were prepared and
PKA determinations were carried out as described under "Materials and
Methods." Data are expressed as fold increase over the basal PKA
activity. The basal activity of nuclear PKA was 3.8 ± 0.5 pmol/min/µg nuclear proteins in cerebellar granule cells
versus 2.7 ± 0.3 pmol/min/µg in cortical cells.
D, cytoplasmic PKA activity in cortical and granule cells
stimulated with 10 µM forskolin or 1 µM
N6,2'-O-dibutyryl-cAMP for 10 min. PKA
determinations were carried out in the cytoplasmic fraction as
described under "Materials and Methods."
|
|
Nuclear C-PKA accumulation, a sensitive marker of cAMP stimulation, was
very efficient in cortical cells compared with granule cerebellar cells
(Fig. 1C), although the cytoplasmic PKA activation was
similar in the two cell types (Fig. 1D). The cortical
enzyme, albeit dissociated poorly, contributed significantly to the
total mass of nuclear PKA because the absolute amount of cytoplasmic PKA was higher in cortical than in granule cerebellar cells (see the
legend of Fig. 1).
These data indicate that the granule cells PKA, stimulated by cAMP,
dissociates efficiently, but does not transmit C-PKA to the nucleus. In
contrast, cortical cells respond very efficiently to cAMP with a
significant increase of nuclear C-PKA activity.
We suggest that the composition of PKA in the two cell types might be
the cause of the different responses to cAMP. Indeed, the regulatory
RII
type is abundantly expressed in cortical cells, is
membrane-bound, binds cAMP with lower affinity and has a longer half-life compared with RI
and RII
(8, 12, 15, 18, 25). These
features suggest that PKAII
might be the sensor of continuous and
persistent cAMP signals.
CREB Phosphorylation in Granule and Cortical Cells--
The
biologically relevant effect of PKA translocation to the nucleus is the
regulation of gene expression mediated by the phosphorylation of the
nuclear transcription factor CREB. CREB is bound to a specific DNA
sequence motif (CRE) and, upon phosphorylation by PKA catalytic subunit
(26) or Ca2+/calmodulin-dependent protein
kinase IV (27), binds the adaptor proteins (CBP and p300). The
association of CREB with these proteins facilitates the assembly of the
transcriptional machinery and leads to the activation of cAMP-induced
genes (28). We assayed CREB phosphorylation in cortical and granule
cells stimulated with forskolin by immunoblot of nuclear proteins with
antibodies that recognize the phosphorylated form of CREB (17). Fig.
2 shows that the phosphorylation of CREB
(PCREB) was strongly induced by forskolin (10 µM, 10 min)
in cortical neurons. In granule cells the PCREB signal was detectable
in basal conditions but was only slightly induced by forskolin.
Forskolin did not induce the de novo synthesis of CREB,
because the total amount of the protein was not modified by forskolin
treatment (Fig. 2). The phosphorylation of CREB was induced by a
cAMP-dependent pathway, because it was mimicked by the cAMP
analogue, 8-bromo-cAMP and was inhibited by 10 µM of the
cell-permeable PKA inhibitor, H89 (14) (data not shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
Forskolin-induced CREB phosphorylation in
cortical and cerebellar granule cells. Immunoblot of nuclear
extracts from cortical and granule cells, stimulated with 10 µM forskolin (FSK) for 15 min, performed using
anti-PCREB and anti-CREB antibodies as described under "Materials and
Methods." Non-phosphorylated CREB is shown in the lower
panels.
|
|
Because the PCREB signal detected by Western blot might originate from
contaminating glial cells present in our cultures, we have investigated
the effect of cAMP-elevating agents in pure type I astrocyte cultures.
Under the conditions described in Fig. 2, stimuli-dependent
cAMP accumulation did not induce CREB phosphorylation (data not shown).
Inhibition of RII
Expression Impairs Nuclear Response to cAMP in
Cortical Neurons--
To test whether the expression of PKAII
affects CREB phosphorylation in cortical cells, we treated cortical
neurons with specific anti-RII
antisense oligonucleotides for
72 h (see "Materials and Methods"). This treatment
specifically reduced RII
protein levels (over 50% reduction, Fig.
3 lane 2), but did not affect the other membrane-bound PKA regulatory subunit RII
, as shown by the
immunoblot with specific anti-RII
or RII
antibodies (Fig. 3). The
reduction of RII
protein was also detected by the ligand binding
assay or overlay (23, 29) in RII
antisense-treated cell extracts.
Also, total PKA activity was not affected by anti-RII
antisense
treatment (data not shown). RII
levels did not change in cells
treated with mismatched oligonucleotides (Fig. 3, lane 1 and
legend).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
Treatment with RII
antisense oligonucleotides reduces RII
content in cortical neurons. Representative immunoblots
of total cellular extracts from cortical neurons treated with
mismatched (1) or anti-RII antisense oligonucleotides
(2) using specific anti-RII (upper panel) or
anti-RII (lower panel) specific antibodies (see
"Materials and Methods"). The lower graph shows the relative amount
of RII and RII derived from the densitometric analysis of the
immunoblots in three experiments. The treatments with specific
oligonucleotides are described under "Materials and Methods."
RII and RII content was not influenced by treatment with
mismatched oligonucleotides. In some experiments we noticed a 25%
reduction of both RII proteins in cells treated with nonspecific
oligonucleotides.
|
|
Cortical cells treated with the RII
antisense oligonucleotide were
stimulated with forskolin and CREB phosphorylation assayed as described
in Fig. 2. In these cells, forskolin-induced CREB phosphorylation was
significantly reduced, whereas total CREB concentration was not
affected (Fig. 4, upper
panel). The decreased cAMP-induced CREB phosphorylation in
anti-RII
oligonucleotide-treated cells was accompanied by a parallel
reduced accumulation of C-PKA in the nucleus (Fig. 4, lower
inset).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Down-regulation of RII
reduces phosphorylated CREB and C-PKA in the nuclei of cortical
neurons. Top panels, immunoblot of phosphorylated CREB
in nuclear extracts of cortical neurons treated with mismatched
oligonucleotides (1) or RII specific antisense (2) and
stimulated with 10 µM forskolin (FSK) as
described in Fig. 2. Total CREB is shown in the lower blot obtained
from the same cell extracts. Lower panels, nuclear C-PKA
accumulation in cortical cells treated with mismatched oligonucleotides
(1) or RII specific antisense (2) and
stimulated with (+) or without ( ) 10 µM forskolin
(FSK) as described in Fig. 2. The basal nuclear C-PKA in
mismatched or RII oligonucleotide-treated cells was 2.8 ± 0.5 pmol/min/µg in cortical cells and 2.7 ± 0.4 pmol/min/µg,
respectively.
|
|
These data indicate that RII
levels influence cAMP-induced nuclear
C-PKA accumulation and CREB phosphorylation and suggest that the
efficient transmission of cAMP signals to the nucleus in cortical cells
might be dependent on the abundance of PKAII
. This observation is
consistent with the finding that neural gene expression and
c-fos induction by cAMP are defective in specific areas of
the central nervous system in RII
/
mice (30, 31).
Expression of RII
and AKAP75 Enhances cAMP-induced Transcription
in Granule Cells--
To test the hypothesis that membrane-bound
PKAII
plays a crucial role in the cAMP-induced transcription, we
microinjected granule cells with expression vectors containing the
cDNAs encoding RII
, the specific brain RII-anchor protein,
AKAP75, and the green fluorescent protein, GFP. Because RII
and
AKAP75 are not expressed in granule cells (8, 12), their synthesis
should result in the formation of new PKAII
bound to AKAP75 as in
cortical cells. We first assayed the expression of RII
and AKAP75 in
microinjected cells. Fig. 5 shows the
staining of microinjected cells with specific antibodies to AKAP75
(1) and to RII
(2 and 3). Both
AKAP75 and RII
were efficiently expressed in microinjected granule
cells. The cells injected with RII
expression vector only showed a
diffuse cytoplasmic staining (Fig.
6A), whereas the cells
microinjected with both AKAP75 and RII
expression vectors showed a
single RII
spot-shaped signal in the cell body (Fig. 6), likely
corresponding to the cytoskeletal and perinuclear localizations of
RII
in neural cells (6).

View larger version (94K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of exogenous
RII and AKAP75 genes in cerebellar granule
cells. Cerebellar granule cells were microinjected with DNA
vectors expressing RII , AKAP75, and green fluorescent protein genes
as described under "Materials and Methods." 24 h later the
cells were fixed and stained with specific antibodies to AKAP75 or
RII . Column A shows the phase-contrast microphotograph of
microinjected cells. Column B shows the staining of
GFP-positive cells, indicated by arrows, with antibodies to
AKAP75 (1) or to RII (2 and 3). The
efficiency of the microinjection was ca. 20%. The expression of the
microinjected gene was maximal at 24-36 h after the injection. In
2B and 3B the faint signal visible in noninjected
is presumably originated by the RII endogenous gene or by
cross-reactivity with RII .
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
RII localization in
granule cerebellar cells expressing RII and
AKAP75. Cells microinjected with RII (A) or RII
and AKAP75 plasmids were stained with anti-RII antibodies as
described under "Materials and Methods."
|
|
To test the effects of the expression of RII
and AKAP75 on
cAMP-induced transcription, a reporter gene (lacZ) driven by
five tandemly linked cAMP-responsive elements, (5×
CRE-lacZ) was co-injected with the plasmids indicated above.
Cells were stimulated for 4 h with 1 µM forskolin,
and the expression of the lacZ gene under control of the CRE
promoter was monitored by immunofluorescence with
anti-
-galactosidase antibodies. Fig. 6 shows a representative picture of granule cells microinjected with the combination
AKAP75-RII
and CRE-lacZ genes. Treatment with forskolin
for 4 h significantly increased the number of lacZ
positive cells.
Table I shows that cells microinjected
with a control plasmid carrying the lacZ fused to a
constitutive non-cAMP-dependent promoter (the long terminal
repeats of Rous sarcoma virus, RSV-lacZ) efficiently
synthesized
-galactosidase. Cells microinjected with the
CRE-lacZ construct did not show
-galactosidase signal
following forskolin stimulation. Injection of RII
or AKAP75
expression vectors stimulated CRE-lacZ expression (3 to 6 and 2.8 to 5 positive cells). Co-injection of RII
and AKAP75
expression vectors resulted in a marked increase in the number of
lacZ-expressing cells (4 to 14 positive cells,
or + forskolin, respectively). The number of positive cells was dramatically
reduced if a mutant version of AKAP75 was co-injected with RII
,
AKAP45, which binds RII but fails to localize it to the membranes (18,
32, 33) or with a vector expressing PKI, the specific PKA inhibitor
(34). Moreover pretreatment of microinjected cells with 10 µM H89, a PKA inhibitor (14), resulted in a significant
reduction of
-galactosidase-expressing cells (data not shown). The
data, derived from six independent experiments, indicate that the
combination of RII
-AKAP expression vectors significantly increased
the number of
-galactosidase positive cells in the presence of
cAMP.
View this table:
[in this window]
[in a new window]
|
Table I
LacZ+ cells were identified by immunofluorescence with
anti- -galactosidase-specific antibodies as described under
"Materials and Methods." In all the samples CRE-LacZ and GFP have
been included.
|
|
 |
DISCUSSION |
Targeting protein kinases and phosphatases in proximity of their
substrates represents an important mechanism to convey intracellular signals to specific cellular sites (4, 7). Two different protein
families bind PKA to the cell compartments: the
Akinase anchor
proteins AKAPs and the microtubule-associated proteins MAP2. AKAPs bind the regulatory subunit RII
with nanomolar affinity and localize PKA to the dendritic cytoskeleton and other cellular compartments (Golgi apparatus, primary branches of dendrites and perikarya), whereas MAP2 predominantly binds the RII
isoform (5).
AKAP 150/75, the rat or the bovine PKA anchor protein, can also bind
RII
but with lower affinity (35-37). The levels of RII
and AKAP
75 differ in cell populations of brain areas and their expression
pattern is strictly correlated. Accordingly, in cortical neurons, which
express AKAP 75, RII
is the most abundant PKA-regulatory subunit;
conversely, in cerebellar granule cells AKAP 75 and RII
has not been
found (5, 8, 12). Because the amounts of RII
and MAP2 are comparable
in both cell populations (8, 12), we decided to test if the expression
of RII
-AKAP might interfere with the cytosolic and nuclear responses
to cAMP in cortical neurons and cerebellar granule cells. PKA activity and CREB phosphorylation were measured as functional correlates of cAMP
stimulation. Although it has been shown that CREB is also a substrate
for other protein kinases (27, 38), we have analyzed only cAMP-induced events.
The response to cAMP signals was different in granule cerebellar and
cortical cells. In granule cerebellar cells, cytoplasmic PKA
dissociated very efficiently, but nuclear C-PKA accumulation induced by
cAMP was rather weak. Cortical cells, on the other hand, did not
activate efficiently cytoplasmic PKA but accumulated PKA in the nuclei
in response to cAMP. The different PKA composition and localization in
the two cell types might account for these different responses. Granule
cells contain mainly type I and type II
PKA, which bind cAMP with
higher affinity compared with type PKAII
(15, 18, 25). These
subunits are soluble (RI
) or partly soluble (RII
) in the cytosol
(10, 12). PKAI and PKAII
dissociate efficiently at low cAMP levels
(Fig. 1B). PKA present in cortical cells, composed mainly of
type II
isoenzyme, dissociates at high cAMP levels and rapidly
reassociates (Fig. 1, A and B). Membrane-bound
PKAII
might be the sensor of persistent cAMP signals and the
preferential source of nuclear C-PKA in cortical cells. This
interpretation is strengthened by the observation that the cAMP-dependent nuclear signaling is inhibited in cortical
cells with lower RII
levels (Fig. 4) or in the RII
-defective
mouse (30, 31).
We have reconstituted the "cortical" PKA by expressing RII
and AKAP75 in the granule cells by microinjection of specific expression vectors. AKAP75 and RII
plasmids were efficiently expressed alone or in combination in microinjected granule cerebellar cells (Fig. 5). The expression of AKAP75 localized RII
in discrete spots inside the cell body close to the nucleus (perikaryon) (Fig. 6B). It is worth noting that expression of AKAP75 in
non-neuronal cells localizes RII under the plasma membrane (33, 39).
Although, we did not identify conclusively the specific compartment in
microinjected granule cells, the data shown indicate that different
partners interacting with AKAP75 are present in neuronal and
non-neuronal cells.
cAMP-induced transcription was stimulated by RII
or AKAP75, but the
simultaneous injection of both genes markedly stimulated cAMP-induced
lacZ expression (Table I). The stimulatory effect on
transcription was inhibited by PKA inhibitors (Table I and Fig.
7) and dependent on AKAP75
membrane-binding domain, because a mutant lacking the segment of the
protein, which localizes AKAP75 to the membrane, inhibited the RII
stimulatory effect (Table I). The expression of both RII
and AKAP
generates high levels of membrane-bound PKAII
, that ultimately might
be responsible for the stimulation of cAMP-dependent
transcription. There is evidence indicating that membrane-bound PKAII
is more stable than cytosolic PKAII, which can be rapidly activated and
degraded. Overexpression of AKAP75 increased RII
levels in kidney
cells (33). Conversely, cytosolic translocation of PKAII, attained by
expression of AKAP45 decreased RII
levels in thyroid, kidney, and
PC12 cells (18, 23, 24).2

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 7.
Coexpression of RII
and AKAP75 genes in cerebellar granule cells stimulates
cAMP-induced transcription. Cells were microinjected with GFP,
RII , AKAP75, and CRE-lacZ as described under "Materials
and Methods." 15 h after the microinjection, the cells were
treated with 50 µM forskolin and 0.5 mM IBMX
for 5 h (indicated in the top panels, +).
Phase-contrast microphotographs and staining with
anti- -galactosidase antibody are shown on the left and
right, respectively.
|
|
Reposition of PKAII in the membranes might protect the enzyme
from degradation and might reduce the basal PKA activation
(33).3 We suggest that
membrane-bound PKA, releasing high levels of C-PKA in proximity of the
nuclear envelope, can promote the entry of C-PKA into the nucleus. We
do not exclude, however, that membrane-bound PKA could also modulate a
Ca2+-dependent component of CRE-driven gene
expression. In cerebellar granule cells the low expression of RII
and AKAP 150/75 probably underlies the weak cAMP-stimulated
transcription and suggests that other mechanisms could be responsible
for CRE-driven gene expression in these cells (40).
Increasing evidence indicate that CREB is a multifunctional
transcription factor that can be activated by cAMP and
Ca2+-dependent transduction pathways (41, 42),
and its activation is critical for long-term memory formation in
different biological systems (43-46). Stimuli that generate
long-lasting long term potentiation have been shown to induce
CRE-mediated gene expression that was reduced by L-type
Ca2+ channel blockers (14), but the cAMP pathway appears to
be necessary for the cellular processes related to long-term memory
(20, 47).
As to the biological correlates of our findings, we suggest that the
differential composition and localization of PKA in discrete populations of neurons might explain the different activation of
cAMP-induced transcription in these cells by the same type of signal.
Consistent with our data, it has been found that in RII
defective
mouse gene induction by cAMP in striatal neurons is significantly
inhibited (31).
 |
ACKNOWLEDGEMENTS |
We thank Dr. D. Grieco for helpful comments
and CEINGE-Advanced Biotechnologies S.R.L. for the use of the
microinjection apparatus. Specifically, we thank Prof. Tina Pietropaolo
and Dr. Giulia Russo for microinjection experiments and Matilde
Recusani, who has been instrumental for the completion of the work
reported here. Special thanks to Charles Rubin (A. Einstein, New York)
who has provided AKAP vectors and specific antibodies to AKAP75 and Dr.
M. V. Barone for the analysis of microinjected cells.
 |
FOOTNOTES |
*
This work was supported by grants from Associazione Italiana
per la ricerca sul Cancro (A. I. R. C.), CNR target Project on Biotechnology, and MURST (Italian Department of University and Research).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.

To whom correspondence should be addressed: Dipartimento di
Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina, Torre Biologica 14 Fl., via S. Pansini, 5, 80131 Napoli, Italy. Tel.:
39-81-7463251; Fax: 39-81-7463252; E-mail: avvedim{at}cds.unina.it.
§§
Present address: INM, Neuromed, Pozzilli, 86077, Isernia, Italy.
¶¶
Present address: Casa di Cura, Villa Chiarugi, Nocera
Inferiore, Italy.
2
Cassano et al., manuscript submitted.
3
A. Feliciello, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, protein kinase
A;
AKAP, a kinase anchor protein;
C-PKA, catalytic subunit of PKA;
CRE, cAMP-responsive element;
CREB, CRE-binding protein;
MAP2:
microtubule-associated protein 2, PCREB, phosphorylated CREB;
GFP, green fluorescent protein;
IBMX, isobutylmethylxanthine;
PKI, PKA
inhibitor protein;
RSV, Rous sarcoma virus.
 |
REFERENCES |
-
Harootunian, A. T.,
Adams, S. R.,
Wen, W.,
Meinkoth, J. L.,
Taylor, S. S.,
and Tsien, R. Y.
(1993)
Mol. Biol. Cell
4,
993-1002[Abstract]
-
Hagiwara, M.,
Brindle, P.,
Harootunian, A. T.,
Armstrong, R.,
Rivier, J.,
Vale, W.,
Tsien, R. Y.,
and Montminy, M. R.
(1993)
Mol. Cell. Biol.
13,
4852-4859[Abstract]
-
Thompson, M. A.,
Ginty, D. D.,
Bonni, A.,
and Greenberg, M. E.
(1995)
J. Biol. Chem.
270,
4224-4235[Abstract/Free Full Text]
-
Rubin, C. S.
(1994)
Biochim. Biophys. Acta
1224,
467-479[Medline]
[Order article via Infotrieve]
-
Cadd, G.,
and McKnight, G. S.
(1989)
Neuron
3,
71-79[Medline]
[Order article via Infotrieve]
-
Glantz, S. B.,
Amat, J. A.,
and Rubin, C. S.
(1992)
Mol. Biol. Cell
3,
1215-1228[Abstract]
-
Mochly-Rosen, D.
(1995)
Science
268,
247-251[Medline]
[Order article via Infotrieve]
-
Faux, M. C.,
and Scott, J. D.
(1996)
Trends Biochem. Sci.
21,
312-315[CrossRef][Medline]
[Order article via Infotrieve]
-
Bregman, D. B.,
Bhattacharyya, N.,
and Rubin, C. S.
(1989)
J. Biol. Chem.
264,
4648-4656[Abstract/Free Full Text]
-
Hirsch, A. H.,
Glantz, S. B.,
Li, Y.,
You, Y.,
and Rubin, C. S.
(1992)
J. Biol. Chem.
267,
2131-2134[Abstract/Free Full Text]
-
Carr, D. W.,
Stofko-Hahn, R. E.,
Fraser, I. D. C.,
Cone, R. D.,
and Scott, J. D.
(1992)
J. Biol. Chem.
267,
16816-16823[Abstract/Free Full Text]
-
Ventra, C.,
Porcellini, A.,
Feliciello, A.,
Gallo, A.,
Paolillo, M.,
Mele, E.,
Avvedimento, V. E.,
and Schettini, G.
(1996)
J. Neurochem.
66,
1752-1761[Medline]
[Order article via Infotrieve]
-
Schinelli, S.,
Paolillo, M.,
and Corona, G. L.
(1994)
J. Neurochem.
62,
944-949[Medline]
[Order article via Infotrieve]
-
Impey, S.,
Mark, M.,
Villacres, E. C.,
Poser, S.,
Chavkin, C.,
and Storm, D. R.
(1996)
Neuron
16,
973-982[Medline]
[Order article via Infotrieve]
-
Hofmann, F.,
Beavo, J. A.,
Bechtel, P. J.,
and Krebs, E. G.
(1975)
J. Biol. Chem.
250,
7795-7801[Abstract]
-
Gallo, A.,
Benusiglio, E.,
Bonapace, I. M.,
Feliciello, A.,
Cassano, S.,
Garbi, C.,
Musti, A. M.,
Gottesman, M. E.,
and Avvedimento, V. E.
(1992)
Gene Dev.
6,
1621-1630[Abstract]
-
Ginty, D. D.,
Kornhauser, J. M.,
Thompson, M. A.,
Bading, H.,
Mayo, K. E.,
Takahashi, J. S.,
and Greenberg, M. E.
(1993)
Science
260,
238-241[Medline]
[Order article via Infotrieve]
-
Feliciello, A.,
Giuliano, P.,
Porcellini, A.,
Garbi, C.,
Obici, S.,
Mele, E.,
Angotti, E.,
Grieco, D.,
Amabile, G.,
Cassano, S.,
Li, Y.,
Musti, A. M.,
Rubin, C. S.,
Gottesman, M. E.,
and Avvedimento, E. V.
(1996)
J. Biol. Chem.
271,
25350-25359[Abstract/Free Full Text]
-
Kaang, B. K.,
Pfaffinger, P. J.,
Grant, S. G. N.,
Kandel, E. R.,
and Furukawa, Y.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
1133-1137[Abstract]
-
Kaang, B. K.,
Kandel, E. R.,
and Grant, S. G. N.
(1993)
Neuron
10,
427-435[Medline]
[Order article via Infotrieve]
-
Mellon, P. L.,
Clegg, C. H.,
Correll, L. A.,
and McKnight, G. S.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
4887-4889[Abstract]
-
Ciardiello, F.,
Tortora, G.,
Kim, N.,
Clair, T.,
Ally, S.,
Salomon, D. S.,
and Cho-Chung, Y. S.
(1990)
J. Biol. Chem.
265,
1016-1020[Abstract/Free Full Text]
-
Ndubuka, C.,
Li, Y.,
and Rubin, C. S.
(1993)
J. Biol. Chem.
268,
7621-7624[Abstract/Free Full Text]
-
Cassano, S.,
Gallo, A.,
Buccigrossi, V.,
Porcellini, A.,
Cerillo, R.,
Gottesman, M. E.,
and Avvedimento, E. V.
(1996)
J. Biol. Chem.
271,
29870-29875[Abstract/Free Full Text]
-
Beebe, S. J.,
and Corbin, J. D.
(1986)
in
The Enzymes (Boyer, P. D., and Krebs, E. G., eds), Vol. 17, pp. 43-111, Academic Press, Orlando, FL
-
Montminy, M. R.,
Gonzalez, G. A.,
and Yamamoto, K. K.
(1990)
Trends Neurosci.
13,
184-188[CrossRef][Medline]
[Order article via Infotrieve]
-
Bito, H.,
Deisseroth, K.,
and Tsien, R. W.
(1996)
Cell
87,
1203-1214[Medline]
[Order article via Infotrieve]
-
Kwok, R. P.,
Lundblad, J. R.,
Chrivia, J. C.,
Richards, J. P.,
Bachinger, H. P.,
Brennan, R. G.,
Roberts, S. G.,
Green, M. R.,
and Goodman, R. H.
(1994)
Nature
370,
223-226[CrossRef][Medline]
[Order article via Infotrieve]
-
Carr, D. W.,
De Manno, D. A.,
Atwood, A.,
Hunzicker-Dunn, M.,
and Scott, J. D.
(1993)
J. Biol. Chem.
268,
20729-20732[Abstract/Free Full Text]
-
Adams, M. R.,
Brandon, E. P.,
Chartoff, E. H.,
Idzerda, R. L.,
Dorsa, D. M.,
and McKnight, G. S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12157-12161[Abstract/Free Full Text]
-
Brandon, E. P.,
Logue, S. F.,
Adams, M. R.,
Qi, M.,
Sullivan, S. P.,
Matsumoto, A. M.,
Dorsa, D. M.,
Wehner, J. M.,
McKnight, G. S.,
and Idzerda, R. L.
(1998)
J. Neurosci.
18,
3639-3649[Abstract/Free Full Text]
-
Glantz, S. B.,
Li, Y,
and Rubin, C. S.
(1993)
J. Biol. Chem.
268,
12796-12804[Abstract/Free Full Text]
-
Feliciello, A.,
Li, Y.,
Avvedimento, V. E.,
Gottesman, M. E.,
and Rubin, C. S.
(1997)
Curr. Biol.
7,
1011-1014[Medline]
[Order article via Infotrieve]
-
Day, R. N.,
Walder, J. A.,
and Maurer, R., A.
(1989)
J. Biol. Chem.
264,
431-436[Abstract/Free Full Text]
-
Leiser, M.,
Rubin, C. S.,
and Erlichman, J.
(1986)
J. Biol. Chem.
261,
1904-1908[Abstract/Free Full Text]
-
Obar, R. A.,
Dingus, J.,
Bayley, H.,
and Vallee, R. B.
(1989)
Neuron
3,
639-645[Medline]
[Order article via Infotrieve]
-
Rubino, H. M.,
Dammerman, M.,
Shaft-Zagardo, B.,
and Erlichman, J.
(1989)
Neuron
3,
631-638[Medline]
[Order article via Infotrieve]
-
Ginty, D. D.
(1997)
Neuron
18,
183-186[Medline]
[Order article via Infotrieve]
-
Li, Y.,
Ndubuka, C.,
and Rubin, C. S.
(1996)
J. Biol. Chem.
271,
16862-16869[Abstract/Free Full Text]
-
Schulman, H.
(1993)
Curr. Opin. Cell Biol.
5,
247-253[Medline]
[Order article via Infotrieve]
-
Sheng, M.,
Thompson, M. A.,
and Greenberg, M. E.
(1991)
Science
252,
1427-1430[Medline]
[Order article via Infotrieve]
-
Ginty, D. D.,
Bonni, A.,
and Greenberg, M. E.
(1994)
Cell
77,
713-728[Medline]
[Order article via Infotrieve]
-
Bourtchuladze, R.,
Frenguelli, B.,
Blendy, J.,
Cioffi, D.,
Schultz, G.,
and Silva, A. J.
(1994)
Cell
79,
59-68[Medline]
[Order article via Infotrieve]
-
Frank, D. A.,
and Greenberg, M. E.
(1994)
Cell
79,
5-8[Medline]
[Order article via Infotrieve]
-
Carew, T. J.
(1996)
Neuron
16,
5-8[Medline]
[Order article via Infotrieve]
-
Deisseroth, K.,
Bito, H.,
and Tsien, R. W.
(1996)
Neuron
16,
89-101[Medline]
[Order article via Infotrieve]
-
Abel, T.,
Nguyen, P. V.,
Barad, M.,
Deuel, T. A. S.,
Kandel, E. R.,
and Bourtchouladze, R.
(1997)
Cell
88,
615-626[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.