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1
,3,§
* Ernest Gallo Research Center,
Departments of Neurology,
§ Cellular and Molecular Pharmacology, Pediatrics, and Neuroscience Graduate Program and Center for Neurobiology of Addiction, University of California San Francisco, San Francisco, California 94110-3518, USA; and
Portland Alcohol Research Center, Department of Behavioral Neuroscience, Oregon Health Science University and Veterans Affairs Medical Center, Portland, Oregon 97201, USA
1Correspondence: Ernest Gallo Research Center, 5858 Horton St., Suite 200, Emeryville, CA 94608, USA. E-mail: dorit{at}itsa.ucsf.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: PKC • activated C kinase receptor • cAMP • signal transduction • ethanol • targeting protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These responses appear to be
modulated at least in part by protein kinase C (PKC) (2)
.
Ethanol alters the normal activities of major neurotransmitter
receptors, including glycine (3)
,
GABAA (4
, 5)
, glutamate
(6)
, and muscarinic receptors (7)
, by
inhibiting or activating the function of PKC. PKC is also required for
ethanol-induced neurite outgrowth (8
, 9)
, and
up-regulation of Ca2+ channels (10
, 11)
. Chronic ethanol intake decreases PKC activity, which is
associated with a reduction in phorbol ester binding in selected rat
brain areas (12)
. In addition, 
PKC knockout mice show
low sensitivity to ethanol (13)
, whereas 
PKC knockout
mice are more sensitive in their GABAA responses
to ethanol (14)
. Although it is clear that ethanol
enhances or decreases PKC function, the mechanism by which ethanol
alters the activity of PKC is not well understood.
The specificity of protein kinase function is regulated in part by
intracellular compartmentalization (15)
. This, in turn, is
regulated by targeting (anchoring, scaffolding, and adaptor) proteins
(15)
. PKC is a family of 10 isozymes that translocate to
distinct intracellular sites upon activation (16)
.
Compartmentalization of the translocated PKC isozymes is mediated in
part by RACKs (receptors for activated C kinase) (17)
.
RACK1 has been cloned and identified as a targeting protein for
activated ßIIPKC (18)
. Among the 10 PKC isozymes, RACK1
is specific for ßIIPKC (19
, 20)
. We have discovered
recently that RACK1 itself is a translocating protein
(20)
. Upon PKC activation, RACK1 colocalizes with, binds
to and translocates with ßIIPKC (20)
. We proposed that
RACK1 is a shuttling protein that brings ßIIPKC to its appropriate
intracellular site upon activation of PKC (20)
.
Recent evidence demonstrates that ethanol causes translocation of
specific kinases such as the catalytic subunit of PKA (21)
and the and 
isozymes of PKC (22)
. Since PKC
compartmentalization is mediated by intracellular targeting, such as
RACK proteins (23)
, one mechanism by which ethanol might
affect kinases is by affecting their corresponding targeting proteins.
In this study we present evidence that in cultured cells and in
vivo, ethanol changes the compartmentalization of RACK1, the
targeting protein for ßIIPKC, thereby altering the physiological
activity of the kinase.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Chinese hamster ovary (CHO) cells stably expressing the long
form of the dopamine D2 receptor (D2L) (24)
were seeded
and grown in Ham’s F12 medium containing 10% fetal bovine serum (FBS)
and 2 mM glutamine. After 48 h, media were replaced with Ham’s
F12 medium containing 5% serum, 25 mM HEPES (pH 7.4), 2 mM glutamine,
50 µg/ml of human transferrin, 10 µg/ml oleic acid (complexed with
2 mg/ml fatty acid-free bovine serum albumin), 25 µg/ml bovine
insulin, and trace elements at the following concentrations: 0.5 nM
MnCl2, 0.5 nM
[NH4]6Mo7O24,
0.25 nM SnCl4, 25 nM
Na3VO4, 5 nM
CdSO4, 0.25 nM NiSO4, 15 nM
H2SeO3, and 25 nM
Na2SiO3. On day 4, the
cells were treated with different reagents as described in
legends to Figs. 1
and 2
.
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C6 glioma cells were seeded and grown in DMEM containing 5% FBS and
Ham’s F12 containing 2 mM glutamine, 20 mM HEPES, 50 µg/ml human
transferrin, and trace elements at concentrations described above.
Cells were seeded in Nalgene Nunc 4-well chamber slides (Fisher,
Pittsburgh, Pa.) and cultures were treated as described in legend of
Fig. 4
.
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Immunostaining and confocal microscopy
CHO/D2L and C6 glioma cells were treated as described in the
figure legends. The cells were then washed with cold phosphate-buffered
saline (PBS), fixed with ice-cold methanol for 3 min, and washed twice
with cold PBS. Cells were incubated for 2 h with 1% normal goat
serum in PBS containing 0.1% Triton X-100, followed by overnight
incubation at 4°C with the appropriate primary antibody (diluted in
PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum albumin).
Cells were then washed three times with PBS containing 0.1% Triton and
incubated for 1.5 h with FITC-conjugated anti-rabbit (IgG)
antibody (1:500, Cappel), Texas red, or Cy5-conjugated anti-mouse (IgM)
antibodies together with the nuclear marker TOTO-3 (1:10,000, Molecular
Probes). Cells were washed an additional three times with cold PBS
containing 0.1% Triton. Slides were mounted using Vectashield and
viewed with a Bio-Rad MRC-1024 laser scanning confocal microscope. The
confocal images were processed using the computer programs NIH Image
version 1.61 (National Institutes of Health) and Adobe PhotoShop
(Adobe Systems Inc., San Jose, Calif.). All the images shown are
individual middle sections of projected Z series.
Localization of RACK1 and ßIIPKC to the Golgi apparatus
The localization of ßIIPKC and RACK1 to the Golgi apparatus
upon PKC activation in CHO/D2L cells was confirmed as described in ref
20
. Briefly, CHO/D2L cells were stained with anti-RACK1
antibodies together with the specific Golgi marker BODIPY-FL
C5-ceramide and anti-ßIIPKC antibodies together
with the Golgi marker anti-mannosidase antibodies.
Brain sections
Adult male WSC outbred mice (Portland Oregon) were injected
intraperitoneally (i.p.) with ethanol (1.5 g/kg) and then exposed to
ethanol via vapor chamber for 3 days. Both control mice (exposed to air
chambers) and ethanol-treated mice were injected i.p. daily with 1
mmol/kg of the alcohol dehydrogenase inhibitor pyrazole-HCl. After 3
days (chronic) exposure to alcohol, the animals were anesthetized and
perfused with 4% paraformaldehyde. Separate mice were injected with
acute ethanol (4 g/kg) or saline and perfused 60 min later. Brains were
removed, saturated with 30% sucrose and sectioned on a freezing
microtome. Slices of 10 µm were blocked in PBS containing 0.3%
Triton X-100 and 5% NGS for 1 h at room temperature. Blocking
solution was aspirated and sections were then stained with monoclonal
anti-RACK antibodies (1:100) and polyclonal anti-ßIIPKC antibodies
(1:100) overnight at 4°C. Antibody was aspirated and sections were
washed in PBS containing 0.3% Triton and 1% NGS. Sections were
incubated for 2 h at RT with secondary antibodies conjugated to
Texas red, FITC, and a nuclear marker TOTO-3. Sections were then washed
and mounted on Fisher Superfrost glass slides. Three mice were used per
treatment. Three slices were mounted and viewed from each mouse brain.
Three images were processed from each slice.
Image analysis
Quantification of colocalization with a nuclear marker
Colocalization of RACK1 and/or ßIIPKC with the nuclear marker
TOTO-3 was determined using NIH 1.61 Image program as described in ref
20
. Briefly, thresholding was used to separate
immunofluorescence pixels from the background and to create binary
images. Pairs of binary images of RACK1 and TOTO-3 or ßIIPKC and
TOTO-3 were multiplied and divided by 255 to generate a final merged
image that could be visualized with an 8 bit gray scale. The percent of
RACK1 or ßIIPKC staining merged with the nuclear marker TOTO-3 was
calculated using the following equation:
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Scoring translocation
Translocation of RACK1 and ßIIPKC in CHO/D2L cells was scored
by counting at least three random fields of cells (total of at least
100 cells) per treatment for staining of RACK1 and/or ßIIPKC. The
percent of translocated RACK1 and/or ßIIPKC was calculated using the
following equation:
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Each image contained 20–100 cells.
Protein–protein interaction assays
In vitro binding assay
Binding assays were done according to Ron et al.
(19)
. Briefly, RACK1-maltose binding fusion protein (1 ml,
10 µg/ml) was loaded on 0.5 ml amylose resin column (New England
Biolabs, Beverly, Mass.) and extensively washed (10 times the column
volume) with column buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM
EDTA, 1 mM sodium azide, and 10 mM ß-mercaptoethanol). ßIIPKC (1
µg purified recombinant SF9 expressed protein) was incubated in
overlay buffer [50 mM Tris-HCl pH 7.4, 0.1% bovine serum albumin, 5
µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor (SBTI), 0.1%
polyethylene glycol, 0.2 M NaCl, 0.1 mM CaCl2,
and 12 mM ß-mercaptoethanol] in the presence of PKC activators 50
µg/ml PS, 0.8 µg/ml DAG, and 1 mM CaCl2. The
mixture of activated ßIIPKC was incubated with RACK1 on the resin for
15 min at room temperature in the presence and in the absence and
presence of 50 mM or 200 mM of ethanol. After washing three times with
overlay wash (50 mM Tris-HCl pH 7.4, 0.1% polyethylene glycol, 0.2 M
NaCl, 0.1 mM CaCl2, and 12 mM
ß-mercaptoethanol) for 10 min each, the complex was eluted with
maltose (10 mM), sample buffer was added, and the sample was resolved
on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gel. Binding of ßIIPKC was detected using anti-ßIIPKC
polyclonal antibodies (1:500), followed by chemiluminescent reaction
(2.5 mM luminol and 400 µM p-coumaric acid).
Overlay assay
CHO/D2L cells were washed once with cold PBS and lysed in 20 mM
Tris-HCl (pH 7.5) containing 10 mM EGTA, 2 mM EDTA, 0.25 M sucrose, 1%
Triton X-100, and 10 µg/ml of the following protease inhibitors: SBTI
(Sigma), aprotinin, phenylmethylsulfonyl fluoride (Sigma), and
leupeptin (Boehringer Mannheim, Mannheim, Germany). Cells were lysed
and incubated at 4°C for 30 min. Lysed cells were then centrifuged at
14,000 g at 4°C for 30 min. Protein determination of the
triton-soluble material (supernatant) was assessed using Bicinchoninic
(BCA) reagent (Pierce, Rockford, Ill.). Approximately 0.5 mg protein
was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose
membrane. The membrane was cut into strips and incubated in overlay
buffer with activated ßIIPKC (as described above) in the presence and
absence of 200 mM ethanol for 15 min at room temperature. The
nitrocellulose membrane strips were washed three times with overlay
wash for 10 min each. Binding of ßIIPKC was detected using
anti-ßIIPKC polyclonal antibodies (1:500), followed by
chemiluminescent reaction (2.5 mM luminol and 400 µM
p-coumaric acid).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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confirms our earlier findings (20)
that RACK1 is localized
to perinuclear structures in unstimulated CHO cells (Fig. 1A-d
, 1B-b
, and ref 20
). However,
exposure to ethanol caused RACK1 to move to the nucleus (Fig. 1A-e, f
, Fig. 1B-d
). Ethanol-induced movement of RACK1 to the nucleus was also observed
in C6 glioma cell lines (see Fig. 4B,C
) and NG108–15
neuroblastoma-glioma (data not shown). We concluded that
ethanol-induced RACK1 to be compartmentalized in the nucleus since
immunofluorescence confocal images are of middle section of projected Z
series, and therefore likely to be in the middle of the nucleus. To
confirm that RACK1 is indeed compartmentalized in the nucleus in
response to ethanol, cells were stained with anti-RACK1 antibodies
together with the nuclear marker TOTO-3. Images were quantified as
described in Materials and Methods. As shown in Fig. 4B
,
more than 50% of RACK1 colocalized with the nuclear marker TOTO-3
after exposure to 200 mM ethanol in C6 glioma cells. Ethanol-induced
nuclear compartmentalization of RACK1 was observed in all three tested
cell lines, but the degree of nuclear compartmentalization varied
between cell types as a function of time and concentration. In CHO
cells for example, ethanol concentrations as low as 5 mM caused RACK1
to move to the nucleus if cells were exposed for 60 min (Fig. 1B-d
).
We next determined whether ethanol alters the intracellular
compartmentalization of ßIIPKC. ßIIPKC was unaffected by ethanol in
any of the tested cell lines (Fig. 1
and data not shown). In
unstimulated CHO cells ßIIPKC is localized to the cytoplasm (Fig. 1A-a
, B-a
, and ref 20
). Exposure to
ethanol did not alter the cytoplasmic staining of ßIIPKC even at
concentrations as high as 200 mM (Fig. 1A-b, c
, Fig. 1B-c
). Furthermore, prolonged incubation with ethanol up to
60 min did not induce ßIIPKC translocation at all tested
concentrations (Fig. 1B-c
and data not shown). Ethanol,
therefore, induced a rapid movement of RACK1 to the nucleus but did not
affect the compartmentalization of ßIIPKC.
There is substantial evidence that RACK1 directs the
compartmentalization and function of PKC (18
19
20
, 25
26
27
28
29
30
31
32
33
34)
.
We recently found that RACK1 is a shuttling protein for ßIIPKC
(20)
. Upon receptor activation, RACK1 binds activated
ßIIPKC and translocates it to another site where ßIIPKC is thought
to phosphorylate its appropriate substrate(s) (20)
.
Therefore, we determined whether ethanol-induced nuclear
compartmentalization of RACK1 affects the translocation of ßIIPKC in
response to receptor activation. We used CHO cells expressing the
dopamine D2 receptors (CHO/D2L) as our model system. We previously
found that activation of the dopamine D2 receptors with the selective
agonist NPA results in the translocation of RACK1 and ßIIPKC
(20)
. Using specific Golgi markers anti-mannosidase
antibodies and BODIPY-FL-C5, the site of
activation-induced translocated RACK1 and ßIIPKC in CHO/D2L cells was
identified as the Golgi apparatus (20)
. Indeed, Fig. 2
shows that in NPA-treated cells, more than 80% of RACK1 and ßIIPKC
are localized to the Golgi apparatus (Fig. 2,
black bars).
We next asked whether exposure to ethanol alters these responses to
NPA. Figures 1
and 2
show that exposure to ethanol induced the movement
of RACK1 to the nucleus (Fig. 2
, hatched bars), but did not affect the
cytosolic compartmentalization of ßIIPKC. On the other hand, when
ethanol was added for 30 min prior to NPA, ßIIPKC translocation to
the Golgi apparatus was inhibited (Fig. 2)
, whereas RACK1 remained
targeted to the nucleus by ethanol despite the addition of NPA (Fig. 2
,
hatched bar). Therefore, ethanol inhibited NPA-induced translocation of
ßIIPKC and changed the direction of RACK1 movement from the Golgi to
the nucleus. Since RACK1 remained in the nucleus in the presence of
ethanol, it is possible that the inhibition of ßIIPKC translocation
by ethanol was due to the miscompartmentalization of RACK1, resulting
in uncoupling of the targeting protein RACK1 from its kinase ßIIPKC.
We next determined whether ethanol-induced nuclear compartmentalization
of RACK1 and the inhibition of NPA-induced ßIIPKC translocation was
due to a direct effect of ethanol on the interaction between ßIIPKC
and RACK1 and/or ßIIPKC to other proteins. Recombinant RACK1 was
immobilized on an amylose resin column, and ßIIPKC binding was tested
in the presence or absence of PKC activators and ethanol. Figure 3A
shows that ethanol concentrations as high as 200 mM did not
affect the interaction of activated ßIIPKC to RACK1. The inability of
ethanol to affect the interaction between RACK1 and ßIIPKC was not
due to excess amounts of RACK1 and/or ßIIPKC, since the PKC
inhibitors calphostin C and DECA were able to inhibit the binding
interaction using same concentration of proteins (20)
. We
also investigated the possibility that ethanol directly affects the
binding of ßIIPKC to other cell proteins. Triton soluble extracts
from CHO cells were run on an SDS gel, transferred to nitrocellulose,
and overlaid with ßIIPKC in the presence of PKC activators with or
without ethanol. Ethanol did not affect the binding of activated
ßIIPKC (Fig. 3B
) or inactive ßIIPKC (data not shown) to
cell extract proteins. These results suggest that ethanol-induced
nuclear compartmentalization of RACK1 and inhibition of ßIIPKC
translocation are not due to a direct effect of ethanol on ßIIPKC
binding to RACK1 or to other proteins.
|
It is possible that ethanol induces RACK1 to move to the nucleus
via a signaling cascade. We found that in C6 glioma cells, the
adenylate cyclase activator forskolin induced RACK1 to move to the
nucleus (Fig. 4A-b
and B
(gray bars). Forskolin
induced RACK1 nuclear compartmentalization in CHO and NG108–15 cells
as well without affecting the compartmentalization of ßIIPKC (data
not shown). If ethanol is mediating RACK1 movement to the nucleus via
the cAMP signaling cascade, then an inhibitory analog of cAMP should
inhibit RACK1 nuclear compartmentalization. Indeed, an inhibitory
analog of cAMP, RpcAMP, inhibited forskolin-induced RACK1 nuclear
localization (data not shown) and completely prevented ethanol-induced
RACK1 nuclear compartmentalization (Fig. 4C
, gray).
Therefore, it is likely that activation of adenylate cyclase and
increase in cAMP levels in the presence of ethanol are mediating the
movement of RACK1 to the nucleus in response to ethanol.
Next, we determined whether ethanol-induced nuclear
compartmentalization of RACK1 also occurs in vivo. Mice were
treated with ethanol acutely (4 g/kg 30 min i.p.) or chronically (3
days vapor in chamber). After acute injection of 4 g/kg ethanol, brain
ethanol concentrations were 3.8 ± 0.1 mg/ml. For mice treated for
3 days of ethanol inhalation by vapor chamber, blood alcohol
concentrations were initially maintained at 1.9 mg/ml and declined
gradually to an average of 1.1 ± 0.1 mg/ml at the time of removal
from the chamber after 72 h. Brain and blood ethanol
concentrations were assayed by a previously published chromatographic
method (25)
. Brain slices were stained for RACK1, and the
nuclear marker TOTO-3. RACK1 was widely expressed in the brain (R. Yaka
and D. Ron, unpublished results). If ethanol induces nuclear
compartmentalization of RACK1 in vivo, then RACK1 should be
found in the nuclei and should be localized with the nuclear marker
TOTO-3 after exposure to alcohol. Indeed, in the CA2/CA3 region of the
hippocampus and granular layer of the cerebellum of ethanol-treated
mice, RACK1 was partially localized to nuclei detected after animals
were exposed to vapor chambers for 3 days (Fig. 5A, B-b, d
and Fig. 5D
). On the other hand, in these regions RACK1 was
not detected in nuclei of control animals (Fig. 5A, B-a, c
).
Ethanol-induced nuclear compartmentalization of RACK1 was specific to
the hippocampus CA2/CA3 region and the cerebellum and was not detected
in any other tested brain region (data not shown). After acute exposure
to ethanol, however, nuclear compartmentalization of RACK1 was observed
only in the cerebellum and not in the hippocampus (Fig. 5D
).
It is noteworthy that RACK1 was highly localized to the nuclei in the
CA1 regions of hippocampus in naive animals and this was not increased
further by alcohol treatment (data not shown). We also tested whether
ethanol alters the compartmentalization of ßIIPKC, or induces
translocation of the kinase to the nucleus. Ethanol did not induce
ßIIPKC translocation to the nuclei in any tested brain region (Fig. 5C, D
and data not shown), nor could we detect any other
changes in kinase compartmentalization after ethanol treatment (Fig. 5C
and data not shown). Therefore, exposure of mice to
ethanol induces the nuclear compartmentalization of RACK1 in specific
brain regions without changing the compartmentalization of ßIIPKC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Our data here suggest that ethanol uncouples RACK1
from ßIIPKC, thereby interfering with this shuttling function. As
depicted in Fig. 6
, our model suggests that when RACK1 is sequestered in the nucleus, it
is not available to shuttle ßIIPKC (Fig. 6C
). As a result
of uncoupling RACK1 from ßIIPKC, therefore, activation-induced
ßIIPKC translocation is inhibited. This model is consistent with the
concept that RACK1 modulates ßIIPKC compartmentalization and function
(20
, 33)
. Furthermore, this model may also account for
non-ethanol-related events in which RACK1 levels are reduced. For
example, in the cortex of adult rat, a 50% reduction in RACK1
expression is associated with inhibition of activation-induced
translocation of PKC (29
, 32)
. Thus, activation-induced
PKC translocation is inhibited when RACK1 is not available for
shuttling. Several studies show that ethanol mediates its effects by
activating PKC (reviewed in ref 2
), and we show a
different mechanism in which ethanol inhibits a PKC isozyme. It is
therefore likely that ethanol will affect different PKC isozymes
differently depending on modes of targeting, receptor, and second
messenger systems.
|
What mechanism might account for the movement of RACK1 to the nucleus?
We find that the adenylate cyclase activator forskolin induces RACK1
movement to the nucleus without affecting the compartmentalization of
ßIIPKC. This is the first demonstration of a shuttling protein moving
to different intracellular sites with different stimuli. RACK1 has
recently been shown to bind to different signaling proteins in addition
to ßIIPKC (28
, 36
37
38)
. Thus, it is possible that
activation of adenylate cyclase results in the shuttling of an
unidentified protein to the nucleus together with RACK1. Alternatively,
it is possible that RACK1 in the nucleus plays a role in events such as
cAMP-mediated gene regulation. We are currently investigating these
possibilities. It is likely that activation of adenylate cyclase by
ethanol induces RACK1 movement to the nucleus, since an inhibitory
analog of cAMP (RpcAMP) inhibits ethanol-induced RACK1 nuclear
compartmentalization. These results are in line with several reports
showing that ethanol increases the activity of adenylate cyclase
(39)
, increases the production of cAMP
(40
41
42)
, and activates the cAMP/PKA signal transduction
cascade (21
, 43)
. It is surprising that ethanol and
forskolin have the same magnitude of an effect on RACK1 nuclear
compartmentalization. It is possible though that low levels of cAMP are
required for RACK1 to move to the nucleus. Another possibility is that
C6 glioma cells secret an activator of cAMP signaling that facilitates
in ethanol’s effects. We also cannot exclude the possibility that
other signaling cascades may be involved in the nuclear
compartmentalization of RACK1.
We found ethanol-induced changes in RACK1 compartmentalization in two
brain areas involved in ethanol-induced abnormalities affecting
learning, memory, and coordination. We found that mice exposed to
ethanol exhibit RACK1 movement to the nuclei in the CA2/CA3 region of
the hippocampus and in the cerebellum. On the other hand, the
compartmentalization of ßIIPKC was unchanged in these regions. Such
miscompartmentalization of RACK1 may contribute to ethanol-induced
changes in PKC activity that could lead to ethanol-induced changes in
brain function. An ethanol-induced decrease in PKC activity has been
previously reported (12
, 44
45)
. Since ßIIPKC is a
major PKC isozyme in the brain (46)
, it is possible that
the uncoupling of the kinase from its targeting protein RACK1 is
responsible for part of the decrease in PKC activity previously
observed with exposure to ethanol. For example, activation of the NMDA
receptor in the hippocampus may be involved in processes such as
learning and memory (47)
. PKC enhances NMDA receptor
activity (48)
by phosphorylation of specific subunits
(49)
. Ethanol, on the other hand, inhibits NMDA receptor
activation (50)
. It is therefore possible that the
miscompartmentalization of RACK1 interferes with PKC phosphorylation of
the NMDA receptor, thereby limiting normal NMDA receptor activity.
Furthermore, a recent report demonstrated that both RACK1 and ßIIPKC
associate with ß1 and ß3 subunits of GABAA
receptors and that ßIIPKC phosphorylates these subunits
(28)
. GABAA receptors are important
targets for ethanol action (51)
. PKC phosphorylation of
the GABAA receptor subunits regulates receptor
function (52)
. It is possible that in response to ethanol,
GABAA phosphorylation on ß1 and ß3 subunits
will be reduced due to the inhibition of ßIIPKC function due to RACK1
miscompartmentalization. We are currently investigating these
possibilities.
In summary, we present here a novel molecular mechanism (Fig. 6)
in
which ethanol via activation of adenylate cyclase induces uncoupling of
the targeting protein RACK1 from its kinase ßIIPKC, thereby
preventing PKC translocation and presumably interfering with normal
activity of the kinase. This model opens a new avenue of investigation
to explain how ethanol modulates signaling cascades. It is therefore of
interest to determine whether ethanol alters the function of other
targeting proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Present address: Furman University, Department
of Psychology, Greenville, SC 29613, USA.
Received for publication March 14, 2000.
Revision received May 15, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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prevents enhancement of nerve growth factor responses by ethanol and phorbol esters. J. Biol. Chem. 272,15028-15035 mediates ethanol-induced up-regulation of L-type calcium channels. J. Biol. Chem. 273,16409-16414. Nat. Neurosci. 2,997-1002[Medline]
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M. R. McMullen, E. Cocuzzi, M. Hatzoglou, and L. E. Nagy Chronic Ethanol Exposure Increases the Binding of HuR to the TNF{alpha} 3'-Untranslated Region in Macrophages J. Biol. Chem., October 3, 2003; 278(40): 38333 - 38341. [Abstract] [Full Text] [PDF]
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R. Yaka, K. Phamluong, and D. Ron Scaffolding of Fyn Kinase to the NMDA Receptor Determines Brain Region Sensitivity to Ethanol J. Neurosci., May 1, 2003; 23(9): 3623 - 3632. [Abstract] [Full Text] [PDF]
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R. Yaka, D.-Y. He, K. Phamluong, and D. Ron Pituitary Adenylate Cyclase-activating Polypeptide (PACAP(1-38)) Enhances N-Methyl-D-aspartate Receptor Function and Brain-derived Neurotrophic Factor Expression via RACK1 J. Biol. Chem., March 7, 2003; 278(11): 9630 - 9638. [Abstract] [Full Text] [PDF]
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 K. R. Birikh, E. H. Sklan, S. Shoham, and H. Soreq Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior PNAS, January 7, 2003; 100(1): 283 - 288. [Abstract] [Full Text] [PDF]
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 A. Young, C. Cefaratti, and A. Romani Chronic EtOH administration alters liver Mg2+ homeostasis Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G57 - G67. [Abstract] [Full Text] [PDF]
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 A. McCahill, J. Warwicker, G. B. Bolger, M. D. Houslay, and S. J. Yarwood The RACK1 Scaffold Protein: A Dynamic Cog in Cell Response Mechanisms Mol. Pharmacol., December 1, 2002; 62(6): 1261 - 1273. [Full Text] [PDF]
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D.-Y. He, A. J. Vagts, R. Yaka, and D. Ron Ethanol Induces Gene Expression via Nuclear Compartmentalization of Receptor for Activated C Kinase 1 Mol. Pharmacol., August 1, 2002; 62(2): 272 - 280. [Abstract] [Full Text] [PDF]
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 M. J. Rebecchi and S. N. Pentyala Anaesthetic actions on other targets:protein kinase C and guanine nucleotide-binding proteins Br. J. Anaesth., July 1, 2002; 89(1): 62 - 78. [Abstract] [Full Text] [PDF]
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