Báo cáo khoa học: RCAN1 (DSCR1 or Adapt78)* stimulates expression of GSK-3b pptx

RCAN1 (DSCR1 or Adapt78)* stimulates expression of
GSK-3b
Gennady Ermak, Cathryn D. Harris, Denis Battocchio and Kelvin J. A. Davies
Ethel Percy Andrus Gerontology Center, and Division of Molecular & Computational Biology, The University of Southern California,
Los Angeles, CA, USA
The RCAN1 (regulator of calcineurin) gene was pre-
viously known as DSCR1 or Adapt78. DSCR1 or
Adapt78 was discovered by two independent laborator-
ies as a chromosome 21 Down syndrome critical
region (DSCR1) gene [1], and as a gene transiently
induced during cellular adaptation (Adapt78) to oxida-
tive stress [2]. The yeast homolog was named RCN1
[3] and the Drosophila homolog was named NEBULA
[4]. Several groups simultaneously reported that
DSCR1 or Adapt78 protein can specifically bind to,
and down-regulate the activity of calcineurin [3,5–7].
Some groups therefore named this protein calcipressin
1, while others called it calcineurin binding protein
(CBP 1) [7] or myocyte-enriched calcineurin interacting
protein (MCIP1) [6]. In yeast, the RCAN1 protein was
called Rcn1p [3], and in Drosophila, this protein was
named sarah [8]. The new name RCAN1 (regulator of
calcineurin) has recently been accepted by the HUGO
Gene Nomenclature Committee for the protein prod-
uct of the RCAN1 gene.
RCAN1 is thought to play a role in number of
physiological processes. It has been associated with
Keywords
Adapt78; calcineurin; DSCR1; GSK-3b;
RCAN1
Correspondence

K. J. A. Davies, Andrus Gerontology Center,
University of Southern California, 3715
McClintock Avenue, Los Angeles,
CA 90089–0191, USA
Fax: +1 213 7406462
Tel: +1 213 7408959
E-mail:
*The new name RCAN1 (regulator of
calcineurin) has recently been accepted by
the HUGO Gene Nomenclature Committee
and the Mouse Genomic Nomenclature
Committee (MGNC) for the gene previously
known as DSCR1 or Adapt78. Similarly,
RCAN1 is the new name for its protein
product, which was previously called
calcipressin1 or MCIP1.
(Received 24 January 2006, revised 1 March
2006, accepted 7 March 2006)
doi:10.1111/j.1742-4658.2006.05217.x
The RCAN1 protein (previously called calcipressin 1 or MCIP1) binds to
calcineurin, a serine ⁄ threonine phosphatase (PP2B), and inhibits its activ-
ity. Here we demonstrate that regulated overexpression of an RCAN1
transgene (this gene was previously called DSCR1 or Adapt78) also stimu-
lates expression of the GSK-3b kinase, which can antagonize the action of
calcineurin. We also show that GSK-3b is regulated by RCAN1 at a post-
transcriptional level. In humans, high RCAN1 expression is found in the
brain, where at least two mRNA isoforms have been reported. Therefore,
we further investigated expression of the various RCAN1 isoforms, result-
ing from differential splicing and alternative promotors in human brain.
We detected at least three distinct RCAN1s: RCAN1-1 Short at 31 kDa

(RCAN1-1S), RCAN1-1 Long at 38 kDa (RCAN1-1 L), and RCAN1-4.
Furthermore, the levels of RCAN1-1S, but not RCAN1-1 L or RCAN1-4
correlated with the levels of GSK-3b. This suggests that RCAN1-1S might
induce production of GSK-3b in vivo. While RCAN1s can regulate cal-
cineurin and GSK-3b, it has also been shown that calcineurin and GSK-3b
can regulate RCAN1s. Here we propose a new model (incorporating all
these findings) in which cells maintain an equilibrium between RCAN1s,
calcineurin, and GSK-3b.
Abbreviations
CBP 1, calcineurin binding protein; GSK, glycogen synthase kinase; MCIP1, myocyte-enriched calcineurin interacting protein; NFATs, nuclear
factors of activated T-cells; PP2B, protein phosphatase 2B.
2100 FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS
Down’s syndrome and Alzheimer’s disease [5,9]. It has
also been demonstrated to inhibit cardiac hypertrophy
[10] and to attenuate angiogenesis and cancer [11].
Exactly how RCAN1 may contribute to human
pathologies, however, is a subject of intense continuing
study. So far, the only established function of the
RCAN1 protein is the inhibition and regulation of
calcineurin, which is protein phosphatase 2B (PP2B).
Calcineurin, however, works together with (or against)
kinases such as glycogen synthase kinase-3b (GSK-3b)
to regulate protein phosphorylation ⁄ dephosphoryla-
tion, and GSK-3b can antagonize calcineurin. There-
fore, here we have investigated whether GSK-3b can
also be regulated by RCAN1.
One of the major cell types expressing RCAN1 in
the human body is neurons [9], suggesting important
roles for the protein in brain function. The RCAN1
(DSCR1, Adapt78) gene consists of seven exons, four

of which (exons 1–4) can be alternatively spliced to
produce a number of different mRNA isoforms. Since
exons 5, 6, and 7 are likely to be common to all
mRNA isoforms, for nomenclature simplification, it
was proposed to assign them (and the proteins they
encode) numbers which correspond to the first exons
they contain [12]. Expression of RCAN1 mRNA in
human brain was previously investigated [9], but not
expression of the protein isoforms. Therefore, we have
now identified which RCAN1s are expressed in human
brain, and we have questioned which isoforms (if any)
may regulate GSK-3b.
It has recently been demonstrated, both in vitro and
in vivo, that RCAN1 can be phosphorylated [13–16].
Phosphorylation may alter RCAN1’s ability to inhibit
calcineurin, as well as RCAN1 degradation rate
[14–16]. Therefore, phosphorylation may represent a
major mechanism for regulating RCAN1. Remarkably,
RCAN1 can be phosphorylated by GSK-3b and
dephosphorylated by calcineurin [13,16] and we now
demonstrate that RCAN1 can also, reversibly, regulate
both enzymes.
Results
Transgenic RCAN1 stimulates expression
of GSK-3b in vitro
To test whether RCAN1 might regulate GSK-3b we
developed a tet-off regulated gene expression system, in
which RCAN1 expression can be regulated by doxy-
cycline [12,17]. To keep the transgene ‘silent’, cells were
continuously grown in the presence of doxycycline, and

inducible RCAN1 transgene expression was achieved
by doxycycline withdrawal from the cell culture med-
ium. RCAN1 was maximally overexpressed about 6 h
after doxycycline withdrawal from the cell medium, but
its levels then declined over the next 48 h (Fig. 1A).
Remarkably, GSK-3 protein levels followed the levels
of RCAN1; they increased following RCAN1 overex-
pression and declined at 48 h Fig. 1A,B. It seems that
both GSK-3b and GSK-3a levels were elevated.
However, the level of GSK-3b was increased much
more significantly ( 2.3-fold after 24 h of RCAN1
overexpression) than was the level of GSK-3a.
GSK-3 activity is regulated by phosphorylation. Par-
ticularly, its activity is inhibited by phosphorylation at
the S9 position [18]. Therefore, we also tested whether
the phosphorylated form of GSK-3 (pGSK-3) was ele-
vated following RCAN1 overexpression. We found no
significant increase in pGSK-3 levels until 48 h after
doxycycline withdrawal. These results indicate that
RCAN1 stimulates production of active GSK-3, which
then phosphorylates the tau protein and promotes
accumulation of hyperphosphorylated tau. It seems,
however, that cells might have a feedback mechanism
that controls GSK-3 activity and, after 48 h, GSK-3
was increasingly phosphorylated (Fig. 1C).
As hypothesized, the levels of phosphorylated tau cor-
related with RCAN1 (Fig. 1A). Tau phosphorylation
increased during RCAN1 overexpression (Fig. 1D),
whereas the levels of total tau protein were unchanged.
When combined, these results further strengthen our

proposal that RCAN1 can induce the active form of
GSK-3. It is interesting that the levels of GSK-3b were
modulated. GSK-3b is closely associated with pTau of
tangle-bearing neurons [19], and it is specifically the
GSK-3b isoform that is increased in pretangle neurons
[20]. GSK-3b levels did not perfectly correlate with
pTau levels; the highest pTau levels were observed
about 6 h after RCAN1 overexpression, and although
GSK-3b levels were increased almost two-fold at this
time point, they did not peak until 24 h (Fig. 1). This
might be due to competition between calcineurin and
GSK-3b, or to activation of other phosphatases after
the 6-h time point. It is also possible that other kinases,
that phosphorylate the tau protein, are also activated
following RCAN1 overexpression but, unlike GSK-3b,
they are activated maximally at the 6 h time point.
GSK-3b appears to be regulated by RCAN1
at a post-transcriptional level
We next tested whether RCAN1 induces transcription
of the GSK-3b gene or if it regulates GSK-3b expres-
sion at a post-transcriptional level. RCAN1 was overex-
pressed as described in Fig. 1 and cells were split into
two portions: one portion was used for western blot
G. Ermak et al. RCAN1 stimulates GSK-3b
FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS 2101
analysis and the other for northern blot analysis. Levels
of GSK-3b and RCAN1 mRNA were evaluated by nor-
thern blot analysis (Fig. 2). To verify that each sample
was equally loaded, the membrane was probed with
GAPDH (a housekeeping gene) probe. Results revealed

that RCAN1 mRNA was elevated as early as 3 h and
was clearly overexpressed at each time point: 6, 24,
and 48 h. GSK-3b mRNA levels, however, remained
unchanged at all time points. These results are also
confirmed by our previous data obtained using micro-
array analysis of genes that are regulated by RCAN1
[17]. In these previous experiments we did not observe
any GSK-3b mRNA transcription level changes follow-
ing RCAN1 down-regulation. Altogether these results
indicate that RCAN1 does not affect the levels of tran-
scription of GSK-3b, but it rather regulates production
of the GSK-3b protein at a post-transcriptional level.
This might be due to either accelerated GSK-3b trans-
lation and ⁄ or slower GSK-3b degradation; the exact
mechanism(s) remain(s) to be addressed.
It is interesting that RCAN1 mRNA levels were not
auto-down-regulated at any time point, while levels of
the RCAN1 protein were slightly down-regulated 24 h
after overexpression, and then returned to basal levels
in 48 h (Fig. 1A, top panel). These results indicate that
RCAN1 expression might be feed-back regulated at a
Fig. 1. RCAN1 stimulates expression of GSK-3. (A) RCAN1 was overexpressed in PC-12 cells as described in Experimental procedures.
Equal amounts of total protein from each sample were loaded and tubulin detection was used to control loading levels. Please note that the
antibody that recognizes GSK-3 binds to both its unphosphorylated and its phoshorylated forms. The amount of phosphorylated GSK-3, how-
ever, is much lower than the unphosphorylated form and therefore is not detected on the blots. Only the antibody that specifically recogni-
zes phosphorylated GSK-3 produced clear signals. (B) X-ray films were quantified using
IPLAB software (Scanalytics) and signals were
adjusted according to the loading. The amount of the loaded protein was controlled using b-tubulin detection. GSK-3 levels are expressed in
arbitrary units (AU) relative to b-tubulin. GSK-3b protein levels were elevated 2.3-fold after 24 h of RCAN1 overexpression, whereas GSK3
protein levels were increased by only 50%. Both elevations were statistically significant at the P £ 0.05 level, as evaluated by the student’s

t-test (one population). (C) Protein levels were quantified as described in (B). pGSK-3 levels are expressed in arbitrary units (AU), relative to
b-tubulin. (D) Protein levels were quantified as described in (B). pTau levels are expressed in arbitrary units (AU) relative to b-tubulin. In (B),
(C), and (D) all bars represent mean values ± standard errors, of three experiments.
RCAN1 stimulates GSK-3b G. Ermak et al.
2102 FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS
post-transcriptional level, most likely, by accelerated
degradation of RCAN1.
At least three RCAN1s are expressed in human
brain
Western blot analysis of RCAN1 expression in human
brain revealed that at least three different RCAN1 pro-
teins are expressed (Fig. 3). The RCAN1 gene consists
of seven exons that can be alternately spliced to
produce a large number of isoforms [12]. Two mRNA
isoforms were previously shown to be expressed in adult
human brain: isoform 1 (encoded by exons 1, 5, 6, and
7) and isoform 4 (encoded by exons 4, 5, 6, and 7). To
determine the structure of each of these RCAN1s we
have developed three different antibodies: an antibody
that recognizes RCAN1 encoded by exon 1 (RCAN1-1),
an antibody that recognizes RCAN1 encoded by exon 4
(RCAN1-4), and an antibody that recognizes all poten-
tial RCAN1 protein isoforms (directed against the
invariant exon 7). Surprisingly, we found that not one
but two RCAN1-1 proteins are expressed in human
brain (Fig. 3A,C): RCAN1-1 ‘Long’ at 38 kDa
(RCAN1-1 L) and RCAN1-1 ‘Short’ at 31 kDa
(RCAN1-1S). Using the complete sequence of chromo-
some 21, we analyzed for RCAN1 potential transcrip-
tion initiation sites and translation codons. Computer

Fig. 2. GSK-3b appears to be regulated by RCAN1 at a post-tran-
scriptional level. (A) RCAN1 mRNA was overexpressed as des-
cribed in Experimental procedures. Equal amounts of total RNA
from each sample were loaded and GAPDH detection was used as
a loading control. GSK-3b mRNA was detected using a labeled
oligonucleotide probe, as described in Experimental procedures.
Oligonucleotides used to prepare labeled probes, and reverse com-
plimentary oligonucleotides, were bound to membranes to control
the specificity of hybridization. Reverse complementary oligonucleo-
tides produced a strong hybridization signal, while the original oligo-
nucleotides did not produce a signal (not shown). (B) Membranes
were scanned and the hybridization signal measured using
IMAGE-
QUANT
software (Molecular Dynamics). Each signal was recalculated
according to the amount of RNA actually loaded on the gels. The
amount of RNA loaded was measured using hybridization with a
GAPDH probe. GSK-3b levels are expressed in arbitrary units (AU)
relative to GAPDH, and bars represent mean values of three experi-
ments ± standard errors. GSK-3b mRNA levels were not signifi-
cantly changed after RCAN1 overexpression.
Fig. 3. RCAN1 expression in human brain. Examples of western
blots analyzing RCAN1 expression in either the cerebral cortex
(‘cortex’), or the hippocampus (‘hc’). (A) The antibody designed
against isoform 1 recognizes RCAN1s of about 31 kDa (RCAN1-1S)
and 38 kDa (RCAN1-1L). (B) The antibody designed against isoform
4 recognizes an RCAN1 protein of about 70 kDa (RCAN1-4). (C)
The antibody designed against all possible RCAN1 isoforms
(containing the invariant exon 7) recognizes three proteins with
molecular weights of approximately 70, 38 and 31 kDa: RCAN1-4,

RCAN1-1 L, and RCAN1-1S, respectively.
G. Ermak et al. RCAN1 stimulates GSK-3b
FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS 2103
analysis revealed that exon 1 has an alternative initi-
ation translation codon, located further upstream
than previously thought, indicating that, indeed, two
RCAN1-1 isoforms can be expressed. This possibility
was also confirmed by our experiments, in which
RCAN1 was overexpressed using the tet-off system
(please see above). The construct used to overexpress
RCAN1 in these experiments carried a DNA fragment
which encodes only RCAN1-1S, and this fragment was
translated only into a 31-kDa protein (Fig. 1).
Unexpectedly, RCAN1-4 was detected as a 70 kDa
protein in human brain (Fig. 3A–C). Based on the
sequence of the RCAN1 gene, the size of the 1–4 iso-
form was expected to be roughly equal to RCAN1-1S,
which is about 31 kDa. This raises questions about
the specificity of our antibodies; however, all three
RCAN1 antibodies, including the RCAN1-4 antibody,
were carefully tested as shown in Fig. 4. The RCAN1-
4 antibody always showed one clear band. In addition,
we further tested our antibodies in substrate competi-
tion experiments, in which the peptide used to develop
each antibody was added to the antibody solutions
used for western blots. In each case, the addition of
the correct peptide significantly reduced the appropri-
ate signal levels (not shown).
It seems that different RCAN1 isoforms can be pro-
duced depending on the cell type, the model system

used, and the particular conditions employed. We have
tested RCAN1 expression in several cell lines and
found that some of them strongly express RCAN1s of
various sizes, while others express very low levels of
RCAN1s (e.g. Fig. 5). In most cases, RCAN1-1L
appears to be the predominant isoform expressed.
GSK-3b correlates with the levels of RCAN1-1S,
but not RCAN1-1L or RCAN1-4 in vivo
We next tried to address whether, similar to our
in vitro results, RCAN1 might influence GSK-3b
expression in vivo. We analyzed 27 randomly chosen
human brain samples from various brain regions: cer-
ebral cortex, cerebellum and hippocampus (Fig. 6).
Interestingly, GSK-3b levels in all samples was tightly
correlated ( r ¼ 0.7, P < 0.001) with the expression of
only one RCAN1 isoform, RCAN1-1S, but not
RCAN1-1L or RCAN1-4. This correlation was inde-
pendent of the brain region analyzed.
RCAN1-1S was the isoform overexpressed in the
experiments described in Fig. 1 above. These results
therefore suggest that, similar to our in vitro observa-
tions (Fig. 1), GSK-3b may also be induced by expres-
sion of RCAN1-1S in human brain in vivo. This study
was not designed to address whether induction of
RCAN1 can actually cause Alzheimer’s disease. The
brain samples examined in our studies were all from
patients  80 years old, some of whom suffered from
Alzheimer’s disease and some of whom did not. Alzhei-
mer’s disease, however, is clearly an age-related disor-
der. Almost half of all humans suffer some degree of

Alzheimer’s disease by this age, and a large proportion
of our population might have developed the disease
without displaying overt symptoms [21]. Therefore,
older patients might have increased expression of
RCAN1 and GSK-3b even if they are not yet diag-
nosed with Alzheimer’s disease. It has actually been
shown that GSK-3b is induced in pretangle neurons
[20]. For these reasons, a completely different approach
will now be needed to address whether induction of
RCAN1 can actually cause Alzheimer’s disease.
Discussion
Previously it has been shown that RCAN1 can down-
regulate the activity of calcineurin, the serine-threonine
phosphatase PP2B [3,5–7]. Here we demonstrate that,
in addition, RCAN1 can also induce GSK-3b, a kinase
that antagonizes calcineurin. Calcineurin and GSK-3b
work in opposition to regulate the phosphorylation ⁄
Fig. 4. Production of the RCAN1-4 antibody. Rabbits were injected with exon 4 peptide, which is described in Experimental procedures.
Serum from ‘immunized’ rabbits was then used for western blots. This serum revealed a new band that was absent in ‘preimmunized’ ani-
mals. Next, the antibody was affinity purified from the serum of immunized animals using columns with the covalently bound exon 4 peptide
that was used for immunization. ‘Purified’ (bound to the column) material detected clear bands that are similar to those observed using
‘immunized’ serum. Such bands were absent in ‘flow-through’ (unbound to the columns) material.
RCAN1 stimulates GSK-3b G. Ermak et al.
2104 FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS
dephosphorylation of proteins and, thus, to regulate
their activities. Because RCAN1 inhibits calcineurin,
its overexpression will lead to protein phosphorylation
and, as RCAN1 induces GSK-3b, its overexpression
should lead to further protein phosphorylation. Thus,
in both cases, phosphorylated forms of proteins will be

produced. The relationship between RCAN1 and cal-
cineurin is, however, extremely complex. It has been
shown, for example, that RCAN1 can inhibit, activate,
or have no effect on calcineurin, depending on its con-
centration and its phosphorylation status [13,14,16].
Therefore, since almost any change in calcineurin
activity could be interpreted in any convenient (or triv-
ial) way, it was not measured in our study.
A number of vital proteins are regulated by GSK-3b
and calcineurin. One of these is tau, a microtubule-
associated protein that is important for cytoskeleton
stabilization and cell growth. Accumulation of hyper-
phosphorylated tau can lead to the formation of
neurofibrillary tangles, and to neurodegeneration asso-
ciated with tauopathies. Hyperphosphorylated forms
of tau are also involved in pathologies such as Down’s
syndrome and Alzheimer’s disease. Importantly, we
observed accumulation of phosphorylated tau protein
following overexpression of RCAN1 in our experi-
ments.
RCAN1 is a stress-inducible gene, and neurodegener-
ative diseases are associated with stresses such as chro-
nic inflammation. For example, it has been shown that
mechanical stress and traumatic brain injury are robust
factors for Alzheimer’s disease [22–25]. Interestingly,
RCAN1 expression has been shown to be inducible by
mechanical stimulus [26], thus providing a possible link
between injury and Alzheimer’s disease. We have also
observed RCAN1 overexpression following mechanical
injury in cell culture models (not shown). Of course, any

event causing inflammation would be expected to induce
RCAN1 expression, since the gene was identified as an
A
B
Fig. 6. Levels of GSK-3b in human brain correlate with RCAN1-1S.
(A) Sample western blots showing GSK-3b and RCAN1 expression.
Equal amounts of total protein from each brain sample were loa-
ded. Three RCAN1 proteins are expressed in brain, but the levels
of GSK-3b only correlate with RCAN1-1S (see samples 4 and 5). A
ponceau stained membrane is shown to demonstrate loading lev-
els. (B) summary of western blot analyses. Twenty-seven samples
originating from different brain areas of 18 patients were analyzed.
X-ray films were quantified using
IPLAB software (Scanalytics,
Fairfax, VA). GSK-3b and RCAN1-1S protein levels are expressed in
arbitrary units (AU). The linear regression line shown revealed a
correlation coefficient (r) of 0.7, P < 0.001.
Fig. 5. Expression of RCAN1 in other cell lines. Examples of west-
ern blots demonstrate RCAN1 expression in two cell culture mod-
els. Our RCAN1 common antibody recognizing exon 7 that is
common to all isoforms was used for the detection. (A) Detection
of RCAN1 protein in human fibroblast cell line WI-38 VA-13. At
least four RCAN1 isoforms (25, 38, 70, and  190 kDa) were
detected. (B) Detection of RCAN1 protein in human neuroblastoma
cell line SK-N-MC. Only one weak 38 kDa band was detected.
G. Ermak et al. RCAN1 stimulates GSK-3b
FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS 2105
oxidative stress adaptive factor [2]. Thus, it is possible
that chronic stress may contribute to neurodegeneration
through induction of RCAN1, which in turn leads to

inhibition of calcineurin and activation of GSK-3b,
both resulting in tau protein hyperphosphorylation.
Besides regulation of tau, both calcineurin and
GSK-3b are key regulators of nuclear factors of activa-
ted T-cells (NFATs), which are important players in a
number of physiological processes. The best character-
ized role of NFATs is in T-cell activation. When
NFATs are dephosphorylated (due to the induced
activity of calcineurin and ⁄ or reduced activity of GSK-
3b) they are transported from the cytoplasm into the
nucleus, and activate T-cells. Therefore, overexpression
of RCAN1 could lead to phosphorylation of NFATs
and T-cell inactivation. NFATs are also important
players in musculoskeletal development, cardiovascular
development and functions [27], and neuronal develop-
ment [28]. Thus, NFATs and RCAN1 may contribute
to the pathologies observed in Down’s syndrome, such
as mental retardation, heart defects, skeletal muscle
abnormalities, and immune system deficiencies. Indeed,
it has been previously shown that activated GSK-3b
phosphorylates NFAT proteins and suppresses cardiac
hypertrophy [29]. On the other hand, it was also
shown that overexpression of RCAN1 can inhibit car-
diac hypertrophy [10], which is consistent with our
hypothesis that overexpression of RCAN1 can activate
GSK-3b, promoting NFATs phosphorylation and sup-
pression of cardiac hypertrophy.
GSK-3b has a number of substrates and is a multi-
functional kinase. Besides Alzheimer’s disease and car-
diac development, its function is closely linked to type

II diabetes, various forms of cancer and other human
pathologies [30]. Our finding that RCAN1 can modu-
late the levels of this enzyme uncovers a possible new
link between stress and human disease. Similarly, cal-
cineurin is involved in many vital pathways [27,31] and
inhibition of this phosphatase by RCAN1 may be
expected to have serious repercussions. Thus, our
results raise the possibility that RCAN1 may play
important roles in a variety of chronic stress, or chro-
nic inflammation-related diseases including (but not
limited to) Alzheimer’s disease, Down’s syndrome, and
other neurodegenerative disorders.
It would seem that RCAN1 overexpression can lead
to dramatic physiological changes, and our results
suggest that cells cope with RCAN1 overexpression
quickly and efficiently. In our experiments, cells recog-
nized high levels of RCAN1-1S and down-regulated its
forced expression to basal levels within 48 h (Figs 1 and
2). We wish to know how cells do this. Current data
suggest that RCAN1 regulation is accomplished by
phosphorylation. While RCAN1 can regulate calcineu-
rin and GSK-3b, calcineurin and GSK-3b can also
regulate the phosphorylation of RCAN1 [13,16,29].
It has been demonstrated that phosphorylation of
RCAN1s increases their degradation rates [15]. There-
fore, both activation of GSK-3b and inactivation of
calcineurin should lead to the same results: increased
RCAN1 phosphorylation and faster degradation
(Fig. 7). Thus, overexpression of RCAN1 will eventu-
ally lead to RCAN1 phosphorylation and removal. It

appears that cells maintain an equilibrium between
RCAN1, calcineurin, and GSK-3b, and serious prob-
lems may arise when this balance is distorted. This
model, however, remains to be tested.
Experimental procedures
Doxycycline-regulated RCAN1 expression system
An RCAN1 DNA fragment corresponding to the RCAN1–
1S protein was produced by long and accurate (LA)
RT-PCR as previously described [12]. The fragment was
inserted into a pTRE vector from Clontech Laboratories,
Inc. (Paolo Alto, CA) PC-12 tet-off cells from Clontech
Laboratories, Inc., stably transfected with ‘regulator plas-
mid’, were next transfected with the pTRE carrying
RCAN1 fragment to finally produce a double-stable cell
line in which the RCAN1 transgene could be turned off by
doxycycline addition. All procedures were performed as
described in the tet-off gene expression user manual from
Clontech Laboratories, Inc.
Fig. 7. RCAN1–calcineurin–GSK-3b equilibrium. Multiple stresses
induce expression of RCAN1. RCAN1 down-regulates the activity
of calcineurin and activates GSK-3b. Both activation of GSK-3b and
inactivation of calcineurin lead to the same results: increased
RCAN1 phosphorylation and faster proteolytic degradation. When
RCAN1 levels are reduced to normal, calcineurin and GSK-3b activ-
ities also return to normal levels.
RCAN1 stimulates GSK-3b G. Ermak et al.
2106 FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS
PC-12 cell culture
PC-12 cells were cultivated in media containing 85% Dul-
becco’s modified Eagle’s medium (DMEM), 10% horse

serum, 5% antibiotic free fetal bovine serum (Clontech
Laboratories, Inc.), 2 mml-glutamine, 100 UÆmL
)1
penicil-
lin, and 100 lgÆmL
)1
streptomycin sulfate. PC-12 tet-off
cells, stably transfected with ‘regulator plasmid’, were main-
tained in media additionally containing 50 lgÆmL
)1
G418.
The double-stable cell line carrying both the ‘regulator plas-
mid’ and the RCAN1 transgene was maintained in media
additionally containing 50 lgÆmL
)1
G418 and 50 lgÆmL
)1
hygromycin. Cells were cultivated in a humidified 10% CO
2
atmosphere at 37°C. Expression of the RCAN1 transgene
was turned off by application of 0.1 lgÆmL
)1
doxycycline
to the media.
Post mortem human brain tissue
Brain tissue ⁄ CSF used in this project was provided by the
Alzheimer’s Disease Research Center, University of South-
ern California. Samples were frozen at )70°C until use.
Every sample was accompanied by a full medical history,
and by detailed neuropathology summaries that included

microscopic exams. Protocols using frozen samples of
human brain were approved by Institutional Review Board.
RCAN1 antibodies
The following three RCAN1 antibodies were designed using
the predicted open reading frame sequence of RCAN1: (1)
an antibody that recognizes RCAN1 encoded by exon 1
(RCAN1-1) was developed using a hypothetical peptide
encoded by the MEEVDLQDLPSAT portion of exon 1; (2)
an antibody that recognizes RCAN1 encoded by exon 4
(RCAN1-4) was developed using a hypothetical peptide
encoded by the VANSDIFSESETR portion of exon 4; and
(3) an antibody that recognizes all potential RCAN1 pro-
tein isoforms was developed using a hypothetical peptide
encoded by the KIIQTRRPEYTPIHLS portion of the exon
7. We added a cysteine to the N-terminus of these peptides
and conjugated them to keyhole limpet hemocyanin. The
peptides were then used to immunize rabbits and polyclonal
antibodies were raised commercially (ProSci, Inc., Poway,
CA). Serum from the immunized rabbits was affinity puri-
fied on columns with the covalently attached RCAN1 pep-
tide used for immunization.
Western blot analysis
Western blot analysis was performed following standard
protocols, using an ECL-detection system from Amersham
Biosciences (Piscataway, NJ). Final dilution of the RCAN1
antibodies for western blot analysis ranged between 1 : 500
and 1 : 1000. Phosphorylated GSK-3 protein (pGSK-3) was
detected using an antibody that specifically recognizes the
GSK-3 protein, phosphorylated at serine 9. Phosphorylated
tau protein (pTau) was detected using an antibody that

specifically recognizes the tau protein, phosphorylated at
threonine 231 (pT[231]). It has been demonstrated that tau
is phosphorylated at threonine 231 by GSK-3 and it can be
dephosphorylated by calcineurin [32].
Anti-tau antibody was obtained from Upstate Biotechno-
logy (catalog #05–348). Anti-tau [pT[231]]-phosphospecific
antibody was from Biosource International (catalog #44–
746) (Camarillo, CA). Anti-GSK-3 antibody was from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) Anti-
GSK-3 [pS[9]] phosphospecific antibody was from Santa
Cruz Biotechnology, Inc. Tubulin antibody was from Santa
Cruz Biotechnology, Inc. Commercial antibodies were dilu-
ted as suggested by the manufacturers.
RNA isolation
Total RNA was extracted using the TRIzol reagent (Life
Technologies, Inc.). RNA concentration was quantified
spectrophotometrically, and relative content was further
confirmed on ethidium bromide-stained gels. Integrity of
the RNA was estimated by agarose gel electrophoresis; only
RNA samples displaying discrete 28S and 18S RNA bands
were used in our experiments.
Northern blot analysis
Samples containing 30 lg of total RNA were subjected to
electrophoresis through 1% agarose formaldehyde gels,
blotted to nylon membranes (Intergen, Purchase, NY) with
HETS (Tel-Test Inc., Friendswood, TX), and cross-linked
by ultraviolet radiation. Three probes were used in this
study: RCAN1, GAPDH, and GSK-3b. RCAN1 and GAP-
DH were synthesized by PCR, and to detect GSK-3b we
used synthetic oligonucleotides. Therefore, hybridization

conditions for these probes were different. For RCAN1 and
GAPDH detection, the membranes were prehybridized for
4 h and hybridized for 15 h in Hybrizol I (Oncor) at 42°C.
After hybridization they were washed with 2 · NaCl ⁄ Cit
plus 0.1% SDS at room temperature for 1 and 10 min, then
with 0.1 · NaCl ⁄ Cit plus 0.1% SDS at 60°C for 10 and
30 min. For hybridization with GSK-3b, we used ULTRA-
hyb–Oligo solution, Ambion Inc. (Austin, TX) followed by
protocol from manufacturer. The membranes were exposed,
developed, and scanned using the phosphorimager system
(Molecular Dynamics, Sunnyvale, CA).
To rehybridize RNA blots, hybridized and labeled probes
were removed by washing the membranes in a solution of
0.1 · NaCl ⁄ Cit, 0.1% SDS, and 10 mm Tris ⁄ HCl (pH 7.0)
at 90°C for 10 min. To quantify the level of DSCR1
(Adapt78) mRNA expression the membranes were scanned
G. Ermak et al. RCAN1 stimulates GSK-3b
FEBS Journal 273 (2006) 2100–2109 ª 2006 The Authors Journal compilation ª 2006 FEBS 2107
and the hybridization signal measured using imagequant
software (Molecular Dynamics). Each signal was recalcu-
lated according to the amount of RNA actually loaded on
the gels. The amount of RNA loaded was controlled by
hybridization with a GAPDH probe.
RCAN1 and GAPDH probes were labeled using
[
32
P]dCTP[aP] and the High Prime system (Boehringer
Mannheim, Indianapolis, IN). A fully cloned hamster
Adapt78 fragment was used to prepare DSCR1 (Adapt78)
probes; and a PCR-fragment consisting of exons 7 and 8

was used to prepare GAPDH probes as we previously des-
cribed [9, 17]. To prepare GSK-3b probe we synthesized
the following oligonucleotide: 5¢-CTGTGGCCTGTCAGG
ACCCTGTCCAGGAGT-3¢.
The reverse complementary oligonucleotide was used as a
negative control for hybridization. This oligonucleotide was
labeled using T4 polynucleotide kinase from Invitrogen Inc.
(Chicago, IL) following a protocol from the manufacturer.
Acknowledgements
Tissues for this study were obtained from the Alzhei-
mer’s Disease Research Center Neuropathology Core,
University of Southern California School of Medicine,
Los Angeles, CA, USA. This work was supported by
PHS grant # AG16256 to K.J.A.D and G.E. from the
NIH ⁄ NIA.
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