MINIREVIEW
TORC-SIK cascaderegulatesCREBactivitythrough the
basic leucinezipper domain
Hiroshi Takemori
1
, Junko Kajimura
1
and Mitsuhiro Okamoto
2
1 Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan
2 Faculty of Contemporary Human Life Science, Tezukayama University, Nara, Japan
Introduction
The cAMP response element-binding protein (CREB)
is a basicleucinezipper (bZIP) transcription factor,
which shares properties with other CREB members,
the CRE-modulator (CREM) and activating tran-
scription factor 1 (ATF1). CREB members are
approximately 70% homologous overall, and are
more than 90% homologous within their bZIPs and
core sequences in the transactivation domain, known
as the kinase inducible domain (KID). Serine residue
133 (Ser133) in the KID of CREB and the equival-
ent residues of CREM ⁄ ATF1 are phosphorylated by
a variety of kinases, whereas the phospho-KID facili-
tates recruitment of the coactivators CREB-binding
protein (CBP) and p300, which enhances CRE-
dependent transcription. The precise mechanisms by
which the KID-coactivator complex activates tran-
scription have been reviewed comprehensively [1–3].
This article summarizes a new insight into bZIP for
the regulation of CREB activity, which is played by
the coactivator transducer of regulated CREB activ-
ity (TORC) and its repressor salt inducible kinase
(SIK).
Importance of bZIP for the action
of CREB
CREB and its cognates bind to the 8-bp CRE sites
that have been characterized as a consensus sequence
Keywords
bZIP; Ca
2
+
; cAMP; coactivator; CRE; CREB;
salt; SIK; TORC; transcription
Correspondence
H. Takemori, Laboratory of Cell Signaling
and Metabolism, National Institute of
Biomedical Innovation, 7-6-8, Asagi, Saito,
Ibaraki, Osaka, 567-0085, Japan
Fax: +81 72 641 9836
Tel: +81 72 641 9834
E-mail: takemori@nibio.go.jp
(Received 29 January 2007, revised 1 May
2007, accepted 7 May 2007)
doi:10.1111/j.1742-4658.2007.05889.x
The transcription factor cAMP response element-binding protein (CREB)
plays important roles in gene expression induced by cAMP signaling and is
believed to be activated when its Ser133 is phosphorylated. However, the
discovery of Ser133-independent activation by the activation of transducer
of regulated CREBactivity coactivators (TORC) and repression by salt
inducible kinase cascades suggests that Ser133-independent regulation of
CREB is also important. The activation and repression are mediated by
the basicleucinezipperdomain of CREB. In this review, we focus on the
basic leucinezipperdomain in the regulation of transcriptional activity of
CREB and describe the functions of TORC and salt inducible kinase.
Abbreviations
AICAR, 5-aminoimidazole-4-carboxamide-riboside; A-loop, activation loop; AMPK, AMP-activated kinase; ATF1, activating transcription
factor 1; bZIP, basicleucine zipper; CBP, CREB-binding protein; CRE, cAMP response element; CREB, CRE-binding protein; CREM, CRE-
modulator; CYP11A1, side chain cleavage cytochrome P450; GFP, green fluorescence protein; ICER, inducible cAMP response element
repressor; KID, kinase inducible domain; MAML2, mastermind-like gene family 2; MECT1, mucoepidermoid carcinoma translocated 1;
PGC1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PKA, protein kinase A; SIK, salt inducible kinase; StAR,
steroidogenic acute regulatory protein; topo II, DNA topoisomerase II; TORC, transducer of regulated CREB activity.
3202 FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS
of TGACGTCA [4,5] and its derivatives: half-sites [6]
and the TRE ⁄ AP-1 sequence [7]. Thedomain in CREB
responsible for binding is bZIP, comprising amino
acids 285–339, which has been shown by a crystal of
homodimer of bZIP with the CRE sequence [8].
The dimer formation is stabilized by hydrophobic
interactions between four pairs of leucine residues,
Lue311, Lue318, Lue325 and Lue332, in the zipper
region. In addition to these hydrophobic interactions,
hydrogen bonds between basic region residue Tyr307
and zipper region residue Glu312 and between
Gln321 and Asn322 residues located in the second
and third leucine repeats are also important for
dimer formation. Theleucine repeat is a common
feature of bZIP family transcription factors, but the
residues that create hydrogen bonds are conserved
only in theCREB family members, which may limit
the partners available for intrafamiliar dimer forma-
tion [8].
A hexahydrate Mg
2+
ion stabilizes the dimmer for-
mation of bZIPs on palindromic CREs [8], but it may
not be required when bZIP binds to half-site CREs [9].
In addition to the DNA binding, regions involved in
the nuclear import and export of CREM have been
mapped in the bZIP domain [10].
The bZIP domain of CREB interacts
with a variety of cellular factors
It is believed that the major transactivation functions
of CREB are encoded in the KID domain, because
A-CREB, one of the dominant negative mutants with
Ala substituted for Ser133, completely inhibits indu-
cible CREB activities [11]. Fusion of reporter CREB
proteins with the yeast Gal4 DNA binding domain
suggests the presence of Ser133-independent activation
of CREB, such as activation in cooperation with
c ⁄ EBPb [12], TAF
II
130 ⁄ 135 [13] and LIM-only protein
[14], which occurs in an N-terminal transactivation
domain, either KID or glutamine-rich regions (Q1 and
Q2). However, it has been reported that, as an excep-
tion to these findings, the bZIP domain also exerts its
transactivation function by interacting with other cellu-
lar factors.
The ring finger protein BARC1 [15,16] and the viral
factor Tax [17,18] associate with the bZIP domain and
recruit coactivators CBP ⁄ p300, which leads to Ser133-
independent activation of CREB.
The Ets-related protein GABPa [19] and replication
factor C p140 [20] have been demonstrated to bind to
the bZIP domains of CREB and its relatives. Over-
expression of these factors leads to a dose-dependent
activation of CRE-containing promoters.
DNA topoisomerase II (topo II) has been shown to
be associated with bZIP family factors, CREB, ATF1
and c-Jun21 [20,21]. A ten-fold excess of CREB relat-
ive to topo II stimulates topo II-mediated decatenation
of CRE-containing promoter DNA, which suggests the
importance of the bZIP domain in the regulation of
topo II activity. Up-regulation of CREB-mediated
transcription by a complex of topo II and RNA heli-
case A has also been reported [22].
The tumor suppressor p53 enhances reporter activit-
ies derived from the Bax promoter or p53-responsive
elements when cAMP signaling is activated [23]. p53
can interact with both CREB and CBP: the former
with the bZIP domain of CREB and the latter with
KIX, the CREB-binding domain of CBP. Because the
cAMP signaling was found to enforce the association
of p53 with the phospho-CREB ⁄ CBP complex, it has
been proposed that phospho-CREB, sandwiched
between p53 and CBP, has an adhesive function. How-
ever, the possibility has not been excluded that the
residual effect of CREs on the p53-dependent promot-
ers independently can activate transcription. These
findings suggest that one of the functions of the bZIP
domain is to interact with other cellular factors.
The bZIP-binding coactivator TORC
High throughput transformation assays of cDNAs,
using EVX-1 and IL-8 promoter-reporters, have identi-
fied a new family of CREB-specific coactivators named
as TORC1-3 (Fig. 1) [24,25]. The N-terminal region of
TORC is expected to form a coiled-coil structure, which
interacts with the bZIP domain of CREB [24]. This
interaction may occur via ionic bonds because it is dis-
rupted under high-salt conditions [26]. Arg314, located
between the first and the second Lue residues in the
zipper region of CREB, is essential for the association
with TORC, and the Arg314 residue is conserved only
in theCREB family. In addition to CREB-binding, the
N-terminal region plays a role in the tetramer forma-
tion of TORC [24], but the physiological function of
the multimeric complex has not been clarified yet.
The C-terminal hydrophobic domain recruits
TAF
II
130 ⁄ 135, which exerts a constitutively active
force [24]. Once TORC is overexpressed in HEK293
cells, CRE-dependent transcriptions are up-regulated
to, or beyond, the levels induced by cAMP. The acti-
vation of CREs by overexpression of TORC requires
CREB, but not Ser133-phosphorylation, indicating
that TORC appears to activate CREB in a phospho-
CREB-independent manner.
TORC1 has been independently identified as a pos-
sible inducer of salivary gland tumors and is known as
H. Takemori et al. Regulation of CREB activity
FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS 3203
mucoepidermoid carcinoma translocated 1 (MECT1)
[27]. The genomic rearrangement of t(11;19), which is
often associated with mucoepidermoid carcinoma, pro-
duces a fusion protein that contains the N-terminal
CREB-binding region (amino acids 1–42) of TORC1 ⁄
MECT1 and the transcriptional activation domain
of another transcription factor, Mastermind-like
gene family 2 (MAML2). The resultant chimeric pro-
tein, MECT1-MAML2, binds to CREB, activates
CRE-mediated transcriptions [24] and induces foci
formation in RK3E cells. Because MAML2 acts as a
carrier for CBP ⁄ p300, MECT1-MAML2 constitutively
up-regulates CREBactivity in a phosphorylation-inde-
pendent manner [28].
TORCs can exert their transactivation activity even
in nonstimulated cells, and high levels of TORC
expression reduce the response of CREs to cAMP,
indicating its possible function as a coactivator for
basal expression. Cytochemical studies of TORC,
however, have demonstrated that the activating sig-
nals that phosphorylate CREB, such as cAMP or
Ca
2+
, also induce the nuclear import of TORC
[26,29]. This suggests that the nucleo-cytoplasmic
shuttling of TORC, as well as CREB Ser133-phos-
phorylation, is an important regulatory mechanism
for CREB activity.
SIK represses CREBactivity via
the bZIP domain
SIK has been identified as a kinase induced in the
adrenal glands of rats fed with a high-salt diet [30,31]
Fig. 1. Cellular factors regulating CREB family members. Players regulating CRE-dependent transcription are depicted. Arrowheads and
blunt-ended lines indicate activation and inhibition, respectively. Although numerous kinases have been reported to activate or initiate CRE-
dependent transcription, the precise mechanism by which the initiators induce dephosphorylation of TORC is not clear (gray arrow). Although
calcineurin (PP2B) is a phosphatase responsible for the Ca
2+
-induced dephosphorylation of TORC, sites dephosphorylated by calcineurin are
not identical to the sites phosphorylated by SIKs. The N-terminal region, coiled coil, of TORC associates with the bZIP domain of CREB,
whereas the C terminal region, constitutive active domain (CAD), interacts with the RNA polymerase II subunits TAF II.
Regulation of CREBactivity H. Takemori et al.
3204 FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS
and in PC12 cells treated with membrane depolariza-
tion [32]. Genome projects revealed that SIK has three
isoforms, SIK1 also known as SNF1LK [33], SIK2
(QIK or SNF1LK2) and SIK3 (Qsk) [34], which
belongs to a family of AMP-activated protein kinases
(AMPK) that play important roles in the regulation of
metabolism during energy stresses [35].
In mouse adrenocortical tumor Y1 cells, the levels
of mRNA, protein and kinase activity of SIK1 were
found to have become elevated within 30 min after the
initiation of cAMP signaling and to have returned to
initial levels in a few hours [36]. The mRNA levels for
sterodiogenic genes, such as those for steroidogenic
acute regulatory protein (StAR) and side chain clea-
vage P450 (CYP11A1), rose as SIK1 expression
declined. Overexpression of SIK1 in Y1 cells lowered
the level of the cAMP-induced expression of the StAR
and CYP11A1 genes [36], suggesting that SIK1 may
function as the negative regulator in cAMP-induced
gene expression.
Reporter analyses of the human CYP11A1 promoter
have demonstrated that SIK1 represses protein kin-
ase A (PKA)-mediated activation of the CYP11A gene
promoter by inhibiting the transcription factor CREB
[37]. Although the kinase activity of SIK is required
for CREB repression, SIK does not phosphorylate
CREB and thus does not alter the level of CREB-
phosphorylation. However, the mapping regions
responsible for SIK1-mediated repression suggest that
SIK represses CREBactivity by acting on its bZIP
domain [37].
Expression of the StAR gene is also inhibited by
overexpression of SIK1, but the time course of its
expression appears to be different from that of the
CYP11A1 gene [38]. Two hours after initiation of
cAMP signaling, StAR mRNA in SIK1-overexpressing
cells had become elevated to a level similar to that in
control cells but the level had become markedly sup-
pressed after 12 h. This suggests that the capability of
SIK1 to repress CREB changes depending on the time
after stimulation of the cells.
PKA attenuates theCREB repressing
activity of SIK1 by phosphorylating
at Ser577
Immunocytochemical analyses have demonstrated that
SIK1 is localized both in the nucleus and in the cyto-
plasm of Y1 cells but, when the cells are stimulated
with cAMP, the nuclear SIK1 rapidly moves to the
cytoplasm. This nucleo-cytoplasmic redistribution of
SIK1 has been confirmed by using an SIK1 protein
tagged with a green fluorescence protein (GFP). Over-
expression of PKA also induces nucleo-cytoplasmic
re-distribution of GFP-SIK1 [38], suggesting that the
cAMP-induced nucleo-cytoplasmic shuttling of SIK1 is
a result of activation of the PKA cascade.
Site-directed mutagenesis for PKA-phosphorylation
motifs indicates that Ser577 is responsible for the nuc-
lear export of SIK1, and western blot analyses using
anti-(phospho-Ser577) serum show that PKA phos-
phorylates Ser577 in the cAMP-stimulated Y1 cells
[38]. The fact that the period when SIK1 is localized in
the cytoplasm correlates with the period when SIK1
does not exert its CREB repression activity suggests
that SIK1 loses its repressive activity in the cytoplasm
when Ser577 is phosphorylated [39].
However, the cytoplasmic localization of SIK2 [40]
and of the SIK1 mutants with impaired nuclear local-
ization signals provide evidence that SIKs can repress
CREB activity even when SIK is localized in the cyto-
plasm [39].
SIK phosphorylates TORC
The location of the site on CREB responsible for the
actions of SIK and TORC implies that both SIK
and TORC regulate CREBactivitythroughthe bZIP
domain in a phospho-Ser133-independent manner.
Moreover, TORC is a shuttling molecule, which is a
prerequisite for the SIK substrate to transmit the SIK
signals from the cytoplasm.
When TORC2 is phosphorylated at Ser171 by SIK1
or SIK2, the resulting phospho-TORC2 recruits the
14-3-3 protein and moves from the nucleus to the cyto-
plasm, which leads to the apparent inactivation of
CREB activity [26,39]. Although the SIK-mediated
intracellular redistribution of TORC1 and TORC3 is
not evident, the coactivation activities of all TORCs
are completely inhibited by SIK1-3 [41].
Additional analyses have suggested that when PKA
activates CREB, it inhibits the TORC-phosphorylation
activity of SIKs [40]. As in the case of cAMP ⁄ PKA
signaling, Ca
2+
signaling also induces dephosphoryla-
tion of TORC, which accelerates its nuclear localiza-
tion and activates CREB-dependent transcription.
Calcineurin, PP2B, is the phosphatase responsible for
the Ca
2+
-dependent dephosphorylation of TORC.
Although the constitutive active TORC2 mutant
(Ser171Ala mutant) shows resistance to the calcineurin
inhibitor cycrosporine A, the level of phospho-Ser171
of the wild-type TORC2 is not affected by either Ca
2+
or cyclosporine A [26,29]. These observations suggest
that the phosphorylation at Ser171 may down-regulate
TORC2 activity in coordination with phosphorylations
at the calcineurin-sensitive sites.
H. Takemori et al. Regulation of CREB activity
FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS 3205
LKB regulatesCREBactivity via
the SIK-TORC system
The phospho ⁄ dephospho regulation of TORC plays an
important role in hepatic gluconeogenesis through
modulation of CREBactivity [42,43]. However, it
remains to be clarified whether this regulation is just
one of several regulatory mechanisms or the cas-
cade indispensable for CREB activity. AMPK family
kinases, including SIK, have flexible activation-loops
(A-loops) near their substrate-binding pockets. The
phosphorylation in the A-loop induces a structural
change in the catalytic site, which then triggers kinase
activation.
The tumor suppressor kinase LKB1 [44] has been
identified as a major upstream activator of AMPK
family kinases, and essential Thr residues in the
A-loops of SIKs are phosphorylated by LKB1 [45].
In LKB1 defective HeLa cells [46], SIK is incapable
of phosphorylating TORC, which results in the con-
stitutive activation of CREB in a Ser133-independent
manner [41]. Moreover, overexpression of LKB1 in
HeLa cells improves CRE-dependent transcriptions in
a regulated manner. Findings obtained with a liver-
specific knockout model targeting the LKB1 gene
also underscores the importance of LKB1 in the regu-
lation of CREBactivity [47]. The loss of LKB1
expression leads to an increase in peroxisome prolifer-
ator-activated receptor gamma coactivator 1-alpha
(PGC1a), apparently due to a decrease in the level of
phosphorylation of TORC followed by the activation
of CREB.
In skeletal muscle cells, however, loss of the LKB1
gene reduces the level of PGC1a gene expression [48]
although the expression has been shown to be
enhanced by overexpression of TORCs [49], suggesting
that unidentified cascades, LKB1-dependent but not
including the SIK-TORC system, regulates PGC1a
gene expression in the muscle.
Inactivation of kinase cascades
up-regulates CREBactivity via
dephosphorylation of TORC
In addition to loss of the LKB1 cascade, inactivation
of kinase cascades by a low dose of staurosporine can
also lead to the constitutive induction of CRE activity
[41]. Staurosporine-induced activation of CREB is not
accompanied by CREB-phosphorylation. These find-
ings suggest that the phospho ⁄ dephospho regulation of
TORC is an indispensable mechanism for CREB activ-
ity. Because a low dose of staurosporine inhibits the
kinase activity of SIK1 without impairment of LKB1
action, the site in the TORC-phosphorylation cascades
blocked by staurosporine may be SIKs.
AMPK against aminoimidazole-4-
carboxamide-1-b-4-ribofuranoside
(AICAR) enhances TORC
phosphorylation
SIKs belong to the AMPK family and share
phosphorylation motifs with AMPK, F-X-B-S ⁄ T-X-
Ser-X-X-X-F (F, hydrophobic residue; B, basic resi-
due; Ser, phosphorylation site). The AMPK agonist
AICAR, a precursor of AMP analogue, is known to
inhibit glyconeogenesis induced by the cAMP-CREB
cascade in the liver [50]. These data suggest that the
mechanism by which AICAR down-regulates glyconeo-
genesis may be a result of TORC phosphorylation by
AMPK.
In fact, AMPK can phoshorylate TORC2 at Ser171
in vitro [41,42]. Treatment of hepatocytes with AICAR
inhibits cAMP-induced dephosphorylation of TORC2
and TORC2-dependent activation of the PGC1a pro-
moter [42]. Overexpression of AMPK, however, failed
to inhibit cAMP-induced CRE activation in COS-7
cells in which kinase domains of SIKs and another
AMPK-related kinase, MARK4, completely inhibit the
activation [41]. Because AICAR is unable to activate
AMPK in COS-7 cells [51], the discrepancy may be
caused by the difference in cell types or the indirect
action of AICAR in hepatocytes. Further analysis is
warranted of the involvement of AICAR in the inhibi-
tion of CREB-mediated glyconeogenesis.
How A-CREB inhibits CREs
Ser133Ala CREB, well known as A-CREB, has a
dominant negative effect on CRE-dependent gene
expression [11], possibly the result of a blockade of
the upstream signals. Interestingly, overexpression of
A-CREB completely inhibits TORC-dependent activa-
tion of CRE [24]. On the other hand, reporter systems
using Gal4-A-CREB show that A-CREB also has the
potential to activate transcription in cooperation with
TORC [26]. To explain this discrepancy, we hypothes-
ized that the overexpressed A-CREB may occupy
TORC, which would result in depletion of TORC
from CREs. If so, the depletion could occur even when
wild-type CREB is overexpressed.
When wild-type CREB was weakly overexpressed as
a result of transformation with 10 ng of plasmid, CRE
activity was enhanced only a little (Fig. 2). However,
transformation with a large amount of plasmids,
100 ng, inhibited the activation of CRE completely.
Regulation of CREBactivity H. Takemori et al.
3206 FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS
Overexpression of the low level of A-CREB had a
minor effect, whereas the high level, as expected, resul-
ted in complete inhibition. These results suggest that
the dominant negative effect of A-CREB may be a
result of not only the blockade of the upstream signals,
but also the depletion of TORC.
Inducible cAMP response element repressor
(ICER), whose mRNA is transcribed from an intron
ahead of exons coding the bZIP domain of CREM,
also represses CREBactivity extensively [52]. The
mechanism of this repression is thought to be similar
to that of A-CREB. Given the fact that the bZIP
domain of CREM acts as an efficient acceptor of
TORC [26], depletion of TORC should be considered
to be one of the mechanisms for the repressive action
of ICER.
Future aspects
Although CREB activates CREs when its Ser133 is
phosphorylated, the level of phospho-Ser133 alone
may not be sufficient to explain theCREB activity.
The discovery of TORC provides us chances to under-
stand complicated regulation of CREB. In mammals,
multiple combinations, CREB ⁄ CREM ⁄ ATF1, coacti-
vators and their phospho- ⁄ dephospho forms, can make
diverse regulation for CRE-dependent transcription
(Fig. 1). Further studies to clarify the contributions of
TORC and phospho-Ser133 to individual gene expres-
sions may lead us clear explanations.
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FEBS Journal 274 (2007) 3202–3209 ª 2007 The Authors Journal compilation ª 2007 FEBS 3209
. regulation of CREB is also important. The activation and repression are mediated by the basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain in the regulation. MINIREVIEW TORC-SIK cascade regulates CREB activity through the basic leucine zipper domain Hiroshi Takemori 1 , Junko Kajimura 1 and Mitsuhiro. located between the first and the second Lue residues in the zipper region of CREB, is essential for the association with TORC, and the Arg314 residue is conserved only in the CREB family. In addition to CREB- binding,