MINIREVIEW TORC-SIK cascade regulates CREB activity through the basic leucine zipper 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 basic leucine zipper (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 CREB activity 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 basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain 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, basic leucine 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]. The domain 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. The leucine repeat is a common feature of bZIP family transcription factors, but the residues that create hydrogen bonds are conserved only in the CREB 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 the CREB 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 CREB activity 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 CREB activity 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 CREB activity 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 CREB activity 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 the CREB 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 CREB activity through the 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 regulates CREB activity via the SIK-TORC system The phospho ⁄ dephospho regulation of TORC plays an important role in hepatic gluconeogenesis through modulation of CREB activity [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 CREB activity [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 CREB activity 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 CREB activity 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 CREB activity 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 the CREB activity. The discovery of TORC provides us chances to under- stand complicated regulation of CREB. 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Biochem Biophys Res Commun 293 , 892–898. 52 Molina CA, Foulkes NS, Lalli E & Sassone-Corsi P (1993) Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75, 875– 886. H. Takemori et al. Regulation of CREB activity 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,