Transducer Of Regulated CREB Activity Research Articles

Berberine Inhibits Gluconeogenesis in Skeletal Muscles and Adipose Tissues in Streptozotocin-induced Diabetic Rats via LKB1-AMPK-TORC2 Signaling Pathway.

The effect and potential molecular mechanisms of berberine on gluconeogenesis in skeletal muscles and adipose tissues were investigated. After adaptive feeding for one week, 8 rats were randomly selected as the normal group and fed on a standard diet. The remaining 32 rats were fed on a high-fat diet and given an intravenous injection of streptozotocin (STZ) for 2 weeks to induce the diabetic models. The diabetic rat models were confirmed by oral glucose tolerance test (OGTT) and randomly divided into 4 groups (n=8 each), which were all fed on a high-fat diet. Berberine (3 g/kg per day) or metformin (183 mg/kg per day) was intragastrically administered to the diabetic rats for 12 weeks, serving as berberine group and metformin group respectively. 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside [AICAR, an agonist of AMP-activated protein kinase (AMPK), 0.5 mg/kg per day] was subcutaneously injected to the diabetic rats for 12 weeks, serving as AICAR group. The remaining 8 diabetic rats served as the model group, which was given a 0.5% carboxyl methylcellulose solution by oral gavage. Fasting serum insulin (FINS), OGTT as well as lipid parameters were tested by commercial kit. The protein levels of liver kinase B1 (LKB1), AMPK, phosphorylated AMP-activated protein kinase (p-AMPK), transducer of regulated CREB activity 2 (TORC2), phosphorylated transducer of regulated CREB activity 2 (p-TORC2), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in skeletal muscles and adipose tissues were examined by Western blotting. The results showed that berberine significantly decreased the body weight, plasma glucose, insulin levels, and homeostatic model assessment for insulin resistance (HOMA-IR) of diabetic rats compared with those in the model group. Meanwhile, the serum total triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were markedly decreased and high-density lipoprotein cholesterol (HDL-C) level was significantly increased after the treatment with berberine. In addition, we found that berberine significantly increased the expression of p-AMPK and LKB1, while decreasing the p-TORC2 levels in skeletal muscles and adipose tissues. Moreover, the expression of PEPCK and G6Pase was significantly down-regulated after the treatment with berberine compared to the model group. It was suggested that the mechanism by which berberine inhibited peripheral tissue gluconeogenesis may be attributed to the activation of the LKB1-AMPK-TORC2 signaling pathway.

PPP1R3C mediates metformin-inhibited hepatic gluconeogenesis

BackgroundMetformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis. MethodsThe differentially expressed genes in primary mouse hepatocytes incubated with 8-Br-cAMP and metformin were analyzed by microarrays. Hepatic glucose production and gluconeogenic gene expressions were detected after adenovirus-mediated overexpression or silence of PPP1R3C in vitro and in vivo. The phosphorylation level and location of transducer of regulated CREB activity 2 (TORC2) were determined by Western blot and immunofluorescence. ResultsMetformin and adenovirus-mediated activation of AMPK suppressed 8-Br-cAMP–stimulated Ppp1r3c mRNA expression in primary mouse hepatocytes. Overexpression of PPP1R3C in primary mouse hepatocytes or the livers of wild-type mice promoted hepatic glucose production and gluconeogenic gene expressions. On the contrary, adenovirus-mediated knockdown of PPP1R3C in primary mouse hepatocytes decreased hepatic gluconeogenesis, with the suppression of cAMP-stimulated gluconeogenic gene expressions and TORC2 dephosphorylation. Notably, Ppp1r3c expression was increased in the liver of db/db mice. After PPP1R3C silence in the livers of wild-type and db/db mice, blood glucose levels and hepatic glucose production were markedly lowered, with decreased expressions of key gluconeogenic enzymes and transcript factors as well as liver glycogen content. ConclusionMetformin-activated AMPK decreases hepatic PPP1R3C expression, leading to the suppression of hepatic gluconeogenesis through blocking cAMP-stimulated TORC2 dephosphorylation. Hepatic specific silence of PPP1R3C provides a promising therapeutic strategy for type 2 diabetes.

Salt-Inducible Kinase Is Involved in the Regulation of Corticotropin-Releasing Hormone Transcription in Hypothalamic Neurons in Rats

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

The molecular physiology of CRH neurons

Corticotropin releasing hormone (CRH) is essential for stress adaptation by mediating hypothalamic-pituitary-adrenal (HPA) axis, behavioral and autonomic responses to stress. Activation of CRH neurons depends on neural afferents from the brain stem and limbic system, leading to sequential CRH release and synthesis. CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevations of CRH and HPA axis activity. Inhibitory feedback mediated by glucocorticoids and intracellular production of the repressor, Inducible Cyclic AMP Early Repressor (ICER), limit the magnitude and duration of CRH neuronal activation. Induction of CRH transcription is mediated by the cyclic AMP/protein kinase A/cyclic AMP responsive element binding protein (CREB)-dependent pathways, and requires cyclic AMP-dependent nuclear translocation of the CREB co-activator, Transducer of Regulated CREB activity (TORC). This article reviews current knowledge on the mechanisms regulating CRH neuron activity.

The Distribution of Messenger RNAs Encoding the Three Isoforms of the Transducer of Regulated cAMP Responsive Element Binding Protein Activity in the Rat Forebrain

Increasing evidence indicates that the cAMP responsive element binding protein (CREB)-dependent transcriptional activation of a number of genes requires the CREB co-activator: transducer of regulated CREB activity (TORC). Because of the central importance of CREB in many brain functions, we examined the topographic distribution of TORC1, 2, and 3 mRNAs in specific regions of the rat forebrain. In situ hybridisation analysis revealed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. All three TORC isoforms were found in a number of brain nuclei, the ventricular ependyma and pia mater. Although high levels of TORC1 were widely distributed in the forebrain, TORC2 was found in discrete nuclei and TORC3 mostly in the ependyma, and pia mater. The relative expression of TORC isoforms was confirmed by quantitative reverse transcriptase-polymerase chain reaction analysis in the hippocampus and hypothalamus. In the paraventricular nucleus of the hypothalamus, TORC1 and 2 mRNAs were abundant in the parvicellular and magnocellular neuroendocrine compartments, whereas TORC3 expression was low. All three isoform mRNAs were found elsewhere in the hypothalamus, with the most prominent expression of TORC1 in the ventromedial nucleus, TORC2 in the dorsomedial and arcuate nuclei, TORCs 1 and 2 in the supraoptic nucleus, and TORC2 in the suprachiasmatic nucleus. These differential distribution patterns are consistent with complex roles for all three TORC isoforms in diverse brain structures, and provide a foundation for further studies on the mechanisms of CREB/TORC signalling on brain function.

Temporal Effect of Adrenocorticotrophic Hormone on Adrenal Glucocorticoid Steroidogenesis: Involvement of the Transducer of Regulated Cyclic AMP-Response Element-Binding Protein Activity

The availability of active steroidogenic acute regulatory protein (StAR) and side-chain cleavage cytochrome P450 (P450scc) are rate-limiting steps for steroidogenesis. Transcription of StAR and P450scc genes depends on cyclic AMP-response element-binding protein (CREB) phosphorylation and CREB co-activator, transducer of regulated CREB activity (TORC), which is regulated by salt-inducible kinase 1 (SIK1). In the present study, we investigated the relationship between TORC activation and adrenocorticotrophic hormone (ACTH)-induced steroidogenesis in vivo, by examining the time-course of the effect of ACTH injection (4 ng, i.v.) on the transcriptional activity of StAR and P450scc genes and the nuclear accumulation of transducer of regulated CREB activity 2 (TORC2) in rat adrenal cortex. ACTH produced rapid and transient increases in plasma corticosterone, with maximal responses between 5 and 15 min, and a decrease to almost basal values at 30 min. StAR and P450scc hnRNA levels increased 15 min following ACTH and decreased toward basal values at 30 min. Concomitant with an increase in nuclear phospho-CREB, ACTH injection induced nuclear accumulation of TORC2, with maximal levels at 5 min and a return to basal values by 30 min. The decline of nuclear TORC2 was paralleled by increases in SIK1 hnRNA and mRNA 15 and 30 min after injection, respectively. The early rises in plasma corticosterone preceding StAR and P450scc gene transcription suggest that post-transcriptional and post-translational changes in StAR protein mediate the early steroidogenic responses. Furthermore, the direct temporal relationship between nuclear accumulation of TORC2 and the increase in transcription of steroidogenic proteins, implicates TORC2 in the physiological regulation of steroidogenesis in the adrenal cortex. The delayed induction of SIK1 suggests a role for SIK1 in the declining phase of steroidogenesis.

Calcineurin Signaling Regulates Human Islet β-Cell Survival

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

Danshen extract 15,16-dihydrotanshinone I functions as a potential modulator against metabolic syndrome through multi-target pathways

Hypertension is a common complication of type 2 diabetes mellitus (T2DM), and is the main cause for T2DM-associated mortality. Although the stringent control of blood pressure is known to be beneficial in reducing the cardiovascular mortality of T2DM patients, drugs with both anti-hypertensive and anti-hyperglycemic effects are seldom reported. The traditional Chinese medicine danshen has long been used for lowering both blood pressure and blood glucose in T2DM patients, shedding lights on the development of such medication. However, the molecular mechanism and active component remain unclear. Here, we report that the lipophilic component, 15,16-dihydrotanshinone I (DHTH) from danshen potently antagonized both mineralocorticoid and glucocorticoid receptors, and efficiently inhibited the expression of their target genes like Na+/K+ ATPase, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK). In addition, DHTH increased AMPKα phosphorylation and regulated its downstream pathways, including increasing acetyl-CoA carboxylase (ACC) phosphorylation, inhibiting transducer of regulated CREB activity 2 (TORC2) translocation and promoting glucose uptake. Such discovered multi-target effects of DHTH are expected to have provided additional understandings on the molecular basis of the therapeutic effects of danshen against the metabolic syndrome.

TORC: A New Twist on Corticotropin-Releasing Hormone Gene Expression

CRH is the principal hypothalamic neurohormone responsible for hypothalamic-pituitary adrenal (HPA) axis activity (1). CRH also serves as an important transsynaptic signal in various neurocircuits in the brain that regulate behavioral and emotional responses to stress (2). Thus, CRH appears to be a key molecule in the coordination of behavioral and neuroendocrine responses to stressful events. Dysregulation of CRH neuronal function is associated with stress-related psychiatric disorders, and abnormal CRH neuropeptide production levels appear to underlie some of this neuronal dysregulation (3). Consequently, there is much interest in determining the regulation of CRH gene expression, with a particular eye on identifying new regulatory proteins that may point to candidate genes for disease susceptibility (4). Studies find that there is high basal CRH gene expression within the medial parvocellular portion of the hypothalamic paraventricular nucleus. However, CRH gene expression is also rapidly increased by various excitatory stimuli that lead to HPA axis activation (5, 6). The coupling of neuronal excitation with CRH gene induction appears to be largely driven by protein kinase A (PKA)-mediated activation of the transcription factor cAMP response element binding protein (CREB) (7, 8). But some recent findings have thrown a wrench in this straightforward understanding of CRH gene expression. Liu et al. (9) noted that, in a hypothalamic-derived neuronal cell line (4B cells) and hypothalamic primary neuronal cultures, treatment with either the phorbol ester phorbol-12-myristate-13-acetate (PMA; protein kinase C activator) or forskolin (adenylate cyclase activator) led to CREB activation (phosphorylation of CREB ser133). Paradoxically, however, only forskolin treatment was sufficient to stimulate CRH gene promoter activity and CRH gene expression, even though this stimulatory activity required CREB activation (9). A related phenomenon has been observed in vivo: microinfusion of forskolin but not phorbol ester in the rat paraventricular nucleus led to increased CRH gene expression, even though both drugs led to increased HPA axis hormone secretion (10). These findings prompted Liu et al. (9) to consider the possible role of a CREB coregulatory factor in the regulation of CRH gene expression. Such a factor would have to meet three conditions. It would have to be rapidly activated by stimulatory intercellular signals, activated by forskolin but not PMA, and dependent on CREB for CRH gene transactivation capability. In the current issue of Endocrinology, Liu et al. (11) explored the possibility that the recently discovered transducer of regulated CREB activity (TORC), which is also known as CREB-regulated transcription coactivator, could be such a factor. Two groups independently discovered this evolutionarily conserved family of CREB coactivators (TORC1, TORC2, and TORC3) by use of high-throughput screening strategies for proteins that potentiated CREB-dependent gene expression (12, 13). Subsequent studies in various peripheral tissue cell lines have found that TORC has a number of interesting biochemical features. These features not only account for TORC’s ability to interact with CREB protein and thereby permit CREB-dependent gene expression, but they also provide for diverse ways in which TORC can be activated or deactivated. TORC binds to the dimerization and DNA binding domain of CREB. Although this interaction does not appear to alter CREB’s DNA binding capability, it enhances CREB’s association with TAFII130, a component of the general transcription

Involvement of Transducer of Regulated cAMP Response Element-Binding Protein Activity on Corticotropin Releasing Hormone Transcription

We have recently shown that phospho-cAMP response element-binding protein (CREB) is essential but not sufficient for activation of CRH transcription, suggesting the requirement of a coactivator. Here, we test the hypothesis that the CREB coactivator, transducer of regulated CREB activity (TORC), is required for activation of CRH transcription, using the cell line 4B and primary cultures of hypothalamic neurons. Immunohistochemistry and Western blot experiments in 4B cells revealed time-dependent nuclear translocation of TORC1,TORC 2, and TORC3 by forskolin [but not by the phorbol ester, phorbol 12-myristate 13-acetate (PMA)] in a concentration-dependent manner. In reporter gene assays, cotransfection of TORC1 or TORC2 potentiated the stimulatory effect of forskolin on CRH promoter activity but had no effect in cells treated with PMA. Knockout of endogenous TORC using silencing RNA markedly inhibited forskolin-activated CRH promoter activity in 4B cells, as well as the induction of endogenous CRH primary transcript by forskolin in primary neuronal cultures. Coimmunoprecipitation and chromatin immunoprecipitation experiments in 4B cells revealed association of CREB and TORC in the nucleus, and recruitment of TORC2 by the CRH promoter, after 20-min incubation with forskolin. These studies demonstrate a correlation between nuclear translocation of TORC with association to the CRH promoter and activation of CRH transcription. The data suggest that TORC is required for transcriptional activation of the CRH promoter by acting as a CREB coactivator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB.

Regulation of TORC-CREB Complex by Salt-inducible Kinases:Implications in Hypertension and Diabetes Mellitus*

Cyclic AMP responsive element-binding protein(CREB) is a transcription factor coactivated in response to a simultaneous increase in intracellular cAMP and Ca2+.The target gene products of CREB involve broad physiological processes such as cell proliferation and survival, carbohydrate and lipid metabolism, steroid synthesis, and learning and memory.The newly-identified transducer of regulated CREB activity(TORC) controls transcription and expression of many CREB target genes by undergoing nucleo-cytoplasmic shuttling.Salt-inducible kinase(SIK) is a group of serine/threonine protein kinase including SIK1, SIK2 and SIK3.These kinases indirectly modulate CREB target gene transcription and expression by affecting TORC phosphorylation and their subsequent distribution between nucleus and cytoplasm.In certain organs and tissues, SIK(SIK1) is also one of the target genes for CREB.Thus, a complete negative feedback regulatory circuit may be formed between SIK and TORC-CREB complex.To date, the latter has been identified in several organs and tissues such as pancreatic β-cell, the liver, adrenal cortex and skeletal muscle where they regulate β-cell survival, hepatic glyconeogenesis, steroid synthesis, and mitochondrial biogenesis and fatty acid oxidation in the skeletal muscle.The feedback regulation of TORC-CREB complex by SIK and its implications in pathogenesis of hypertension and diabetes mellitus are focused on.

The Induction of IL-10 by Zymosan in Dendritic Cells Depends on CREB Activation by the Coactivators CREB-Binding Protein and TORC2 and Autocrine PGE2

Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated kappaB-binding activity, but inhibition of NF-kappaB was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE(2), and inhibitors of PKA, COX, and glycogen-synthase kinase-3beta showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE(2) and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3beta, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.

Inactivation of HDAC5 by SIK1 in AICAR-treated C2C12 Myoblasts

Salt inducible kinase (SIK) 1, a member of the AMP-activated kinase (AMPK) family, is activated by the AMPK-activator LKB1 which phosphorylates SIK1 at Thr182. The activated SIK1 then auto-phosphorylates its Ser186 located at the +4 position of Thr182. The phospho-Ser186 is essential for sustained activity of SIK1, which is maintained by sequential phosphorylation at Ser186-Thr182 by glycogen synthase kinase (GSK)-3beta. Meanwhile, SIK1 represses the transcription factor cAMP-response element binding protein (CREB) by phosphorylating its co-activator transducer of regulated CREB activity (TORC). Recently, histone deacetylase (HDAC) 5 was identified as a new substrate of SIK1. Inhibition of SIK1 or AMPK results in the stimulation of glyconeogensis in the liver by enhancing dephosphorylation of TORC2 followed by up-regulation of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha gene expression. However, expression of the PGC-1alpha gene has been found to be repressed in LKB1-defective muscle cells. Our findings show that the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-dependent expression of PGC-1alpha is diminished by inhibitors of GSK-3beta or SIKs in C2C12 myoblasts. Treatment with AICAR or the overexpression of SIK1 induces nuclear export of HDAC5 followed by the activation of myogenic transcription factor (MEF)-2C. The levels of phosphorylation at Thr182 and Ser186 of SIK1 in AICAR-treated C2C12 cells are elevated, and GSK-3beta enzyme purified from AICAR-treated cells shows enhanced phosphorylation activity of SIK1 in vitro. These observations suggest that GSK-3 beta and SIK1 may play important roles in the regulation of PGC-1alpha gene expression by inactivating HDAC5 followed by activation of MEF2C.

TORCing Up Metabolic Control in the Brain

Transducer of regulated CREB activity 2 (TORC2) is a coactivator of CREB and an important regulator of energy balance in mammals through control of gluconeogenesis in the liver. In this issue of Cell Metabolism, Wang and coworkers (2008) report an intriguing role for Drosophila TORC in the neuronal regulation of metabolism.

The Insulin-Regulated CREB Coactivator TORC Promotes Stress Resistance in Drosophila

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.

Regulation of CREB-mediated gene expression by salt inducible kinase

Salt inducible kinase (SIK) was identified as a molecule induced in the adrenal glands of rats fed with a high-salt diet. A major downstream of SIK is regulation of camp-responsive element (CRE)-dependent gene expression. SIK represses the activity of CRE-binding protein (CREB) by phosphorylating a CREB-specific co-activator transducer of regulated CREB activity (TORC). When TORC is dephosphorylated it activates CREB in a CREB-phosphorylation independent manner. The importance of the dephosphorylation of TORC has been suggested by the fact that a kinase inhibitor staurosporine induces dephosphorylation of TORC and upregulates the gene expression of CYP11A, CYP11B1, CYP11B2 and StAR in adrenocortical cells. The identification of SIK caused a stir in the field of CREB studies and led to disclosure of cascades hidden behind the classical mechanism for CREB activity.

Transcription factor cAMP response element‐binding protein CREB

The cAMP response element-binding protein (CREB) was identified almost 20 years ago. Since then, CREB has become one of the most extensively studied transcription factors. Currently, the importance of CREB for numerous physiological events has been confirmed by the number of targets: up to 4000 genes or up to 6000 loci. In addition, it is well known that numerous signals, not only the cAMP signal, but also Ca, cellular stresses and cell cycle systems, regulate CREB activity in a phosphorylation-dependent manner. In mice, knockout models targeting CREB and its cognate factors have disclosed essential functions of the CREB family in cell survival from early embryo to germ cells. By contrast, the CREB family factors are found only in multicellular animals, although cAMP⁄ stressdependent gene regulation plays an important role in a variety of organisms, such as Escherichia coli and yeast, indicating that CREB may regulate physiological events acquired by animals during their evolution. In view of these findings, we summarize a new insight of CREB regulation actualized through its basic leucine zipper domain and three physiological events regulated by CREB: ischemia, tumorigenesis and memory. Hiroshi Takemori, Junko Kajimura and Mitsuhiro Okamoto describe transactivation activities encoded by the basic leucine zipper (bZIP) domain. Studies of protein–protein interaction suggest the presence of CREB’s transactivation activities outside of its transactivation domain. This notion was confirmed by the discovery of the CREB specific coactivator: transducer of regulated CREB activity (TORC). TORCs are also regulated by phosphorylation, but they are active in their dephospho-forms. The dephosphorylation of TORCs occurs concomitantly with the phosphorylation of CREB. The combination of phosphorylation and dephosphorylation of CREB and TORC allows CREB to regulate CREdependent transcription in a variety of spectra. Kazuo Kitagawa reports on the importance of CREB and CRE-mediated gene expression after ischemic stresses. When nerve cells are subjected to sublethal conditions, kinase cascades increase CREB phoshorylation and upregulate CRE-mediated expression of genes, such as Bcl-2 and brain derived neurotrophic factor, which enhance cell survival. Recent studies also provide evidence that CREB agonists, inhibitors of phosphodiesterase and acetylcholinesterase, protect neurons from stress, thus indicating the neuro-protective function of CREB. Hao Wu, Yang Zhou and Zhi-Qi Xiong deal with a mechanism by which CREB maintains memory and learning in the brain. Neuronal actions ⁄ signals are classified into two categories, nongenomic actions and genomic actions. The latter occurs in the late phase of signal transduction, and memory is established through long-term synaptic potentiation. However, CREB phosphorylation is mediated by nongenomic actions that occur in the acute phase and diminish in the late phase. TORC1 is abundantly expressed in the hippocampus and plays an important role in the prolonged activation of CREB under the regulation of cAMP ⁄Ca signaling. Yeung-Tung Siu and DongYan Jin describe the involvement of CREB in oncogenesis. Escape from cell death is an essential mechanism for cells to become immortalized. Excessive and prolonged activation of CREB then maintains cell proliferation. Some of the gene-realignments that produce chimeric proteins of oncogenic factors with CREB and its related factors induce constitutive activation of CRE-dependent transcription followed by onsets of tumorigensis. Viruses also utilize CREB for their transcription and replication, and some viral proteins consistently keep CREB activated, which then becomes a factor in virus-mediated tumorigensis. The identification and clarification of the physiological roles of CREB and its regulatory mechanisms disclosed by these studies can be expected to lead to an acceleration in related discoveries in the not too distant future.

CREB − a real culprit in oncogenesis

The cAMP response element-binding protein (CREB) is a stimulus-induced transcription factor that responds rapidly to phosphorylation and/or coactivator activation. Regulated activation of CREB has a significant impact on cellular growth, proliferation and survival. To overturn the cellular control of these processes, tumor cells have developed various mechanisms to achieve constitutive activation of CREB, including gene amplification, chromosome translocation, interaction with viral oncoproteins, and inactivation of tumor suppressor genes. These mechanisms converge on the phosphorylation of CREB and/or the activation of transducer of regulated CREB activity (TORC) coactivators to effect uncontrolled proliferation of cells. This minireview summarizes the different lines of existing evidence that support a direct role of CREB in oncogenesis.

Dephosphorylation of TORC initiates expression of the StAR gene

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.