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CREB mammalian homologs

Mammalian CREB, CREM and ATF-1 gene products are close matches to CrebB-17A (Yin 1994). Two other Drosophila proteins have homology to mammalian CREB but the relation to mammalian genes is more distant (Yin 1994).

Activation of CREB by phosphorylation

CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. A nuclear protein of M(r)265K, CBP, binds specifically to the PKA-phosphorylated form of CREB. CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1. Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes (Kwok, 1994).

Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. The stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic subunit (C subunit) into the nucleus. PKA is activated in a stimulus-dependent fashion that leads to nuclear entry of C subunit over a 30-min period. The degree of CREB phosphorylation correlates with the amount of PKA liberated. The time course of phosphorylation closely paralleled the nuclear entry of the catalytic subunit. There is a linear relationship between the subsequent induction of the cAMP-responsive somatostatin gene and the degree of CREB phosphorylation, suggesting that each event--kinase activation, CREB phosphorylation, and transcriptional induction--was tightly coupled to the next. In contrast to other PKA-mediated cellular responses which are rapid and quantitative, the slow, incremental regulation of CREB activity by cAMP suggests that multifunctional kinases like PKA may coordinate cellular responses by dictating the kinetics and stoichiometry of phosphorylation for key substrates like CREB (Hagiwara, 1993).

The single phosphorylation of the cyclic AMP response element binding factor (CREB) at Ser-133 is sufficient for the transcriptional activation by cAMP-mediated pathways. Previous in vivo studies investigating this point have relied upon transfection of cyclic AMP-dependent kinase (cAPK) or its activation by treatment of cells with cell-permeable cAMP analogs. However, as numerous cellular proteins, including CREB, are substrates for activated cAPK, the possibility remains that cAPK substrates other than CREB are required for the transcriptional activity of CRE-containing promoters. The activity of recombinant CREB phosphorylated on Ser-133 were studied in both cell-free transcription assays and in vivo after introduction of the same preparations into fibroblasts by microinjection. The activity of phosphorylated CREB, nonphosphorylated CREB, and a mutant form of CREB, containing Ala substituted for Ser at position 133, was found to be nearly identical in cell-free in vitro transcription assays. In contrast, only the phosphorylated CREB microinjected into fibroblasts results in the stimulation of expression of CRE-regulated genes. These results suggest that phosphorylation of CREB on Ser-133 directly stimulates its ability to transactivate gene expression in intact cells (Alberts. 1994).

A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase reveals that it is identical to a member of the pp90(RSK; see Drosophila RSK) family, RSK2. RSK2 mediates growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression (Xing, 1996).

A-kinase anchor protein 75 (AKAP75) binds regulatory subunits (RIIalpha and RIIbeta) of type II protein kinase A (PKAII) isoforms and targets the resulting complexes to sites in the cytoskeleton that abut the plasma membrane. Co-localization of AKAP75-PKAII with adenylate cyclase and PKA substrate/effector proteins in cytoskeleton and plasma membrane effects a physical and functional integration of up-stream and downstream signaling proteins, thereby ensuring efficient propagation of signals carried by locally generated cyclic AMP (cAMP). An important, but previously untested, prediction of the AKAP model is that efficient, cyclic nucleotide-dependent liberation of diffusible PKA catalytic subunits from cytoskeleton-bound AKAP75-PKAII complexes will also enhance signaling to distal organelles, such as the nucleus. This idea was tested by using HEK-A75 cells, in which PKAII isoforms are immobilized in cortical cytoskeleton by AKAP75. The abilities of HEK-A75 and control cells (with cytoplasmically dispersed PKAII isoforms) to respond to increases in cAMP content were compared. Cells with anchored PKAII exhibit a threefold higher level of nuclear catalytic subunit content and 4 to10 fold greater increments in the phosphorylation of a regulatory serine residue in cAMP response element binding protein (CREB) and in phosphoCREB-stimulated transcription of the c-fos gene. Each effect occur more rapidly in cells containing targeted AKAP75-PKAII complexes. Thus, the anchoring of PKAII in actin cortical cytoskeleton increases the rate, magnitude and sensitivity of cAMP signaling to the nucleus (Feliciello, 1997).

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases (Sun, 1994).

Phosphorylation of CREB by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, Ca(2+)-mediated activation of a CRE-containing promoter can be conferred by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation can also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. These results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction (Matthews, 1994).

The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Regulated TCRalpha enhancer activity has been reconstituted in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCRalpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhances transcription 10-fold in vitro, and this effect is abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhances the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain is sufficient to form a higher-order complex with AML1/Ets-1, but exhibits only partial activity in transcription. It is concluded that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCRalpha enhancer in a chromatin environment (Mayall, 1997).

Activation by growth factors of the Ras-dependent signaling cascade result in the induction of p90 ribosomal S6 kinases [p90(rsk)]. These are translocated into the nucleus upon phosphorylation by mitogen-activated protein kinases, with which p90(rsk) is physically associated in the cytoplasm. In humans there are three isoforms of the p90(rsk) family: Rsk-1, Rsk-2, and Rsk-3; all are products of distinct genes. Although these isoforms are structurally very similar, little is known about their functional specificity. Recently, mutations in the Rsk-2 gene have been associated with the Coffin-Lowry syndrome (CLS). A fibroblast cell line established from a CLS patient that bears a nonfunctional Rsk-2 has been studied. In CLS fibroblasts there is a drastic attenuation in the induced Ser-133 phosphorylation of transcription factor CREB (cAMP response element-binding protein) in response to epidermal growth factor stimulation. The effect is specific, since response to serum, cAMP, and UV light is unaltered. Furthermore, epidermal growth factor-induced expression of c-fos is severely impaired in CLS fibroblasts despite normal phosphorylation of serum response factor and Elk-1. Finally, coexpression of Rsk-2 in transfected cells results in the activation of the c-fos promoter via the cAMP-responsive element. Thus, a link in the transduction of a specific growth factor signal to changes in gene expression via the phosphorylation of CREB by Rsk-2 has been established (De Cesare, 1998).

A novel mitogen- and stress-activated protein kinase (MSK1) has been identifed that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of both unstimulated and stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB (Deak, 1998).

The p38/stress-activated protein kinase2 (p38/SAPK2) is activated by cellular stress and proinflammatory cytokines. In human Jurkat T-cells, induction of the early growth response gene-1 (egr-1) by anisomycin is completely inhibited by SB203580, a specific inhibitor of p38/SAPK2a and p38/SAPK2b. Northern blot and reporter gene experiments indicate that this block is at the level of mRNA biosynthesis. Using mutants of the egr-1 promoter, it has been demonstrated that a distal cAMP-responsive element (CRE; nucleotides -134 to -126) is necessary to control egr-1 induction by p38/SAPK2. Pull-down assays indicate that phospho-CRE binding protein (CREB) and phospho-activating transcription factor-1 (ATF1) bind to this element in a p38/SAPK2-dependent manner. In response to anisomycin, two known CREB kinases downstream of p38/SAPK2 [MAPKAP kinase 2 (MK2) and mitogen- and stress-activated kinase 1 (MSK1)] show increased activity. However, in MK2 -/- fibroblasts derived from mice carrying a disruption of the MK2 gene, the phosphorylation of CREB and ATF1 and the expression of egr-1 reach levels comparable with wild type cells. This finding excludes MK2 as an involved enzyme. It is concluded that egr-1 induction by anisomycin is mediated by p38/SAPK2 and probably by MSK1. Phosphorylated CREB and ATF1 then bind to the CRE of the egr-1 promoter and cause a stress-dependent transcriptional activation of this gene (Rolli, 1999).

Although BTK plays multiple roles in the life of a B cell, its functional role in neuronal cells has not been elucidated. In the present study, BTK is shown to activate the transcription factor CREB and subsequent CRE-mediated gene transcription during basic fibroblast growth factor (bFGF)-induced neuronal differentiation in immortalized hippocampal progenitor cells (H19-7). The kinase activity of BTK is also induced by bFGF, and BTK directly phosphorylates CREB at Ser-133 residue, indicating that BTK has a dual protein kinase activity. In addition, blockading BTK activation significantly inhibits CREB phosphorylation as well as the neurite outgrowth induced by bFGF in H19-7 cells. These results suggest that the activation of BTK and the subsequent phosphorylation of CREB at Ser-133 are important in the neuronal differentiation of hippocampal progenitor cells (Yang, 2004).

Aplysia miR-124 functions in constraining synaptic plasticity through CREB

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, massive parallel sequencing was carried out to profile the small RNAs of Aplysia californica. 170 distinct miRNAs were identified, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. Direct evidence is presented that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs, and a role is presented for miR-124 in long-term plasticity of synapses in the mature nervous system (Rajasethupathy, 2009).

miR-124 serves as a negative constraint on serotonin-induced long-term facilitation, since increased or decreased miR-124 levels in sensory neurons leads to a significant inhibition or enhancement, respectively, of synaptic facilitation. In particular, the inhibition of miR-124 confers to sensory-motor synapses a greater sensitivity for serotonin, since just one pulse of serotonin is sufficient to cause long-term facilitation. These physiology data also suggest that miR-124 inhibition is just one of many 5HT-mediated events that activate CREB to induce long-term facilitation, since the inhibition of miR-124 alone, in the absence of 5HT, does not lead to long-term facilitation. Therefore, while the observed effects of the miR-124 manipulations on LTF are of a significant magnitude, it is likely that these effects would be even greater if there were a coordinated manipulation of several miRNAs that act together in parallel pathways during synaptic plasticity. The observation that miR-124 levels affect facilitation both at 24 and 48 hr after exposure to spaced pulses of serotonin suggests that miR-124 regulation is required not only for the induction phase but that it is also critical for the maintenance phase of synaptic facilitation. Since miR-124 levels return back to baseline within 12 hr after exposure to serotonin, the initial drop in miR-124 during this time window appears to be sufficient enough to upregulate the relevant transcripts to allow for facilitation for up to 48 hr after exposure to serotonin. Indeed, the upregulation of many plasticity-related transcripts are transient and fall into this initial time window. The data also suggest that miR-124 does not significantly affect or contribute to serotonin-independent processes such as basal and constitutive synaptic activity. However, since all of the experiments were conducted on several-day-old cultures, at which point the cells and synapses are fully mature and stable, these studies leave open the possibility that miR-124 contributes to serotonin-independent processes in immature neurons such as neurite out-growth and synapse formation (Rajasethupathy, 2009).

The negative constraint that miR-124 imposes on synaptic facilitation is mediated, at least in part, by its direct regulation of CREB. The fact that miR-124 inhibition significantly and specifically increases CREB1 levels, along with immediate downstream genes such as UCH, C/EBP, and KHC, that miR-124 serotonin kinetics parallels the CREB1 serotonin kinetics, and that miR-124 inhibition can provide the switch necessary to convert short-term facilitation into long-term facilitation all strongly support the conclusion that miR-124 can tightly control CREB and CREB-mediated signaling during plasticity. CREB has been extensively studied over the years for its regulation by kinase-dependent posttranslational modifications, such as phosphorylation by PKA and MAPK. The present study, however, is one of the first to address posttranscriptional regulation of CREB. While this additional level of regulation might appear redundant, for example by paralleling the function of CREB2, it is likely that miR-124 inhibition allows for more rapid and transient control over CREB expression, as well as the opportunity for CREB to be drawn into various distinct downstream pathways once activated. It was also noticed that CREB, in turn, may be able to regulate miR-124 expression levels since there are several putative CREB binding sites in the presumed promoter region upstream of the Aplysia mir-124 gene. Although Aplysia and mammalian systems have clear differences in the complexities of their CNS, and also even in the types of neurotransmitters used during long-term memory processes, the underlying calcium-induced signaling pathways (including cAMP, PKA, MAPK, and CREB) and their functions are very much shared. It is therefore very likely that miR-124 is activity-regulated in the mammalian hippocampus and regulates CREB in much the same way as observed in this study, especially in light of the fact that the mammalian CREB1 UTR bears a conserved miR-124 target site as predicted by targetscan, which was recently confirmed as a site directly bound by Argonaute in mouse brain (Rajasethupathy, 2009).

In summary, this study has identified a comprehensive set of brain-enriched miRNAs in Aplysia, many of which can be regulated by the neuromodulator serotonin, signifying potential roles in learning-related synaptic plasticity. Specifically, it was demonstrated that brain-specific miR-124 responds to serotonin by derepressing CREB and enhancing serotonin-dependent long-term facilitation. This initial study compels the exploration of how neuromodulators act through small RNAs during various forms of plasticity and whether some act locally at synapses. This study also provides evidence that some 5HT-regulated Aplysia miRNAs regulate plasticity-related genes involved in local protein synthesis at the synapse. The likelihood of a coordinated set of miRNAs combinatorially regulating events at the synapse makes possible a new and rich layer of computational complexity that could be responsible for the emergence of discrete and long-lasting states of activity at the synapse (Rajasethupathy, 2009).

γCaMKII shuttles Ca(2+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression

Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at voltage gated CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. This study reports a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin (see Drosophila Calmodulin) from the surface membrane to the nucleus. This study shows that the shuttle protein is γCaMKII (see Drosophila CaMKII), its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN (see Drosophila Calcineurin) triggers its nuclear translocation. Both betaCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders (Ma, 2014).

Table of contents

CrebB-17A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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