Gene name - Cyclic-AMP response element binding protein B
Synonyms - CREB, dCREB2
Cytological map position - 17A
Function - transcription factor
Symbol - CrebB
Genetic map position -
Classification - basic leucine zipper
Cellular location - nuclear
|Recent literature||Kuntz, S., Poeck, B. and Strauss, R. (2017). Visual working memory requires permissive and instructive NO/cGMP signaling at presynapses in the Drosophila central brain. Curr Biol [Epub ahead of print]. PubMed ID: 28216314
The gaseous second messenger nitric oxide (NO) has been shown to regulate memory formation by activating retrograde signaling cascades from post- to presynapse that involve cyclic guanosine monophosphate (cGMP) production to induce synaptic plasticity and transcriptional changes. This study analyzed the role of NO in the formation of a visual working memory that lasts only a few seconds. This memory is encoded in a subset of ring neurons that form the ellipsoid body in the Drosophila brain. Using genetic and pharmacological manipulations, NO signaling was shown to be required for cGMP-mediated CREB activation, leading to the expression of competence factors like the synaptic homer protein. Interestingly, this cell-autonomous function can also be fulfilled by hydrogen sulfide (H2S) through a converging pathway, revealing for the first time that endogenously produced H2S has a role in memory processes. Notably, the NO synthase is strictly localized to the axonal output branches of the ring neurons, and this localization seems to be necessary for a second, phasic role of NO signaling. Evidence is provided for a model where NO modulates the opening of cGMP-regulated cation channels to encode a short-term memory trace. Local production of NO/cGMP in restricted branches of ring neurons seems to represent the engram for objects, and comparing signal levels between individual ring neurons is used to orient the fly during search behavior. Due to its short half-life, NO seems to be a uniquely suited second messenger to encode working memories that have to be restricted in their duration.
|Lee, P. T., Lin, G., Lin, W. W., Diao, F., White, B. H. and Bellen, H. J. (2018). A kinase-dependent feedforward loop affects CREBB stability and long term memory formation. Elife 7. PubMed ID: 29473541
In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, Trojan exons encoding T2A-GAL4 were integrated into genes encoding putative kinases and genes expressed in MB were selected for. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. A novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human) was identified, the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. PKA phosphorylates MP and both proteins synergize in a feedforward loop to control CREBB levels and LTM.
|Palacios-Munoz, A. and Ewer, J. (2018). Calcium and cAMP directly modulate the speed of the Drosophila circadian clock. PLoS Genet 14(6): e1007433. PubMed ID: 29879123
Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. This question was investigated by focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. Genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. These results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.
Memory involves synthesis of new proteins. In organisms as diverse as Aplasia, Drosophila and humans, the enzyme cyclic-AMP response element binding protein (CREB) is directly involved in the establishment of the capacity for long term memory (Bailey, 1994). CREB is so named because of the part it plays in binding the cyclic-AMP response element in the promoter of the mammalian proto-oncogene Jun. CREB was originally described as a factor stimulating the transcription of genes in response to growth factors and phorbol esters.
Suppose a Superfly were to exist, one that could learn from a single experience without need for repetition. In fact, such an animal has been created. Precocious long term memory has been observed after a single cycle of training when a transgene for a PKA-responsive activator form of CREB is expressed before training takes place (Yin, 1995b). This activator is one isoform found in multiple splicing of CREB.
It seems that flies need to rest during training periods in order to establish long term memory. Mass training, that is, repeated experience without rest, fails to produce long term memory. Spaced training, training with rest periods between repeated tests, produces two functionally independent forms of consolidated memory: long term memory (LTM) and anesthesia resistant memory (ARM). LTM is disrupted by cyclohexamide, a drug that blocks protein synthesis, in contrast to ARM, which remains unaffected. LTM, and not ARM is disrupted by induced expression of a dominant negative transgene of CREB (Yin, 1995b).
Understanding of gene activation in the memory pathway comes from an analysis of transcriptional regulation of the Jun gene. A classic model for the formation of heterodimers is the Jun/Fos heterodimer, the predominant form of the gene regulator AP-1 (activator protein-1). Analysis of the Drosophila Jun promoter reveals binding sites for AP-1, CREB and DTF1, a protein that acts as an enhancer binding factor of the D2 promoter of Antennapedia (Perkins, 1988a and b). Thus Jun is regulated by JUN itself combined with FOS and by two other proteins: DTF1 and CREB. Three of these proteins (FOS, JUN and CREB) are responsive to the phosphorylation cascade that results from cAMP signaling. Additional information on the role of cyclic-AMP in fly memory (Davis, R. L., 1996) will be found in the dunce and rutabaga sites.
The role of dCREB2 (CrebB-17A) in circadian rhythms has been examined. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. It is proposed that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila (Belvin, 1999).
To measure dCREB2 activity in vivo, transgenic Drosophila lines were constructed carrying the luciferase reporter gene driven by an enhancer element comprised of consensus CREB binding sites. Three cAMP response elements (CREs), 5'-TGACGTCA-3', were placed upstream of the TATA box region of the hsp70 gene promoter, followed by the luciferase reporter gene. This sequence was flanked by the scs and scs' insulator elements to reduce potential positional effects caused by the random insertion site of the transgene. The transfected lines are referred to as CRE-luc lines. A mutant CRE-luc reporter construct (mCRE-luc) was also generated in which the consensus CRE sites were mutated to TGAAATCA. dCREB2 protein binds this mutant CRE site with at least 20-fold lower affinity in gel shift experiments. This construct is otherwise identical to wild-type CRE-luc (Belvin, 1999).
The expression of luciferase in the wild-type CRE-luc flies oscillates in a 24 hr rhythm, both in a light:dark cycle and in constant darkness. The main peak of activity occurs just after lights out, with the nadir just before the main peak. Since this rhythmic transcription pattern is sustained in constant darkness, it is regulated by the circadian system, rather than simply being a response to light. In light:dark conditions, a second peak is observed in the middle of the day; however, these two peaks gradually blend together under conditions of constant darkness. This pattern is very similar to that seen for per activity. The per-luc reporter also exhibits a similar secondary peak under light:dark conditions, even though per RNA peaks only once per cycle. It is likely that the secondary peaks of both reporters, which occur during the day, are due to a light response of luciferase rather than a circadian response. The expression level of the mCRE-luc reporter is drastically reduced relative to the wild-type reporter, indicating that the CRE sites mediate the high-level expression of the wild-type reporter (Belvin, 1999).
These experiments show that dCREB2 activity is under circadian control, but they do not show whether dCREB2 plays a role in maintaining the rhythms or is just responsive to them. The A dCREB2 mutation, S162, one that allows for survival of a few flies to adulthood, was used to address this question. To test for behavioral effects of the mutation, S162 escaper males were assayed for circadian locomotor activity. The flies were tested for 10 days in constant darkness to determine whether they display normal circadian fluctuations in activity. Of the 34 S162 mutants tested, 13 (38%) were arrhythmic while the 21 that were rhythmic had a short period averaging 22.8 hr. None of these flies had a wild-type 24 hr rhythm. The high percentage of arrhythmicity is typical of mutations that affect period length. To verify that this behavioral phenotype is specific for the S162 mutation, the phenotype was rescued by induction of the hs-dCREB2-10 transgene. To induce the transgene, larvae and pupae were subjected to a daily 60 min heat pulse of 37°C during development. All of the rescued flies (12/12) were rhythmic, and they all displayed normal circadian locomotor rhythms of 23.5-24 hr. This demonstrates that the short period phenotype is caused by the S162 mutation rather than a second site mutation elsewhere on the chromosome. It also shows the involvement of dCREB2 in the timing of the clock (Belvin, 1999).
If S162 is indeed acting in the clock, then it should affect the per clock gene. The effects of S162 on two different per-dependent reporters were examined. The first is a transcriptional fusion with a 4.2 kb fragment of the per promoter upstream of the luciferase reporter gene, referred to as per-luc. The second is a translational fusion containing the same promoter fragment, plus the 5' untranslated region and the first 2.4 kb of the per coding region fused in frame to the luciferase gene, referred to as BG-luc. When the expression of these reporters was compared, it was found that the BG-luc reporter cycles much more robustly than the per-luc reporter, consistent with the interpretation that there are at least two mechanisms contributing to the cycling of Per: one mediated by the promoter, and the other(s) mediated by sequences in either the per transcript or Per protein itself. S162 affects the two reporters differently. The S162 mutation reduces both the expression level and cycling pattern of the per-luc reporter. However, its effect on the BG-luc reporter is weaker. In S162 flies, the BG-luc reporter maintains a robust cycling pattern, although its expression level and amplitude are reduced. The peak in the mutant background also occurs in advance of the peak in wild-type flies, consistent with the short period phenotype of these flies (Belvin, 1999).
In order to demonstrate a direct effect of the S162 mutation on the clock, its effects on the Per protein itself were examined. Wild-type and S162 flies were entrained on a 12 hr light:12 hr dark cycle and aliquots were frozen every 2 hr throughout the cycle. Head extracts were prepared and analyzed by Western blot using an antibody directed against Per. Per is present at very low levels at ZT 6 and ZT 8 (Zeitgeber time; 6 and 8 hours after lights on), increasing through the lights of period to peak levels before lights on, which occurs at ZT 0. A corresponding change in phosphorylation, and protein mobility, accompanies the change in absolute levels, with Per becoming more highly phosphorylated as it accumulates. This temporal pattern of Per is altered in the S162 mutant background, where Per is present at more equal levels throughout the circadian cycle. At the peak time, ZT 20, the amount of Per is at least comparable to that in wild-type flies; however, it decreases less at ZT 6 and ZT 8, when Per is virtually absent in wild-type flies. At these trough periods of Per expression, a discrete doublet protein band persists in S162, perhaps representing preservation of certain phosphorylated forms. There also seems to be a general increase in the amount of Per protein throughout the cycle in the mutant flies. The change in both per-luc expression and Per protein levels in the S162 mutant background demonstrates that Per activity is under the influence of the dCREB2 gene. The effects of S162 on Tim protein were assayed in the same experiment. The effect of S162 on Tim is much more subtle than the effect on Per. The Tim protein appears to accumulate slightly sooner in the mutant than in the wild type (ZT 12 versus ZT 14); however, its overall oscillation remains fairly normal (Belvin, 1999).
What is the biological significance of dCREB2's participation in the clock? Since a mutation in the dCREB2 gene affects the clock, the possibility exists that stimuli that activate CREB could affect circadian rhythmicity. This is clearly true for light pulses that reset the clock when delivered at the appropriate times during the nighttime period and have been shown to induce CREB phosphorylation in mammals. What about 'noncircadian' stimuli? One example of this phenomenon is work showing that rats can learn an association between air puffs delivered to the eye (conditioned stimulus) and light (unconditioned stimulus). After acquisition, the conditioned stimulus alone, when delivered during the nighttime period, phase shifts the clock. This suggests that there may be much more overlap in the conditioning circuitry and the circadian circuitry than is generally appreciated. Since the molecules within all of these neurons are similar, the molecular pathways may also overlap. CREB-mediated transcription occurs in response to stimuli that induce stress, long-term memory formation, and growth factor responses. What is the significance of the fact that this transcription factor also responds to circadian signals, which happen regularly over a 24 hr period? One speculation is that the cyclical pattern in CREB activity means that there are optimal periods during the 24 hr cycle for CREB-responsive physiological processes. One physiological process for which there is evidence that the nighttime period is important is the consolidation of long-term memory formation. Over the years, a large number of experiments, primarily on rodents and humans, suggest a possible involvement of some aspect of nighttime sleep in the consolidation of memory. A unifying interpretation of the physiological and behavioral data is that the brain utilizes the sleep period to 'replay' plasticity-related events, thereby ensuring their total consolidation and maintenance. This replay occurs during sleep, when there is minimal external input into the brain. It is speculated that the circadian system controls some of the neuronal activity that occurs during the sleep period. This activity in turn leads to activation of CREB-responsive transcription, which may be important in consolidating and maintaining preexisting circuits and memories. Experiments to test these types of ideas are underway (Belvin, 1999 and references).
CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the 'enhancing molecule' unclear. This report shows that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. The results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement (Tubon, 2013).
This report has shown that a 28 kDa protein initiates from the internal ATG2 codon, that it functions as a CRE-dependent transcriptional activator both in vitro and in vivo, and is responsible for the original report of memory enhancement. Although ATG2 is infrequently used, and the resulting protein is expressed at low levels, its existence has been shown using multiple antibodies and different two-step enrichments (EMSA supershifts and Western identification of proteins on EMSA complexes) (Tubon, 2013).
he ATG2 codon is also used on endogenous dCREB2-encoded mRNAs, since all of the sequenced dCREB2 cDNAs contain ATG1 and ATG2 on the same molecule. Interestingly, internal translation initiation is also used on both of the mammalian CREM and CREB genes. 'Intronic' or internal ATGs can become positioned to be the first start codons through alternative promoter usage and alternative splicing. The mammalian CREB β isoform is a minority species that becomes upregulated upon deletion of the α and Δ isoforms. This study has not caracterized the transcriptional regulation of the dCREB2 gene, so it is possible that ATG2 is the first initiation codon on a minor, currently uncharacterized, dCREB2 transcript (Tubon, 2013).
A number of related issues have complicated molecular analysis of dCREB2-encoded protein isoforms, and are likely to be relevant in the characterization of these proteins in all species. First, the number and variety of posttranslational modifications that occur on dCREB2-encoded proteins is large. The KID region contains up to 7 phosphorylation sites, and other modifications, including O-GlcNac glycosylation, SUMOylation, ubiquitylation, and cysteine oxidation and/or nitrosylation, occur elsewhere on CREB proteins. These posttranslational modifications can dramatically affect the apparent mobility of protein species, and make it difficult to determine whether Western blots that contain many bands are due to a nonspecific or specific recognition of dCREB2-encoded proteins. A related observation is that these modifications can alter the binding affinity of many of the antibodie, suggesting that any given antibody reports a specialized subpool of protein. Finally, the blocker (40 kDa doublet) and activator (22-35 kDa cluster) species seem to be differentially modified, further complicating detailed analysis. It is likely that combinations of modifications are used to regulate the complex subcellular localization and activity of dCREB2 protein isoforms (Tubon, 2013).
Various parameters contribute to the inconsistency of memory enhancement. The expression of the 28 kDa protein off of the original 572 transgene is low but detectable. However, this modest level of expression is not responsible for inconsistent enhancement of olfactory avoidance memory, since the 807 transgenic fly (which has consistently higher levels of expression) also sporadically enhances memory formation. Instead, the limited duration of dCREB2-mediated transcriptional activation can place serious timing constraints on the requisite interval between transgene induction and behavioral training (the temporal window). A second temporal parameter is the time-of-day when induction and behavioral training occur. There is a growing awareness that the time-of-day-of training can affect memory formation, and this literature highlights the importance of circadian/sleep-related physiological processes and their relationship with the neuroanatomy and molecular machinery of memory formation. Careful control of these different timing issues greatly increases the reproducibility of behavioral enhancement using the olfactory avoidance assay. The consistent enhancement of the courtship behavior reinforces the original observation that the 28 kDa protein can enhance memory formation (Tubon, 2013).
Why does 807 enhance memory of courtship suppression reliably, but affects memory of olfactory avoidance less consistently? Comparing two diverse behavioral paradigms is difficult, since there are many parameters that differ. However, this type of approach is necessary, and will be useful. Another behavioral paradigm was developed using conditioned place preference. In the place preference behavioral assay, the 807 transgene enhances memory formation consistently, reinforcing the conclusion that the 28 kDa protein can have important effects on memory formation. Current experiments are directed at determining what behavioral factor(s) differ between courtship suppression and place preference (where consistent enhancement is seen) and olfactory avoidance (where enhancement is less consistent). One possibility is that enhancement in flies specifically requires a 'behavioral state' that is difficult to control experimentally, and which can be epistatic to the other parameters such as expression levels, activity windows, and the time-of-day of training. This behavioral state appears to be an 'all-or-nothing' group effect, with all of the flies in a given experiment affected similarly (Tubon, 2013).
Recent work using acute interventions in mice and other systems has shown that increasing CREB activity increases the intrinsic excitability of neurons, while interfering with CREB activity has the opposite effect (see for example Liu, 2011 and Suzuki, 2011). The CREB-dependent increase in excitability is correlated with memory enhancement, and vice versa. If dCREB2 enhances memory formation partially through affecting the excitability of relevant neurons, then the 'excitable state' of those neurons at the time of training might determine whether additional dCREB2 protein has enhancing potential or not (Benito, 2010). Since excitability is saturable, there are two simple outcomes, depending upon the state of the neurons at the time of training. If neurons are more quiescent, dCREB2 induction can increase excitability, and enhancement will occur in response to training (relative to equally quiescent neurons that just receive training). However, if the neurons are already excitable at the time of training, then extra dCREB2 will not have any effect, since excitability is saturable. The pretraining handling and housing of flies differs between various behaviors, and is somewhat variable even with the same behavior. These parameters could affect the baseline excitability of the flies, and indirectly affect enhancement. The behavioral data are consistent with this view, since enhancement usually becomes significant when the control fly population has lower memory scores, rather than the experimental population having higher memory scores. The effect of the time-of-day on enhancement also is consistent with this general hypothesis, since excitability is known to vary across the circadian cycle, at least for certain neurons. This possibility and its relevance has important implications for the role that dCREB2 plays in memory formation are currently being tested in a non-transgenic fly (Tubon, 2013).
The transcription factor CREB is an important regulator of many adaptive processes in neurons, including sleep, cellular homeostasis, and memory formation. The Drosophila dCREB2 family includes multiple protein isoforms generated from a single gene. Overexpression of an activator or blocker isoform has been shown to enhance or block memory formation, but the molecular mechanisms underlying these phenomena remain unclear. In this study isoform-specific antibodies and new transgenic flies were generated to track and manipulate the activity of different dCREB2 isoforms during memory formation. It was found that nuclear accumulation of a dCREB2 activator-related species, p35+, is dynamically regulated during memory formation. Furthermore, various dCREB2 genetic manipulations that enhance or block memory formation correspondingly increase or decrease p35+ levels in the nucleus. Finally, it was shown that overexpression of S6K can enhance memory formation and increase p35+ nuclear abundance. Taken together, these results suggest that regulation of dCREB2 localization may be a key molecular convergence point in the coordinated host of events that lead to memory formation (Fropf, 2013).
Surprisingly, the antibodies showed that the majority of dCREB2 protein, regardless of structure and apparent mobility on protein gels, is localized in the cytoplasmic compartment. This finding is true for both the blocker and activator isoforms. The blocker primarily consists of the 'backbone' structure (exons 1, 3, 5 and 7), which migrates as a doublet species around 38-40 kD. When the alternative exons (2, 4 and/or 6) are spliced into this backbone, the apparent mobility of the resulting proteins does not change dramatically. The activator-related molecular species (p24, p28, p30, p35+, p60; Tubon, 2013) also are enriched in the cytoplasm, and this is most clearly demonstrated when nuclear and cytoplasmic samples are loaded on a per head-equivalent basis. All of the dCREB2 protein isoforms contain the amino acid residues RRKKK in their basic regions, and this sequence has been shown to be necessary and sufficient to localize mammalian CREB proteins into the nucleus. Since the mammalian CREB protein is mostly found in the nucleus, the cytoplasmic enrichment for dCREB2 protein is unexpected. This localization is maintained even when transgenes are induced and different dCREB2 protein isoforms are overexpressed. These data suggest that a significant fraction of dCREB2-encoded proteins are tethered in the cytoplasmic compartment through an unknown mechanism (Fropf, 2013).
Since dCREB2 is a transcription factor, its primary function occurs in the nuclear compartment. Previous studies have demonstrated that the majority of dCREB2 protein exists with serine 231 (the fly equivalent to the mammalian serine 133 residue) phosphorylated in the basal state. This contrasts sharply with the mammalian CREB protein, which exists in the nucleus in a largely unphosphorylated state, awaiting activity-dependent signaling mechanisms that activate it. Taken together, the cytoplasmic localization of dCREB2 protein and the phosphorylation of serine 231 in the basal state suggest strongly that the rate-limiting step in activation of dCREB2 probably involves increasing the nuclear abundance of the activator species. This could at least partially occur through an increase in nuclear entry (Fropf, 2013).
A number of results support the view that p35+ is an endogenous, dCREB2-specific protein product. Different dCREB2-specific antibodies recognize it on western blots. p35+ is decreased or missing when head extracts are made from the S162 mutant fly. When blocker-encoding transgenes are induced, they affect the nuclear levels of p35+. Finally, when the 807 (activator-encoding) transgene is induced, p35+ increases. Although the 807-encoded protein is transgenic and not endogenous, the primary product has an apparent mobility of 24 kD, but when expressed in flies increases the levels of both p28 and p35+ bands (Fropf, 2013).
Three independent lines of evidence support the interpretation that p35+ represents an activator-related species. First, there is a correlation between memory formation and nuclear abundance of p35+. Forward, but not backward, paired training produces long-term memory and results in a greater than 3-fold increase in nuclear p35+. Weaker training (5xS instead of 10xS), which produces less robust memory, produces a smaller, non-significant trend towards an increase. Second, there is a strong correlation between memory formation and a qualitative increase in p35+ in the nucleus. Genetic manipulations of dCREB2 that enhance memory formation (807 induction) increase nuclear abundance of p35+, ones that interfere with long-term memory formation (induction of 17-2 and 936) prevent an increase, while a control manipulation (induction of 581) does not interfere with either memory formation or affect p35+ levels. Third, manipulating a totally different protein (S6K) results in a parallel set of correlations. Increasing the amount of the S6K protein enhances memory formation and increases p35+ nuclear abundance (Fropf, 2013).
What is the nature of the p35+ isoform? Mammalian CREB has been shown to be SUMOylated and o-glycosylated, and these mod ifications decrease the apparent mobility of the resulting proteins on gels. This study has evidence that dCREB2 protein can be o-glycosylated, and one of those species migrates around 35 kD. Future experiments will be needed to determine if is p35+ o-glycosylated, and how this would affect protein interactions, subcellular localization, or transcriptional activity (Fropf, 2013).
Since a nuclear increase in p35+ correlates so well with memory formation, it is believed that p35+ (or its predecessor species p24, p28 and/or p30) enter the nucleus from the cytoplasm, where it is tethered in a heterodimer with other dCREB2 proteins. Transgenic experiments support this view. The 807 transgene makes p24/p28, overexpression of which increases nuclear abundance of p35+ and can enhance memory formation. The 936 transgene makes a protein that differs in two of the five residues that constitute the nuclear localization signal, which renders the induced protein exclusively cytoplasmic. Induction of 936 decreases nuclear p35+ levels, and blocks memory formation. Taken together, these data are consistent with the view that 936 blocks memory formation through a cytoplasmic tethering mechanism, where the overexpressed protein binds to, and prevents the nuclear entry of, the endogenous activator species. The induced 17-2 protein is able to form heterodimers with the activator, but is less efficient at staying in the cytoplasm. However, it is still able to interfere with memory formation, since a heterodimeric species (activator:17-2) is less transcriptionally potent. The 17-2 and 581 transgenes differ in two leucine residues in the leucine zipper. These amino acid changes are known to disrupt the ability of the mutant (581) protein to form dimers. Therefore, the overexpressed 581 protein cannot form a dimer with the activator, does not prevent a nuclear increase in p35+ after training, and does not affect memory formation. Finally, the overexpressed 568 protein is mostly in the nucleus, and it is speculated that it can outcompete activator-containing dimers for binding to CRE sites. Therefore, it blocks memory formation but does not decrease nuclear p35+ abundance. This mechanism of action is identical to the actions of the ICER and S-CREM blockers (Fropf, 2013).
Although the data from the transgenic experiments supports the hypothesis that nuclear entry of the activator is rate-limiting for transcription and memory formation, other possibilities. For example, it is possible that there exists a constant rate of activator import and export from the nucleus, and that memory formation transiently decreases export, thus increasing nuclear abundance. Another possibility is that behavioral training could transiently increase the nuclear stability of the activator protein. More detailed experimentation would be needed to determine the relative contribution of each of these possible mechanisms. However, transgenic experiments argue that regulating nuclear import of the activator is at least partially involved in controlling the strength and duration of dCREB2-responsive transcription (Fropf, 2013).
This study has shown that inducing S6K stimulates nuclear abundance of p35+ and enhances memory formation. These data are the first demonstration that S6K overexpression can enhance long-term memory. Although the correlation between memory formation and an increase in the nuclear abundance of p35+ is clear, the underlying molecular mechanisms are not known. Behavioral training could stimulate translation of the activator protein or decrease its turnover. In addition, training might stimulate nuclear entry and/or decrease nuclear export of the activator protein. Similarly, it is not clear if induction of S6K stimulates translation (as this pathway is known to do), affects nuclear entry/export, or modulates protein stability. The kinetics of the increase in dCREB2 protein in both the nuclear and cytoplasmic compartments cannot distinguish among these possibilities. However, taken together with transgenic experiments, the simplest interpretation is that behavioral training and S6K at least affect nuclear entry of an activator isoform (Fropf, 2013).
One question that arises from the data is why the different antibodies do not recognize a more common pattern of bands. There are nine different dCREB2 protein isoforms reported in the literature. Six out of the nine isoforms contain the bZIP region, while the remaining three use alternative splice sites that result in termination of translation prior to the bZIP region (Tubon, 2013). In addition, there is strong evidence that internal translation initiation can occur at one or more of the three late, in-frame ATG codons just upstream of the bZIP (near the C-terminus of the protein). Therefore, the current estimate is that there are at least six to nine distinct coding isoforms made off of this gene that would contain the bZIP region (Fropf, 2013).
There are seven known, identified phosphorylation sites that are conserved between the mammalian CREB and dCREB2 proteins. In addition, mammalian CREB has been shown to be acetylated, SUMOylated, o-glycosylated, and subject to oxidation-reduction responsive modifications. Almost all of these sites that have been identified on the mammalian protein are conserved on the fly molecules. The possible combinations between protein coding capacity and modification state is very large. For reasons that are not clear, each of seven antibodies that were made, and one mammalian antibody that that this study has used extensively, recognizes some common bands, and some unique ones. It is believe that the combinatorial, [coding + post-translational state] of the protein affects antibody affinity, but the bases for these differences are unclear. However, over the years, the general impression is that certain antibodies recognize certain subsets of bands, and that comparisons need to be made across all of the antibodies to validate specific bands as belonging to dCREB2. Therefore, it is likely that the total number of bands that can be resolved on protein gels is extremely large, and somewhat limited by the availability and relative affinity of each individual antibody (Fropf, 2013).
This report has demonstrated that dCREB2 protein species are enriched in the cytoplasmic compartment. However, at least one species, p35+, increases in nuclear abundance in response to behavioral signals and S6K induction. Moreover, genetic manipulations that block nuclear entry of p35+ also block memory formation. The correlation between behavioral training that produces long-term memory and an increase in p35+ nuclear abundance is consistent with the possibility that nuclear entry is the rate-limiting step for dCREB2-responsive transcription (Fropf, 2013).
CrebB-17A consists of seven isoforms (alternatively spliced forms). The isoforms fall into two categories. In one category, four of the seven isoforms have the basic leucine zipper domain of exon 7. In the second category the b zip domain is absent. The basic zip domain consists of a basic activation domain followed by a leucine zipper domain. A short amino acid motif is a target for phosphorylation by kinases (P-box). The P-box is located carboxyl terminal to a glutamate-rich region (Yin 1994).
date revised: 10 July 99
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