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Phylogenetic tree of PKA catalytic subunits. An unrooted phylogenetic tree (phenogram) was generated from a ClustalW multiple alignment of honeybee (Apis) catalytic subunits with their Drosophila (Dmel) and human (Hsapiens) homologues. The branches are balanced to average the distances between ancestors in the tree. Dotted lines indicate a negative branch length.
Source publication
The cAMP-dependent kinase (PKA) plays a crucial part in long-term memory formation in the honeybee (Apis mellifera). One of the putative substrates of the PKA activity is the cAMP response element binding protein (CREB), a transcription factor in the bZIP protein family. We searched the honeybee genome to characterize genes from the CREB/CREM and t...
Citations
... Biologically meaningful overactive kinases were also identified in the other three cell lines. This included CSKN2A in PML-RARA driven NB4 cells [36], several kinases involved in the PRK/CREB-signaling in MLLdriven THP1 cells [37,38] and kinases of the AKT1pathway in the mutated NPM1-driven OCI-AML1 cells [39] (Suppl. Fig. 1). ...
Background
Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance.
Methods
We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4–11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors.
Results
Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4–11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4–11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks.
Conclusions
We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples.
Graphical abstract
... Activation of ERK/MAPK is required for the formation of contextual and spatial memories in mammals, with impairments to this signaling cascade likely contributing to cognitive decline. Analysis of the honey bee genome has revealed genes encoding PKA subunits (both regulatory and catalytic) and a gene encoding CREB, providing further evidence that this pathway occurs in bees (Eisenhardt et al., 2006). The pathway linking cAMP to CREB gene transcription may also regulate functions inside the mitochondria, as cAMP, PKA, and CREB bound to mitochondrial DNA (mtDNA) have been detected in mammals (Valsecchi et al., 2013). ...
Ongoing honey bee (Apis mellifera) colony losses are of significant international concern because of the essential role these insects play in pollinating crops. Both chemical and non-chemical stressors have been implicated as possible contributors to colony failure; however, the potential role(s) of commonly-used neonicotinoid insecticides has emerged as particularly concerning. Neonicotinoids act on the nicotinic acetylcholine receptors (nAChRs) in the central nervous system to eliminate pest insects. However, mounting evidence indicates that neonicotinoids also may adversely affect beneficial pollinators, such as the honey bee, via impairments on learning and memory, and ultimately foraging success. The specific mechanisms linking activation of the nAChR to adverse effects on learning and memory are uncertain. Additionally, clear connections between observed impacts on individual bees and colony level effects are lacking. The objective of this review was to develop adverse outcome pathways (AOPs) as a means to evaluate the biological plausibility and empirical evidence supporting (or refuting) the linkage between activation of the physiological target site, the nAChR, and colony level consequences. Potential for exposure was not a consideration in AOP development and therefore this effort should not be considered a risk assessment. Nonetheless, development of the AOPs described herein has led to the identification of research gaps which, for example, may be of high priority in understanding how perturbation of pathways involved in neurotransmission can adversely affect normal colony functions, causing colony instability and subsequent bee population failure. A putative AOP network was developed, laying the foundation for further insights as to the role of combined chemical and non-chemical stressors in impacting bee populations. Insights gained from the AOP network assembly, which more realistically represents multi-stressor impacts on honey bee colonies, are promising toward understanding common sensitive nodes in key biological pathways and identifying where mitigation strategies may be focused to reduce colony losses.
... The transcription factor cAMP-response element-binding protein (CREB) is widespread in the animal kingdom. It was cloned in various vertebrate and invertebrate animals, including honeybees (Apis mellifera), where it is termed Apis mellifera CREB (AmCREB) (Gonzalez et al. 1989;Eisenhardt et al. 2003Eisenhardt et al. , 2006Sadamoto et al. 2004;Song et al. 2009;van den Berg et al. 2010). CREB is activated following post-translational modifications such as phosphorylation by protein kinase A (PKA). ...
The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.
... However, this does not mean that insects should learn and remember information from all possible experiences they encounter. Studies on diverse insect species have revealed the daunting complexity of different forms of memory each with different stabilities, including short-term memory, mid-term memory, anesthesia-resistant memory and long-term memory (STM, MTM, ARM and LTM), for example [3][4][5][6]. In Figure 1 we provide a basic description of these memory forms and their abbreviations. ...
... MTM in flies refers to a late phase of STM [70]. STM, ARM and LTM have further been subdivided in early and late forms by other specific inhibitors, see, for example, [4][5][6]. From the combined results of control, cold shock and protein synthesis inhibitor experiments, memory type graphs can be prepared as shown in (c) (left panel). Here the memory type is shown where first STM is formed (orange), followed by ARM (blue) and LTM (green), with the sum of all memories (observed without inhibiting treatments) depicted as yellow. ...
The ability to learn and form memories is widespread among insects, but there exists considerable natural variation between species and populations in these traits. Variation manifests itself in the way information is stored in different memory forms. This review focuses on ecological factors such as environmental information, spatial aspects of foraging behavior and resource distribution that drive the evolution of this natural variation and discusses the role of different genes and neural networks. We conclude that at the level of individual, population or species, insect learning and memory cannot be described as good or bad. Rather, we argue that insects evolve tailor-made learning and memory types; they gate learned information into memories with high or low persistence. This way, they are prepared to learn and form memory to optimally deal with the specific ecologies of their foraging environments.
... Both could also be possible target candidates for miRNAs in the honeybee. There were eight isoforms of CREB described to exist in the honeybee (Eisenhardt et al. 2003;Eisenhardt et al. 2006). Anyway, this hypothesis at first has to be proven by different target prediction and validation methods which will be discussed later in more detail. ...
... The honeybee and several ant species can be discriminated from solitary species based on the abundance of binding sites for the transcription factor cAMP-dependent response element binding protein (CREB) (Simola et al., 2013a). In addition, CREB is suggested to be involved in polyethism: In ants, a coactivator of CREB-dependent transcription (CREB-binding protein [CBP]) is involved in caste differentiation (Simola et al., 2013b), and the Apis mellifera CREB homolog AmCREB (Eisenhardt et al., 2003(Eisenhardt et al., , 2006 was identified as a potential regulator of age-dependent behavior in honeybees (Chandrasekaran et al., 2011;Ament et al., 2012;Khamis et al., 2015). However, these results stem from bioinformatic analyses of genomic and transcriptomic data. ...
... Therefore, CREB phosphorylation at this PKA phosphorylation site is regarded as an indicator for activated CREB (Mayr and Montminy, 2001). The KID including the PKA consensus-phosphorylation site is highly conserved between mammals and invertebrates (Bartsch et al., 1995;Eisenhardt et al., 2006;Song et al., 2007;van den Berg et al., 2010). Therefore, we here analyzed the localization of AmCREB phosphorylated at this conserved PKA consensus sequence (in honeybees located around S142) in the honeybee brain and asked whether the level of phosphorylated AmCREB (pAmCREB) is associated with age. ...
... AmCREB shares principle protein domains with human CREB such as the KID that contains a PKA phosphorylation site at S133 (ScanProsite Tool, release 20.87, http://prosite.expasy.org/ scanprosite; see also Eisenhardt et al., 2003Eisenhardt et al., , 2006. A BLAST analysis revealed that the amino acid sequence around this phosphorylation site differs only at two positions between human CREB1 (S133, isoform AmCREB1, accession number CAD23079.1) ...
Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP‐response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age‐dependent division of labor. In honeybee brains, CREB‐dependent genes are regulated in an age‐dependent manner, indicating that there might be a role for neuronal honeybee CREB ( Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB‐dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB‐dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age‐dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age‐dependent increase of pAmCREB.
Our results demonstrate the IC to be candidate neurons involved in age‐dependent division of labor. We hypothesize that the IC display a high level of CREB‐dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior. J. Comp. Neurol. 524:1165–1180, 2016. © 2015 Wiley Periodicals, Inc.
... AmCREB, a CREB homolog containing the KID domain and the highly con- served DNA binding and dimerization domain, the basic region- leucin zipper (bZIP), has been isolated from honeybee brain. This suggests that AmCREB is a possible target of learning-dependent alterations of cAMP and Ca 2+ ( Eisenhardt et al. 2003Eisenhardt et al. , 2006). ...
The honeybee (Apis mellifera) has long served as an invertebrate model organism for reward learning and memory research. Its capacity for learning and memory formation is rooted in the ecological need to efficiently collect nectar and pollen during summer to ensure survival of the hive during winter. Foraging bees learn to associate a flower's characteristic features with a reward in a way that resembles olfactory appetitive classical conditioning, a learning paradigm that is used to study mechanisms underlying learning and memory formation in the honeybee. Due to a plethora of studies on appetitive classical conditioning and phenomena related to it, the honeybee is one of the best characterized invertebrate model organisms from a learning psychological point of view. Moreover, classical conditioning and associated behavioral phenomena are surprisingly similar in honeybees and vertebrates, suggesting a convergence of underlying neuronal processes, including the molecular mechanisms that contribute to them. Here I review current thinking on the molecular mechanisms underlying long-term memory (LTM) formation in honeybees following classical conditioning and extinction, demonstrating that an in-depth analysis of the molecular mechanisms of classical conditioning in honeybees might add to our understanding of associative learning in honeybees and vertebrates.
... Hierzu wurden zwei unterschiedlichen IP-Elutionspuffer getestet, ein keine DNA Bindungsdomäne aufweist, [214] zeigt nur in der Region mit cis-Elementen (+) eine sehr schwache Bande. Diese könnte auf unspezifisch gebundene DNA-Fragmente beruhen oder die PKA bindet indirekt an die DNA, eventuell über die ...
... Bindung an Creb. [112,214,215] Somit konnte gezeigt werden, dass die ChIP-Methode funktioniert. Da bei der PKA unklar ist, ob sie an die DNA binden kann, wurde für nachfolgende Versuche als Negativkontrolle ein unspezifischer IgG-Antikörper verwendet, der an kein Protein binden soll und bereits in anderen ChIP-Experimenten zur Anwendung kam. ...
Zur Analyse der Genregulation durch die Bindungen von Transkriptionsfaktoren an DNA, wurde in dieser Arbeit zum ersten Mal die Chromatin-Immunopräzipitation (ChIP) für Gewebe in der Biene etabliert. Hierbei konnte durch gezielte Optimierung, bei gleicher Effizienz der CHIP-Methode, die Menge des notwendigen Gewebes um den Faktor 10 bis 50 reduziert werden. An den Creb- und Jun-like-Promotoren wurden Trankriptionsfaktor-Bindungsstellen (TFBS) identifiziert und die Funktionalität der Methode demonstriert. Für den weitergehenden Einsatz der ChIP-Methode wurden Antikörper gegen den Transkriptionsfaktor Creb für die Biene hergestellt. Die Anwendung der ChIP-Methode in Verbindung mit der Hochdurchsatzsequenzierung (ChIP-seq) liefert erste Hinweise auf genomweite Bindungsstellen des Activating-Transcription-Factor-2 (Atf-2) in neuronalen Zellen des Bienengehirns. Die Untersuchungen von TFBS aus der ChIP-seq und der Korrelation mit der mRNA Expression zeigen, dass für aussagekräftige Erklärungen zur genomweiten Genregulation, Experimente in einem viel größerem Maßstab notwendig sind. Die Grundlagen für solche zukünftigen Untersuchungen an kleinen Gewebeproben sind jetzt durch diese Arbeit geschaffen.
... This gene is especially active in the mushroom body, and works together with several other genes including dunce and rutabaga to regulate cAMP levels (Gervasi et al. 2010 ). In honey bees, cAMP-dependent protein kinase c2 (GB16164) is one of three genes encoding catalytic subunits of cAMP-dependent kinase (PKA), which plays a crucial part in long term memory formation by contributing to long term synaptic plasticity (Eisenhardt et al. 2006). Interestingly, previous research on honey bee defensive behavior identified the first catalytic subunit of cAMP-dependent kinase (PKA) within a QTL region for colony-level defense (Hunt 2007). ...
... Interestingly, previous research on honey bee defensive behavior identified the first catalytic subunit of cAMP-dependent kinase (PKA) within a QTL region for colony-level defense (Hunt 2007). This subunit is also unique in insects, as it has no counterpart in mammals (Eisenhardt et al. 2006). The guarding QTL also contained a sequence of homeobox genes controlling early development. ...
In order to identify genes that are influencing defensive behaviors, we have taken a new approach by dissecting colony-level defensive behavior into individual behavioral measurements using two families containing backcross workers from matings involving European and Africanized bees. We removed the social context from stinging behavior by using a laboratory assay to measure the stinging response of individual bees. A mild shock was given to bees using a constant-current stimulator. The time it took bees to sting in response to this stimulus was recorded. In addition, bees that were seen performing guard behaviors at the hive entrance were collected. We performed QTL mapping in two backcross families with SNP probes within genes and identified two new QTL regions for stinging behavior and another QTL region for guarding behavior. We also identified several candidate genes involved in neural signaling, neural development and muscle development that may be influencing stinging and guarding behaviors. The lack of overlap between these regions and previous defensive behavior QTL underscores the complexity of this behavior and increases our understanding of its genetic architecture.
... mellifera), and CREBa (Mus musculus and Homo sapiens). The exon borders and functional domains for CgCREB are based on the AmCREB5 amino acid sequence (Eisenhardt et al., 2003(Eisenhardt et al., , 2006. The Cotesia CREB amino acid sequences share 83% identity with AmCREB5, 28% identity with dCREB2a, and 31% identity with mouse CREBa. ...
... Elucidation of CREB expression and its role in learning and memory formation is inherently complex. For example, in mammals three members of the CREB family of transcription factors have been identified: CREB, CREM and ATF-1 (Hoeffler et al., 1988;Rehfuss et al., 1991;Foulkes et al., 1992) and alternative splicing of these genes results in the expression of several splice The exons are numbered E1-E10, based on (Eisenhardt et al., 2003(Eisenhardt et al., , 2006. (See also Table 5 for the number of clones found of each of these transcripts). ...
... Patterns of CREB expression comparable to those in mammals have been found in other organisms such as the sea hare A. californica (Bartsch et al., 1995(Bartsch et al., , 1998 and the pond snail Lymnaea stagnalis (Sadamoto et al., 2004b), both of which have more than one CREB gene. In the insect Ap. mellifera, however, there is only one CREB gene, AmCREB that plays a role in memory formation (Eisenhardt et al., 2006). Even so, in Drosophila only one member of the CREB/ CREM family was found in a search for bZIP proteins in the drosophila genome dCREB2 (Yin et al., 1995a), whereas another previously reported CREB gene (dCREB-A) (Smolik et al., 1992) appears to be related to the human Oasis gene but not to CREB/CREM (Fassler et al., 2002). ...
The cAMP/PKA signalling pathway and transcription factor cAMP response element-binding protein (CREB) play key roles in long-term memory (LTM) formation. We used two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, which were previously shown to be different in LTM formation, and sequenced at least nine different CREB transcripts in both wasp species. The splicing patterns, functional domains and amino acid sequences were similar to those found in the CREB genes of other organisms. The predicted amino acid sequences of the CREB isoforms were identical in both wasp species. Using real-time quantitative PCR we found that two low abundant CREB transcripts are differentially expressed in the two wasps, whereas the expression levels of high abundant transcripts are similar.
