Gene name - Protein kinase, cAMP-dependent, catalytic subunit 1
Synonyms - DC0, PKA, cAMP-dependent protein kinase 1
Cytological map position - 30C1--2
Function - Signal transduction
Symbol - Pka-C1
Genetic map position - 2-
Classification - cAMP-dependent protein kinase A, serine/threonine kinase
Cellular location - cytoplasmic
|Recent literature||Pavot, P., Carbognin, E. and Martin, J. R. (2015). PKA and cAMP/CNG channels independently regulate the cholinergic Ca(2+)-response of Drosophila mushroom body neurons. eNeuro 2 [Epub ahead of print]. PubMed ID: 26464971
This work investigated the role of mushroom bodies (MBs) in olfactory learning and memory. Advantage was taken of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. Neuronal Ca(2+)-activity was imaged continuously, over a long time period, and the nicotine-evoked Ca(2+)-response was caracterized. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, it was first shown that the Ca(2+)-response is proportional to the levels of cAMP. Second, it was reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. These results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.
|Gonzales, E. D., Tanenhaus, A. K., Zhang, J., Chaffee, R. P. and Yin, J. C. (2015). Early onset sleep defects in Drosophila models of Huntington's Disease reflect alterations of PKA/CREB Signaling. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26604145
Huntington's Disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. This study reports that Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in the HD models. It was asked whether aberrations in PKA/CREB signaling were responsible for these early onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway was shown to suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, these data suggests that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention.
|Getahun, M. N., Thoma, M., Lavista-Llanos, S., Keesey, I., Fandino, R. A., Knaden, M., Wicher, D., Olsson, S. B. and Hansson, B. S. (2016). Intracellular regulation of the insect chemoreceptor complex impacts odor localization in flying insects. J Exp Biol 219(Pt 21):3428-3438. PubMed ID: 27591307
Flying insects are well-known for airborne odor tracking, and evolved diverse chemoreceptors. While ionotropic receptors (IRs) are found across Protostomes, insect odorant receptors (ORs) have only been identified in winged insects. It was therefore hypothesized that the unique signal transduction of ORs offers an advantage for odor localization in flight. Using Drosophila, expression and increased activity of the intracellular signaling protein, PKC, in was found antennal sensilla following odor stimulation. Odor stimulation also enhances phosphorylation of the OR coreceptor, Orco, in vitro, while site directed mutation of Orco or mutations in PKC subtypes reduces sensitivity and dynamic ranges of OR-expressing neurons in vivo, but not IRs. It was ultimately shown that these mutations reduce competence for odor localization of flies in flight. We conclude that intracellular regulation of OR sensitivity is necessary for efficient odor localization, which suggests a mechanistic advantage for the evolution of the OR complex in flying insects.
|Richlitzki, A., Latour, P. and Schwarzel, M. (2017). Null EPAC mutants reveal a sequential order of versatile cAMP effects during Drosophila aversive odor learning. Learn Mem 24(5): 210-215. PubMed ID: 28416632
This study defines a role of the cAMP intermediate EPAC in Drosophila aversive odor learning by means of null epac mutants. Complementation analysis revealed that EPAC acts downstream from the rutabaga adenylyl cyclase and in parallel to protein kinase A. By means of targeted knockdown and genetic rescue, mushroom body Kenyon cells (KCs) were identified as a necessary and sufficient site of EPAC action. Mechanistic insights were provided by analyzing acquisition dynamics and using the "performance increment" as a means to access the trial-based sequential organization of odor learning. Thereby it was shown that versatile cAMP-dependent mechanisms are engaged within a sequential order that correlate to individual trials of the training session.
|Chen, P., Zhou, Z., Yao, X., Pang, S., Liu, M., Jiang, W., Jiang, J. and Zhang, Q. (2017). Capping enzyme mRNA-cap/RNGTT regulates Hedgehog pathway activity by antagonizing Protein kinase A. Sci Rep 7(1): 2891. PubMed ID: 28588207
Hedgehog (Hh) signaling plays a pivotal role in animal development and its deregulation in humans causes birth defects and several types of cancer. Protein Kinase A (PKA) modulates Hh signaling activity through phosphorylating the transcription factor Cubitus interruptus (Ci) and G protein coupled receptor (GPCR) family protein Smoothened (Smo) in Drosophila, but how PKA activity is regulated remains elusive. This study identified a novel regulator of the Hh pathway, the capping-enzyme mRNA-cap, which positively regulates Hh signaling activity through modulating PKA activity. Genetic and biochemical evidence is provided that mRNA-cap inhibits PKA kinase activity to promote Hh signaling. Interestingly, regulation of Hh signaling by mRNA-cap depends on its cytoplasmic capping-enzyme activity. In addition, the mammalian homolog of mRNA-cap, RNGTT, can replace mRNA-cap to play the same function in the Drosophila Hh pathway, and knockdown of Rngtt in cultured mammalian cells compromised Shh pathway activity, suggesting that RNGTT is functionally conserved. This study makes an unexpected link between the mRNA capping machinery and the Hh signaling pathway, unveils a new facet of Hh signaling regulation, and reveals a potential drug target for modulating Hh signaling activity.
|Vagnoni, A. and Bullock, S. L. (2018). A cAMP/PKA/Kinesin-1 axis promotes the axonal transport of mitochondria in aging Drosophila neurons. Curr Biol 28(8): 1265-1272.e1264. PubMed ID: 29606421
Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms, and has been proposed to contribute to the broader decline in neuronal function that occurs during aging. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. This study provides evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, the results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. This study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons.
Drosophila research is currently uncovering the role G-protein coupled receptors play in development. G-protein coupled receptors have been extensively characterized for their involvement in adult physiology (see The Learning Pathway). In mammals for example, G-protein coupled receptors are commonly found for hormones, neuropeptides and neurotransmitters. However, the involvement of G-protein coupled receptors in developmental pathways is a fairly new area of inquiry.
Cyclic AMP is the classic second messenger (see Cyclic AMP Second Messenger System for more information). It is used in Drosophila to transduce external signals sensed by G protein coupled receptors (e.g., Smoothened) or as a signal transducer of cytoplasmic events such as a change in Ca++ levels, for example, sensed by Calmodulin, which then interacts with Rutabaga).
Mammalian cyclic AMP-dependent protein kinase A (PKA) may serve here as a model for its Drosophila protein counterpart. The mammalian protein is a tetramer composed of two regulatory subunits and two catalytic subunits. In the holoenzyme, the enzymatic activity of the catalytic subunit, coded for by the gene described here, PKA-C1) is latent because of the inhibition conferred by the inhibitory domain of the regulatory subunit. When two mammalian cAMP molecules bind per regulatory subunit, the affinity of the regulatory subunit for the catalytic subunit decreases 10,000-100,000 fold, the tetramer dissociates into dimeric regulatory subunits, two monomers of the catalytic subunit are released, and inhibition is relieved. The catalytic subunit is subject to autophosphosphorylation that may stabilize the catalytic site and foster optimal alignment of peptide substrate within the site for phosphotransfer. Likewise, the catalytic subunit phosphorylates the regulatory subunit in an autoinhibitory domain (Francis, 1994).
Mutation in the catalytic subunit of PKA mimics the phenotype generated by ectopic expression of hedgehog in the anterior compartment of wing and leg discs and ahead of the morphogenetic furrow in the developing eye. That is, either PKA mutation or hedgehog ectopic expression induce ectopic expression of decapentaplegic, wingless and patched. Based on this evidence, it has been suggested that PKA is a component of a signaling pathway that represses dpp, wg and ptc expression and that hh antagonizes this pathway to maintain target gene expression at the anterior-posterior compartment border of appendages and in the morphogenetic furrow (Pan, 1995 and Li, 1995).
With the recent purification of Smoothened, the HH receptor, these findings can now be placed in a broader context. Smoothened is a 7 pass transmembrane receptor, characteristic of G-protein coupled receptors. As mentioned above, these receptors have well characterized roles as both hormone and neurotransmitter signal transducers, but Smoothened has a developmental role as HH receptor (Alcedo, 1996). Like other members of the serpentine receptor family, Smoothened is also coupled to G proteins. The presumed target of SMO is Adenylate cyclase (See Rutabaga), an enzyme that converts ATP to cyclic AMP. In turn, cAMP regulates Protein kinase A.
The target of PKA in segmentation is not yet known, although recent work suggests that this target is likely to be Cubitus interruptus (see below). In the learning pathway the target is CREB, the cyclic AMP response element binding protein, a transcription factor activated by PKA phosphorylation. Likewise, PKA targeted CREB is implicated in the regulation of proliferation of anterior pituitary somatotropic cells. Blocking CREB function in transgenic mice prevents proliferation of somatotropic cells, resulting in pituitary atrophy and dwarfism (Struthers, 1991).
PKA has a another documented role in Drosophila, in addition to signal transduction in segmentation and learning. PKA is associated with germ cell membranes and involved in establishment of oocyte polarity, acting to transduce a signal for microtubule reorganization involved in oocyte mRNA localization (Lane, 1994 and 1995).
PKA also controls quantal size in the Drosophila neuromuscular junction (NMJ). The NMJ is a glutamatergic synapse (the neurotransmitter is glutamate) with ultrastructural similarities to glutamatergic central synapses of vertebrates. Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. The gene encoding a second muscle-specific glutamate receptor, DGluRIIB, has been cloned and is closely related to the previously identified DGluRIIA, which is located adjacent to it in the genome. Both are non-NMDA type but cannot be classified as either AMPA or kainate type receptors. Both genes are expressed in muscle from stage 12, and neither gene is expressed in the nervous system. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal, because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function (Petersen, 1997).
Two distinct mechanisms regulate synaptic efficacy at the Drosophila neuromuscular junction: a PKA-dependent modulation of quantal size and a retrograde regulation of presynaptic release. Postsynaptic expression of a constitutively active PKA catalytic subunit decreases quantal size, whereas overexpression of a mutant PKA regulatory subunit (inhibiting PKA activity) increases quantal size. Increased PKA activity also decreases the response to direct iontophoresis of glutamate onto postsynaptic receptors. The PKA-dependent modulation of quantal size requires the presence of the muscle-specific glutamate receptor DGluRIIA, since PKA-dependent modulation of quantal size is lost in viable homozygous DGluRIIA- mutants. The DGluRIIA sequence contains an optimal PKA consensus phosphorylation site on the C-terminal tail (RRXS), believed to be located in the cytoplasmic portion of the protein. Elevated postsynaptic PKA reduces the quantal amplitude and the time constant of miniature excitatory junctional potential (mEJP) decay to values that are nearly identical to those observed in DGluRIIA mutants. PKA modulation of quantal size is sensitive to the copy number of DGluRIIA. Larvae heterozygous for a deletion of DGluRIIA show significantly less modulation by PKA than wild-type controls. This suggests that PKA-dependent modulation of receptor function may be influenced by the subunit composition of postsynaptic receptors. PKA activity appears to constitutively regulate synaptic function at the wild-type synapse. The demonstration that inhibition of PKA leads to a large increase in quantal size suggests that there is a high basal phosphorylation of DGluRIIA at the wild-type synapse. The PKA-dependent reduction in quantal size is accompanied developmentally by an increase in presynaptic quantal content, indicating the presence of a retrograde signal that regulates presynaptic release. A retrograde regulation of presynaptic transmitter release may serve to maintain postsynaptic excitation during the developmental growth of this synapse (Davis, 1998).
Analysis of protein kinase A (PKA) has been initiated in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. GAL4/UAS-regulated transgenes were constructed in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. Evidence is provided that the wild-type transgenes are active, and a summary is given of the phenotypes produced by a number of GAL4 enhancer-detector strains. The effects of transgenes encoding PKI(1-31) are compared with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits (Kiger, 1999).
Phenotypes produced by PKI(1-31) and PKAr* are surprisingly different. The phenotypic effects of PKI(1-31) appear to represent a subset of those of PKAr*. Both retard or otherwise block larval growth. PKAr* alone affects patterning in embryos and imaginal discs by activating Hedgehog signaling, and it alone causes abnormal differentiation in imaginal discs (which may reflect minor aberrations in patterning). The origin of this difference might reside in some fundamental difference in the biological properties of PKI(1-31) and PKAr* or perhaps in their relative stabilities in different cell types. However, PKI(1-31) is demonstrably active in wing imaginal discs and in other tissues since it is capable of inhibiting ectopic PKAcF (epitope tagged PKA catalytic subunit). Regardless of the origin of the difference, it would appear that PKI(1-31) specifically targets larval growth (Kiger, 1999).
Newly hatched larvae consist of two cell types: (1) mitotic cells composing the imaginal discs, gonad, and some neuroblasts, and (2) endoreplicating cells making up the exclusively larval tissues. These latter cells do not divide after hatching, but they increase in size as the larva grows, being maintained by cycles of DNA replication without nuclear division. These two cell types are regulated in fundamentally different ways, as demonstrated by their responses to nutritional deprivation. It would appear that mitotic cells are not sensitive to expression of PKI(1-31), but only to expression of PKAr*, whereas endoreplicating cells are sensitive to both. Both proteins are effective inhibitors of the catalytic site of PKAc, possessing a pseudosubstrate binding site with a pair of adjacent Arg residues that interact with the catalytic site. PKAr, which is larger than PKI(1-31) or full-length PKI(1-77), makes additional contacts with PKAc that make the PKAr:PKAc complex more stable than the PKI(1-77):PKAc complex. PKI(5-24) and PKI(1-77) bind to PKAc with the same affinity, and PKI(1-31) is probably no different. The C terminus of PKI(5-24) is not involved in binding to PKAc, so the FLAG epitope at the C terminus of PKI(1-31) should not interfere with its inhibitory function. Free PKAc and PKI(1-77) are small enough to enter the nucleus by diffusion. PKI(1-77) possesses a nuclear export signal (residues 35-49) that is hidden until PKAc is bound, whereupon the PKI(1-77):PKAc complex is extruded from the nucleus. Moreover, the expression and intracellular distribution of PKI(1-77) is regulated during the cell cycle and is necessary for cell cycle progression. PKI(1-31) would be expected to inhibit both nuclear and cytoplasmic PKAc. In addition, PKI(1-31) could compete with a Drosophila homolog of PKI(1-77) for nuclear PKAc and block PKAc export. In contrast, PKAr is cytoplasmic whether or not it is complexed with PKAc because it is too large to enter the nucleus. In addition, anchoring proteins have been identified that bind PKAr to the membrane or cytoskeleton. PKAr* would be expected to inhibit cytoplasmic PKAc and to deplete nuclear PKAc by forming a cytoplasmic sink for PKAc that diffuses from the nucleus (Kiger, 1999 and references).
With regard to Hedgehog signaling, a possible target of PKAr* and PKI(1-31) in the cytoplasm would be the complex responsible for the proteolysis of the transcription factor Ci, where PKAr* and PKI(1-31) would inhibit phosphorylation of the PKA sites necessary for proteolysis of Ci155 to the repressor form Ci75. The ability of PKAr* to interact with anchoring or other proteins might give it greater access to this complex than PKI(1-31), accounting for the failure of the latter to activate Hedgehog target genes (Kiger, 1999).
Another possible explanation for the different actions of PKAr* and PKIF(1-31) is that free PKAr* (and by implication free PKAr) has a target other than PKAc through which it activates Hedgehog signaling. Precedent for such a role exists. In Dictyostelium, free PKAr binds and activates a cAMP-specific phosphodiesterase that is postulated to have functional homology to the cAMP-specific phosphodiesterase encoded by dunce. The Dictyostelium phosphodiesterase is also activated by bovine PKAr1a, and a synthetic monomeric form of this regulatory subunit is a more potent activator than the dimeric form. (The Dictyostelium PKAr protein lacks a dimerization domain, and its PKA exists as a heterodimer). In this scenario, in the absence of a cAMP signal, PKAc would bind to PKAr, inhibiting this novel activity. Reduction in the level of PKAc, e.g., in a mitotic clone of cells homozygous for a lethal allele of DC0, would lead to free PKAr that would activate Hedgehog signaling (Kiger, 1999 and references).
In an alternative scenario, the effect of PKAr* on Hedgehog target genes could be caused by its ability to deplete nuclear PKAc, a role that cannot be fulfilled by PKI(1-31). Since the normal role of PKI(1-77) is not only to inhibit, but to export, nuclear PKAc, it is possible that PKAc plays another critical role in the nucleus in addition to its catalytic role in phosphorylation. For example, PKAc might function as a corepressor with Ci75 to block transcription of Hedgehog target genes. Consistent with this hypothesis PKI(1-60) has been shown to activate Ci-mediated chloramphenicol acetyltransferase transcription from a model Gli enhancer in Drosophila Kc cells, a finding can be attribute solely to inhibition of proteolysis of cytoplasmic Ci155. It may be that PKAc can function as a corepressor even if its catalytic site is occupied by PKI(1-31). Corepression by PKI(1-31):PKAc and Ci75 might block transcription of target genes, even in the presence of Ci155 produced by concommitant inhibition of Ci155 proteolysis in the cytoplasm. Small changes in the ratio of Ci155 and Ci75 are believed to be critical for activation of Hedgehog target genes. In addition, PKI(1-77) may differ from PKI(1-31) because only the former reduces basal transcription from cAMP-stimulated promoters. If PKAc has such an additional role, then the R224 mutant must have lost this function, as well as its ability to bind PKAr* and PKIF, since PKAcR224F produces no abnormal phenotypes and has no effect on viability. On the other hand, the hypothesized nuclear role of PKAc might be catalytic if nuclear PKAc is in some way inaccessible to nuclear PKI(1-31) (Kiger, 1999 and references).
These considerations suggest that normal Hedgehog signal transduction may require both inhibition of cytoplasmic PKAc activity and export of nuclear PKAc. The Drosophila homolog of PKI(1-77) would be a good candidate for carrying out these functions. The fact that PKI(1-77) seems to play some role in regulating the cell cycle may help to explain why PKI(1-31) has different effects on endoreplicating cells and mitotic cells. Resolving the nature of the roles played by PKAc in the cytoplasm and in the nucleus may lead to simultaneous understanding of the effects seen here on pattern formation and on cell growth (Kiger, 1999 and references).
Direct comparisons of the effects of PKI(1-31) and of PKI(1-77) are needed to provide more insight into how different PKAc inhibitors are functioning. PKAc transgenes with specific catalytic site mutations should provide evidence for or against a noncatalytic nuclear role for PKAc. PKAr* transgenes with domain-specific mutations should provide insight into the role of PKAR* in Hedgehog signaling. Identification of a Drosophila homolog of PKI(1-77) and study of its regulation will be important to achieve a clear understanding of the roles of PKAc. From a practical standpoint, PKI(1-31) transgenes should provide a useful tool for investigating the role of PKA in larval growth regulation, independent of PKA's effects on pattern formation. Mutations that permit larvae to survive the effect of PKI(1-31) and develop to adults should help to identify elements controlling larval growth. Conversely, mutations that sensitize adults or embryos to PKI(1-31) may reveal elements important for pattern formation (Kiger, 1999).
The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).
Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).
Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).
GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).
There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).
Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).
Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).
The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).
Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).
At least four RNA species with differing sizes derive from the catalytic subunit gene. The relative abundance of these RNA forms varies at different times during development; the shortest species is particularly prominent in embryos and the longest in adults. The size differences among these RNA species occur principally at the 3' ends, perhaps by the use of different sites of polyadenylation (Kalderon, 1988).
Exons - 2
Pka-C1 is 82% identical to the mammalian catalytic subunit of the cAMP-dependent protein kinase (Kalderon, 1988).
Since a basic surface on the catalytic (C) subunit of mammalian cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Seven specific carboxylates in RIalpha have been identified that are protected from chemical modification in the holoenzyme; each was then replaced with alanine. Of these, mutated rRI amino acids E15, E106, D107, E105, D140, E143, and D258 all are defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha, were mutated to Ala. Replacement of Glu101 has no effect while rRII E99 is very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, D170A and K242A, inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability (Gibson, 1997).
Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. The active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core. Also, the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis. In addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal "tail" (Shaltiel, 1998).
date revised: 21 APR 97
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