cAMP-dependent protein kinase 1


Table of contents

PKA as a transcription factor

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in the catabolite repressor protein and the regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. It is here reported that the RIIbeta subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIbeta to a CRE is enhanced by cAMP; in addition, RIIbeta exhibits transcriptional activity as a Gal4-RIIbeta fusion protein. These experiments identify RIIbeta as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells (Srivastava, 1998).

PKA, Vesicular Budding and Transport

The role played by protein kinase A (PKA) was examined in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step is inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurs at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlates with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied in vitro using vesicular stomatitis virus-infected, permeabilized cells. Vesicle release is stimulated by the addition of C-PKA to this assay; it is suppressed by the PKA inhibitory peptide, H89, and antibody against C-PKA. Vesicle release is decreased when PKA-depleted cytosol is used, and it is restored by the addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN (Muniz, 1997).

Melanophores, cells specialized for regulated organelle transport, were used to study signaling pathways involved in the regulation of transport. Immortalized Xenopus melanophores were transfected with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogs of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus that increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. It is concluded that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A (Reilein, 1998).

Synaptic vesicle traffic is supported by transient actin filaments and regulated by PKA and NO

Synaptic vesicles (SVs) can be pooled across multiple synapses, prompting questions about their dynamic allocation for neurotransmission and plasticity. This study found that the axonal traffic of recycling vesicles in primary cultures of hippocampal neurons is not supported by ubiquitous microtubule-based motility but relies on actin instead. Vesicles freed from synaptic clusters undergo ~1 microm bouts of active transport, initiated by nearby elongation of actin filaments. Long distance translocation arises when successive bouts of active transport were linked by periods of free diffusion. The availability of SVs for active transport can be promptly increased by protein kinase A, a key player in neuromodulation. Vesicle motion is in turn impeded by shutting off axonal actin polymerization, mediated by nitric oxide-cyclic GMP signaling (see Nitric oxide synthase) leading to inhibition of RhoA. These findings provide a potential framework for coordinating post-and pre-synaptic strength, using retrograde regulation of axonal actin dynamics to mobilize and recruit presynaptic SV resources (Chenouard, 2020).

Focusing on presynaptic vesicles that undergo activity-dependent fusion/retrieval, this study revealed that their axonal movements are longitudinally bidirectional, depend on actin, and do not require nocodazole-sensitive microtubules. SV AT was not detectably affected by nocodazole or colchicine, a microtubule-disrupting agent with a distinct mechanism. In contrast, the motion of vesicular transport packets, labeled via VAMP2/synaptobrevin but not endocytosed markers, was confirmed to be strongly directionally-biased and microtubule-dependent. These distinctions extend current views of axonal membrane traffic that emphasize microtubules and recall photobleaching experiments that first implicated actin in interbouton SV motion (Chenouard, 2020).

To clarify the structural determinants of SV dynamics, two-color imaging was used to monitor the motion of recycling vesicles while simultaneously tracking actin elongation. The initiation of vesicle movement corresponded spatiotemporally with the arrival of growing actin structures. Rather than actin-driven propulsion, the experiments support repeated binding and unbinding of SV to the newly laid actin track together with motor-mediated processive motion, in line with myosin V inhibition experiments. F-actin nucleation and elongation preceded SV transport, rather than being triggered by it as in Rab11a transport in oocytes. The formation of fresh actin tracks dictated the rate of SV AT occurrence and the speed of filament elongation put a ceiling on vesicle velocity. Actin filament polymerization and SV AT were slowed to a similar degree by exposure to latrunculin A, or by manipulating NO-cGMP-RhoA signaling. Unlike actin-based transport of axonal organelles in invertebrate axons, which persists after phalloidin-based actin stabilization, actin stabilization abolished SV traffic. Beyond differences in how actin polymerization is controlled or exploited, a common feature is that vesicle motion depends on actomyosin interaction. Continuously tracking QD-containing SVs, it was affirmed that they can travel for long distances along axons, doing so by combining episodes of directed AT, interspersed with diffusive motion. AT frequently started and ended at the periphery of synaptic clusters, but never in their core, confirming that AT reflected extra- rather than intra-synaptic mobility. Others have implicated SV-actin interactions in sub-diffusive motion, oscillating states, and mobility within synaptic clusters, possibly via acto-myosin2 complexes. In contrast, a major role for myosin II in extrasynaptic SV mobility was not apparent from a pharmacological intervention. Myosin V, a processive motor, seems better suited to carry out repetitive AT along axons. Interspersed diffusive pauses began when vesicles ceased to associate with nearby actin structures; directed motion resumed both without and with the arrival of a new growing actin structure as if temporarily disengaged vesicles can either re-engage with the original track or with a fresh one (Chenouard, 2020).

SVs moved bi-directionally, without an obviously preferred direction under basal conditions, enabling axonal re-distribution of SVs among synaptic sites. Dynamic actin filaments are suited for this function in growing in both axonal directions without strong bias. In contrast, axonal microtubules offer greater stability and singular polarization, befitting efficient and unidirectional fast transport between distant, pre-determined sites (soma to remote synaptic sites or vice-versa) (Chenouard, 2020).

By revealing checkpoints for SV trafficking, this work suggested scenarios by which neuromodulation could regulate interbouton exchange in association with learning. Intracellular cAMP can be elevated by dopamine-, serotonin- or activity-dependent activation of adenylate cyclase. cAMP elevation with forskolin enriched vesicles in the periphery of synaptic clusters, likely mediated by PKA phosphorylation of vesicular synapsin, liberating vesicles from synaptic confinement. Alternatives were considered such as PKA-mediated modulation of myosin V or myosin light chain kinase. Freeing vesicles from cytoskeletal constraints with PKA would make more vesicles eligible for interbouton trafficking, enlarging the vesicular 'superpool'. Indeed, it was observed that PKA stimulation increased the abundance of AT events without changing actin elongation or AT unitary properties (Chenouard, 2020).

Postsynaptic NMDA receptor activation locally enhances post- and presynaptic function in certain forms of LTP. Presynaptic enhancement is reflected by elevation of mini frequency, of uptake of presynaptic vesicular tags, of the number of synaptophysin-immunopositive puncta and of interbouton vesicle traffic. It has been shown that SV proteins accumulate at potentiated synapses by means of signaling via NO, cGMP, PKG, and RhoA. The observations of this study‚Äč provide a way to link plasticity-inducing signals to the recruitment of presynaptic vesicular resources. It is propose dthat retrograde signaling by NO drives a local cascade of NO > ↑cGMP > ↑PKG > ↑RhoA to inhibit actin elongation and stall SV transport, enabling recruitment of stalled vesicles into synaptic pools at presynaptic sites destined for potentiation. This study found that raising NO or cGMP, key players in retrograde signaling, dampened actin filament elongation, and vesicle transport, involving both mDia1- and ROCK-dependent branches of signaling between RhoA and actin dynamics (Chenouard, 2020).

Such local recruitment of SVs could operate in tandem with a generalized PKA-dependent mobilization of SVs to concentrate SVs at potentiated postsynaptic sites, thus coordinating pre- and postsynaptic capabilities. By supporting short-haul SV traffic, actin-based SV movement facilitates local retrograde control, serving an altogether different function than microtubule-based, long-range, movements of transport vesicles (Chenouard, 2020).

PKA and axon pathfinding

The role of intracellular protein kinase A (PKA) signaling in the axonal pathfinding of olfactory sensory neurons has been examined in transparent zebrafish embryos. Microinjection of an appropriate vector directs the expression of both the dominant form of PKA and green fluorescent protein fused with the microtubule-associated protein tau in the same olfactory neurons. The dominant-negative form of PKA enhances the turning of olfactory neuron axons in the olfactory placode, whereas the disturbance effect of the constitutively active form on the axonal pathfinding is prominent in the olfactory bulb. Consistently, forskolin treatment severely inhibits axonal extension in the olfactory bulb, but not in the olfactory placode. These results suggest that the switching of PKA signaling in developing olfactory sensory neurons is important for axonal pathfinding through the boundary between the olfactory placode and the olfactory bulb in vivo. It is thus proposed that the regulation of PKA signaling plays a key role in the long-distance axonal pathfinding through intermediate guideposts (Yoshida, 2002).

PKA and CREB regulation of odorant receptor axonal projection

In mammals, odorant receptors (ORs) direct axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. G protein-mediated cAMP signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, Gs, protein kinase A and a transcription factor, CREB, shifted the axonal projection sites along the anterior-posterior axis in the olfactory bulb. Thus it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determines the target destinations of OSNs (Imai, 2006).

Each olfactory sensory neuron (OSN) in the mouse expresses only one functional odorant receptor (OR) gene out of ~1,000 members. Axons from OSNs expressing a given OR converge onto a specific site, glomerulus, in the olfactory bulb. It has been proposed that OR molecules at axon termini may directly recognize guidance cues on the olfactory bulb and mediate homophilic interactions of like-axons. OR molecules are G protein-coupled receptors (GPCRs) that transduce the odorant-binding signals by activating the olfactory-specific heterotrimeric G protein (Golf) expressed in mature OSNs. Activation of Golf stimulates adenylyl cyclase type III, generating cAMP, which opens cyclic nucleotide-gated (CNG) channels. Mice deficient for Golf and CNGA2 are anosmic, but form a normal glomerular map, suggesting that a G protein, other than Golf, may aid targeting OSNs independent of CNG channels (Imai, 2006).

OR molecules are rhodopsin-like type A GPCRs that contain a conserved tripeptide motif, Asp-Arg-Tyr (DRY), at the cytoplasmic end of transmembrane domain III, which is required for coupling of GPCRs to the partner G proteins. To examine whether the G protein signaling is involved in guidance of OSN axons, a DRYmotif mutant (D126R/R127D) was generated for the rat OR gene, I7, and it was expressed using a transgenic system. Axons from OSNs expressing the wild-type I7, I7(WT), converged to a specific site in the olfactory bulb, while those expressing the DRY-motif mutant, I7(RDY), remained in the anterior region of the olfactory bulb, failing to converge onto a specific glomerulus. The I7(RDY)-expressing axons never penetrated the glomerular layer, but stayed within the olfactory nerve layer. These axon termini were devoid of synaptotagmin (presynaptic marker) and MAP2 (dendritic marker) immunoreactivities, and thus likely did not form synapses. OSNs expressing a nonfunctional OR gene can activate other OR genes and will fail to converge onto a single glomerulus. However, the inability of I7(RDY) axons to converge on a specific glomerulus was not due to the co-expression of other OR genes; OSNs expressing the I7(RDY) transgene expressed no other OR genes. OSNs expressing I7(WT) all showed Ca2+ signals in response to octanal (agonist of I7 receptor), whereas those expressing I7(RDY) did not. Thus the I7(RDY) mutant is deficient in both axon targeting and G protein coupling (Imai, 2006).

Both Go and Gs genes are expressed in immature mouse OSNs. Although the Gs knock-out mutation is embryonic lethal, the Go deficient mouse shows no obvious anatomical defect in the olfactory system. Since the DRY-motif mutant was assumed to be incapable of coupling with G proteins, whether the constitutively-active Gs mutant (caGs) would rescue the defective phenotype of I7(RDY) in axonal projection was examined. The caGs gene was inserted into the I7(RDY) construct with an internal ribosome entry site (IRES), generating the I7(RDY)-caGs. In OSNs expressing this construct, cAMP signals should be generated constitutively by caGs in a receptor-independent manner. Axons expressing I7(RDY)-caGs (cyan) converged to a specific site in the olfactory bulb, whereas axons expressing I7(RDY) did not. YFP-positive and CFP-positive axons did not intermingle or co-converge, suggesting that homophilic interaction of OR molecules is unlikely. Axons expressing I7(RDY)-caGs were found within a glomerular structure, and were immunoreactive for synaptotagmin. Gs stimulates adenylyl cyclase to produce cAMP, which in turn activates protein kinase A (PKA). A constitutively-active (ca) PKA rescued the defective phenotype of I7(RDY) in OSN projection and glomerular formation, although a few projection sites were found in the posterior region in the olfactory bulb. When the I7(RDY) was coexpressed with a constitutively-active variant of CREB, a PKA-regulated transcription factor, axon termini were found within glomerular structures although with incomplete convergence. These results confirm the role of G proteins in OSN axon targeting, and suggest involvement of cAMP in transcriptional regulation of axon guidance molecules (Imai, 2006).

To study cAMP signaling in OSN projection, the effect of caGs on OSNs expressing the wild-type OR was examined. Two transgenic constructs, I7(WT)-Cre and I7(WT)-caGs were analyzed. The Cre recombinase gene was assumed not to affect the Gs-mediated signaling. Axons from OSNs expressing I7(WT)-Cre or I7(WT) converged in similar regions, whereas those expressing I7(WT)-caGs projected to more posterior regions. Note that additional cAMP signals are generated by caGs. In OSNs expressing I7(WT)-caGs, cAMP signals are generated by both the transgenic caGs and endogenous Gs, whereas in OSNs expressing I7(RDY)-caGs, generation of cAMP signals by endogenous Gs is blocked. The glomerulus for I7(WT)-caGs showed a smaller posterior shift from that for I7(RDY)-caGs. Thus, the signaling level of the endogenous Gs appears to be relatively low, when coupled with the wild-type OR. Whether decreased levels of cAMP signals would affect the OSN projection was also tested. Axons expressing a dominant-negative (dn) PKA with the wild-type OR converged to the anterior part of the olfactory bulb. Unlike axons carrying I7(RDY), axons expressing the I7(WT)-dnPKA generated glomerular structures. These transgenic experiments indicate that increased or decreased levels of cAMP signals shift the glomerular target of OSNs posteriorly or anteriorly, respectively (Imai, 2006).

To examine the effect of excessive cAMP signals on OSN projection, the transgenic construct, caGshi, where the OR coding sequence has been replaced with the caGs, was generated. More caGs was translated from the cap-dependent caGshi than from the IRES-mediated I7(RDY)-caGs. Althougha posteriorly shifted, but scattered pattern of projection with caGshi was expected, only one or a few glomeruli were detected. Projection sites driven by caGshi were located posterior to the I7(RDY)-caGs glomeruli. In situ hybridization and single-cell RT-PCR indicate that OSNs expressing the caGshi express multiple OR species. In the double transgenic mouse carrying CFP-tagged I7(WT) and YFP-tagged caGshi, a few I7(WT)- expressing axons that probably also expressed caGshi projected to the caGshi glomerulus. Thus the caGshi glomerulus represent a heterogeneous population of axons expressing different ORs. It is possible that caGshi produces saturated levels of cAMP signals and generates a distinct glomerular structure regardless of the OR species (Imai, 2006).

In contrast to Golf, Gs is expressed early in OSN differentiation. These experiments suggest involvement of a PKA-regulated transcription factor, CREB, in OSN projection. Microarray and RT-PCR analyses was used to screen for genes with expression levels correlated with cAMP signals. cDNA libraries were prepared from single OSNs from four different transgenic mice, and gene expression profiles were compared between caGshi and I7(RDY), and between I7(WT) and I7(WT)-dnPKA. Among the genes differentially expressed were some encoding axon guidance molecules, e.g., Neuropilin-1 (Nrp1). Nrp1 was expressed in the caGshi OSNs (where cAMP signals might be high), but not in the I7(RDY)-expressing OSNs (where cAMP signaling is blocked). Immunostaining demonstrated a gradient of Nrp1 expression, with low expression in the anterior and high expression in the posterior of the olfactory bulb. In the I7(WT) / I7(WT)-dnPKA mouse, the I7(WT) glomerulus was Nrp1-positive, and the I7(WT)-dnPKA glomerulus was Nrp1-negative. Nrp1 has been implicated in guidance of OSN axons because disruption of the Sema3A gene, which encodes a repulsive ligand for Nrp1, alters glomerular arrangements along the anterior-posterior axis. It is suggested that Gs-mediated cAMP signals regulate transcription of genes encoding axon guidance molecules, which in turn guide positioning of glomeruli (Imai, 2006).

These results explain some puzzling observations about OSN targeting. The α2-adrenergic receptor (α2-AR) but not a vomeronasal receptor (V1rb2), can substitute for an OR in OR-instructed axonal outgrowth and glomerular formation. The explanation may be that the α2-AR can couple to Gs, but the V1rb2 can not. This is consistent with the idea that the Gs-mediated cAMP levels set by the receptors determine the target sites of OSN axons. Another puzzling observation is that alterations in OR expression levels can affect OSN projection. The level of cAMP signals may be affected by both OR identity and amount of OR protein, which would be a factor of transcription and translation parameters. OR-instructed Gs signals are not dependent on odorants, and disruption of Golf or CNGA2 genes did not affect positioning of glomeruli, which suggests that Gs-mediated cAMP signaling is distinct from that mediated by odor-evoked neuronal activity. It has been thought that ORs at axon termini may recognize guidance cues on the olfactory bulb and mediate the homophilic interactions of like-axons. However, the results favor a model in which cAMP signals posterior axis. These results complement previous studies indicating that the dorsal-ventral arrangement of glomeruli is determined by the locations of OSNs within the olfactory epithelium. It is proposed that a combination of dorsal-ventral patterning based on anatomical locations of OSNs and anterior-posterior patterning based on OR-derived cAMP signals establish olfactory bulb topography. After OSN axons reach their approximate destinations in the olfactory bulb, further refinement of the glomerular map may occur through fasciculation and segregation of axon termini in an activity-dependent manner (Imai, 2006).

PKA and neural facilitation (Short and long term potentiation - a model for learning): Studies in Aplysia

One of the hallmarks of long-term memory in both vertebrates and invertebrates is the requirement for new protein synthesis. In sensitization of the gill-withdrawal reflex in the mollusc Aplysia, this requirement can be studied on the cellular level. Here, long-term but not short-term facilitation of the monosynaptic connections between the sensory and motor neurons requires new protein synthesis and is reflected in an altered level of expression of specific proteins regulated through the cAMP second-messenger pathway. Using gene transfer into individual sensory neurons of Aplysia, it can be shown that serotonin (5-HT) induces transcriptional activation of a lacZ reporter gene driven by the cAMP response element (CRE) and that this induction requires CRE-binding proteins (CREBs) (See DCREB2). The induction by 5-HT does not occur following a single pulse, but becomes progressively more effective following two or more pulses. Moreover, expression of GAL4-CREB fusion genes shows that 5-HT induction requires phosphorylation of CREB on Ser119 by protein kinase A. These data provide direct evidence for CREB-modulated transcriptional activation with long-term facilitation (Kaang, 1993).

The switch from short- to long-term facilitation induced by behavioral sensitization in Aplysia involves CREB-like target proteins of PKA, as well as the immediate-early gene ApC/EBP. Using the bZIP domain of ApC/EBP in a two-hybrid system, ApCREB2 has been cloned. It is a transcription factor constitutively expressed in sensory neurons that resembles human CREB2 and mouse ATF4. ApCREB2 represses ApCREB1-mediated transcription in F9 cells. Injection of anti-ApCREB2 antibodies into Aplysia sensory neurons causes a single pulse of serotonin (5-HT), which induces only short-term facilitation lasting minutes, to evoke facilitation lasting more than 1 day. This facilitation has the properties of long-term facilitation: it requires transcription and translation, induces the growth of new synaptic connections, and occludes further facilitation by five pulses of 5-HT (Bartsch, 1995).

Increases in activity of both protein kinase A (PKA) and protein kinase C (See Drosophila PKC) contribute to short-term facilitation of Aplysia sensorimotor synapses evoked by serotonin (5-HT). Increasing levels of cAMP in sensory neurons evokes increases in both synaptic efficacy and in the number of sensory neuron varicosities contacting the major axons of motor cell L7 at intermediate times (3 hr) that persist for 24 hr. Treatment with phorbol esters results in a large transient increase in synaptic efficacy that is accompanied by a large transient increase in the number of sensory neuron varicosities with the newest varicosities most susceptible to elimination. The reversal of the synaptic facilitation and the structural changes does not appear to be the result of long-term inhibitory actions of persistent PKC activation by phorbol esters, since changes in synaptic efficacy can be evoked by additional applications of either phorbol esters or 5-HT. The short-lived changes in structure evoked by phorbol esters occur in preexisting sensory neurites and not by new growth, since increases in PKC activity with phorbol esters lead to reductions in neurite extension and to retractions by sensory neuron growth cones. The action of phorbol esters on growth cone extension is reversible with washout. The results suggest that increases in PKA and PKC activities by 5-HT contribute to short (minutes) and intermediate (hours) forms of facilitation of sensorimotor synapses while increases in PKA activity also mediate long-term (days) maintenance of synaptic facilitation (Wu, 1995).

Protein phosphorylation plays important roles in the mechanisms underlying serotonin (5-HT)-induced presynaptic facilitation of Aplysia sensory neurons. To study mechanisms involved in facilitation, the pattern of protein phosphorylation in sensory neurons was investigation as a function of different durations of 5-HT. Two minutes and 1.5 hr treatments with 5-HT alters the phosphorylation of 5 and 10 proteins, respectively. These different duration treatments with 5-HT produce unique effects on the phosphorylation of different sets of proteins. This result suggests that cells may encode and measure the duration of a stimulus by the pattern of specific proteins that are phosphorylated or dephosphorylated. In addition, because the changes in phosphorylation produced by 2 min treatments with 5-HT were not observed after 25 min treatments with 5-HT, mechanisms must exist for the transient phosphorylation of some proteins even when the 5-HT treatment persists. Anisomycin, an inhibitor of protein synthesis, blocked the effect of 1.5 hr treatments with 5-HT on the phosphorylation of six proteins but had no effect on the phosphorylation change of four other proteins. Both CPT-cAMP (an activator of protein kinase A) and PDAc (an activator of protein kinase C) mimicked the effects of 5-HT on four proteins. Interestingly, the effect of 5-HT on these four proteins did not require protein synthesis. CPT-cAMP, but not PDAc, mimicked the effect of 5-HT on one protein (L55) and, the effect of 5-HT on this protein appeared to require protein synthesis. Because both activation of PKA and protein synthesis are involved in the induction of long-term facilitation, protein L55 is a good candidate for a protein that might play a key role in long-term facilitation. The effects of 5-HT on four proteins were not mimicked by either CPT-cAMP or PDAc. This finding raises the interesting possibility that some effects of 5-HT are mediated by second-messenger systems other than PKA or PKC (Homayouni, 1995).

Enhancement of the defensive withdrawal reflex of Aplysia involves a prolongation of the action potentials of mechanosensory neurons, which contributes to facilitation of transmitter release from these cells. Recent reports have suggested that whereas cAMP-dependent modulation of K+ current increases sensory neuron excitability, a cAMP-independent decrease in K+ current may increase the action potential duration and, thus, facilitate transmitter release. cAMP plays a major role in the spike-broadening effects of facilitatory transmitter; however, broadening requires higher levels of activation of the cAMP-dependent kinase than does increasing excitability. A steeply voltage-dependent transient K+ current, termed IKV,early, and the slowly activating S-type K+ (S-K+) current are both reduced by activation of the cAMP cascade, although with different sensitivities to the second messenger, enabling excitability and spike duration to be regulated independently. Differences in cAMP sensitivity also suggested that the originally described S-K+ current actually consists of two independent components, a slowly activating component and a time-independent, "steady-state" current that is activated at rest (Goldsmith, 1992).

In the sensory neurons of Aplysia, 5-HT acts through cAMP to reduce current flow through two classes of K+ channels, the S-K + channel and a transient K+ channel (Ikv). In addition, 5-HT increases a voltage-dependent, nifedipine-sensitive Ca2+ current. While the effect on the S-K+ channel is mediated exclusively by cAMP, the effect on the Ca2+ current can be mimicked by phorbol as well as by intracellular injection of cAMP. Specific blockers of protein kinase C (PKC) and the cAMP-dependent protein kinase A (PKA) were used to examine the roles of PKC and PKA in mediating the effect of 5-HT on the nifedipine-sensitive Ca2+ current. H-7, a kinase inhibitor that appears to inhibit PKC more effectively than PKA in intact Aplysia neurons, reverses the increase in the Ca2+ current produced by PDBu. Moreover, H-7 partially blocks the effect of 5-HT on the Ca2+ current without affecting the decrease in the S-K+ current. A more specific PKC inhibitor (the 19-31 pseudosubstrate of PKC) also partially blocks the increase in the Ca2+ current produced by 5-HT, suggesting that this increase is mediated by PKC. Rp-cAMPS, a specific blocker of PKA, does not block the increase in the Ca2+ current produced by 5-HT, suggesting that the effect of 5-HT on this current may be mediated to only a small extent by PKA. The effect of 5-HT on the S-K+ current and the Ca2+ current can also be separated on basis of the time course of their appearance. The fact that the decrease in the S-K+ current precedes the increase in Ca2+ current suggests that there may be a temporal difference in the activation of the two kinase systems (Braha, 1993).

cAMP-dependent protein kinase (PKA) is an important participant in neuronal modulation: the ability of neurons to change their properties in response to external stimuli. In Aplysia mechanosensory neurons, PKA plays roles in both short and long term presynaptic facilitation, which is a simple model for learning and memory. PKA in Aplysia is a collection of structurally and functionally diverse regulatory and catalytic (C) subunits. This diversity may in part account for the ability of the enzyme to take part in neuronal events that are spatially and temporally separated. C subunits of Aplysia PKA with alternative N termini target different substrates in subcellular fractions from Aplysia neurons, despite their similar actions on synthetic peptide substrates. Purified recombinant CAPL-AN1A1, which has an N terminus that is homologous to the myristylated sequence described in mammals, catalyzes the formation of two phosphoproteins of 24 and 8 kDa more rapidly than CAPL-AN2A1, which has a distinct N terminus weakly related to that of the yeast TPK1 gene product. The 24-kDa phospoprotein, but not the 8-kDa species, is detected in taxol-stabilized microtubules, suggesting that it is associated with the cytoskeleton. CAPL-AN2A1, in contrast, generates a 55-kDa phosphoprotein that is not observed with CAPL-AN1A1. The 55-kDa species is found in the detergent supernatant of the cytoskeleton fraction. Differential targeting of substrates by C subunits of PKA may therefore contribute to the ability of this kinase to play multiple roles in neuronal modulation (Panchall, 1994).

FMRFamide evokes long-term inhibition of the sensorimotor connection of Aplysia that includes structural alterations in the presynaptic sensory cell. FMRFamide also evokes a down-regulation of the adhesion molecule apCAM from the surface of the postsynaptic motor cell L7. The second messenger pathways were examined that mediate the long-term actions of FMRFamide on both the pre- and postsynaptic cells. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism, but not the cyclooxygenase pathway, blocks the long-term changes in the presynaptic sensory cell evoked by FMRFamide. The down-regulation of apCAM in L7 appears to be mediated by cAMP-dependent activation of protein kinase A. Blocking the cAMP-dependent changes also blocks FMRFamide-induced long-term functional and structural changes. These results suggest that the expression of long-term heterosynaptic inhibition in Aplysia may require concomitant presynaptic and postsynaptic changes, each transduced by specific second messenger systems (Wu, 1994).

Tyrosine kinases modulate synaptic plasticity and ion channel function. Tyrosine kinases can also modulate both the baseline excitability state of Aplysia tail sensory neurons (SNs) as well as the excitability induced by the neuromodulator serotonin (5HT). The effects of increasing and decreasing tyrosine kinase activity in the SNs were examined. Tyrosine kinase inhibitors decrease baseline SN excitability in addition to attenuating the increase in excitability induced by 5HT. Conversely, functionally increasing cellular tyrosine kinase activity in the SNs by either inhibiting opposing tyrosine phosphatase activity or by direct injection of an active tyrosine kinase (Src) induces increases in SN excitability in the absence of 5HT. With respect to the enhancement of SN excitability, an examination was made of the interaction between protein kinase A (PKA), which is known to mediate 5HT-induced excitability changes in the SNs, and tyrosine kinases. The tyrosine kinases function downstream of PKA activation since tyrosine kinase inhibitors reduce excitability induced by activators of PKA. The role of tyrosine kinases in other forms of 5HT-induced plasticity in the SNs was examined. While tyrosine kinase inhibitors attenuate excitability produced by 5HT, they have no effect on short-term facilitation (STF) of the SN-motor neuron (MN) synapse induced by 5HT. Thus tyrosine kinases modulate different forms of SN plasticity independently. Such differential modulation would have important consequences for activity-dependent plasticity in a variety of neural circuits (Purcell, 2001).

PKA, synaptic plasticity, and protein degradation in Aplysia

In Aplysia, behavioral sensitization of defensive reflexes and the underlying presynaptic facilitation of sensory-to-motor neuron synapses lasts for several minutes (short term) or days to weeks (long term). Short-term sensitization has been explained by modulation of ion-channel function through cAMP-dependent protein phosphorylation. Long-term facilitation requires additional molecular changes including protein synthesis. A key event is the persistent activation of the cAMP-dependent protein kinase at baseline concentrations of cAMP. This activation is due to selective loss of regulatory (R) subunits of PKA without any change in catalytic (C) subunits. To understand the molecular mechanisms that produce the loss of R subunits in long-term facilitation, how R subunits are degraded in extracts of Aplysia nervous tissue and in rabbit reticulocyte lysates has been investigated. Degradation of Aplysia R subunits requires ATP, ubiquitin, and a particulate component that appears to be the proteasome complex. Degradation is blocked by hemin, which causes the accumulation of high molecular weight derivatives of R subunits that are likely to be ubiquitin conjugates of R subunits and intermediates in the degradative pathway. Vertebrate RI and RII subunits can be degraded through the ubiquitin pathway. It is suggested that degradation is initiated by cAMP, which causes the holoenzyme to dissociate and, further, that the altered R-to-C ratio in Aplysia sensory neurons is maintained in long-term facilitation by newly synthesized proteins that help target R subunits for accelerated degradation (Hedge, 1993).

In response to the facilitating neurotransmitter serotonin (5-HT), the cAMP-dependent protein kinase (PKA) acquires a special mnemonic characteristic in Aplysia sensory neurons. PKA becomes persistently activated at basal cAMP concentrations owing to a decreased regulatory to catalytic (R to C) subunit ratio. Ubiquitin-mediated proteolysis has been implicated in this selective loss of R. Ubiquitin (Ub), Ub-conjugates and proteasomes are present in the cell bodies, axon, neuropil and nerve terminals of Aplysia neurons. Because R subunits are not decreased in muscle exposed to 5-HT, a comparison of the two tissues provides a tractable approach to determine how the Ub pathway is regulated. The structure of M1, the muscle-specific R isoform, is compared to that of N4, a major neuronal R isoform, to rule out the possibility that the differences in their stability result from differences in structure. Evidence indicates that N4 and M1 are encoded by identical transcripts; they also behave similarly as protein substrates for the Ub pathway in extracts of the two tissues. Nervous tissue contains 20-times more free Ub, but evidence is presented that the susceptibility of R subunits to degradation in neurons relative to muscle results from the greater capacity of neurons to degrade ubiquitinated proteins through the proteasome. Thus, factors that regulate the activity of proteasomes could underlie the enhanced degradation of R subunits in long-term sensitization (Chain, 1995).

The switch from short-term to long-term facilitation of the synapses between sensory and motor neurons mediating gill and tail withdrawal reflexes in Aplysia requires CREB-mediated transcription and new protein synthesis. Several downstream genes have been isolated, one of which encodes a neuron-specific ubiquitin C-terminal hydrolase. This rapidly induced gene encodes an enzyme that associates with the proteasome and increases its proteolytic activity. This regulated proteolysis is essential for long-term facilitation. Inhibiting the expression or function of the hydrolase blocks induction of long-term but not short-term facilitation. It is suggested that the enhanced proteasome activity increases degradation of substrates that normally inhibit long-term facilitation. Thus, through induction of the hydrolase and the resulting up-regulation of the ubiquitin pathway, learning recruits a regulated form of proteolysis that removes inhibitory constraints on long-term memory storage (Hedge, 1997).

The degradation of proteins by the ubiquitin-proteasome pathway has been implicated in regulating several important cellular processes, including transcriptional activation, differentiation and growth and synaptogenesis. What is the function of ubiquitin-mediated proteolysis in sensory neurons, and how does it operate? One possible substrate of proteolysis is the regulatory (R) subunit of PKA. Each R subunit contains two tandem cAMP-binding sites, one (B) a high-affinity site close to the carboxyl terminus, and the other (A) a low-affinity site near the domain masked by the C subunit in the holoenzyme. Activation of the kinase occurs when the second messenger, cAMP, binds sequentially and cooperatively to these two sites, causing the C subunit to dissociate. Degradation of R subunits results in an excess of C and would generate a persistently active and autonomous kinase. In support of this idea, several unidentified proteins remain phosphorylated for at least 24 hr in stimulated sensory neurons undergoing long term faciliations (LTF). These observations raise two questions that can be addressed: (1) is a persistently active PKA essential for LTF, and (2) is proteolysis of R required to establish a persistently active kinase and to produce LTF (Chain, 1999)?

The injection of bovine PKA catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase (Ap-uch). The mechanism through which PKA becomes persistently activated is described; R subunit degradation requires at least two overlapping mechanisms, both caused by the elevation of cAMP in response to 5-HT. (1) The binding of cAMP produces a conformation of R that is particularly susceptible to degradation. (2) By activating PKA and initiating its importation into the nucleus, cAMP promotes gene induction, leading to the expression of Ap-uch. In turn, the hydrolase, which is fully induced by 0.5 hr after the end of the 5-HT treatment, increases the degradative capacity of proteasomes. Only early in the development of LTF do these two mechanisms -- cAMP binding to R and the induction of hydrolase -- overlap (Chain, 1999).

Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. This study identifies a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrates that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory (Sutton, 2001).

Expression of ITF is abolished when PKA activity is blocked, and the synapse returns to a facilitated level when the PKA inhibition is relieved. Blocking PKA activity reversibly disrupts ITM expression. The fact that ITM recovers following removal of the PKA inhibitor suggests that the underlying processes responsible for the persistent PKA activation, once induced, are maintained in the face of PKA inhibition. These results demonstrate that a persistent activation of PKA underlies the expression of ITM (Sutton, 2001).

While the relationship between ITM and LTM in Aplysia remains to be determined, the persistent activation of PKA that underlies the expression of ITM is an excellent candidate mechanism for playing a role in LTM consolidation. For example, by prolonging the activation of downstream molecular events required for LTM, the persistent activation of PKA that underlies the expression of ITM may also act to promote the induction of LTM. Related to this possibility, the induction of LTF at SN-MN synapses requires transcriptional activation via the cAMP response element binding protein, which is a target of PKA in Aplysia SNs. Moreover, exposure of cultured SN-MN synapses to a cAMP analog for a duration that is similar to the time course of ITM is sufficient to induce LTF. These studies in Aplysia suggest that the persistent activation of PKA underlying ITM expression may also have a critical role in the consolidation of LTM. Results from other systems also implicate specific temporal phases of PKA activation in the consolidation of LTM. In particular, a time-limited role for activators of PKA (8-bromo-cAMP and forskolin) has been demonstrated in the consolidation of LTM for step-down avoidance training. Interestingly, while these agents effectively enhance LTM when administered into the CA1 region of hippocampus 3 and 6 hr after training, they are ineffective when administered either immediately after or 9 hr after training. Moreover, upregulation of PKA in the CA1 region of hippocampus produces L-LTP, and the PKA-dependent intermediate phase of LTP (I-LTP) in this region gates the full expression of L-LTP. Finally, multiple olfactory conditioning trials produce a prolonged activation of PKA in the antennal lobes of the honeybee, and prolonging the normally transient activation of PKA in this region after a single conditioning trial enhances the induction of LTM. Collectively, these results suggest that intermediate synaptic and molecular events, in addition to contributing to the induction and expression of intermediate phases of memory, may play important roles in the transition to more long-lasting forms of memory as well (Sutton, 2001).

PKA activation bypasses the requirement for UNC-31 in the docking of dense core vesicles from C. elegans neurons

The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. This technical limitation was addressed by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, the docking and fusion of single dense core vesicles (DCVs) was monitoring employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, direct evidence is required that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis (Zhou, 2007).

Neuropeptides are packed into dense core vesicles (DCVs) and play critical roles in synaptic signaling. The many differences in the properties of DCVs and synaptic vesicles (SVs) suggest that these two classes of secretory organelles employ different molecules during secretion. Relatively little is known, however, about the factors that differentially participate in SV and DCV release. Ca2+-dependent activator protein for secretion (CAPS; see Drosophila Caps) was proposed to function specifically in DCV exocytosis. Surprisingly, a genetic study in mouse chromaffin cells suggests an unpredicted function for CAPS1 in regulating the uptake or storage of monoamines in DCVs. However, the exact role of CAPS1 cannot be directly inferred in the presence of the redundant CAPS2. In C. elegans, in which a single gene (unc-31) encodes the ortholog of mammalian CAPS, recent studies have revealed a remarkable reduction in peptide release but not in SV recycling. Despite the controversy over the role(s) of CAPS in vesicular exocytosis, the challenge remains to elucidate where and how CAPS acts in DCV exocytosis (Zhou, 2007).

Vesicular exocytosis is subject to regulation by various second messenger pathways, e.g., PKA, PKC, diacylglycerol (DAG) and its analogs, the phorbol esters, which have been shown to enhance the readily releasable pool (RRP) of both SVs and DCVs in various cell types. Although an array of exocytotic proteins have been identified in vitro as targets for these regulatory mechanisms, conclusive tests of these hypotheses in vivo with the aid of genetic manipulations are still necessary. As an example, the employment of Munc13-1 knockout mice has identified Munc13 proteins, in parallel to PKC, as major targets of DAG in neurotransmitter and peptide hormone release. Interestingly, activating the Gas pathway, which turns on cAMP and PKA, restored the paralysis phenotype in the unc-31 null mutant to hyperactive, coordinated locomotion. It has been proposed that UNC-31 acts upstream of the Gas pathway via regulation of DCV exocytosis. Little is known, however, of how Gas activation rescues the uncoordinated phenotype in the absence of UNC-31 (Zhou, 2007).

Conclusive testing of various hypotheses in DCV exocytosis will greatly benefit from the fast genetics of C. elegans. An obstacle in this promising direction is the lack of suitable functional assays for DCV exocytosis in C. elegans. One recent piece of progress is the ability of quantifying GFP-labeled peptide release by its endocytic accumulation in coelomocyte cells. This method, however, does not provide the resolution to distinguish multiple stages of DCV trafficking along the exocytotic pathway. Hence, it is desirable to develop direct assays to monitor the release process at high resolution. Only with these tools can the power of genetic manipulation in C. elegans model systems be fully exploited to decipher the complex network that regulates DCV exocytosis and endocytosis (Zhou, 2007).

The small size of most somatic worm cells makes high-resolution cellular assays technically challenging. Electrophysiological analysis has been applied to only limited numbers of neurons and muscle cells in C. elegans. Direct monitoring of the release process using high resolution membrane capacitance (Cm) and optical measurements has not been performed in C. elegans cells. This study describes the first attempt to apply these methods to monitor exocytosis and endocytosis in primary cultured C. elegans neurons. The identity of the recorded cells is assured by cell-specific GFP reporters. A Cm measurement combined with Ca2+-uncaging stimuli was performed to monitor both the amplitudes and the kinetics of exocytosis. The release kinetics of single vesicles was monitored using refined carbon fiber amperometry. Through employing total internal reflection fluorescence microscopy (TIRFM), it has been possible to quantify the docking and fusion of single DCVs labeled with a DCV-specific GFP marker. Through a combination of these methods, the action site of UNC-31 in DCV exocytosis was investigated. The results suggest that UNC-31 is required for the docking of DCVs and that PKA activation can abrogate this requirement for UNC-31. Furthermore, a functional interaction was demonstrated between UNC-31 and UNC-13 in augmenting DCV exocytosis (Zhou, 2007).

Fast cAMP modulation of neurotransmission via neuropeptide signals and vesicle loading

Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA; see Drosophila PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, optogenetic stimulation of cAMP signaling by Beggiatoa-photoactivated adenylyl cyclase (bPAC) was used in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size. cAMP enhanced synaptic vesicle (SV) fusion by increasing mobilization and docking/priming. cAMP further evoked dense core vesicle (DCV) release of neuropeptides, in contrast to channelrhodopsin (ChR2) stimulation. cAMP-evoked DCV release required UNC-31/Ca2+-dependent activator protein for secretion (CAPS). Thus, DCVs accumulated in unc-31 mutant synapses . bPAC-induced neuropeptide signaling acts presynaptically to enhance vAChT-dependent SV loading with acetylcholine, thus causing increased miniature postsynaptic current amplitudes (mPSCs) and significantly enlarged SVs (Steuer Costa, 2017).

PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. This study shows that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, it was found that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, it was shown that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, these results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels (Wang, 2013).

PKA, long-term depression and long-term excitability

Hippocampal N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic depression (LTD) is associated with a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at a site (Ser-845) phosphorylated by cAMP-dependent protein kinase (PKA). Dephosphorylation of a postsynaptic PKA substrate may be crucial for LTD expression. PKA activators inhibit both AMPA receptor dephosphorylation and LTD. Injection of a cAMP analog into postsynaptic neurons prevents LTD induction and reverses previously established homosynaptic LTD without affecting baseline synaptic transmission. Moreover, infusing a PKA inhibitor into postsynaptic cells produces synaptic depression that occludes homosynaptic LTD. These findings suggest that dephosphorylation of a PKA site on AMPA receptors may be one mechanism for NMDA receptor-dependent homosynaptic LTD expression (Kameyama, 1998).

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by either (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)]; (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy fails to alter the induction of LTH by each form of axotomy, although the inhibitor antagonizes hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor is not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush fails to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH is reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury (Liao, 1999).

PKA and response to cocaine

Using a balanced conditioned place preference (CPP) paradigm, the role of protein kinases A (PKA) and C (PKC) on the acquisition, consolidation and expression of cocaine place conditioning was studied. H7, a non-selective inhibitor of protein kinases, was administered intracerebroventricularly at 1 and 10 micrograms/10 microliters. The higher dose significantly reduces the time spent by rats in the cocaine compartment when given immediately after each conditioning session (consolidation), whereas it had no effect when administered before cocaine during the training phase (acquisition) or before testing for place preference in the absence of cocaine (expression). The same effect was found on administering immediately after each training session 3 micrograms/10 microliters chelerythrine, a selective PKC inhibitor, or 10 micrograms/10 microliters H89, a selective PKA inhibitor, suggesting that both kinases contribute to the consolidation of stimulus-reward association which determines rats' behavior in the cocaine CPP. Changes in the activity of PKA and PKC may thus be part of the cascade of events that contribute to enhancing synaptic responses in the consolidation phase of cocaine CPP and determine rats' behavior associated with the memory of the rewarding effect of cocaine during cocaine CPP expression. These findings may have implications for the study of cocaine 'craving' and relapse (Cervo, 1997).

Adaptations in neurons of the midbrain periaqueductal gray (PAG) induced by chronic morphine treatment mediate expression of many signs of opioid withdrawal. The abnormally elevated action potential rate of opioid-sensitive PAG neurons is a likely cellular mechanism for withdrawal expression. Opioid withdrawal in vitro induces an opioid-sensitive cation current that is mediated by the GABA transporter-1 (GAT-1) and requires activation of protein kinase A (PKA) for its expression. Inhibition of GAT-1 or PKA also prevents withdrawal-induced hyperexcitation of PAG neurons. These findings indicate that GAT-1 currents can directly increase the action potential rates of neurons and that GAT-1 may be a target for therapy to alleviate opioid-withdrawal symptoms (Bagley, 2004).

Opioids are intensely addictive, and cessation of their chronic use is associated with a withdrawal syndrome consisting of severe early physical symptoms and late features such as craving. Relapse into drug-taking behaviors often occurs as a result of this withdrawal syndrome, which is thought to result from neuronal adaptations that develop to restore homeostasis during chronic opioid exposure. On cessation of opioid administration, persistent counteradaptations in critical brain regions are unmasked and cause the withdrawal syndrome. A rebound increase of adenylyl cyclase/protein kinase A (PKA) signaling is one counteradaptation that has been implicated in the expression of opioid-withdrawal signs (Bagley, 2004 and references therein).

The midbrain periaqueductal gray (PAG) is crucial for expression of many somatic signs of opioid withdrawal. Studies using microinjections of opioid antagonists and agonists indicate that the PAG is involved in the expression of physical withdrawal signs such as teeth chattering, wet-dog shakes, and eye twitch, as well as escape behaviors and place aversion. While opioid agonists acutely inhibit adenylyl cyclase activity in the brain and specifically in the PAG, there is a compensatory increase in adenylyl cyclase signaling during chronic treatment with morphine in vitro and in vivo resulting in rebound hyperactivity of this cascade during withdrawal. Microinjections of PKA inhibitors into the PAG attenuate a spectrum of opioid-withdrawal behaviors similar to those induced by microinjections of opioid antagonists. One cellular mechanism by which elevated adenylyl cyclase signaling could produce behavioral signs of withdrawal is the hyperexcitation of opioid-sensitive PAG neurons. Increased adenylyl cyclase signaling has also been proposed to result in the increased action potential rates of neurons in other brain regions during opioid withdrawal (Bagley, 2004 and references therein).

Adaptations in PAG neurons that result in hyperexcitation of action potential rate during withdrawal have been examined, along with whether PKA is involved in this process. Whole-cell, cell-attached, and perforated patch recordings of PAG neurons from mice chronically treated with morphine were used to demonstrate that opioid withdrawal increases a GABA transporter-1 (GAT-1) cation current. The increased GAT-1 current is dependent on PKA activity and is associated with the withdrawal-induced elevated action potential rate of PAG neurons. Thus, this study has established that opioid withdrawal stimulates a GAT-1 current that increases the excitability of PAG neurons and is dependent on PKA activation (Bagley, 2004).

PKA in ERK-dependent activation of MSK1 during fear conditioning

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. This study reports that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. It is concluded that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory (Sindreu, 2007).

One of the major objectives of this study was to identify which MAPK-activated CREB kinase is stimulated during memory formation. Furthermore, it was important to define the relationship between MAPK and cAMP signaling following training for contextual fear conditioning, and to determine why Ca2+-stimulated adenylyl cyclase activity is required for contextual memory. There are several mechanisms by which cAMP could contribute to memory, including regulation of AMPA receptor trafficking and MAPK activation. No increased PKA phosphorylation of AMPA receptors was detected following contextual fear conditioning. Consequently, focus was placed on the role of cAMP signaling in MAPK activation because of the central role played by MAPK during memory formation. Confocal imaging was used to identify individual hippocampal cells in which PKA, MAPK, and CREB kinases are activated after contextual fear conditioning. It has not been previously shown that contextual fear conditioning activates PKA, nor was it known that PKA and MAPK are activated in the same neurons in the hippocampus. Furthermore, there was no evidence for activation of specific CREB kinases following fear conditioning (Sindreu, 2007).

Training for contextual memory caused a 5- to 6-fold increase in MAPK activation in approximately 10% of CA1 pyramidal neurons in two distinct intracellular pools: a nuclear pool and a postsynaptic pool. Furthermore, PKA was activated in the same subset of neurons as MAPK, and both showed increased nuclear activities after training. MAPK activation strongly correlated with activation of MSK1, a CREB kinase. Most importantly, the training-induced increases in MAPK, PKA, and MSK1 activities were ablated in mice lacking Ca2+-stimulated adenylyl cyclase activity. It is concluded that one of the major roles of cAMP signaling in memory is to support the activation and nuclear translocation of MAPK in CA1 pyramidal neurons (Sindreu, 2007).

Signal transduction pathways are usually implicated in memory formation because they are activated in specific areas of the brain by training and inhibition of the pathway blocks memory. For example, MAPK activity is stimulated in area CA1 following training for hippocampus-dependent memory, and administration of MEK inhibitors blocks both training-induced increases in MAPK and memory formation. Ca2+-stimulated adenylyl cyclase and PKA activities are required for memory formation, suggesting that either basal PKA activity is necessary or that an increment in PKA activity contributes to memory. Using an antibody that recognizes phosphorylated PKA substrates (pPKA-s), it was discovered that PKA is not only activated in area CA1 following contextual fear conditioning, but there is also a strong correlation between neurons showing MAPK activation and those in which PKA is activated. In keeping with this, increased nuclear levels of the PKA catalytic α subunit was observed in pERK+ neurons after training. The increase in pPKA-s was readily blocked by inhibitors of PKA and lost in mice lacking Ca2+-stimulated adenylyl cyclase activity, thus validating the use of the pPKA-s antibody to monitor PKA activation (Sindreu, 2007).

The observation that fear conditioning activates MAPK selectively in area CA1 agrees with other evidence that stimulation of transcription in this area of the hippocampus is particularly important for contextual memory formation. Much less was known, however, about the identity and size of the cellular population activated during training for contextual memory, and the intracellular compartments in which MAPK is stimulated. Although this analysis focused on the role of MAPK in the nucleus because of its importance for CREB-mediated transcription, MAPK was simultaneously activated in dendrites and at distal synapses following fear conditioning. It is noteworthy that MAPK regulates a number of other proteins, including dendritic K+ channels and glutamate receptors, and it may also control dendritic protein synthesis. Thus, the parallel activation of synaptodendritic and somatonuclear pools of MAPK supports the general hypothesis that memory formation depends on several MAPK-regulated events, including synaptic activity, dendritic protein synthesis, and transcription (Sindreu, 2007).

Although CREB-mediated transcription is necessary for memory formation and depends on MAPK signaling, the CREB kinase activated by MAPK following training for contextual memory was not certain. It was particularly interesting to determine if training for contextual fear activates RSK2 or MSK1 because studies using cultured neurons have implicated both kinases in CREB-mediated transcription through the phosphorylation of transcription factors and histones. This study discovered that fear conditioning activates MSK1, but not RSK2, in CA1 neurons, and that activation of MAPK and MSK1 is tightly correlated on a cell-by-cell basis. Furthermore, the activation of MSK1 induced by training is abrogated in mice lacking Ca2+-stimulated adenylyl cyclases or by post-training inhibition of MEK1/2. This strongly implicates MSK1 in MAPK-dependent CREB phosphorylation during formation of contextual memory. The identification of MSK1, and not RSK2, as the activated CREB kinase emphasizes that signaling mechanisms inferred from cultured neuron studies do not necessarily apply in vivo. Definitive evidence as to the relative importance of both CREB kinases during memory formation may come from the use of conditional mutant mice or novel MSK1 antagonists (Sindreu, 2007).

In summary, the data indicates that stimulation of MAPK in dendrites and the nucleus following training for contextual memory depends on Ca2+-stimulated adenylyl cyclase activity and leads to the activation of the CREB kinase MSK1. Furthermore, signaling elements for CREB-mediated transcription, starting with the initial cAMP signal, and including PKA, MAPK, MSK1, and CREB, are all activated in the same subset of neurons after training. It is concluded that one of the major roles of adenylyl cyclase activity in memory is to support the activation of MAPK, MSK1, and CREB in hippocampal neurons (Sindreu, 2007).

PKA and aging

Activation of the cAMP/protein kinase A (PKA) pathway has been proposed as a mechanism for improving age-related cognitive deficits based on studies of hippocampal function. However, normal aging also afflicts prefrontal cortical cognitive functioning. This study reports that agents that increase PKA activity impair rather than improve prefrontal cortical function in aged rats and monkeys with prefrontal cortical deficits. Conversely, PKA inhibition ameliorates prefrontal cortical cognitive deficits. Western blot and immunohistochemical analyses of rat brain further indicate that the cAMP/PKA pathway becomes disinhibited in the prefrontal cortex with advancing age. These data demonstrate that PKA inhibition, rather than activation, is the appropriate strategy for restoring prefrontal cortical cognitive abilities in the elderly (Ramos, 2003).

Table of contents

cAMP-dependent protein kinase 1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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