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Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk,Aplysia californica. Moreover, different types of long-term facilitation (LTF) in culturedAplysiasensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix “handle” in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF inAplysia.SIGNIFICANCE STATEMENTLong-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in theAplysiasensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a “handle” helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over-expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin-tagged Aplysia glutamate receptor measured using live imaging after intermediate- or long-term facilitation. Over-expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate-term facilitation, but did block increases in synaptic strength seen during long-term facilitation. There was no effect of over-expression of this form of ApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long-term maintenance of synaptic strength. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems including the hermaphrodite marine mollusk, Aplysia californica. Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform was required, the kidney and brain expressed adaptor protein (KIBRA) was also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant negative (DN)-PKMs to erase LTF is correlated with isoform specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an alpha-helix ‘handle’ in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia.Significance StatementLong lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs in not isoform specific, isoform specificity is defined by a ‘handle’ helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk, Aplysia californica. Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix “handle” in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia. SIGNIFICANCE STATEMENT Long-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a “handle” helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that dominant-negative PKMs show isoform-specificity. A dominant-negative PKM from the classical PKC Apl I blocks activity-dependent intermediate-term facilitation (a-ITF) when expressed in the sensory neuron, while a dominant-negative PKM from the atypical PKC Apl III does not. Consistent with a specific role for PKM Apl I in activity-dependent facilitation, live imaging FRET-based cleavage assays reveal that activity leads to cleavage of the classical PKC Apl I, but not the atypical PKC Apl III in the sensory neuron varicosities of Aplysia. In contrast, massed intermediate facilitation (m-ITF) induced by 10 min of 5HT is sufficient for cleavage of the atypical PKC Apl III in the motor neuron. Interestingly, both cleavage of PKC Apl I in the sensory neuron during a-ITF and cleavage of PKC Apl III in the motor neuron during m-ITF are inhibited by a dominant-negative form of a penta-EF hand containing classical calpain cloned from Aplysia. Consistent with a role for PKMs in plasticity, this dominant-negative calpain also blocks both a-ITF when expressed in the sensory neuron and m-ITF when expressed in the motor neuron. This study broadens the role of PKMs in synaptic plasticity in two significant ways: (i) PKMs generated from multiple isoforms of PKC, including classical isoforms, maintain memory traces; (ii) PKMs play roles in the presynaptic neuron.

At the sensory-motor neuron synapse ofAplysia, either spaced or continuous (massed) exposure to serotonin (5-HT) induces a form of intermediate-term facilitation (ITF) that requires new protein synthesis but not gene transcription. However, spaced and massed ITF use distinct molecular mechanisms to maintain increased synaptic strength. Synapses activated by spaced applications of 5-HT generate an ITF that depends on persistent protein kinase A (PKA) activity, whereas an ITF produced by massed 5-HT depends on persistent protein kinase C (PKC) activity. In this study, we demonstrate that eukaryotic elongation factor 2 (eEF2), which catalyzes the GTP-dependent translocation of the ribosome during protein synthesis, acts as a biochemical sensor that is tuned to the pattern of neuronal stimulation. Specifically, we find that massed training leads to a PKC-dependent increase in phosphorylation of eEF2, whereas spaced training results in a PKA-dependent decrease in phosphorylation of eEF2. Importantly, by using either pharmacological or dominant-negative strategies to inhibit eEF2 kinase (eEF2K), we were able to block massed 5-HT-dependent increases in eEF2 phosphorylation and subsequent PKC-dependent ITF. In contrast, pharmacological inhibition of eEF2K during the longer period of time required for spaced training was sufficient to reduce eEF2 phosphorylation and induce ITF. Finally, we find that the massed 5-HT-dependent increase in synaptic strength requires translation elongation, but not translation initiation, whereas the spaced 5-HT-dependent increase in synaptic strength is partially dependent on translation initiation. Thus, bidirectional regulation of eEF2 is critical for decoding distinct activity patterns at synapses by activating distinct modes of translation regulation.

Multiple kinase activations contribute to long-term synaptic plasticity, a cellular mechanism mediating long-term memory. The sensorimotor synapse ofAplysiaexpresses different forms of long-term facilitation (LTF)—nonassociative and associative LTF—that require the timely activation of kinases, including protein kinase C (PKC). It is not known which PKC isoforms in the sensory neuron or motor neuron L7 are required to sustain each form of LTF. We show that different PKMs, the constitutively active isoforms of PKCs generated by calpain cleavage, in the sensory neuron and L7 are required to maintain each form of LTF. Different PKMs or calpain isoforms were blocked by overexpressing specific dominant-negative constructs in either presynaptic or postsynaptic neurons. Blocking either PKM Apl I in L7, or PKM Apl II or PKM Apl III in the sensory neuron 2 d after 5-hydroxytryptamine (5-HT) treatment reversed persistent nonassociative LTF. In contrast, blocking either PKM Apl II or PKM Apl III in L7, or PKM Apl II in the sensory neuron 2 d after paired stimuli reversed persistent associative LTF. Blocking either classical calpain or atypical small optic lobe (SOL) calpain 2 d after 5-HT treatment or paired stimuli did not disrupt the maintenance of persistent LTF. Soon after 5-HT treatment or paired stimuli, however, blocking classical calpain inhibited the expression of persistent associative LTF, while blocking SOL calpain inhibited the expression of persistent nonassociative LTF. Our data suggest that different stimuli activate different calpains that generate specific sets of PKMs in each neuron whose constitutive activities sustain long-term synaptic plasticity.SIGNIFICANCE STATEMENTPersistent synaptic plasticity contributes to the maintenance of long-term memory. Although various kinases such as protein kinase C (PKC) contribute to the expression of long-term plasticity, little is known about how constitutive activation of specific kinase isoforms sustains long-term plasticity. This study provides evidence that the cell-specific activities of different PKM isoforms generated from PKCs by calpain-mediated cleavage maintain two forms of persistent synaptic plasticity, which are the cellular analogs of two forms of long-term memory. Moreover, we found that the activation of specific calpains depends on the features of the stimuli evoking the different forms of synaptic plasticity. Given the recent controversy over the role of PKMζ maintaining memory, these findings are significant in identifying roles of multiple PKMs in the retention of memory.

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.

J. Neurochem. (2012) 122, 1108–1117. Abstract Rapamycin-insensitive companion of TOR (Rictor) is a conserved component of target of rapamycin complex 2 (TORC2), a complex implicated in phosphorylation of a number of signal transduction-related kinases, including protein kinase Cs (PKCs) at their ‘hydrophobic’ site in the carboxy-terminal extension domain. In the marine mollusk, Aplysia californica, an increase in phosphorylation of the novel PKC, Apl II, at the hydrophobic site is associated with a protein synthesis-dependent increase in synaptic strength seen after continuous application of serotonin. To determine if Rictor plays a role in this increase, we cloned the Aplysia ortholog of Rictor (ApRictor). An siRNA-mediated decrease in ApRictor levels in Aplysia sensory neurons led to a decrease in the phosphorylation of PKC Apl II at the hydrophobic site suggesting a role for ApRictor in hydrophobic site phosphorylation. However, over-expression of ApRictor was not sufficient to increase phosphorylation of PKC Apl II. Continuous application of serotonin increased phosphorylation of PKC Apl II at the hydrophobic site in cultured sensory neurons, and this was blocked by Torin, which inhibits both TORC1 and TORC2. Over-expression of ApRictor did not lead to change in the magnitude of serotonin-mediated phosphorylation, but did lead to a small increase in the membrane localization of phosphorylated PKC Apl II. In conclusion, these studies implicate Rictor in phosphorylation of a novel PKC during synaptic plasticity and suggest an additional role for Rictor in regulating the localization of PKCs.

Novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. We have previously shown that the Aplysia novel PKC Apl II's C2 domain inhibits binding of diacylglycerol (DAG) to the C1 domain and that this inhibition is removed by phosphatidic acid (PA) binding to the C1b domain. Another model for C2 domain regulation of nPKCs suggests that the C2 domain binds to receptors for activated C kinase (RACKs) to assist in kinase translocation and activation. In the present study, we examined how a pharmacological peptide derived from RACK-binding site in the vertebrate novel PKCɛ regulates translocation of PKC Apl II from the cytosol to the plasma membrane. We found that a C2 domain-derived inhibitor peptide inhibited PKC Apl II translocation. This inhibition was removed by R273H mutation in the C1b domain and by phosphatidic acid, which can both remove C2-domain mediated inhibition suggesting that the peptide can regulate C1–C2 domain interactions.

J. Neurochem. (2010) 115, 994–1006. Abstract Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT2, 5HT4, and 5HT7 families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT1Apl(a), 5HT2Apl, and 5HT7Apl were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT2Apl could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT2 receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT1Apl(a) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

Learning is highly regulated by the pattern of training. InAplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (5HT)-mediated activation of the novel PKC Apl II. Here, we demonstrate that activation of PKC Apl II is highly sensitive to the pattern of 5HT application. Spaced applications downregulate PKC translocation through PKA signaling, whereas massed applications lead to persistent translocation of PKC. Differential regulation of PKC translocation is mediated by competing feedback mechanisms that act through protein synthesis. These studies elucidate a fundamental molecular difference between spaced and massed training protocols.

In vertebrates, a brain-specific transcript from the atypical protein kinase C (PKC) zeta gene encodes protein kinase M (PKM) zeta, a constitutively active kinase implicated in the maintenance of synaptic plasticity and memory. We have cloned the atypical PKC from Aplysia, PKC Apl III. We did not find a transcript in Aplysia encoding PKMzeta, and evolutionary analysis of atypical PKCs suggests formation of this transcript is restricted to vertebrates. Instead, over-expression of PKC Apl III in Aplysia sensory neurons leads to production of a PKM fragment of PKC Apl III. This cleavage was induced by calcium and blocked by calpain inhibitors. Moreover, nervous system enriched spliced forms of PKC Apl III show enhanced cleavage. PKC Apl III could also be activated through phosphorylation downstream of phosphoinositide 3-kinase. We suggest that PKM forms of atypical PKCs play a conserved role in memory formation, but the mechanism of formation of these kinases has changed over evolution.

Protein kinase Cs (PKCs) are a family of lipid-activated kinases that mediate a wide variety of cellular processes, including the regulation of synaptic strength in the nervous system (23, 42, 48). In Aplysia californica, behavioral sensitization is mediated in part by an increase in the strength of the connections between mechanoreceptor sensory neurons and motor neurons (19). This increase, called synaptic facilitation, is mediated by the neurotransmitter serotonin (5HT), which induces facilitation in isolated ganglia as well as in cocultures of sensory neurons and motor neurons (5, 6). Sensitizing stimulation causes the translocation of PKC to neuronal membranes (39, 53). In the Aplysia nervous system, there are two phorbol ester-regulated PKCs: PKC Apl I, which is homologous to the Ca2+-activated PKC family in vertebrates (α, β1, β2, and γ) that are called conventional or classical PKCs (cPKCs), and PKC Apl II, which is homologous to the Ca2+-independent epsilon family of PKC in vertebrates (ɛ and η) that are called novel PKCs (nPKCs) (21, 42, 43). PKC Apl I and PKC Apl II translocate under different conditions to mediate distinct types of synaptic facilitation (53). PKC Apl II, but not PKC Apl I, is translocated by the application of 5HT to sensory neurons and is required for 5HT-mediated facilitation at synapses that previously have been depressed (24). In contrast, combining 5HT and the firing of the sensory neuron leads to the additional translocation of PKC Apl I, and PKC Apl I is required for the intermediate-term facilitation induced by the combination of sensory neuron firing and 5HT (53). Both cPKCs and nPKCs contain two C1 domains and one C2 domain. However, the C2 domain of nPKCs is located N terminal to the C1 domains and lacks critical aspartic acid residues involved in coordinating Ca2+ ions in cPKCs (29). The C2 domain of cPKCs mediates Ca2+-dependent binding to the membrane lipid phosphatidylserine (PS) (30, 31). This binding transiently recruits the enzyme to the membrane, where its physiological activator, diacylglycerol (DAG), resides. Then, in conjunction with the C1 domain interacting with DAG, the binding of the C2 domain to PS induces a conformational change that activates the enzyme (26, 32). The function of the C2 domain of nPKCs is less clear. The C2 domain of PKCδ binds to phosphotyrosine, allowing for the regulation of the kinase (2, 41, 51). However, the structure of the C2 domain of the epsilon family of PKCs including PKC Apl II is considerably different (11, 15, 33, 35, 45), and these C2 domains do not bind phosphotyrosine (10, 42). It has been reported that the C2 domain of PKCɛ can bind to phospholipids, especially phosphatidic acid (PA), and that PA binding to the C2 domain of PKCɛ is required for translocation (11, 18). However, the amount of PA required to bind to the C2 domain is high compared to that for the Ca2+-dependent binding of the C2 domains of cPKCs to PS (11, 16, 25, 45). In PKC Apl II, the C2 domain binds poorly to lipids; however, the phosphorylation of the C2 domain increases binding (38). While lipid binding suggests a positive role for the C2 domain in translocation, the removal of the C2 domain of PKC Apl II allows for the activation of the enzyme at lower concentrations of PS, implicating the C2 domain as a negative regulator of kinase regulation (44). Moreover, the C2 domain of PKC Apl II lowers the affinity of phorbol esters to bind to the C1 domains, and this is removed by PA (37). To clarify the role of PA and the C2 domain in the translocation of PKC Apl II, we examined the translocation of fluorescent protein-labeled PKC constructs for PKC Apl II, PKC Apl II ΔC2, and a PKC Apl II-C1b mutant that does not respond to PA. Our results demonstrate an important role for PA in the physiological translocation of PKC Apl II.

In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.

Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability.

The defensive withdrawal reflexes of Aplysia are important behaviors for protecting the animal from predation. Habituation and dishabituation allow for experience-dependent tuning of these reflexes and the mechanisms underlying these forms of behavioral plasticity involve changes in transmitter release from the sensory to motor neuron synapses through homosynaptic depression and the serotonin-mediated recovery from depression, respectively. Interestingly, dishabituation is reduced in older animals with no corresponding change in habituation. Here we show that the cultured sensory neurons of heavier animals (greater than 120 g) that form synaptic connections with motor neurons have both reduced recovery from depression and reduced novel PKC Apl II activation with 5HT. The decrease in the recovery from depression correlated better with the size of the animal than the age of the animal. Much of this change in PKC activation and synaptic facilitation following depression can be rescued by direct activation of PKC Apl II with phorbol dibutyrate, suggesting a change in the signal transduction pathway upstream of PKC Apl II activation in the sensory neurons of larger animals.

In the marine mollusk Aplysia californica, serotonin initiates three phases of translational regulation: an initial decrease in translation, followed by a transient increase in protein synthesis, both of which are independent of transcription, followed by a later increase in protein synthesis that is dependent on transcription. These increases in protein synthesis may underlie translation-dependent changes in synaptic plasticity. We have characterized the second messenger pathways that underlie these changes in the pleural ganglia of Aplysia. Activation of protein kinase C was both necessary and sufficient for the initial decrease in translation. Protein kinase C, cyclic AMP-dependent protein kinase, and a tyrosine kinase were all required for the second phase, a transient increase in protein synthesis. The late increase in protein synthesis required both protein kinase A and spaced applications of serotonin. Rapamycin, a specific inhibitor of a downstream translational regulator, blocked the transient increase in protein synthesis (second phase), suggesting that this drug may be useful in determining the specific physiological consequences of this translational regulation. Indeed, we used rapamycin to demonstrate that one type of intermediate form of synaptic plasticity induced by serotonin did not require the rapamycin-sensitive increase in translation.

Activation of protein kinase C (PKC) increases microtubule (MT) growth lifetimes, resulting in extension of a nocodazole-sensitive population of MTs in Aplysia growth cones. We examined whether the two phorbol ester-activated PKCs inAplysia, the Ca2+-activated PKC Apl I and the Ca2+-independent PKC Apl II, are associated with these MTs. Phorbol esters translocated PKC to the Triton X-100-insoluble fraction, and a significant portion of this translocated pool was sensitive to low concentrations of nocodazole. Low doses of nocodazole had no effect on the amount of PKC in the Triton X-100-insoluble fraction in the absence of phorbol esters, whereas higher doses of nocodazole reduced basal levels of PKC Apl II. The F-actin cytoskeletal disrupter, latrunculin A, removed both PKCs from the Triton X-100-insoluble fraction in both control and phorbol ester-treated nervous systems. PKC Apl II also directly interacted with purified MTs. In detergent-extracted cells, both PKCs immunolocalized predominantly with MTs. PKCs were associated with newly formed MTs invading the actin-rich peripheral growth cone domain after PKC activation. Our results are consistent with a central role for PKCs in regulating MT extension.

The Ca 2+ -independent protein kinase C (PKC) Apl II, but not the Ca 2+ -activated PKC Apl I, becomes autonomously active during intermediate periods of facilitation in Aplysia neurons. We examined the ability of superoxide formed by the enzymatic reaction of xanthine with xanthine oxidase (X/XO) to induce autonomous activity of PKCs in Aplysia . X/XO stimulated autonomous PKC activity in Aplysia nervous system homogenates, but this activity resulted solely from activation of PKC Apl I. PKC Apl I is also more sensitive to activation by X/XO when expressed in insect cells. Our results suggest that oxidation can autonomously activate PKC Apl I in the Aplysia nervous system, but that the activation of PKC Apl II during synaptic facilitation is not due to oxidation of the enzyme.

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant negative atypical PKM selectively reversed associative LTF, while a dominant negative classical PKM selectively reversed non-associative LTF. While both PKMs are formed from calpain-mediated cleavage of PKCs, each form of LTF is sensitive to a distinct dominant negative calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dominant negative classical calpain, while non-associative LTF is blocked by dominant negative small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories.

Protein kinase Cs (PKCs) are activated by translocating from the cytoplasm to the membrane. We have previously shown that serotonin-mediated translocation of PKC to the plasma membrane in Aplysia sensory neurons was subject to desensitization, a decrease in the ability of serotonin to induce translocation after previous application of serotonin. In Aplysia, changes in the strength of the sensory-motor neuron synapse are important for behavioral sensitization and PKC regulates a number of important aspects of this form of synaptic plasticity. We have previously suggested that the desensitization of PKC translocation in Aplysia sensory neurons may partially explain the differences between spaced and massed training, as spaced applications of serotonin, a cellular analog of spaced training, cause greater desensitization of PKC translocation than one massed application of serotonin, a cellular analog of massed training. Our previous studies were performed in isolated sensory neurons. In the present study, we monitored translocation of fluorescently-tagged PKC to the plasma membrane in living sensory neurons that were co-cultured with motor neurons to allow for synapse formation. We show that desensitization now becomes similar during spaced and massed applications of serotonin. We had previously modeled the signaling pathways that govern desensitization in isolated sensory neurons. We now modify this mathematical model to account for the changes observed in desensitization dynamics following synapse formation. Our study shows that synapse formation leads to significant changes in the molecular signaling networks that underlie desensitization of PKC translocation.

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.

At nondepressedAplysiasensory to motor synapses, serotonin (5-HT) facilitates transmitter release primarily through a protein kinase A pathway. In contrast, at depressedAplysiasensory to motor synapses, 5-HT facilitates transmitter release primarily through a protein kinase C (PKC)-dependent pathway. It is known that only two phorbol ester-activated PKC isoforms, the Ca2+-dependent PKC Apl I and the Ca2+-independent PKC Apl II, exist in theAplysianervous system. For the first time, we have now been able to functionally determine which isoform of PKC is involved in a particular form of plasticity. We microinjected cultured sensorimotor pairs of neurons with various PKC constructs tagged with the enhanced green fluorescent protein as a reporter for successful plasmid expression. Our results demonstrate that short-term facilitation of depressed synapses is mediated by PKC Apl II. Dominant-negative PKC Apl II, but not dominant-negative PKC Apl I, disrupted the normal kinetics of 5-HT-induced facilitation by completely blocking its rapid onset. This effect was specific to depressed synapses, because dominant-negative PKC Apl II did not inhibit 5-HT-mediated facilitation of nondepressed synapses. Our results suggest that not only different signal transduction pathways but also different isoforms of a specific cascade may mediate physiological responses according to the state of a synapse.

Manseau, Frédéric, Wayne S. Sossin, and Vincent F. Castellucci. Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons. J. Neurophysiol. 79: 1210–1218, 1998. Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4β-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.

The nervous system of Aplysia californica has three isoforms of protein kinase C (PKC): the conventional PKC Apl I, the novel PKC Apl II, and the atypical PKC Apl III. Each isoform has distinct requirements for activation and distinct downstream roles in synaptic plasticity. PKCs can be cleaved by calpains into constitutively active forms, called protein kinase Ms (PKMs). Multiple forms of plasticity in Aplysia are mediated by PKMs, and these may be due to cleavage of distinct isoforms of PKC. PKCs also interact in complex ways with other second messenger pathways. The diversity of PKC isoforms allows for this family of kinases to play important roles in decoding extracellular stimuli into the formation of distinct molecular memory traces.

Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT) underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF) receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF)-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs) or other molecules must also be involved in translocation of PKC Apl II.

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist inAplysia californicadespite the fact that inhibitors of PKMζ erase memory inAplysiain a fashion similar to rodents. We have previously shown that, inAplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.

Rapamycin-insensitive companion of TOR (Rictor) is a conserved component of target of rapamycin complex 2 (TORC2), a complex implicated in phosphorylation of a number of signal transduction-related kinases, including protein kinase Cs (PKCs) at their 'hydrophobic' site in the carboxy-terminal extension domain. In the marine mollusk, Aplysia californica, an increase in phosphorylation of the novel PKC, Apl II, at the hydrophobic site is associated with a protein synthesis-dependent increase in synaptic strength seen after continuous application of serotonin. To determine if Rictor plays a role in this increase, we cloned the Aplysia ortholog of Rictor (ApRictor). An siRNA-mediated decrease in ApRictor levels in Aplysia sensory neurons led to a decrease in the phosphorylation of PKC Apl II at the hydrophobic site suggesting a role for ApRictor in hydrophobic site phosphorylation. However, over-expression of ApRictor was not sufficient to increase phosphorylation of PKC Apl II. Continuous application of serotonin increased phosphorylation of PKC Apl II at the hydrophobic site in cultured sensory neurons, and this was blocked by Torin, which inhibits both TORC1 and TORC2. Over-expression of ApRictor did not lead to change in the magnitude of serotonin-mediated phosphorylation, but did lead to a small increase in the membrane localization of phosphorylated PKC Apl II. In conclusion, these studies implicate Rictor in phosphorylation of a novel PKC during synaptic plasticity and suggest an additional role for Rictor in regulating the localization of PKCs.

More than two decades ago, the discovery of the first C2 domain in conventional Protein Kinase Cs (cPKCs) and of its role as a calcium-binding motif began to shed light on the activation mechanism of this family of Serine/Threonine kinases which are involved in several critical signal transduction pathways. In this chapter, we review the current knowledge of the structure and the function of the different C2 domains in PKCs. The C2 domain of cPKCs is a calcium sensor and its calcium-dependent binding to phospholipids is crucial for kinase activation. While the functional role of the cPKC C2 domain is better understood, phylogenetic analysis revealed that the novel C2 domain is more ancient and related to the C2 domain in the fungal PKC family, while the cPKC C2 domain is first associated with PKC in metazoans. The C2 domain of novel PKCs (nPKCs) does not contain a calcium-binding motif but still plays a critical role in nPKCs activation by regulating C1-C2 domain interactions and consequently C2 domain-mediated inhibition in both the nPKCs of the epsilon family and the nPKCs of the delta family. Moreover, the C2 domain of the nPKCs of the delta family was shown to recognize phosphotyrosines in a novel mode different from the ones observed for the Src Homology 2 (SH2) and the phosphotyrosine binding domains (PTB). By binding to phosphotyrosines, the C2 domain regulates the activation of this subclass of PKCs. The C2 domain was also shown to be involved in protein-protein interactions and binding to the receptor for activated C-kinase (RACKs) thus contributing to the subcellular localization of PKCs. In summary, the C2 domain is a critical player that can sense the activated signaling pathway in response to external stimuli to specifically regulate the different conventional and novel PKC isoforms.

Long-term memory formation is sensitive to the pattern of training sessions. Training distributed over time (spaced training) is superior at generating long-term memories than training presented with little or no rest interval (massed training). This spacing effect was observed in a range of organisms from invertebrates to humans. In the present paper, we discuss the evidence supporting cyclic-AMP response element-binding protein 2 (CREB), a transcription factor, as being an important molecule mediating long-term memory formation after spaced training. We also review the main upstream proteins that regulate CREB in different model organisms. Those include the eukaryotic translation initiation factor (eIF2α), protein phosphatase I (PP1), mitogen-activated protein kinase (MAPK), and the protein tyrosine phosphatase corkscrew. Finally, we discuss PKC activation and protein synthesis and degradation as mechanisms by which neurons decode the spacing intervals.

More than two decades ago, the discovery of the first C2 domain in conventional Protein Kinase Cs (cPKCs) and of its role as a calcium-binding motif began to shed light on the activation mechanism of this family of Serine/Threonine kinases which are involved in several critical signal transduction pathways. In this chapter, we review the current knowledge of the structure and the function of the different C2 domains in PKCs. The C2 domain of cPKCs is a calcium sensor and its calcium-dependent binding to phospholipids is crucial for kinase activation. While the functional role of the cPKC C2 domain is better understood, phylogenetic analysis revealed that the novel C2 domain is more ancient and related to the C2 domain in the fungal PKC family, while the cPKC C2 domain is first associated with PKC in metazoans. The C2 domain of novel PKCs (nPKCs) does not contain a calcium-binding motif but still plays a critical role in nPKCs activation by regulating C1-C2 domain interactions and consequently C2 domain-mediated inhibition in both the nPKCs of the epsilon family and the nPKCs of the delta family. Moreover, the C2 domain of the nPKCs of the delta family was shown to recognize phosphotyrosines in a novel mode different from the ones observed for the Src Homology 2 (SH2) and the phosphotyrosine binding domains (PTB). By binding to phosphotyrosines, the C2 domain regulates the activation of this subclass of PKCs. The C2 domain was also shown to be involved in protein-protein interactions and binding to the receptor for activated C-kinase (RACKs) thus contributing to the subcellular localization of PKCs. In summary, the C2 domain is a critical player that can sense the activated signaling pathway in response to external stimuli to specifically regulate the different conventional and novel PKC isoforms.

We examined the activation of protein kinase C (PKC) produced by phorbol esters inAplysia nervous tissue. Translocation of PKC in intact ganglia requires higher concentrations of phorbol esters than would be expected from: (1) their affinity forAplysia PKCs measured in vitro; (2) their physiological effects on culturedAplysia neurons; and (3) their actions on PKC in synaptosomes. Although phorbol esters enter intact ganglia slowly, delayed access to neurons is insufficient to account for the high concentrations needed for translocation. Increasing accessibility to the neural components of ganglia increases the rate at which translocation occurs, but does not affect the concentration of phorbol ester required. We suggest that this might best be explained by the presence of a competitive inhibitor at the binding site for phorbol esters in PKC. An indication for an inhibitor is that the concentration of phorbol esters needed for translocation in homogenates of nervous tissue is markedly decreased by diluting the extract. Preliminary characterization shows that the inhibitory activity is unusual: in addition to being competitive with lipid activators, it is soluble and tissue-specific. This type of inhibitor may be an important regulator of protein phosphorylation by PKC in neurons.

We investigated activation of the two major neuronal protein kinase C (PKC) isoforms in Aplysia, Ca(2+)-activated Apl I and Ca(2+)-independent Apl II, during the induction and maintenance of behavioral sensitization of Aplysia defensive reflexes. Activation of PKC occurred during the training stimulus and persisted for at least 2 hr thereafter but was not maintained for 24 hr. The persistent activation required protein synthesis and was blocked by cyproheptidine, an agent that also blocked the initial activation of PKC. Persistent activation involved both an increase in membrane-associated Apl I and an increase in an autonomous kinase activity that may be related to a post-translational modification of Apl II. These results are consistent with the hypothesis that in addition to its role in producing the presynaptic facilitation of mechanosensory-motor neuron synapses that underlie short-term facilitation, PKC is needed for maintaining synaptic changes in an intermediate period that precedes the modifications accompanying consolidation of memory.

Expression of the 5-HT1Apl(a)receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses ( Nagakura et al. 2010 ). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT1Apl(b)receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

By molecular cloning Kruger et al. (Kruger, K. E., Sossin, W. S., Sacktor, T. C., Bergold, P. J., Beushausen, S., and Schwartz, J. H. (1991) J. Neurosci. 11, 2303-2313) provided evidence for three isoforms of protein kinase C (PKC) in Aplysia, two of which, Apl I and Apl II, are expressed abundantly in the nervous system. We resolve two major kinase activities from nervous tissue by column chromatography on DEAE-cellulose and hydroxylapatite (HAP), one Ca(2+)-activated and the other Ca(2+)-independent. We show that these two activities correspond to the previously cloned Apl I and II. These two isoforms appear to be the only major PKCs present in nervous tissue. The Apl I kinase is strongly activated by cis-fatty acids but only in the presence of Ca2+. Thus functionally, Apl I is more like the alpha and beta isoforms of vertebrate PKC, than to the vertebrate neural gamma isoform. The Apl II kinase is Ca(2+)-independent and resembles vertebrate PKC epsilon. The simplicity of the PKC isoform distribution in Aplysia makes this mollusc an attractive animal for understanding the differential regulation and physiological activities of Ca(2+)-activated and Ca(2+)-independent PKCs.

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

To explore the role of both Aplysia cell adhesion molecule (ApCAM) and activity of specific protein kinase C (PKC) isoforms in the initial formation of sensory neuron synapses with specific postsynaptic targets (L7 but not L11), we examined presynaptic growth, initial synapse formation, and the expression of the presynaptic neuropeptide sensorin following cell-specific reduction of ApCAM or of a novel PKC activity. Synapse formation between sensory neurons and L7 begins by 3 h after plating and is accompanied by a rapid accumulation of a novel PKC to sites of synaptic interaction. Reducing ApCAM expression specifically from the surface of L7 blocks presynaptic growth and initial synapse formation, target-induced increase of sensorin in sensory neuron cell bodies and the rapid accumulation of the novel PKC to sites of interaction. Selective blockade of the novel PKC activity in L7, but not in sensory neurons, with injection of a dominant negative construct that interferes with the novel PKC activity, produces the same actions as down regulating ApCAM; blockade of presynaptic growth and initial synapse formation, and the target-induced increase of sensorin in sensory neuron cell bodies. The results indicate that signals initiated by postsynaptic cell adhesion molecule ApCAM coupled with the activation of a novel PKC in the appropriate postsynaptic neuron produce the retrograde signals required for presynaptic growth associated with initial synapse formation, and the target-induced expression of a presynaptic neuropeptide critical for synapse maturation.

Ca(2+)-activated and Ca(2+)-independent protein kinase Cs (PKCs) are present in the nervous system of the marine mollusk Aplysia californica (Kruger et al., 1991). Sensitizing stimuli or application of the facilitatory transmitter 5-HT to intact isolated ganglia produces the presynaptic facilitation of sensory-to-motor neuron synapses that underlies behavioral sensitization, which is a simple form of learning. Activation of PKC can also produce this presynaptic facilitation (Braha et al., 1990). To determine which type of PKC is activated, we developed a sensitive and selective assay to measure both Ca(2+)- activated and Ca(2+)-independent PKC activities in crude supernatant and membrane fractions of nervous tissue. This assay is based on the specific binding of the Ca(2+)-activated PKCs to phosphatidylserine vesicles in the presence of Ca2+ and makes use of a novel synthetic peptide with sequences conforming to phylogenetically conserved pseudosubstrate regions of the Ca(2+)-independent kinases. We provide evidence that the presynaptic facilitation is produced by a Ca(2+)- activated isoform: application of 5-HT increases the amount of the Ca(2+)-activated PKC activity associated with the membrane. Under these conditions, no increase in Ca(2+)-independent kinase activity is seen.

The protein kinase C (PKC) and the cAMP-dependent kinase (protein kinase A; PKA) pathways are known to play important roles in behavioral plasticity and learning in the nervous systems of a wide variety of species across phyla. We briefly review the members of the PKC and PKA family and focus on the evolution of the immediate upstream activators of PKC and PKA i.e., phospholipase C (PLC) and adenylyl cyclase (AC), and their conservation in gastropod mollusks, taking advantage of the recent assembly of the Aplysiacalifornica and Lottia gigantea genomes. The diversity of PLC and AC family members present in mollusks suggests a multitude of possible mechanisms to activate PKA and PKC; we briefly discuss the relevance of these pathways to the known physiological activation of these kinases in Aplysia neurons during plasticity and learning. These multiple mechanisms of activation provide the gastropod nervous system with tremendous flexibility for implementing neuromodulatory responses to both neuronal activity and extracellular signals.

Serotonin (5-HT) mediates learning-related facilitation of sensorimotor synapses inAplysia californica. Under some circumstances 5-HT-dependent facilitation requires the activity of protein kinase C (PKC). One critical site of PKC's contribution to 5-HT-dependent synaptic facilitation is the presynaptic sensory neuron. Here, we provide evidence that postsynaptic PKC also contributes to synaptic facilitation. We investigated the contribution of PKC to enhancement of the glutamate-evoked potential (Glu-EP) in isolated siphon motor neurons in cell culture. A 10 min application of either 5-HT or phorbol ester, which activates PKC, produced persistent (> 50 min) enhancement of the Glu-EP. Chelerythrine and bisindolylmaleimide-1 (Bis), two inhibitors of PKC, both blocked the induction of 5-HT-dependent enhancement. An inhibitor of calpain, a calcium-dependent protease, also blocked 5-HT's effect. Interestingly, whereas chelerythrine blocked maintenance of the enhancement, Bis did not. Because Bis has greater selectivity for conventional and novel isoforms of PKC than for atypical isoforms, this result implicates an atypical isoform in the maintenance of 5-HT's effect. Although induction of enhancement of the Glu-EP requires protein synthesis (Villareal et al., 2007), we found that maintenance of the enhancement does not. Maintenance of 5-HT-dependent enhancement appears to be mediated by a PKM-type fragment generated by calpain-dependent proteolysis of atypical PKC. Together, our results suggest that 5-HT treatment triggers two phases of PKC activity within the motor neuron, an early phase that may involve conventional, novel or atypical isoforms of PKC, and a later phase that selectively involves an atypical isoform.

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)- independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.

Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used Aplysia as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented physiological roles for PKC in synaptic plasticity in this system. In particular, we have shown that distinct isoforms mediate distinct types of synaptic plasticity induced by the same neurotransmitter: The novel calcium-independent PKC Apl II is required for actions mediated by serotonin (5-HT) alone, while the classical calcium-dependent PKC Apl I is required for actions mediated when 5-HT is coupled to activity. We will discuss the reasons for PKC isoform specificity, assess the tools used to uncover isoform specificity, and discuss the implications of isoform specificity for understanding the roles of PKC in regulating synaptic plasticity.

Activation of phosphokinase C (PKC) can increase transmitter release at sensory–motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)–coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP–SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at naïve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT–mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory–motor neuron synapses by a number of distinct processes.

Protein kinase Cs (PKCs) are important effectors of synaptic plasticity. InAplysia, there are two major phorbol ester-activated PKCs, Ca2+-activated PKC Apl I and Ca2+-independent PKC Apl II. Functional Apl II, but not Apl I, in sensory neurons is required for a form of short-term facilitation induced at sensorimotor synapses by the facilitatory transmitter serotonin (5-HT). Because PKCs are activated by translocating from the cytoplasm to the membrane, we used fluorescently tagged PKCs to determine the isoform and cell-type specificity of translocation in livingAplysianeurons. In Sf9 cells, low levels of diacylglycerol translocate Apl II, but not Apl I, which requires calcium for translocation at low concentrations of diacylglycerol. Accordingly, application of 5-HT toAplysiasensory neurons in the absence of neuronal firing translocates Apl II, but not Apl I, consistent with the role of Apl II in short-term facilitation. This translocation is observed in sensory neurons, but not in motor neurons. Apl I translocates only if 5-HT is coupled to firing in the sensory neuron; firing alone is ineffective. Because combined 5-HT and firing are required for the induction of one type of intermediate-term facilitation at these synapses, we asked whether this form of synaptic plasticity involves activation of Apl I. We report here that dominant-negative Apl I, but not Apl II, blocks intermediate-term facilitation. Thus, different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity: Ca2+-independent Apl II is involved in short-term facilitation, and Ca2+-dependent Apl I contributes to intermediate-term facilitation.

We discovered a novel alternatively spliced form of synaptotagmin I (Syt I). This splicing event is conserved over evolution and, inAplysia, results in a two amino acid insert in the juxtamembrane domain of Syt I (Syt IVQ). Both Syt I and Syt IVQare localized to synaptic vesicles; however, we also observed punctae that contained one or the other spliced products. Both Syt I and Syt IVQare phosphorylated at the adjacent PKC site. Overexpression of Syt IVQ,but not of Syt I, inAplysianeurons blocked the ability of serotonin to reverse synaptic depression. This effect is upstream of PKC activation, because neither Syt IVQnor Syt I blocked the effects of phorbol esters on reversing synaptic depression or the effects of serotonin on facilitating nondepressed synapses. Our results demonstrate a physiological role for splicing in the juxtamembrane domain of Syt I.