Phosphoinositide-dependent kinase phosphorylation of protein kinase C Apl II increases during intermediate facilitation in aplysia

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine mollusk Aplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation.     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

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.     

Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKC, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.     

Epsilon protein kinase C in pathological myocardial hypertrophy. Analysis by combined transgenic expression of translocation modifiers and Galphaq

The epsilon isoform of protein kinase C (PKC) has a critical cardiotrophic function in normal postnatal developing heart as demonstrated by cardiac-specific transgenic expression of epsilonPKC-selective translocation inhibitor (epsilonV1) and activator (psiepsilonRACK) peptides (Mochly-Rosen, D., Wu, G., Hahn, H., Osinska, H., Liron, T., Lorenz, J. N., Robbins, J., and Dorn, G. W., II (2000) Circ. Res. 86, 1173-1179). To define the role of epsilonPKC signaling in pathological myocardial hypertrophy, epsilonV1 or psiepsilonRACK were co-expressed in mouse hearts with Galpha(q), a PKC-linked hypertrophy signal transducer. Compared with Galpha(q) overexpression alone, co-expression of psiepsilonRACK with Galpha(q) increased epsilonPKC particulate partitioning by 30 +/- 2%, whereas co-expression of epsilonV1 with Galpha(q) reduced particulate-associated epsilonPKC by 22 +/- 1%. Facilitation of epsilonPKC translocation by psiepsilonRACK in Galpha(q) mice improved cardiac contractile function measured as left ventricular fractional shortening (30 +/- 3% Galpha(q) versus 43 +/- 2% psiepsilonRACK/Galpha(q), p < 0.05). Conversely, inhibition of epsilonPKC by epsilonV1 modified the Galpha(q) nonfailing hypertrophy phenotype to that of a lethal dilated cardiomyopathy. These opposing effects of epsilonPKC translocation activation and inhibition in Galpha(q) hypertrophy indicate that epsilonPKC signaling is a compensatory event in myocardial hypertrophy, rather than a pathological event, and support the possible therapeutic efficacy of selective epsilonPKC translocation enhancement in cardiac insufficiency.     

Stimulation of an Insulin Receptor Activates and Down-Regulates the Ca2+-Independent Protein Kinase C, Apl II, Through a Wortmannin-Sensitive Signaling Pathway in Aplysia

Abstract: Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca²⁺-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca²⁺-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca²⁺-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC₁₆phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca²⁺-independent protein kinase C isoforms.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II

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.     

Decline in the Recovery from Synaptic Depression in Heavier Aplysia Results from Decreased Serotonin-Induced Novel PKC Activation

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 120g) 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.     

Rictor regulates phosphorylation of the novel protein kinase C Apl II in Aplysia sensory neurons

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.     

Protein kinase C isoforms are translocated to microtubules in neurons

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 in Aplysia, the Ca(2+)-activated PKC Apl I and the Ca(2+)-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.     

Binding of Protein Kinase C Isoforms to Actin in Aplysia

Abstract: Recently, two of the 10 vertebrate protein kinase C (PKC) isoforms, PKCβII and PKCε, have been shown to bind specifically to actin filaments, suggesting that these kinases may regulate cytoskeletal dynamics. Here, we present evidence that two PKCs from the marine mollusk Aplysia californica , PKC Apl I, a Ca²⁺-activated PKC, and PKC Apl II, a Ca²⁺-independent PKC most similar to PKCε and η, also bind F-actin. First, they both cosedimented with purified actin filaments in a phorbol ester-dependent manner. Second, they both translocated to the Triton-insoluble fraction of the nervous system after phorbol ester treatment. PKC Apl II could also partially translocate to actin filaments and associate with the Triton-insoluble fraction in the absence of phorbol esters. Translocation to purified actin filaments was increased in the presence of a PKC inhibitor, suggesting that PKC phosphorylation reduces PKC bound to actin. Although both kinases bound F-actin, actin was not sufficient to activate the kinases. In support of a physiological role for actin-PKC interactions, immunochemical localization of PKC Apl II in neuronal growth cones revealed a striking colocalization with F-actin. Our results are consistent with a role for actin-PKC interactions in regulating cytoskeletal dynamics in Aplysia .     

State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation

Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.     

Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces

Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on epsilonPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of epsilonPKC. These peptides were used to identify the role of epsilonPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of epsilonPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in epsilonPKC knock-out cells revealed that only a subset of the peptides were selective for epsilonPKC over other PKC isozymes. These epsilonPKC-selective peptides were also protective of the myocardium from ischemic injury, an epsilonPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of epsilonPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from epsilonPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within epsilonPKC and/or between epsilonPKC and other partner proteins; some of these interactions are unique to epsilonPKC, and others are common to other PKC isozymes.     

Beta- and alpha-adrenergic cross-signaling for L-type Ca current is impaired in transgenic mice with constitutive activation of epsilonPKC

It is well established that beta-adrenoceptor stimulation activates PKA and alpha(1)-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of alpha(1)-adrenoceptors inhibits beta-adrenoceptor stimulated L-type Ca current (I(Ca-L)) and direct activation of epsilonPKC leads to I(Ca-L) inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of epsilonPKC alters I(Ca-L) and its response to adrenergic stimulation. Therefore, we investigated the interaction between beta- and alpha(1)-adrenoceptors vis-à-vis I(Ca-L) in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of epsilonPKC (epsilonPKC agonist). Whole-cell I(Ca-L) was recorded from epsilonPKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) beta-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus alpha(1)-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of epsilonPKC leads to reduced basal I(Ca-L) density. beta-adrenoceptor activation of I(Ca-L) is blunted in epsilonPKC agonist TG mice. alpha-adrenoceptor cross-talk with beta-adrenoceptor signaling pathways vis-à-vis L-type Ca channels is impaired in epsilonPKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in epsilonPKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure.     

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.     

Importance of C1B domain for lipid messenger-induced targeting of protein kinase C

The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated epsilonPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, epsilonPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of epsilonPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of epsilonPKC, we determined that the epsilonC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from epsilonPKC in the context of deltaPKC (delta(epsilonC1B)) and vice versa (epsilon(deltaC1B)), were generated and tested for their translocation in response to ceramide and AA. delta(epsilonC1B) translocated upon treatment with both ceramide and AA; epsilon(deltaC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of epsilonPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.     

A novel PIP2 binding of epsilonPKC and its contribution to the neurite induction ability

Protein kinase C-epsilon (epsilonPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the epsilonPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding of epsilonPKC and its correlation with the neurite extension. We found that full length epsilonPKC bound to PIP(2) in a 12-omicron-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of epsilonPKC (epsilonRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from epsilonRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact epsilonRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP(2) binding ability of the V3-deleted mutant was weakened. Those PIP(2) bindings of epsilonPKC, epsilonRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cdelta and the V3 region of epsilonPKC revealed that PIP(2) binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, epsilonPKC directly binds to PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.