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.     

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.     

Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm.

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.     

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.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

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 .     

Live-imaging of PKC Translocation in Sf9 Cells and 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 Ca²⁺ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca²⁺-independent. The atypical PKCs are activated by neither Ca²⁺ 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.Download video file.^{(60M, mov)}     

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.     

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.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

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.     

Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOIP (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKCalpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to downregulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling.     

Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions: Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Molecular Cloning and Expression of Starfish Protein Kinase C Isoforms

To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCδ, ι, and α isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells.     

The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.     

Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.