MINIREVIEW Protein kinase Ce: function in neurons Yasuhito Shirai, Naoko Adachi and Naoaki Saito Biosignal Research Center, Kobe University, Japan Expression of PKC- in the brain Northern- and immunoblot analyses of the brain have revealed the predominant presence of protein kinase Ce (PKCe) mRNA and protein, respectively [1,2]; the subtype has been cloned from rat brain [3]. Immuno- histochemistry of PKCe reveals the most abundant expression of the enzyme in the hippocampus, olfac- tory tubercle and Calleja’s islands, with moderate expression in the cerebral cortex, anterior olfactory nuclei, accumbens nucleus, lateral septal nuclei and caudate putamen (Table 1). The distribution of PKCe in the brain is consistent with results of in situ hybrid- ization [4]. Interestingly, the immunoreactivity of the protein is evident in the nerve fibers, and precise obser- vation using electron microscopy reveals its presy- naptic localization [4,5]. These findings suggest the involvement of PKCe in neurite outgrowth and presyn- aptic functions such as neurotransmitter release. As little is known about the role of PKCe in brain development, we measured changes in the amount of PKCe protein in the rat brain from birth to day 28. Substantial amounts of PKCe were already detected at birth but substantial increases were detected in the forebrain between days 5 and 7, and in the hindbrain between days 7 and 14 (Fig. 1). This remarkable increase suggests the importance of PKCe for neural network construction because its timing is coincident with synapse formation in the brain. Neurite outgrowth Overexpression of PKCe promotes nerve growth factor-induced neurite outgrowth, whereas its down- regulation inhibits this [6]. Interestingly, the phenome- non is independent of its catalytic activity because expression of the regulatory domain alone (eRD) induces neurite outgrowth [7]. Larsson et al. [8] dem- onstrated that the actin binding site between the C1A and C1B is important for morphological change of neurons. They also reported that the PKCe-induced neurite outgrowth is blocked by active Ras homolog Keywords alcohol; ischemia; LTP; neurite outgrowth; pain Correspondence Y. Shirai, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe 657-8501, Japan Fax: +81 78803 5971 Tel: +81 78803 5962 E-mail: shirai@kobe-u.ac.jp (Received 26 December 2007, revised 26 May 2008, accepted 16 June 2008) doi:10.1111/j.1742-4658.2008.06556.x Protein kinase Ce is expressed at higher levels in the brain compared to other tissues such as the heart and kidney, suggesting that it plays an important role in the nervous system. Several neural functions of PKCe, including neurotransmitter release and ion channel regulation, have been identified using PKCe knockout mice. In this review, we focus on the involvement of protein kinase Ce in neurite outgrowth, presynaptic regula- tion, alcohol actions, ischemic preconditioning and pain. Abbreviations ERK, extracellular signal-regulated kinase; GABA A , c-aminobutyrate type A; KO, knockout; LTP, long-term potentiation; NMDA, N-methyl- D-aspartate; PIP 2 , phosphatidylinositol 4,5-bis phosphate; PKCe, protein kinase Ce; RhoA, Ras homolog gene family, member A; ROCK, Rho-associated coiled-coil-containing protein kinase. 3988 FEBS Journal 275 (2008) 3988–3994 ª 2008 The Authors Journal compilation ª 2008 FEBS gene family, member A (RhoA) and led by inhibition of the RhoA effector Rho-associated coiled-coil- containing protein kinase (ROCK), indicating that attenuation of the RhoA-ROCK pathway is involved in the process [9]. Additionally, activation of Cdc42 is implicated in PKCe-induced neurite outgrowth [10]. How PKCe regulates RhoA and Cdc42 is still unknown. We have recently shown that PKCe binds to phosphatidylinositol 4,5-bis phosphate (PIP 2 ) and that the PIP 2 binding is necessary for PKCe-induced neurite induction and its membrane localization [11]. The PIP 2 binding of PKCe may influence the function of actin binding proteins, leading to actin rearrange- ment and neurite induction. In addition, the binding of PKCe to PIP 2 may contribute towards inhibition of the RhoA-ROCK pathway. For example, the binding of PKCe to PIP 2 may reduce the level of free PIP 2 that can activate RhoA by regulating the open state of the RhoA ⁄ RhoGDI complex [12]. Alternatively, PKCe may attenuate RhoA via RhoGAP, which is reported to bind to PKCe and induce neurite outgrowth [13]. Interestingly, the binding of full length PKCe to Rho- GAP and PIP 2 is dependent on 12-O-tetradecanoyl- phorbol 13-acetate, but eRD can bind to PIP 2 without 12-O-tetradecanoylphorbol-13-acetate [11,13]. These results suggest that the open conformation is impor- tant for the binding of PKCe to RhoGAP and PIP 2 . Actin binding to PKCe may regulate the neurite cyto- sckelecton as well as induce the open conformation of PKCe, permitting its interaction with RhoGAP and ⁄ or PIP 2 . Taken together, these studies suggest a model for the involvement of PKCe in neurite function. Hor- mones and neurotransmitters, including nerve growth factor, activate PKC, resulting in a conformational change that accompanies translocation to the plasma membrane. Activated PKCe on the plasma membrane interacts with actin, RhoGAP and PIP 2 , resulting in neurite outgrowth by inhibiting the RhoA-ROCK pathway, activation of Cdc42 and cytoskeletal rear- rangement (Fig. 2). Although the C1B and V3 regions of PKCe play a function in the induction of neurite outgrowth, the mechanism by which this occurs has yet to be determined [11,14]. Presynaptic functions Long-term potentiation (LTP) is at least one com- ponent in the complex mechanism of learning and Table 1. Relative densities of PKCe immunoreactivity in the rat brain. 0, no immunoreactivity; 1, faint immunoreactivity; 2, lower immunoreactivity; 3 moderate immunoreactivity; 4, moderately dense immunoreactivity; 5, most dense immunoreactivity. Modified from Table 1 of [4]. Region Region Olfactory bulb Thalamus Olfactory nerve layer 0 Reticular nucleus 1 External plexiform layer 3 Dorsal nuclei 1 Internal granular layer 2 Ventroposterior nucleus 1 Glomerular layer 2 Lateral geniculate nuclei 1 Mitral cell layer 1 Medial geniculate nuclei 2 Anterior olfactory nuclei 3 Cerebellar cortex Amygdara 2 Molecular layer 2 Caudate-putamen 3 Purkinje cell layer 0 Globus pallidus 2 Granular layer 1 Accumbens nucleus 3 Substantia nigra Olfactory tubercle 4 Pars compacta 1 Callrja’s island 4 Pars reticulate 2 Septal area Mammilary nuclei 2 Laternal septal nucleus 3 Suprior colicullus 1 Medial septal nucleus 1 Inferior colicullus 1 Diagonal band 1 Pontine nuclei 1 Septo-hippocampal nucleus 4 Locus coeruleus 1 Habnulla Mesencephalic trigeminal nucleus 2 Medial 0 Inferior olive 2 Lateral 0 Vestbular nuclei 0 Neocortex Cochlear nucleus 0 Layer I 3 Solitary nucleus 0 Layer II 3 Gracile nucleus 0 Layer III 1 Cuneate nucleus 0 Layer IV 2 Spinal cord Layer V 2 Substantia gelatinosa 2 Layer VI 2 Vental horn 1 Hippocampus White matter CA1 4 Optic nerve 0 CA2 4 Anterior commissure 0 CA3 5 Corpus callosum 0 Dentate gyrus Pyramidal tract 2 Hilus 4 Facial nerve 1 Granular layer 2 Cochlear nerve 1 Molecular layer 3 Cerebral peduncle 1 Spinal trigeminal tract 1 Medial longitudinal fasciculus 1 Inferior cerebellar peduncle 1 P1 P3 P5 P7 P14 P28 F H H HFHF H H PKC Actin FFF Fig. 1. The ontogeny of PKCe in the rat brain as assessed by immunoblot analysis. The brain was dissected from rats of various ages and cut at the level of the caudal end of the inferior colliculus. The rostal half (F) and the caudal half (H) were homogenized and the homogenates (50 lg of protein) were subjected to SDS ⁄ PAGE, followed by a western blot for PKCe. Y. Shirai et al. Function of PKCe in neurons FEBS Journal 275 (2008) 3988–3994 ª 2008 The Authors Journal compilation ª 2008 FEBS 3989 memory. There is general agreement that calmodulin- dependent kinase II plays an essential role in this phe- nomenon. PKC is also thought to be necessary, but not sufficient, to induce LTP based on the findings that phorbol esters mimic LTP and PKC inhibitors prevent it [15]. The mechanisms underlying LTP have been most extensively studied in the hippocampus, although LTP occurs in a number of brain regions. There are two types of LTP in the hippocampus [15]; one is LTP in the pathway from the Schaffer collaterals to CA1 (SC- CA1) and the other is the pathway from the mossy fibers to CA3 (MF-CA3). The former is calcium- dependent and involves N -methyl-d-aspartate (NMDA) receptor phosphorylation by PKCc in the postsynaptic neurons [16]. The latter appears to be mediated by presynaptic events. PKCe is present at the terminals of neurons and is localized at the presynaps- es of the mossy fibers, consistent with a role for PKCe in LTP at MF-CA3 [3,4]. Indeed, PKCe at the nerve terminal is involved in phorbol ester-induced enhance- ment of glutamate exocytosis [17] and in phorbol ester-induced synaptic potentiation [19]. Thus, PKCe at the nerve terminal would be expected to contribute to the MF-CA3 LTP by increasing presynaptic neuro- transmitter release. Generally, sustained activation of PKC is needed for the presynaptic regulation of neural plasticity [4]. The importance of the actin binding site in both the sustained activation of PKCe and enhanced exocytosis has been reported [18]. However, how PKCe is activated presynaptically during LTP is not fully understood. One attractive mechanism is that of the ‘retrograde messenger’. Arachidonic acid pro- duced at a postsynaptic site could diffuse to the presynaptic terminal to activate PKCe. Indeed, arachi- donic acid is released from cultured neurons by activa- tion of NMDA receptors [19] and subtype-specifically activates PKCe [20]. Presynaptic PKCe is also important for synaptic maturation. It is well known that co-culture of purified neurons with astrocytes facilitates synaptogenesis and synapse maturation. Hama et al. [21] reported that contact of neurons with astrocytes enhances the excit- atory postsynaptic potential and induces excitatory synapses, and that facilitated excitory synaptogenesis is blocked by inhibitors of PKC [19]. Of the several PKC subtypes present in the presynaptic neurons, Hama et al. [19] propose that PKCe plays a key role in the phenomenon because arachidonic acid production by phospholipase A 2 is necessary for PKC activation and synaptogenesis [19]. These results strongly suggest that PKCe is involved in presynaptic modulation and regu- lation, although there is no consensus on the involve- ment of PKCe in LTP. Actions of alcohol PKCe is thought to mediate several actions of ethanol. For example, ethanol stimulates the translocation of PKCe in NG108-15 cells [22] and chronic ethanol Activation Conformational change Membrane localization PKC RhoGAP PIP 2 F-actin RhoA/ ROCK inhibition Cdc42 Actin binding Protein Neurite outgrowth ? Fig. 2. Proposed model for neurite induc- tion by PKCe. Activation of PKCe results in an open conformation and translocation to the plasma membrane. The PKCe on the plasma membrane interacts with actin, RhoGAP and PIP 2 via the actin binding site C1 and V3 regions, resulting in neurite outgrowth by inhibiting the RhoA-ROCK pathway, activation of Cdc42, and cytoskeletal rearrangement. Function of PKCe in neurons Y. Shirai et al. 3990 FEBS Journal 275 (2008) 3988–3994 ª 2008 The Authors Journal compilation ª 2008 FEBS exposure increases the amount of PKCe in NG108-15 and PC cells [23,24]. More directly, PKCe null mice show a higher sensitivity than their wild-type litter- mates to the acute behavioral effects of ethonal, and demonstrate a marked reduction in ethanol self-admin- istration [25]. The involvement of PKCe in the actions of alcohol was confirmed in PKCe transgenic mice [26]. Conditional expression of PKCe in the basal fore- brain, amygdala and cerebellum of PKCe null mice rescues the hypersensitivity and restores ethanol consumption. Also, doxycycline-induced reduction of PKCe expression results in a knockout (KO) mice-like phenotype [26]. As the conditional transgenic mice do not express PKC e in the hippocampus, these effects of PKCe on the response to ethanol are unlikely to be the result of responses in the hippocampus. The hypersensitivity to and avoidance of ethanol in PKCe KO mice appears to be mediated by the c-ami- nobutyrate type A (GABA A ) receptor. This is based on studies demonstrating that KO mice showed a greater increase in locomotor activity than wild-type mice in response to pentobarbital and diazepam, which are allosteric activators of the GABA A receptor [25,26]. GABA A receptors are ligand-gated Cl – chan- nels that are considered to be an important target of ethanol. GABA A receptors are pentameric proteins complexes comprising eight different classes. Most GABA A receptors are compossed of two a, two b and one c 2 subunit. Recently, it has shown that PKCe directly phosphorylates S327 in the large intracellular loop of the c 2 subunit and that mutation of this site enhanced the ethanol-induced GABA-stimulated cur- rent [27]. These results confirm that PKCe regulates the sensitivity of GABA A receptors to ethanol via direct phosphorylation. Finally, chronic ethanol exposure up-regulates N-type calcium channels. Because this up-regulation can be inhibited by a selective inhibitor of PKCe [28], ethanol induces N-type calcium channel expression in a PKCe-dependent manner. These results suggest that inhibition of PKCe might comprise a viable treatment for alcoholism. Ischemic preconditioning Subleathal and mild ischemic insult, or ‘precondition- ing’, promotes tolerance against more severe subse- quent ischemic insults in organs such as the heart and brain. This phenomenon involves many factors, includ- ing PKC [29–31]. Involvement of PKCe in the precon- ditioning has been well established in cardiac cells using PKCe-specific peptide activators and inhibitors [29,31] and has been confirmed using PKCe KO mice [32]. Unlike wild-type and heterozygous mice, precon- ditioning in PKCe KO mice does not reduce infarct size caused by ischemia reperfusion, implicating the involvement of PKCe in preconditioning. These results were obtained from a non-neural system but PKCe acts in a similar way in neurons. Indeed, the role of PKCe in neural preconditioning has been investigated using hippocampal slices [33,34] and primary cultured neurons [35,36] as well as PKCe- specific peptide activators and inhibitors. According to these studies, NMDA and adenosine receptor-mediated neural preconditioning require PKCe activation. Although the mechanism of PKCe-mediated neural preconditioning is not fully understood, inhibition of extracellular signal-regulated kinase (ERK) attenuated the adenosine receptor-mediated neural precondition- ing, implicating the involvement of ERK in precondi- tioning [34]. These findings suggest that PKCe may have a protective role in stroke. Recently, Shimomura et al. [37] demonstrated that the levels of PKCe are markedly lower in the core of focal cerebral ischemia and that this loss was prevented by hypothermia, which is known to be neuroprotective [37]. How hypotheramia alters levels of PKCe is currently unknown. Pain PKCe also localizes and functions in peripheral neurons such as nociceptive neurons. The nociceptive sensory neurons express the capsaicin receptor TRPV1, which is a nonselective cation channel activated not only by capsaicin, but also by heat (> 43 °C). TRPV1 is essential for the sensation of thermal and inflammatory pain [38] and pro-inflammatory signals, including ATP and bradykinin, enhance TRPV1 activ- ity in a PKC-dependent manner [39,40]. Among the PKC subtypes, PKCe has been reported to be predom- inantly and specifically involved in nociceptor sensiti- zation [39,40]. Indeed, PKCe directly phosphorylates Ser502 and Ser800 of TRPV1 [41]. It has also been shown that desensitization of TRPV1 is regulated by PKCe-mediated phosphorylation at Ser800 [42]. Fur- thermore, phosphorylation by PKCe appears to con- tribute to the proteinase-activated receptor 2-mediated potentiation of TRPV1 [43]. These findings suggest that PKCe may be a therapeutic target for regulating TRPV1 and pain. Other functions PKCe also modulates the Na + channel in hippocam- pal neurons [44]. Acetylcholine binding to muscarinic Y. Shirai et al. Function of PKCe in neurons FEBS Journal 275 (2008) 3988–3994 ª 2008 The Authors Journal compilation ª 2008 FEBS 3991 receptors activates G-proteins, phospholipase C and PKCe, resulting in a reduction of the peak of the Na + current in hippocampal neurons. This reduction is not observed in PKCe KO mice, implicating PKCe in the regulation of Na + channels. Such modulation of Na + channels by PKCe is likely to affect integration of depolarizing inputs in dendrites and the threshold and frequency of firing of action. In addition, PKCe is involved in phorbol ester-induced secretion of b-amy- loid precursor protein and the reduction of b-amyloid (Ab) peptides [45]. Neural overexpression of PKCe decreased Ab levels via endothelin-coverting enzyme [46], suggesting that PKCe is one of the regulators of a-secretase and Ab production. PKCe in the non-neural system is sometimes related to the neuronal system. For example, astrocytes have traditionally been considered as passive bystanders in the formation and operation of the neural network, but accumulating evidence argues against such a model, and instead supports a model in which astro- cytes play a critical role in the creation and control of synapses. Interestingly, differentiation of astrocytes, in part, is regulated by PKCe. Sterinhart et al. [47] dem- onstrated that 4b-phorbol 12-myristate 13-acetate and PKCe overexpression induces the differentiation of multipotential neural precursor cells to astrocytes, and that this induction is inhibited by a kinase negative mutant of PKCe [47]. They also demonstrated the involvement of Notch in this processes. Perspective As described above, PKCe plays several important roles in neurons. However, its precise function and mechanism of action in neurons is yet to be fully understood compared to the other functions deter- mined for this enzyme [48]. One of the reasons is that several PKC isoforms exist in the same neuron [3,4] and different PKC subtypes can have opposing func- tions. For example, PKCc is also involved in GABA A receptor regulation in response to ethanol but, in contrast to PKCe, PKCc plays an inhibitory role in ethanol-induced enhancement of GABA A receptor- mediated inhibitory postsynaptic currents in CA1 [49]. Ethanol enhanced inhibitory postsynaptic currents in wild-type mice, but not in PKCc null mice. By con- trast, these currents are potentiated in PKCe KO mice. In addition, unlike the protective effect of PKCe on reperfusion injury, PKCd exacerbates injury [30]. The PKCe KO mice have been an invaluable model for discriminating between the effects of ePKC and other PKCs. Notably, the importance of PKCe in the ethanol sensitivity and in Na + channel regulation have been clearly demonstrated using KO mice. By contrast, the involvement of PKCe in the LTP or its protective effect on stoke have not been validated using PKCe KO mice. It is possible that such experiments are ongoing or that the KO mice might be somehow different from wild-type mice due to compensation by other PKC subtype(s). Because PKCe obviously has important functions in neurons, more specific inhibi- tors and activators would be useful to define the pre- cise and subtype-specific functions of PKCe. With respect to delivery to the brain, development of a chemical inhibitor of PKCe that crosses the blood– brain barrier is necessary because the PKCe inhibitors used so far are peptides that cannot be employed effec- tively for in vivo neural studies. These PKCe-specific inhibitors or activators are expected to be utilized as a drug for stroke, Alzheimer’s disease and pain because the functions of the enzyme have been reported as descibed above. 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J Pharmacol Exp Ther 305, 264–270. Function of PKCe in neurons Y. Shirai et al. 3994 FEBS Journal 275 (2008) 3988–3994 ª 2008 The Authors Journal compilation ª 2008 FEBS . PIP 2 binding of PKCe may in uence the function of actin binding proteins, leading to actin rearrange- ment and neurite induction. In addition, the binding. phosphatidylinositol 4,5-bis phosphate; PKCe, protein kinase Ce; RhoA, Ras homolog gene family, member A; ROCK, Rho-associated coiled-coil-containing protein kinase. 3988