Pharmacological Research 54 (2006) 317–325 Review The different facets of protein kinases C: old and new players in neuronal signal transduction pathways Marialaura Amadio a , Fiorenzo Battaini b , Alessia Pascale a,∗ a Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy b Department of Neuroscience, University of Roma “Tor Vergata”, Roma, Italy Accepted 8 August 2006 Abstract Signal transduction pathways are crucial for cell-to-cell communication. Various molecular cascades allow the translation of distinct stimuli, targeting the cell, into a language that the cell itself is able to understand, thus elaborating specific responses. Within this context, a strategic role is played by protein kinases which catalyze the phosphorylation of specific substrates. The serine/threonine protein kinase C (PKC) enzymes family (at least 10 isoforms) is implicated in the transduction of signals coupled to receptor-mediated hydrolysis of membrane phospholipids. Within this molecular pathway, protein–protein interactions play a critical role in directing the distinct activated PKCs towards selective subcellular compartments, in order to guarantee spatio-temporal and localized cellular responses. A space-specific modulation of biochemical events is particularly important during learning. Among the various mechanisms, the modulation of mRNA decay appears to be an efficient post-transcriptional way of controlling gene expression during learning, allowing changes to take place in selected neuronal regions, in particular at synaptic level. To this regard, recent studies have pointed out that PKC activation is also involved in a novel signalling cascade leading to the stabilization of specific mRNAs. This review will especially focus the attention on the implication of PKC in memory trace formation and how alterations within this molecular cascade may have consequences on physiological and pathological neuronal aging (i.e. Alzheimer’s disease). © 2006 Elsevier Ltd. All rights reserved. Keywords: PKC; RACK1; ELAV proteins; Memory; Aging and Alzheimer’s disease Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PKC family and the importance of the interaction with scaffolding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of PKCs within some cellular functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Nuclear activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Neurotransmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Hypotheses on the involvement of ELAVs in memory and on their relationship with PKC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Physiological and pathological aging target PKC pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction The flow of information from the outside to the inside of the cell and vice versa allows each cell to adapt to continual changes ∗ Corresponding author. Tel.: +39 0382 987 963; fax: +39 0382 987 405. E-mail addresses: [email protected] (F. Battaini), [email protected] (A. Pascale). 1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.08.002 317 318 320 320 321 321 322 323 323 323 and requests coming from the external environment, and to maintain a general equilibrium. Among the multiple molecular systems belonging to the so-called signal transduction process, the proteins phosphorylation and dephosphorylation represent a versatile mechanism to mediate cellular responses. The two main classes of enzymes controlling these processes are on one side the protein kinases, which catalyse the phosphotransfer reaction, and on the other side the phosphatases, which are implicated in 318 M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 the subsequent dephosphorylating hydrolysis reaction. Several signalling networks, operating on both kinases and phosphatases, orchestrate these phosphorylation/dephosphorylation events. Anchoring proteins also participate in this complex machinery that has as a final goal the regulation of specific cellular functions, through adding/removing phosphate group(s) on selected substrates present in distinct subcellular sites . Localized responses are in fact crucial to guarantee that specific neuronal regions become theatre of key changes, some of them leading to synaptic remodelling occurring during learning. 2. PKC family and the importance of the interaction with scaffolding proteins The term protein kinase C (PKC) defines a serine/threonine family of phosphorylating enzymes ubiquitously expressed and implicated in multiple cellular functions. These kinases transduce signals involved in short-term processes (ion fluxes, neurotransmitter release), mid-term process (receptor modulation), as well as long-term processes (cell proliferation, synaptic remodelling and gene expression). According to sequence homology and sensitivity to activators, at least 10 isoforms (encoded by nine different genes) have been described. The various PKCs are grouped in three distinct classes: (1) calcium-dependent or classical cPKCs (␣, ␤I, ␤II and ␥); (2) calcium-independent or novel nPKCs (␦, , and ); and finally (3) atypical aPKCs ( and /) (for a review see [2,3]). The general structure of the different PKCs includes conserved domains (C1–C4) (see Fig. 1) separated by variable sequences (V1–V5). C1–C2 represent the regulatory portion of each enzyme, where the specific activators interact with, while C3–C4 form the catalytic region responsible for both the substrate binding and the kinase activity. The catalytic domain is characterized by a high degree of homology among the various kinases. The C1 domain bears one (in atypical) or two (in conventional and novel) cysteine-rich domains, located near the amino-terminal region, that represent the docking-sites for phosphatidylserine (PS) and the physiological activator diacylglycerol (DAG) as well as for the analogous phorbol esters (i.e. PMA), which activate all the different PKCs, with the exception of the atypical isoforms (that are sensitive/activated by PS and also ceramide and phosphatidylinositol-3,4,5-trisphosphate). The C2 region contains the Ca2+ binding-site and it is present in both the conventional and the novel isoenzymes (as a “C2like” sequence). However, the nPKCs are not responsive to Ca2+ because of the absence in the C2-like domain of amino acids residues essential for the Ca2+ binding: recruitment to membrane is dependent on a C1 region with higher affinity for phospholipids (in comparison with cPKCs) . Finally, C3 bears the ATP binding lobe, while C4 contains the substrate docking sequence . All known PKCs, at the amino-terminal side, are also characterized by a pseudosubstrate or autoinhibitory region, adjacent to C1, which keeps each isoform in an inactive conformation, as indicated in Fig. 1 as a “folded conformation”. The interaction with the specific activators allows the activation of the enzyme by opening its folded conformation: these cofactors are in fact able to decrease the affinity of the pseudosubstrate domain for the catalytic site that can exert its phosphorylating activity. This activation mechanism is related to the translocation of PKCs to different intracellular sites , and the subsequent phosphorylation of specific substrates by the translocated enzyme. The translocation/activation mechanism of each PKC from one to another subcellular compartment was firstly thought to reflect only the interaction between PKC and lipids. However, several proteins can interact with inactive and active PKCs dictating enzyme location in both basal and stimulated conditions, underlining the additional relevance of protein–protein interactions in PKCs homeostasis [7,8]. A relevant example is the importance of receptors for activated C kinase (RACKs) in the localization of activated PKCs close to pertinent substrates [9–11] (see also Fig. 2). RACKs are intracellular scaffold proteins belonging to the tryptophan-aspartate 40 (WD40) repeat family. They are homologous to the ␤-subunit of heterotrimeric G-proteins. These anchoring proteins appear to be isoform-specific; so far, four RACK-like proteins have been described: RACK1 preferentially binds to PKC␤II ; RACK2 (␤ -COP) interacts with PKC , PRKCBP1 is a putative anchoring protein for PKC␤I  while p32 (gC1qBP) can bind activated PKC␦ . RACK1, cloned from a rat brain cDNA library , is the best described member of this family of scaffolding proteins. RACK1 comprises seven WD40 repeats and several studies indicate that bears various independent binding-sites . The individual repeats can simultaneously interact with diverse signalling molecules, suggesting that RACK1 can participate to distinct intracellular cascades [10,17]. A direct correlation between PKC and RACK1 has been demonstrated in cardiac myocytes where phorbol ester-activated PKC␤ colocalizes with RACK1 , confirming the key implication of RACKs in PKC-mediated functions. In addition, PKC␤II and RACK1 redistribute synchronously after activation with both phorbol esters and dopamine in CHO cells overexpressing the long isoform of the dopamine receptor type 2 . In astrocytes, translocation of PKC from the cytosol to the plasma membrane has been reported to precede and to be related to the transformation of polygonal-shaped astrocytes into processbearing cells [20,21]. It is interesting to note that such changes in astrocyte morphology are documented not only during brain development, but also in pathological conditions such as reactive gliosis . We found that in rat astrocytes, following phorbol esters exposure, PKC-␤II was efficiently translocated from the cytosol to the membrane compartment . However, RACK1 did not redistribute synchronously with PKC-␤II to the plasma membrane, as instead reported in studies on activated CHO (expressing dopamine D2 receptors) and in neuroblastomaglioma hybrid NG108-15 cells . These data may indicate that the requirement for an asynchronous relocation of PKC-␤II and its anchoring protein is specific in astrocytes. On the other hand, we observed that the integrity of the actin-cytoskeleton (but not of tubulin-cytoskeleton) was required in these cells, since only the disruption of the actin-based cytoskeleton deranged the translocation process of PKC-␤II. Additional functional data on chromaffin cells indicate that the interaction of F-actin with activated PKC␣ and ␤ through the binding to RACK1 is M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 319 Fig. 1. Signal transduction PKC cascades: the interaction of calcium-dependent PKCs with selective activators [diacylglycerol (DAG), phosphatidylserine (PS) and calcium (Ca2+ )] reduces the affinity of the pseudosubstrate domain for the catalytic site thus opening the folded conformation and allowing the activation of the enzyme. The activated PKC can be directed to specific subcellular compartments via anchoring proteins (i.e. RACKs). PKC is also involved in the phosphorylation/activation of the mRNA-stabilizing ELAV proteins, especially important for gene expression regulation. The correct activation of these molecular pathways triggers specific responses in the cell; in contrast, changes in any of these steps may affect cellular responses and be implicated in aging-associated physiological or pathological alterations (i.e. Alzheimer’s disease). [ARE: Adenine-uridine rich element; ELAV: embryonic lethal abnormal vision; PKC: protein kinase C; RACKs: receptors for activated C kinase.] essential for the potentiation by phorbol ester of catecholamine release (Ohara-Imaizumi, Battaini and Kumakura, in preparation). According to all these results, we can hypothesize that, within some transduction pathways, PKC may translocate, via actin filaments, to different pools of RACKs located in distinct compartments. RACKs may then act not only as shuttling proteins for PKCs, but also as protein acceptors. We also propose that, in some cells, rapidly occurring changes of actin cytoskeleton organization may be implicated in the fast reprogramming of PKCs targeting to specific subcellular compartments where these enzymes can phosphorylate appropriate substrates. A fascinating role proposed for RACK1 is that it may also assemble signalling complexes on the ribosomes (for a review see ). Within this context, it has been reported that the PKC-␤II-mediated phosphorylation of the initiation factor eIF6 allows the functional assembling of the ribosome. In fact, the unphosphorylated form of eIF6 binds to the large subunit of the ribosome, thus preventing its association with the smaller ribosomal subunit, while the phosphorylated eIF6 dissociates from the large ribosomal subunit allowing its association with the smaller subunit . One of the most exciting aspects of PKC/RACKs interactions, having relevant pharmacological implications, is the observation that peptides that specifically inhibit or stimulate PKC binding to its RACK, inhibit or stimulate the activity of that specific isozyme in vitro . These peptides with appropriate carriers in vivo can be delivered to different organs, including brain, where that PKC isozyme is specifically affected . 320 M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 Fig. 2. PKC translocation-dependent pathways. The activation of Gq-coupled metabotropic or tyrosine kinase receptors leads to the hydrolysis of membrane phospholipids (i.e. PIP2, phospatidyl-inositol-4,5-bisphosphate) via phospholipase C activation and the generation of the two second messengers: diacylglycerol (DAG) and inositol trisphophate (IP3). IP3 induces the following release of calcium (Ca2+ ) from the intracellular store—the endoplasmic reticulum. Ca2+ and DAG activate PKC. Activated PKC can either bind to soluble RACK and being shuttled to subcellular sites or bind RACK directly close to relevant substrates where it can exert its specific function (i.e. on synaptic remodelling processes associated with learning; on proliferation and differentiation mechanisms; on translation control, etc.; see text for more details). Besides the already mentioned RACKs, proteins that interact with C kinase (PICKs) and substrates that interact with C kinase (STICKs) have been reported to be also implicated in PKCs compartmentalization. Although through slightly different mechanisms, the common function of these anchoring proteins is to recruit the various PKCs within specific signalling networks where they can affect selected cellular functions . 3. Role of PKCs within some cellular functions 3.1. Nuclear activities Although most of the biological functions of PKC have been related to events occurring within the membrane/cytoplasm compartments, a growing body of evidence point out on a role of PKC in nuclear functions as DNA replication, RNA synthesis and processing, gene expression . Different PKCs have been mapped in the nucleus in various tissues and especially at brain level, where PKC is activated in response to a variety of mitogenic stimuli. Within this context, several studies have tried to link the presence of nuclear PKCs with cell proliferation. The most direct finding has come from investigations in HL60 cells on the nuclear envelope component laminin B, a major PKC substrate at this level . Laminin B is phosphorylated by PKC-␤II following its translocation to the nucleus after the treatment with a DAG-mimicking compound (i.e. Bryostatin). Functionally, this phosphorylation triggers laminin B solubilization. These results indicate that PKC-␤II stimulation is required for nuclear laminin phosphorylation and disassembly leading to the entry into mitosis. In parallel, during the G2 –M transition, increased levels of nuclear DAG have been also documented . Increasing evidences indicate that the translocation to the nucleus of the various PKCs may be implicated in cell differentiation, mainly of hematolymphopoietic lineage (reviewed in ). However, functional studies addressed to how PKCs may affect cellular differentiation are lacking. One possibility is that nuclear PKCs may mediate transcriptional changes of genes involved in this process. In contrast to necrosis, which is a form of cell death that results from acute cellular injury, apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms and it is carried out in an ordered and highly regulated fashion. Within this context, a role in such an important mechanism has also been reported for different PKCs, being either proapoptotic or antiapoptotic depending on PKC isozyme and cell type (for a review see ). A direct link between PKC and apoptosis was documented in HL60 cells. In these cells, induced to apoptosis, the PKC␣-mediated M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 phosphorylation of laminin B preceded both laminin proteolytic degradation and DNA fragmentation . However, although some information have been added within the field of nuclear PKCs, more details need to be still collected in order to better understand the physiological significance of the phosphorylation events taking place in the nucleus. As far as RACK1 is concerned, although this protein is not constitutively expressed in nuclei, at least at brain level , it can move to nuclei upon acute ethanol exposure in cultured neurons and in vivo mouse brain areas involved in memory (hippocampus CA2/3 and cerebellum)  and then regulates gene expression, at least in glioma cells . 3.2. Neurotransmission Release of neurotransmitters from presynaptic terminals requires a highly specialized mechanism leading to the fusion of synaptic vescicles with the plasma membrane. This multiplesteps process is regulated by various protein kinases; actually, all the elementary steps implicated in the exocytosis mechanism can be modulated by phosphorylation. Within this context, in PC12 cells the SNARE protein family plays a key role in promoting membrane fusion; indeed, SNAP-25 has been reported to be a PKC substrate . Growth associated protein-43 (GAP43), a presynaptic PKC target, also seems to interact directly with the components of the synaptic release machinery, including the SNARE complex, and some of these interactions seem to depend on PKC-mediated GAP-43 phophorylation . Additionally, an intriguing partner for RACK1 is the SNARE protein Syntaxin1A which binds the dopamine transporter (DAT). DAT is implicated in the regulation of the extent and duration of the dopamine receptor activation. This process depends on DAT phosphorylation by PKC, stressing the involvement of PKC in neuronal exocytosis . Moreover, RACK1 also interacts with the NR2B subunit of the NMDA receptor, the major excitatory system in the brain. Modulation of the neuronal NMDA is critical within synaptic transmission; in fact, the regulation of channel gating and trafficking via PKCs is intimately related to neuronal synaptic plasticity . RACK1 has also been reported to participate in the functioning of GABA receptors , the primary inhibitory system in the mammalian brain. Furthermore, it has been documented that, in adrenal chromaffin cells, PKC-dependent phosphorylation of myristoylated Alanine-rich C-kinase substrate (MARCKS)  and of GAP-43  are involved in the regulation of the rapid phase of catecholamine release. Finally, PKC inhibitors suppress phorbol-ester mediated potentiation of neurotransmitter release . 3.3. Hypotheses on the involvement of ELAVs in memory and on their relationship with PKC Memory processes allow organisms to keep and recall experiences. Persistent changes in the efficacy of cell-to-cell communication are assumed to reflect long-lasting biochemical and morphological modifications implicated in encoding mechanisms. 321 Investigations on animal models and patients suggest that memory is not a single entity; rather, different kinds of memory can be dissected and involved in distinct neuronal systems in the brain (reviewed in ). Within this context, several families of protein kinases have been reported to play a key role in learning and memory processes. In particular, growing evidence documents the importance of PKC (see also ). The implication of long term potentiation (LTP) in memory storage has been widely explored and is a well established model for learning and memory. In this synaptic model of memory, brief trains of high-frequency stimuli to monosynaptic excitatory pathways trigger a sustained increase in the efficiency of synaptic transmission (for a review see [46,47]). Several studies have indicated a role of PKC isoforms both in the induction and in the maintenance phases of LTP  as well as of the constitutive active PKM (deriving from endogenous proteolysis of PKC) specifically in the maintenance step . The prominent involvement of PKC in neuronal changes related to associative learning was initially pointed out by investigations on the marine snail Hermissenda crassicornis . Pavlovian conditioning was also shown to trigger PKC redistribution in rabbit hippocampus , and following behavioural studies reported that the kinase C pathway is also implicated in spatial memory [51,52]. In fact, poor spatial learners present decreased PKC activity in the hippocampus , as well as a reduced amount of the neuron-specific PKC␥ . Moreover, intraventricular injections of phorbol esters improve spatial learning abilities in rodents , while kinase inhibitors administration impairs the same performances . Furthermore, pharmacological investigations proved that nootropic drugs, known to facilitate cognitive mechanisms, target PKC [56–58]. Generally speaking, it should be taken into account that learning is a complex mechanism associated with specific biochemical changes taking place in selected neuronal regions. These changes reflect both transcriptional and post-transcriptional controls of gene expression, although only post-transcriptional mechanisms permit a localized modulation of biochemical events occurring, at synaptic level, during learning. Within this context, literature data suggest the importance of messenger RNA stabilization and the consequent increase of the amount of a given gene product. The best characterized RNA binding proteins reported to act post-transcriptionally as positive controllers of gene expression are the ELAV proteins. ELAVs are highly conserved proteins that preferentially interact with adenine and uridine-rich elements (AREs). These ARE motifs are present in the 3 -untranslated region (3 -UTR) of a subset of mRNAs, including those of many early responsive genes, which through their 3 -UTR are targeted for rapid degradation . Following the binding of ARE-containing mRNAs to ELAV proteins, there is an increase in their cytoplasmic stability and rate of translation . Of relevance to this aspect, we showed that in vivo PKC activation is related to an upregulation of the neuron-specific ELAV proteins (nELAVs) in the hippocampal regions of rat brain . Studies performed by our group previously demonstrated that nELAVs play a critical physiological role in control- 322 M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 ling gene expression during memory trace formation. Indeed, only animals that had learned a spatial task showed an upregulation of nELAVs in hippocampal pyramidal cells . This change was accompanied by enhanced expression of GAP-43 , a gene known to be implicated in synaptic plasticity and whose mRNA is a nELAVs target . The strategic implication of nELAVs in memory was confirmed by experiments showing a significant impairment in the radial maze performance of mice treated with an antisense specifically raised against with one of the nELAVs . Finally, we showed a specific increase, both in vitro and in vivo, of the expression of the nELAV HuD subsequent to treatment with PKC activators (e.g. DAG-mimicking compounds such as bryostatins) , which were also reported to improve cognitive abilities in Alzheimer’s disease transgenic mice . All these findings indicate that PKC signalling and nELAV proteins recruitment and activation belong to the same molecular pathway that ultimately can affect the expression of genes essential for synaptic remodelling. It remains to be explored whether RACK1 protein also participates within this cellular cascade anchoring PKCs to the specific ELAV substrates. This new scenario opens novel pharmacological approaches focused to modulate learning and memory processes, and to eventually counteract age-related alterations. 3.4. Physiological and pathological aging target PKC pathway A growing body of evidence clearly indicates that memory deteriorates with aging. Some of the prominent changes of the brain reflect alterations involving a number of neurotransmitter systems leading to modified interneuronal communications. Taking into account that protein kinases play a key role in numerous signal transduction cascades, alterations in the function of these enzymes may be directly related to neuronal dysfunctions taking place in aging. Considering that PKC stimulation strictly depends on an increase of intracellular free calcium levels and on cell membrane lipid composition, age-related changes in these parameters may affect the cellular functions mediated by this kinase family. In order to avoid the influence of these variables, we explored the functional response of PKC during senescence in terms of enzyme translocation. We found that, following phorbol esters exposure, calcium-dependent brain PKC translocation was preserved up to middle-age (8 months old), but was impaired in aged rodents (more than 24 months) in both cortex and hippocampus, this effect being strain-independent as opposed to kinase activity [66–68]. Additionally, PKC translocation process was blunted also in terms of calcium-independent activity in cortical tissues of senescent Wistar rats . These findings indicate that the age-related alteration of this mechanism is independent of eventual changes in neurotransmitter release, receptor(s) availability and second messenger(s) production, since phorbol esters challenge bypass receptor activation and the subsequent IP3 /DAG production. Further results indicate that the amount of the various isoenzymes (both calcium-dependent and -independent) are preserved during senescence, stressing the concept that only the enzyme activation undergoes age-related changes irrespective of the strain investigated [67,70]. Taking into account the presumable implication of RACK proteins in directing the activated PKCs to relevant targets, we explored whether the age-mediated impairment observed in PKC relocation could be associated with additional changes in protein–protein interactions. Following researches were thus aimed to assess RACK1 content during senescence. We found that, in comparison with adult and middle-aged animals, old Wistar rats presented a reduced amount of RACK1 in the brain cortex (roughly 50%) . Further results obtained in aged Sprague–Dawley rats confirmed a decrease in cortical RACK1 protein content, coupled with a parallel reduction in mRNA levels, also in this rodent strain . Overall these data stress the involvement of RACKs in PKC translocation and indicate that changes occurring in senescence may affect those cellular components essential for PKCs full activation and function. Alzheimer’s disease (AD), the most common form of senile dementia, is characterized by gradual deterioration of cognitive functions and memory, associated with widespread neuronal death [72–74]. Several in vitro studies demonstrated a direct implication of the PKC transduction cascade also in brain neurodegeneration occurring in AD. For the sporadic form of the disease, the highest risk factor is represented by aging. The growing attention devoted to this dramatic pathology is related to the increase of span-life during these last decades. Neurofibrillary tangles and neuritic plaques are distinct markers of AD. Intracellular neurofibrillary tangles are characterized by paired helical filaments that are forms of abnormally phosphorylated tau protein, while neuritic plaques, which are largely extracellular lesions, are mainly composed of a 40–42 amino acids protein referred to as ␤-amyloid peptide (A␤). A␤ is produced via an aberrant proteolytic cleavage of a larger precursor named APP (amyloid precursor protein). The amyloidogenic cleavage of APP occurs through ␤-secretase that firstly originates a C-terminal fragment (CTF␤), which is subsequently cut by ␥-secretase to generate A␤. The non-amyloidogenic pathway of APP involves, instead, the ␣-secretase enzyme that cuts within the A␤ sequence leading to the secretion of a soluble APP fragment (sAPP) . Several in vitro researches point out a direct role of PKC in the regulation of the nonamyloidogenic metabolism of APP . Relevantly to our previous findings, PKC activation is deficient in AD brain in terms of marked loss of translocation of cortical PKC from the cytosolic compartment to the membrane in response to phorbol esters and K+ depolarization . Moreover, according to our data obtained from rat brains during senescence, also in Alzheimer’s disease the expression of RACK1 was reduced in AD human frontal cortex when compared to agematched controls . In analogy with our previous results from old rodents (see ), no pathology-related changes were documented in PKC-␤II basal protein contents in AD tissues . These data strongly indicate that AD is not associated with a reduced expression of PKCs, rather, the mechanism of kinase activation-anchoring undergoes pathology-dependent alterations. M. Amadio et al. / Pharmacological Research 54 (2006) 317–325 4. Conclusion Literature data clearly indicate that PKC certainly plays a critical role in several signal transduction pathways. Within the multiple functions in which the PKC signalling cascade is implicated, recent investigations indicate also its involvement in a new neuronal pathway leading to mRNA stabilization (see Fig. 1). However, how the cell is able to selectively elaborate a precise response in a distinct neuronal district remains a still partially unsolved problem. During the last years a more complex scenario is emerging, where different cellular components seem to be involved in order to assess specific and localized responses. A primary role within this complex machinery is played by the so-called anchoring proteins, able to direct the various PKCs in distinct compartments of the cell (see Fig. 2). Memory and learning processes represent the best examples where localized responses play a key importance. In fact, molecular changes taking place at synaptic level are essential for neuronal plasticity and remodelling mechanisms to occur. Changes in any of these cellular components or a disruption of the PKC system may then have consequences in cell alterations, such as those documented in physiological or pathological aging (i.e. Alzheimer’s disease) or in other nervous system-related abnormal states . A better understanding of the whole picture may lead to new pharmacological interventions aimed to improve or modulate those signal transduction pathways affected during senescence or age-associated pathologies that are responsible for memory disturbances. A first step in this direction is the observation that activation of PKC with the non tumour promoter bryostatin decreases brain beta amyloid accumulation, premature death and improves behavioural outcomes in mice models of Alzheimer’s disease . Acknowledgments The authors would like to thank Annamaria Pascale-Proksch for carefully reviewing the manuscript, and Dr. Miriam Duchen for her precious and constant support. This work was partially sponsored by a grant from Italian Ministero Sanità/Regione Lazio (Progetto Alzheimer) to F.B. References  Bauman AL, Scott JD. Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Nat Cell Biol 2002;4:E203–6.  Newton AC. Regulation of the ABC kinases by phosphorylation: PKC as a paradigm. Biochem J 2003;370:361–71.  Nishizuka Y. The protein kinase C family and lipid mediators for transmembrane signaling and cell regulation. Alcohol Clin Exp Res 2001;25(5 Suppl. ISBRA):3S–7S.  Giorgione JR, Lin JH, McCammon JA, Newton AC. Increased membrane affinity of the C1 domain of protein kinase C delta compensates for the lack of involvement of its C2 domain in membrane recruitment. J Biol Chem 2006;281:1660–9.  Newton AC, Johnson JE. 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