PKMζ is necessary and sufficient for synaptic clustering of PSD-95

The persistent activity of protein kinase Mzeta (PKMζ), a brain-specific, constitutively active protein kinase C isoform, maintains synaptic long-term potentiation (LTP). Structural remodeling of the postsynaptic density is believed to contribute to the expression of LTP. We therefore examined the role of PKMζ in reconfiguring PSD-95, the major postsynaptic scaffolding protein at excitatory synapses. In primary cultures of hippocampal neurons, PKMζ activity was critical for increasing the size of PSD-95 clusters during chemical LTP (cLTP). Increasing PKMζ activity by overexpressing the kinase in hippocampal neurons was sufficient to increase PSD-95 cluster size, spine size, and postsynaptic AMPAR subunit GluA2. Overexpression of an inactive mutant of PKMζ did not increase PSD-95 clustering, and applications of the ζ-pseudosubstrate inhibitor ZIP reversed the PKMζ-mediated increases in PSD-95 clustering, indicating that the activity of PKMζ is necessary to induce and maintain the increased size of PSD-95 clusters. Thus the persistent activity of PKMζ is both necessary and sufficient for maintaining increases of PSD-95 clusters, providing a unified mechanism for long-term functional and structural modifications of synapses.     

How does PKMζ maintain long-term memory?

Most of the molecular mechanisms contributing to long-term memory have been found to consolidate information within a brief time window after learning, but not to maintain information during memory storage. However, with the discovery that synaptic long-term potentiation is maintained by the persistently active protein kinase, protein kinase Mζ (PKMζ), a possible mechanism of memory storage has been identified. Recent research shows how PKMζ might perpetuate information both at synapses and during long-term memory.     

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130 ^-/- ) and gp130 floxed (gp130 ^fl/fl ) mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130 ^fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130 ^-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.     

Involvement of Spinal PKMζ Expression and Phosphorylation in Remifentanil-Induced Long-Term Hyperalgesia in Rats

Up-regulation of GluN2B-containing N-methyl-d-aspartate receptors (NMDARs) expression and trafficking is the key mechanism for remifentanil-induced hyperalgesia (RIH), nevertheless, the signaling pathway and pivotal proteins involved in RIH remain equivocal. PKMζ, an isoform of protein kinase C (PKC), maintains pain memory storage in neuropathic pain and inflammatory pain, which plays a parallel role regulated by NMDARs in long-term memory trace. In the present study, Zeta Inhibitory Peptide (ZIP), a PKMζ inhibitor, and a selective GluN2B antagonist Ro-256981 are injected intrathecally before remifentanil infusion (1 μg kg^?1 min^?1 for 1 h, iv) in order to detect whether GluN2B contributes to RIH through affecting synthesis and activity of PKMζ in spinal dorsal horn. Nociceptive tests are measured by Paw withdrawal mechanical threshold (PWT) and paw withdrawal thermal latency (PWL). The L₄–L₆ segments of dorsal horn taken from rats with RIH are for determining expression of PKMζ and pPKMζ by Western blot and immunohistochemistry. Our data suggest that remifentanil infusion causes an increase of PKMζ in expression and phosphorylation in rats with nociceptive sensitization, beginning at 2 h, peaked at 2 days, and returned to basal level at 7 days. ZIP (10 ng) could block behavioral sensitization induced by remifentanil. Ro25-6981 dosage-dependently attenuated mechanical and thermal hyperalgesia and reversed expression of PKMζ and pPKMζ, indicating that GluN2B-containing NMDA receptor facilitates development of RIH through mediating expression and activity of spinal PKMζ in rats. Although detailed mechanisms require further comprehensive study, the preventive role of Ro25-6981 and ZIP provide novel options for the effective precaution of RIH in clinics.     

PKMζ Differentially Utilized between Sexes for Remote Long-Term Spatial Memory

It is well established that male rats have an advantage in acquiring place-learning strategies, allowing them to learn spatial tasks more readily than female rats. However many of these differences have been examined solely during acquisition or in 24h memory retention. Here, we investigated whether sex differences exist in remote long-term memory, lasting 30d after training, and whether there are differences in the expression pattern of molecular markers associated with long-term memory maintenance. Specifically, we analyzed the expression of protein kinase M zeta (PKMζ) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2. To adequately evaluate memory retention, we used a robust training protocol to attenuate sex differences in acquisition and found differential effects in memory retention 1d and 30d after training. Female cohorts tested for memory retention 1d after 60 training trials outperformed males by making significantly fewer reference memory errors at test. In contrast, male cohorts tested 30d after 60 training trials outperformed females of the same condition, making fewer reference memory errors and achieving significantly higher retention test scores. Furthermore, given 60 training trials, females tested 30d later showed significantly worse memory compared to females tested 1d later, while males tested 30d later did not differ from males tested 1d later. Together these data suggest that with robust training males do no retain spatial information as well as females do 24h post-training but maintain this spatial information for longer. Males also showed a significant increase in synaptic PKMζ expression and a positive correlation with retention test scores, while females did not. Interestingly, both sexes showed a positive correlation between retention test scores and synaptic GluA2 expression. Furthermore, the increased expression of synaptic PKMζ, associated with male memory but not with female memory, identifies another potential sex-mediated difference in memory processing.     

PKMζ Maintains Spatial,Instrumental, and Classically Conditioned Long-Term Memories

How long-term memories are stored is a fundamental question in neuroscience. The first molecular mechanism for long-term memory storage in the brain was recently identified as the persistent action of protein kinase Mzeta (PKMζ), an autonomously active atypical protein kinase C (PKC) isoform critical for the maintenance of long-term potentiation (LTP). PKMζ maintains aversively conditioned associations, but what general form of information the kinase encodes in the brain is unknown. We first confirmed the specificity of the action of zeta inhibitory peptide (ZIP) by disrupting long-term memory for active place avoidance with chelerythrine, a second inhibitor of PKMζ activity. We then examined, using ZIP, the effect of PKMζ inhibition in dorsal hippocampus (DH) and basolateral amygdala (BLA) on retention of 1-d-old information acquired in the radial arm maze, water maze, inhibitory avoidance, and contextual and cued fear conditioning paradigms. In the DH, PKMζ inhibition selectively disrupted retention of information for spatial reference, but not spatial working memory in the radial arm maze, and precise, but not coarse spatial information in the water maze. Thus retention of accurate spatial, but not procedural and contextual information required PKMζ activity. Similarly, PKMζ inhibition in the hippocampus did not affect contextual information after fear conditioning. In contrast, PKMζ inhibition in the BLA impaired retention of classical conditioned stimulus–unconditioned stimulus (CS-US) associations for both contextual and auditory fear, as well as instrumentally conditioned inhibitory avoidance. PKMζ inhibition had no effect on postshock freezing, indicating fear expression mediated by the BLA remained intact. Thus, persistent PKMζ activity is a general mechanism for both appetitively and aversively motivated retention of specific, accurate learned information, but is not required for processing contextual, imprecise, or procedural information.     

Specific involvement of atypical PKCζ/PKMζ in spinal persistent nociceptive processing following peripheral inflammation in rat

Background

Central sensitization requires the activation of various intracellular signalling pathways within spinal dorsal horn neurons, leading to a lowering of activation threshold and enhanced responsiveness of these cells. Such plasticity contributes to the manifestation of chronic pain states and displays a number of features of long-term potentiation (LTP), a ubiquitous neuronal mechanism of increased synaptic strength. Here we describe the role of a novel pathway involving atypical PKCζ/PKMζ in persistent spinal nociceptive processing, previously implicated in the maintenance of late-phase LTP.

Results

Using both behavioral tests and in vivo electrophysiology in rats, we show that inhibition of this pathway, via spinal delivery of a myristoylated protein kinase C-ζ pseudo-substrate inhibitor, reduces both pain-related behaviors and the activity of deep dorsal horn wide dynamic range neurons (WDRs) following formalin administration. In addition, Complete Freund's Adjuvant (CFA)-induced mechanical and thermal hypersensitivity was also reduced by inhibition of PKCζ/PKMζ activity. Importantly, this inhibition did not affect acute pain or locomotor behavior in normal rats and interestingly, did not inhibited mechanical allodynia and hyperalgesia in neuropathic rats. Pain-related behaviors in both inflammatory models coincided with increased phosphorylation of PKCζ/PKMζ in dorsal horn neurons, specifically PKMζ phosphorylation in formalin rats. Finally, inhibition of PKCζ/PKMζ activity decreased the expression of Fos in response to formalin and CFA in both superficial and deep laminae of the dorsal horn.

Conclusions

These results suggest that PKCζ, especially PKMζ isoform, is a significant factor involved in spinal persistent nociceptive processing, specifically, the manifestation of chronic pain states following peripheral inflammation.

     

PKMζ Inhibition Reverses Learning-Induced Increases in Hippocampal Synaptic Strength and Memory during Trace Eyeblink Conditioning

A leading candidate in the process of memory formation is hippocampal long-term potentiation (LTP), a persistent enhancement in synaptic strength evoked by the repetitive activation of excitatory synapses, either by experimental high-frequency stimulation (HFS) or, as recently shown, during actual learning. But are the molecular mechanisms for maintaining synaptic potentiation induced by HFS and by experience the same? Protein kinase Mzeta (PKMζ), an autonomously active atypical protein kinase C isoform, plays a key role in the maintenance of LTP induced by tetanic stimulation and the storage of long-term memory. To test whether the persistent action of PKMζ is necessary for the maintenance of synaptic potentiation induced after learning, the effects of ZIP (zeta inhibitory peptide), a PKMζ inhibitor, on eyeblink-conditioned mice were studied. PKMζ inhibition in the hippocampus disrupted both the correct retrieval of conditioned responses (CRs) and the experience-dependent persistent increase in synaptic strength observed at CA3-CA1 synapses. In addition, the effects of ZIP on the same associative test were examined when tetanic LTP was induced at the hippocampal CA3-CA1 synapse before conditioning. In this case, PKMζ inhibition both reversed tetanic LTP and prevented the expected LTP-mediated deleterious effects on eyeblink conditioning. Thus, PKMζ inhibition in the CA1 area is able to reverse both the expression of trace eyeblink conditioned memories and the underlying changes in CA3-CA1 synaptic strength, as well as the anterograde effects of LTP on associative learning.     

Cellular and subcellular localization of PKMζ

In contrast to protein kinases that participate in long-term potentiation (LTP) induction and memory consolidation, the autonomously active atypical protein kinase C isoform, protein kinase Mzeta (PKMζ), functions in the core molecular mechanism of LTP maintenance and long-term memory storage. Here, using multiple complementary techniques for light and electron microscopic immunolocalization, we present the first detailed characterization of the cellular and subcellular distribution of PKMζ in rat hippocampus and neocortex. We find that PKMζ is widely expressed in forebrain with prominent immunostaining in hippocampal and neocortical grey matter, and weak label in white matter. In hippocampal and cortical pyramidal cells, PKMζ expression is predominantly somatodendritic, and electron microscopy highlights the kinase at postsynaptic densities and in clusters within spines. In addition, nuclear label and striking punctate immunopositive structures in a paranuclear and dendritic distribution are seen by confocal microscopy, occasionally at dendritic bifurcations. PKMζ immunoreactive granules are observed by electron microscopy in cell bodies and dendrites, including endoplasmic reticulum. The widespread distribution of PKMζ in nuclei, nucleoli and endoplasmic reticulum suggests potential roles of this kinase in cell-wide mechanisms involving gene expression, biogenesis of ribosomes and new protein synthesis. The localization of PKMζ within postsynaptic densities and spines suggests sites where the kinase stores information during LTP maintenance and long-term memory.     

Nuclear relocation of DGKζ in cardiomyocytes under conditions of ischemia/reperfusion

Diacylglycerol (DG) and phosphatidic acid (PA) are generated under various conditions, such as ligand stimulation and several stresses. They serve as second messengers to respond to pathophysiological conditions. DG kinase (DGK) catalyzes DG to produce PA. It is regarded as a regulator of these lipid messengers. Previous studies show that DGKζ, a nuclear isozyme, translocates from the nucleus to the cytoplasm in hippocampal neurons under transient ischemia and never relocates to the nucleus after reperfusion. This study examined whether a similar phenomenon is observed in cardiomyocytes, which represent another type of postmitotic, terminally differentiated cell. We performed immunostaining on ischemic hearts induced by occlusion of the left anterior descending coronary artery and on primary cultured cardiomyocytes under oxygen-glucose deprivation (OGD). In the animal model, 10 min ischemia is sufficient to cause DGKζ to disappear from the nucleus in cardiomyocytes. However, DGKζ is observed again in the nucleus at 10 min following reperfusion after 10 min ischemia, which contrasts sharply with ischemic hippocampal neurons. Similar results were obtained from experiments using primary cultured cardiomyocytes under OGD conditions, except that DGKζ relocates autonomously, if at all, to the nucleus, even under continuous OGD conditions. Results suggest that DGKζ is involved in the acute phase of cellular response to ischemic stress in cardiomyocytes in a similar, but not identical, manner to that of neurons.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.     

Down-regulation of PKCζ in renal cell carcinoma and its clinicopathological implications

Background

Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.

Methods

PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.

Results

PKCζ expression was significantly higher in normal than in cancerous tissues (P < 0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P = 0.04), but no such association was found in TNM stage (P = 0.13). Tumors with higher PKCζ expression were associated with tumor size (P = 0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.

Conclusions

PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.     

PLCζ and its role as a trigger of development in vertebrates

A major unresolved issue in developmental biology is the precise mechanism whereby the sperm activates the oocyte. With the discovery that calcium signals are the primary trigger for oocyte activation, a key remaining question became the identification of the signaling protein that mediates such calcium signals at fertilization. A major step forward came in 2002 with the discovery of a sperm-specific mammalian phospholipase C called phospholipase C zeta (PLCζ), which had the expected properties of the mammalian oocyte activation factor and was subsequently identified in other vertebrate groups. Most recently, defects in PLCζ have been shown to be linked to certain types of male infertility in humans. Despite these advances, many questions remain about the precise mechanism of action of PLCζ and the extent of its role during oocyte activation in the vertebrate kingdom. In this review, we will look at the current state of understanding of PLCζ's mechanism of action and physiological role in mammals and other vertebrates, and identify areas of uncertainty that still remain to be resolved.     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.Key words: protein kinase C, chemoresistance, oxidative stress, nuclear translocation, apoptosis     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.     

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.     

Comparative Analysis of PKСα and PKCζ Activities in Rat and Lamprey Erythrocytes of Different Ages

The present study aimed to find out a link between ageing of rat and lamprey erythrocytes and activity of two isoforms of protein kinase C (PKC), РKСα and РKСζ. The whole cell population was separated into fractions of different ages in Percoll density gradient. The validity of separation was confirmed by the number of immature erythrocytes, reticulocytes. PKC activity was analyzed in cytosolic and membrane cell fractions. Rat erythrocytes express both PKC isoforms, РKСα and РKСζ, whereas lamprey erythrocytes express only РKСζ. РKСα is identified as a major band at ~ 80 kDa and minor bands at ~ 55–65 kDa; РKСζ is represented by a single band at ~ 80 kDa. In young rat erythrocytes, РKСα is detected mainly in cytosolic fractions, while in membrane fractions its level is by far lower. As cells age, PKCα is translocated from the cytosol to membranes and undergoes proteolytic degradation due to repeated cycles of activation. As a result, in aged erythrocytes relative total PKCα expression (as a sum of expressions in the cytosol and membranes per total protein level) diminishes, indicating a depletion of the PKCα pool and a decline in its functional activity. In both animal species, a highest PKCζ level is observed in the cytosol of young erythrocytes. Erythrocyte ageing is accompanied by a gradual decrease in expression of free cytosolic PKCζ and concurrent increase in the level of its membrane-bound forms. However, in contrast to PKCα, PKCζ is not proteolyzed; its total level in cells and perhaps functional activity do not change throughout the erythrocyte lifespan.     

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.