Two-dimensional surface plasmon resonance imager: An approach to study neuronal differentiation

There is a growing demand for the development of a new bioanalytical technique that is capable of monitoring neuronal differentiation noninvasively, in real time, and without any fluorescent probes. In a previous article, we demonstrated that a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager was very useful to monitor cell response on chemical stimulation in which protein kinase C (PKC) translocation was related. In the current study, we focused on developing a new method for monitoring neuronal differentiation and examined the application of the high-resolution 2D–SPR imager to monitor neuronal differentiation noninvasively and by a label-free format. We successfully monitored the intracellular signal transduction, which was mainly translocation of PKC in PC12 cells by the 2D–SPR imager, and found that the cells treated with a differentiation factor, nerve growth factor (NGF), showed a remarkable enhancement of 2D–SPR response to muscarine, carbachol, and acetylcholine stimulation. The results demonstrated that 2D–SPR sensing is applicable to in situ assessment of neuronal differentiation and to studying the expression state of the specific receptors in the living state.     

Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.     

Chronic exposure to ammonia induces isoform-selective alterations in the intracellular distribution and NMDA receptor-mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.     

Protein kinase C (PKC) isozyme-specific substrates and their design

Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, appears to be involved in the signal transduction response to many hormones and growth factors; there are 11 different PKC isozymes. Because PKC isozymes directly and/or indirectly participate in signal transduction pathways of normal and transformed cells through phosphorylation of target proteins, it is critical to understand the diversity of the intracellular signaling pathways regulated by each PKC isozyme. Thus, PKC isozyme-specific substrates are useful to understand the characterization of the intracellular signaling pathways for each PKC isozyme. Consensus sequences and sequence information obtained from PKC target proteins are very important to design PKC isozyme-specific peptide substrates. Moreover, computational prediction programs of phosphorylation sites using a library of peptide substrates aid in the fast design of PKC isozyme-specific peptide substrates. Although a large number of target proteins and synthetic peptides for PKCs are known, only two peptide substrates (peptide 422–426 of murine elongation factor-1α and Alphatomega peptide) have been reported as PKC isozyme-specific peptide substrates. This discussion will review the literature concerning these native and synthetic PKC isozyme-specific peptide substrates and their design.     

Stimulation of hyaluronan synthesis by interleukin-1beta involves activation of protein kinase C betaII in fibroblasts from patients with Graves' ophthalmopathy

Hyaluronan accumulation in the retroorbital connective tissue is one of the pathological features of Graves' ophthalmopathy. Interleukin-1beta (IL-1beta) is known to stimulate hyaluronan synthesis in orbital fibroblasts. In the present study, the intracellular signal transduction pathways involved in this stimulatory effect were investigated in cultured human retroorbital fibroblasts from patients with Graves' ophthalmopathy. IL-1beta-induced hyaluronan synthesis was significantly inhibited by pretreatment of the cells with two protein kinase C (PKC) inhibitors, chlerythrine chloride and H-7. In addition, treatment with phorbol 12-myristate 13-acetate (PMA), a direct PKC activator, also resulted in increased hyaluronan production. IL-1beta- or PMA-stimulated hyaluronan synthesis was blocked by the protein synthesis inhibitor, cycloheximide. Moreover, the intracellular Ca(2+) concentration of the orbital fibroblasts was also involved in the IL-1beta induced transduction pathway, the effect being completely inhibited by BAPTA, an internal calcium chelator. In addition, A23187, a calcium ionophore, increased hyaluronan synthesis in unstimulated cells. These results suggest that the Ca(2+)-dependent PKC signal transduction pathway plays an important role in the IL-1beta-induced hyaluronan synthesis. Moreover, IL-1beta treatment resulted in increased PKC activity and the rapid translocation of PKC betaII from the cytoplasm to the plasma membrane. These results indicate that cytosolic Ca(2+) and PKC betaII are involved in IL-1beta-induced hyaluronan synthesis in cultured orbital fibroblasts from patients with Graves' ophthalmopathy.     

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µ M TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µ M ). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.     

Barbituric acid derivative BAS 02104951 inhibits PKCε, PKCη, PKCε/RACK2 interaction, Elk-1 phosphorylation in HeLa and PKCε and η translocation in PC3 cells following TPA-induction

Protein kinase C (PKC) is a family of at least 10 isozymes involved in the activation of different signal transduction pathways. The exact function of these isozymes is not known at present. Isozyme-selective inhibitors would be important to explain the function of the different PKCs and are anticipated to have pharmaceutical potential. Here we report that the small organic molecule BAS 02104951 [5-(1,3-benzodioxol-5-ylmethylene)-1-(phenylmethyl)-2,4,6(1H,3H,5H)-pyrimidinetrion], a barbituric acid derivative, inhibited PKCη and PKCε in vitro (IC(50) 18 and 36 μM, respectively). BAS 02104951 also inhibited the interaction of PKCε with its adaptor protein receptor for activated C-kinase 2 (RACK2) (IC(50) 28.5 μM). BAS 02104951 also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Elk-1 phosphorylation in HeLa cells, translocation of PKCε and PKCη to the membrane following treatment of PC3 cells with TPA. The compound did not inhibit the proliferation of PC3 and HeLa cells. BAS 02104951 can be used as selective inhibitor of PKCε in cells not expressing PKCη and may serve as a basis for the rational development of a selective inhibitor of PKCε or PKCη, or for an inhibitor of the PKCε/RACK2 interaction.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

Signal transduction events in human basophils. A comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine.

We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.     

T-cell activation

Activation of T lymphocytes results in immediate intracellular biochemical changes, including increases in cytosolic Ca(2+) levels, stimulation of protein kinase C (PKC) and regulation of protein tyrosine kinases (PTKs). This review describes recent advances in the study of the signalling steps downstream of PKC and PTKs in T cells. A model is presented in which the GTP-binding protein p21(ras) acts as an integrator of the signal transduction pathways controlled by the T-cell antigen receptor.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.     

胰岛素影响自发性高血压大鼠血管平滑肌细胞增殖及肌动蛋白分布的丝裂原激酶的机制探讨

血管平滑肌细胞增殖的同时伴有细胞内肌动蛋白的改变,这种改变受PKC-MAPK信号转导途径调控,但目前机制尚不清楚。为探讨胰岛素对PKC-MAPK信号转导途径参与调控血管平滑肌细胞增殖及细胞内肌动蛋白分布的影响,本研究用PKC抑制剂预处理SHR在鼠体外培养的血管平滑肌细胞,观察预处理的血管平滑肌细胞经胰岛素刺激后细胞内DNA的合成、MAPK的活性、表达及细胞内肌动蛋白的分布。发现,胰岛素刺激后可使血管平滑肌细胞增殖,同时伴有[^3H]TdR掺入增加、MAPK活性及表达与对照组比较明显升高。这些作用可被PKC抑制剂阻断。胰岛素在刺激血管平滑肌细胞增殖的同时也使细胞内肌动蛋白重新分布,这一效应也可被PKC抑制剂阻断。 上述结果提示,胰岛素使血管平滑肌细胞增殖的效应可能与MAPK信号转导途径有关。     

Mitogen-Activated Protein Kinase-Dependent and Protein Kinase C-Dependent Pathways Link the m1 Muscarinic Receptor to β-Amyloid Precursor Protein Secretion

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21^ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans,trans -farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

Effect of the Tumor Promoter Phorbol 12-Myristate 13-Acetate (PMA) on Proliferation of Young and Senescent WI-38 Human Diploid Fibroblasts

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event [64], was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.     

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor-Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-α-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP₃ level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

Activity of novel protein kinase C and distribution of protein kinase C theta in subcellular fractions of normal and Duchenne muscular dystrophic muscle

Phospholipid-dependent, Ca(2+)-independent isoenzymes termed novel protein kinase C or nPKC, include PKC delta, epsilon, eta, theta and mu. Status and role of nPKC and PKC theta in Duchenne muscular dystrophic (DMD) condition is unknown. In the present study, we have shown that most of the nPKC isoforms are translocated to the membrane fraction of DMD tissue specimen. It is well established that translocation plays a key role in signal transduction by individual PKC isoforms. In our experiment, the increased association of nPKC isoform PKC theta to membrane was further confirmed by Western blot. Increased expression of PKC theta mRNA was identified by dot blot analysis. The above results suggest that, the alterations in nPKC location and increased expression of PKC theta observed is a result of modification of PKC-mediated signal transduction and cell function.     

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

To define the molecular bases of growth factor-induced signal transduction pathways, antibodies known to block the activity of either protein kinase C (PKC) or the fos protein were introduced into PC12 cells by microinjection. The antibody against PKC significantly inhibited neurite outgrowth when scored 24 h after microinjection and exposure to nerve growth factor (NGF). Microinjection of antibodies to fos significantly increased the percentage of neurite-bearing cells after exposure to either NGF or basic fibroblast growth factor (bFGF) but inhibited the stimulation of DNA synthesis by serum, suggesting that in PC12 cells, fos is involved in cellular proliferation. Thus, activation of PKC is involved in the induction of neurite outgrowth by NGF, but expression of the fos protein, which is induced by both NGF and bFGF, is not necessary and inhibits neurite outgrowth.