Intracellular Translocation Of Protein Kinase Research Articles

Two-Dimensional Surface Plasmon Resonance Imaging System for Cellular Analysis.

Optical biosensors based on surface plasmon resonance (SPR) phenomenon have received a great deal of attention in cellular analysis applications. Sensitive and high-resolution SPR imaging (SPRi) platforms are very useful for real-time monitoring and measurement of individual cell responses to various exogenous substances. In cellular analysis, mainstream SPR-based sensors have potential for investigations of cell responses under ambient conditions. Evaluations that account only for the average response of cell monolayers mask the understanding of precise cell-molecular interactions or intracellular reactions at the level of individual cells. SPR/SPRi technology has attracted a great deal of attention for detecting the response of cell monolayers to various substances cultivated on the gold sensor chip. To unleash the full strength of SPRi technology in complex cell bio-systems, the applied SPR imaging system needs to be sufficiently effective to allow evaluation of a compound's potency, specificity, selectivity, toxicity, and effectiveness at the level of the individual cell. In our studies, we explore the utility of high-resolution 2D-SPR imaging for real-time monitoring of intracellular translocation of protein kinase C (PKC), and detection of neuronal differentiation in live cells at the level of individual cells. The PC12 cell line, which is one of the most commonly used neuronal precursor cell lines for research on neuronal differentiation, was chosen as a nerve cell model. Two dimensional SPR (2D-SPR) signals/images are successfully generated. We have found that cells treated with the differentiation factor nerve growth factor (NGF) showed a remarkable enhancement of SPR response to stimulation by muscarine, a nonselective agonist of the muscarinic acetylcholine receptor.

Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells.

The spatio-temporal changes of signaling molecules in response to G protein-coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady-state distribution of green fluorescent protein (GFP)-tagged PKD indicated that in non-stimulated IEC-18 cells, GFP-PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP-PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)-mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin-induced GPCR activation.

Transforming growth factor-β 1 regulation of surfactant protein B gene expression is mediated by protein kinase-dependent intracellular translocation of thyroid transcription factor-1 and hepatocyte nuclear factor 3

The transforming growth factor-β (TGFβ) polypeptides control a variety of cellular processes including organogenesis and cellular proliferation and differentiation. In the developing lung, TGFβ 1 treatment inhibits airway branching and expression of the genes for surfactant proteins (SP). Many effects of TGFβ are mediated at the level of gene transcription but there is limited information regarding signaling pathways and target transcription factors. In this study with human pulmonary adenocarcinoma H441 cells, we investigated TGFβ 1 effects on SP-B, a protein which is essential for normal function of pulmonary surfactant. TGFβ 1 (10 ng/ml) reduced SP-B mRNA content in a time-dependent fashion, and transient transfection studies localized responsiveness to the region of the SP-B promoter (−112/−72 bp) containing binding sites for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor 3 (HNF3), transcription factors that are important enhancers of SP gene expression. Using electrophoretic mobility shift assay and immunofluorescence, we demonstrated rapid accumulation of these transcription factors in the cytoplasm and subsequent loss from the nucleus on TGFβ 1 treatment of both adenocarcinoma cells and cultured human fetal lung. TGFβ 1 treatment caused intracellular translocation of protein kinase C and effects of TGFβ 1 were mostly abrogated in the presence of the protein kinase inhibitor calphostin C. We conclude that TGFβ 1, acting via protein phosphorylation, blocks nuclear translocation of TTF-1 and HNF3 which results in down-regulation of the SP-B gene and presumably other pulmonary genes which are transactivated by these factors.

Developmental changes in glutamate receptor-activated translocation of protein kinase C in cerebellar granule neurons

Developmental changes in glutamate receptor agonist-produced enhancement of 4-β-[ 3H]phorbol-12,13-dibutyrate binding ([ 3H]-PDBu binding), indicative of an intracellular translocation of protein kinase C (PKC), were investigated in cerebellar granule cells. Our observations demonstrate that the magnitude of glutamate-, NMDA-, and kainate-produced enhacemenn of PKC translocation was dramatically decreased between 2 and 12 DIV, whereas there was only a minor reduction in the corresponding response caused by the non-NMDA receptor agonist, AMPA. The maximally enhanced stimulation of PKC translocation caused by glutamate and NMDA was significantly reduced already at 4 DIV, whereas a significant reduction of the kainate-induced enhancement of [ 3H]PDBu binding was not observed until 8 DIV. Glutamate- and NMDA-induced responses were effectively blocked by the specific NMDA receptor antagonists MK-801 (1 μM) and APV (100 μM) as well as by the addition of Mg 2+ into assay media. In contrast, the non-NMDA receptor antagonist, CNQX (10 μM), effectively blocked the kainate-induced enhancement of [ 3H]PDBu binding, but had no effect on the NMDA- and glutamate-induced stimulation of PKC translocation. The metabotropic glutamate receptor agonist, ACPD (up to 250 μM), had no effect on the translocation of PKC. Taken together, our data support the working hypothesis that the rapidly occurring changes in the glutamate receptor agonist-produced translocation of PKC are most likely due to a differential maturation of glutamate ionotropic receptor subtypes and/or to development-dependent alterations in mechanisms responsible for the coupling between the glutamate receptor subtypes and the activation of PKC translocation in cerebellar granule neurons.

Imaging of intracellular translocation of protein kinase C

Imaging of intracellular translocation of protein kinase C

Cell genetic evidence of correlation of intracellular translocation of protein kinase C (PKC) and PKC-mediated phosphorylation of 80-kDa protein with mitogenic action of tumor promoters

Recently, we isolated a series of 3T3-L1 cell variants that are unable to respond to mitogenic stimulation by the tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA). Since protein kinase C (PKC) is the major receptor for TPA and appears to play a key role in cellular proliferation, we have examined the distribution of PKC in the parental 3T3-L1 cells and the variant VT-1 cells. PKC was located predominantly in the cytosol of growth-arrested confluent 3T3-L1 cells, and upon TPA treatment it was rapidly translocated into the plasma membrane. In contrast, PKC was located predominantly in the plasma membrane of confluent VT-1 variant cells and was no longer activated by TPA. Two-dimensional gel analysis showed that a Mr 80,000 acidic protein (80-kDa protein) was rapidly phosphorylated in 3T3-L1 cells upon TPA treatment, whereas phosphorylation of this protein was barely detected in VT-1 cells. In growing cultures, the majority of PKC was found in the plasma membrane of both cell lines, and no change occurred upon TPA treatment. Hydroxyapatite column chromatography revealed the presence of alpha-type PKC as the major component in both cell lines. These results suggest that the intracellular translocation of alpha-type PKC and the PKC-mediated phosphorylation of the 80-kDa protein may be involved in the mechanism of mitogenic signal transfer.

Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. Differences between the effects of insulin and phorbol esters.

To determine whether insulin activates protein kinase C in BC3H-1 myocytes, we evaluated changes in protein phosphorylation, protein kinase activities, and the intracellular translocation of protein kinase C activity in response to insulin and phorbol esters. Phorbol 12-myristate 13-acetate (PMA), but not insulin, stimulated the phosphorylation of an acidic Mr 80,000 protein which has been shown to be an apparently specific marker for protein kinase C activation. In addition, PMA, but not insulin, stimulated the rapid association of protein kinase C activity with a cellular particulate fraction. In contrast to these differences, both insulin and PMA stimulated the phosphorylation of ribosomal protein S6 and activated a ribosomal protein S6 kinase in cell-free extracts from cells exposed to these agents. In cells exposed to high concentrations of PMA for 16 h, protein kinase C activity and immunoreactivity were abolished, without changes in cellular morphology. Under these conditions, insulin, but not PMA, stimulated phosphorylation of the ribosomal protein S6 in intact cells and activated the S6 kinase in cell-free extracts derived from insulin-treated intact cells. We conclude that: insulin does not appear to activate protein kinase C in BC3H-1 myocytes, at least as assessed by phosphorylation of the Mr 80,000 protein; both insulin and PMA activate an S6 protein kinase in these cells; and insulin can promote S6 phosphorylation and activate the S6 kinase normally in protein kinase C-deficient cells. Activation of the S6 kinase by insulin and PMA, although apparently proceeding through different mechanisms, may explain some of the similar biological actions of these compounds in BC3H-1 myocytes.

Differential mechanisms of translocation of protein kinase C to plasma membranes in activated human neutrophils

Three classes of activators of human neutrophils that induce the intracellular translocation of protein kinase C from the cytosol to the particulate fraction were compared for their effects on the properties of the particulate (membrane-bound) enzyme. In cells stimulated with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) the particulate enzyme is almost fully active in the absence of added Ca 2+ or phospholipids and this activity is not released by the Ca 2+-chelator EDTA. In contrast, binding of protein kinase C to the particulate fraction in cells treated with the chemotactic factor f-Met-Leu-Phe (fMLF) or with the ionophore A-23187 plus Ca 2+ is observed only when the cells are lysed in the presence of 1 mM Ca 2+. With these stimuli the particulate enzyme retains a nearly absolute requirement for Ca 2+ and phospholipids. Thus only the full intercalation of protein kinase C caused by PMA, which is resistant to removal by chelators stabilizes an active form of protein kinase C in the neutrophil membrane. In confirmation of this conclusion, in isolated plasma membranes loaded with partially purified protein kinase C by incubation with 5 μM Ca 2+ further incubation with PMA, but not with fMLF, caused a significant fraction of the bound PKC to become resistant to removal by chelators, and to be nearly fully active in the absence of added activators.

A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters.

The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.

Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C.

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14CO2 production from [1-14C]glucose and a fivefold increase in the rate of lipid synthesis from [14C]glucose. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate increased the rate of 86Rb+ uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-O-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondrial and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.