Thyroid cell responses to thyrotropin and 12-O-tetradecanoyl-phorbol-13-acetate: translocation of protein kinase C and phosphorylation of thyroid cell polypeptide substrates

Not all of the effects of thyroid-stimulating hormone (TSH) on the thyroid are mediated by activation of the adenylate cyclase-cyclic AMP system, indicating that other control systems must also exist. Although a calcium-phospholipid-dependent protein kinase (protein kinase C) and specific substrates had been identified in thyroid tissue, their responsiveness to TSH and other stimulators has not been determined. In thyroid cells which had been preloaded with [32P]orthophosphate, TSH and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increased the phosphorylation of a 33K polypeptide substrate within 5 min in a dose-dependent fashion. The effect was observed with 1 mU/ml TSH and 3 nM TPA and was maximal with 100 mU/ml TSH and 100 nM TPA. The biologically inactive analog of TPA, 4 alpha-phorbol, had no effect. Isobutylmethylxanthine (IBMX) decreased the phosphorylation of the 33K polypeptide and inhibited the effect of TSH and TPA, indicating that the phosphorylation is not mediated by cyclic AMP. TSH and IBMX, but not TPA, augmented phosphorylation of a 38K polypeptide, suggesting involvement of cyclic AMP. In contrast TPA, but not TSH, increased the phosphorylation of 58K and 28K polypeptides. TSH, but not TPA or 4 alpha-phorbol, elevated the cyclic AMP level of thyroid slices. Incubation of thyroid slices with TSH or TPA significantly decreased protein kinase C activity in the 100,000g cytosol fraction and increased it in an extract of plasma membranes. The effect was present within 5 min and was maximal by 30 min. The effect was observed with 100 mU/ml TSH or 1 nM TPA. The stimulation by TSH or TPA of protein kinase C and its translocation from the cytosol to the plasma membranes of thyroid tissue may provide another mechanism for control of thyroid cell metabolism.     

Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Activation signals in human lymphocytes: interleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C

The mitogenic activation of resting T lymphocytes involves two distinct cellular events, the synthesis of the ultimate mitogen interleukin 2 and the synthesis and expression of receptors for it. In order to get more detailed information on the mechanisms associated with these activating steps (the effects of different stimuli, leading to activation of protein kinase C were investigated in human lymphocytes). The anti-T-cell receptor (TCR) and anti-CD3 monoclonal antibodies (BMA 031 and BMA 030, respectively), as well as the combination of the phorbol ester, TPA, with a calcium ionophore-induced interleukin 2 synthesis and subsequent proliferation in human peripheral blood lymphocytes. Incubation of cells with synthetic diacylglycerols and calcium ionophores proved to be effective in expression of high affinity interleukin receptors, no detectable amounts of interleukin 2 were, however, synthetized. When diacylglycerols were, however, added repetitively, interleukin 2 was also produced. Both anti-TCR/CD3 antibodies and TPA or DiC8 caused activation and translocation of protein kinase C from the cytosol to the plasma membrane. Significant differences, however, were observed between the time kinetics of the translocation of the enzyme. In plasma membranes of TPA-stimulated cells activation of protein kinase C was detectable up to 4 hr. In contrast, the highest specific activity of protein kinase C was measured in the plasma membranes after 15 min of DiC8 addition to cells. Anti-CD3 monoclonal antibodies activated protein kinase C in a biphasic manner. Shortly after binding of BMA 030 to the T cell antigen receptor/CD3 complex the activity of protein kinase C was increased in the plasma membrane, then it declined to control levels followed by a second long-lasting activation of the enzyme up to 4 hr. These results suggest different signal requirements for different activation steps. While for synthesis and expression of interleukin 2 receptors a short term activation of protein kinase C is sufficient, long-term activation of the enzyme is necessary for interleukin 2 synthesis in human lymphocytes.     

Acetylcholine-induced phosphorylation and membrane translocation of CPI-17 in bronchial smooth muscle of rats

A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca2+ sensitization in smooth muscle contraction. Therefore, it is possible that a downstream target of PKC, CPI-17 [PKC-potentiated inhibitory protein for heterotrimeric myosin light chain (MLC) phosphatase of 17 kDa], might also be translocated to membrane when activated. To confirm this hypothesis, cytosolic and membrane CPI-17 was measured in acetylcholine (ACh)- and high-K+ depolarization-stimulated bronchial smooth muscle of rats. An active form of CPI-17, i.e., Thr38-phosphorylated CPI-17, was also measured in cytosolic and membrane fractions. Immunoblot analyses demonstrated a translocation of CPI-17 from cytosolic to membrane fraction by ACh, but not high-K+ depolarization, stimulation in time- and concentration-dependent manners. Interestingly, phosphorylated CPI-17 was detected only in membrane fractions in the ACh-stimulated tissues. However, in the high-K+ depolarization-stimulated tissues, phosphorylated CPI-17 was not detected both in membrane and cytosolic fraction. To estimate downstream of activated CPI-17, immunoblotting for phosphorylated MLC was performed in ACh- or high-K+ depolarization-stimulated tissues. ACh- and high-K+ depolarization-induced phosphorylation of MLC was observed in its contraction-dependent manner. In conclusion, we, for the first time, suggested that CPI-17 is translocated and phosphorylated by ACh, but not high-K+ depolarization, in rat bronchial smooth muscle. ACh-induced translocation and phosphorylation of CPI-17 might be caused via the activation of muscarinic receptor.     

Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor-Mediated Calmodulin Redistribution in SK-N-SH Neuroblastoma Cells

Abstract: Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 nM TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 µM. In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the α-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the ε-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.     

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.     

Altered regulation of a major substrate of protein kinase C in rat 6 fibroblasts overproducing PKC beta I.

We have shown previously that the stable overproduction of protein kinase C beta I (cPKC beta I) in rat 6 (R6) embryo fibroblasts results in multiple cellular growth abnormalities. To characterize the pathways through which cPKC beta I acts to exert its effects, we have undertaken a biochemical analysis of the cell line R6-PKC3. The subcellular distribution of cPKC beta I in unstimulated R6-PKC3 cells was approximately 80% cytosolic and approximately 20% membrane bound, and treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in translocation and down-regulation of an appreciable fraction of the cPKC beta I enzyme. However, long term TPA treatment was not sufficient to down-regulate all of the overproduced enzyme from both the cytosolic and membrane fractions. Two-dimensional gel analysis of 32P-labeled cellular phosphoproteins from either untreated or TPA-treated cultures revealed only minor qualitative differences between R6-PKC3 cells and a vector control cell line, R6-C1. On the other hand, several quantitative differences in the level of phosphorylation of discrete protein spots were seen. The most prominent phosphoprotein was a previously described 80/87-kDa protein designated MARCKS (myristoylated alanine-rich C kinase substrate). Compared with R6-C1 cells, R6-PKC3 cells exhibited a 2-3-fold increase in the basal level of phosphorylation of MARCKS and after treatment with TPA, displayed a dramatic prolongation in phosphorylation of this protein. Additionally, treatment of R6-PKC3 cells with TPA led to a prolonged increase in both the cytosolic and total cellular level of the MARCKS protein and a pronounced decrease in the level of MARCKS mRNA. Taken together, these results indicate that overproduction of cPKC beta I markedly alters several parameters of the MARCKS protein which may be responsible, at least in part, for the altered phenotype of these cells.     

Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C

The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.     

Activation of Protein Kinase C-α and Translocation of the Myristoylated Alanine-Rich C-Kinase Substrate Correlate with Phorbol Ester-Enhanced Noradrenaline Release from SH-SY5Y Human Neuroblastoma Cells

Abstract: The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the α-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-ε from cytosolic and membrane-associated to cytoskeleton- and membrane-associated TPA had no effect on the cytosolic location of PKC-ζ. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 µ M ). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 n M ) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-α (but not -ε or -ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-α.     

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes

A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.     

Translocation of diacylglycerol kinase in response to chemotactic peptide and phorbol ester in neutrophils

When neutrophils were stimulated by the chemotactic peptide, fMLP, a rapid, transient increase in the activity of diacylglycerol(DG) kinase in the membrane fraction was detected. DG kinase in cytosol, on the contrary, showed a transient decrease. The total activity in homogenates was not affected. Tetradecanoylphorbol acetate(TPA) and 1-oleoyl-2-acetylglycerol(OAG) also caused an increase in DG kinase activity in the membrane fraction. Km value of DG kinase in membranes was not changed by the treatment of fMLP or TPA, though Vmax was increased. Considering these results, DG kinase may translocate from cytosol to membranes on stimulation by fMLP, TPA or OAG in neutrophils. The translocation may play important roles in regulation of protein kinase C activity, since DG kinase competes with protein kinase C for DG, which is formed by receptor-activation.     

Immunocytochemical evidence for phorbol ester-induced protein kinase C translocation in HL60 cells

The ability of tumor promoting 12-O-tetradecanoylphorbol-13-acetate (TPA) to redistribute protein kinase C in human promyelocytic leukemic HL60 cells was investigated. It was found that TPA caused a rapid translocation (within 10 min) of protein kinase C from the cytosolic (soluble) fraction to the particulate (membrane) fraction, as determined indirectly by assaying for the enzyme activity or by immunoblotting of the enzyme protein in the isolated subcellular fractions. Immunocytochemical localization of the enzyme demonstrated directly that the TPA caused an enzyme translocation t the plasma membrane. These findings suggest that translocation to the plasma membrane of the enzyme may represent initial events related to the TPA effect on terminal differentiation of HL60 cells to monocytes/macrophages.     

Phorbol ester and 1,2-diolein are not fully equivalent activators of protein kinase C in respect to phosphorylation of membrane proteins in vitro

Phosphorylation of liver plasma proteins by protein kinase C was studied by using the two best known activators of the enzyme, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,2-diolein. While the effects of TPA and diolein were almost identical on two proteins and similar in magnitude on four proteins, the phosphorylation of an additional four proteins was increased only by TPA. We conclude that in respect to phosphorylation of membrane proteins, TPA and diglycerides are not fully equivalent activators of kinase C.     

Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C

We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.     

Transmembrane signaling: tumor promoter distribution

Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) localization acts in concert with substrate targeting to create spatial restriction for phosphorylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) acts in diverse cell types by phosphorylating proteins with key calcium-dependent functions such as synaptic plasticity, electrical excitability, and neurotransmitter synthesis. CaMKII displays calcium-dependent binding to proteins in vitro and translocation to synaptic sites after glutamatergic activity in neurons. We therefore hypothesized that subcellular targeting of CaMKII can direct its substrate specificity in an activity-dependent fashion. Here, we examined whether activity-dependent colocalization of CaMKII and its substrates could result in regulation of substrate phosphorylation in cells. We find that substrates localized at cellular membranes required CaMKII translocation to these compartments to achieve effective phosphorylation. Spatial barriers to phosphorylation could be overcome by translocation and anchoring to the substrate itself or to nearby target proteins within the membrane compartment. In contrast, phosphorylation of a cytoplasmic counterpart of the substrate does not require CaMKII translocation or stable protein-protein binding. Cytosolic phosphorylation is more permissive, exhibiting partial calcium-independence. Localization-dependent substrate specificity can also show more graded levels of regulation within signaling microdomains. We find that colocalization of translocated CaMKII and its substrate to lipid rafts in the plasma membrane can modulate the magnitude of phosphorylation. Thus, dynamic regulation of both substrate and kinase localization provides a powerful and nuanced way to regulate CaMKII signal specificity.     

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.     

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.