Rapid modulation of tumor necrosis factor membrane receptors by activators of protein kinase C

Tumor necrosis factor membrane receptors are rapidly down-regulated upon treatment of activated T lymphocytes with various activators of protein kinase C. Loss of binding-capacity was half maximal after 2 min. incubation in 10 ng/ml of phorbol 12-myristate 13-acetate. A similar modulation could be induced with either the calcium ionophore A 23187 or the protein kinase C activator 1-oleyl-2-acetyl glycerol, whereas 1,2-diolein and dibutyryl cAMP were ineffective. Protein kinase C inhibitor H7 antagonizes the phorbol ester-induced TNF receptor modulation. These data suggest an important role of protein kinase C in the control of TNF responsiveness by regulation of TNF binding-capacity possibly via direct phosphorylation of specific receptor proteins.     

Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.     

Direct modulation of Na+ currents by protein kinase C activators in mouse neuroblastoma cells

We investigated the effects of different protein kinase C (PKC) activators on Na⁺ currents using the conventional whole-cell and the inside-out macropatch voltage-clamp techniques in mouse neuroblastoma cells (N1E-115). Two different categories of PKC activators were investigated: the cis-unsaturated fatty acids (CUFAs): oleic (cis-9-octadecenoic), linoleic (cis-9-12-octadecadienoic), and linolenic acid (cis-9-12-15-octadecatrienoic), and, the diacylglycerol (DAG) derivative 1-2-dioctanoyl-sn-glycerol (DOG). These substances caused the following alterations on Na⁺ currents: (i) Na⁺ currents were attenuated as a function of voltage. While DOG attenuated both inward and outward Na⁺ currents in a monotonic and continuous voltage-dependent manner, CUFAs preferentially attenuated inward currents; (ii) the steady-state activation curve of Na⁺ currents shifted to more depolarized voltages; (iii) opposite to the activation curve, the steady-state inactivation curve of Na⁺ channels (h curve) shifted to more hyperpolarized voltages; (iv) the time course of inactivation development was accelerated by PKC activators, while the recovery from inactivation was not affected; (v) substances that inhibit different metabolic pathways (PKC activation, cyclooxygenase, lipooxygenase, and P-450 pathways) did not prevent the effects of PKC activators on Na⁺ currents. One fully saturated fatty acid (octadecanoic acid), a trans-unsaturated fatty acid (trans-9-octadecenoic), and different phorbol esters did not affect Na⁺ currents; (vi) effects of different PKC activators on Na⁺ currents were completely reversible. These observations suggest that PKC activators might interact with Na⁺ channels directly. These direct effects must be taken into consideration in evaluating the overall effect of PKC activation on Na⁺ channels. Moreover, it is likely that this direct interaction could account, at least in part, for the diversity of effects of PKC activators on Na⁺ channels.This work was supported in part by a grant-in-aid from the American Heart Association (National Center).     

Phosphorylation of bovine cardiac calcium-activated neutral protease by protein kinase-C

Protein kinase C prepared from rat brain was used to phosphorylate a calcium-activated neutral protease, purified from bovine cardiac muscle. Attempts to phosphorylate the enzyme in the presence of calcium were unsuccessful, unless the protease inhibitor leupeptin was also present. Phosphorylation of the 74K subunit of the protease was completely inhibited in the absence of phosphatidylserine and diolein, indicating that phosphorylation of the enzyme was catalysed by the calcium and phospholipid-dependent protein kinase C.     

Negative allosteric modulation of the glucagon receptor by RAMP2

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Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism

Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.     

Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C

Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.     

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k_cat for PKN1 and PKN2, and a decrease in peptide K_M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K_i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.     

Granulocyte colony-stimulating factor-induced activation of protein kinase-C in myeloid cells

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, differentiation, and activation of myeloid cells. It binds to a high affinity receptor (G-CSF-R) expressed on myeloid cells, for which the signal transduction mechanisms other than protein tyrosine kinase (PTK) activation have not been completely identified. We explored the potential involvement of protein kinase-C (PKC) in G-CSF-R signal transduction. In this report, we provide direct evidence of PKC activation by G-CSF-R. G-CSF treatment of peripheral blood neutrophils, granulocytic cell lines (HL-60, NFS-60, KG-1), and monocytic cell lines (WEHI-3B,U-937) resulted in PKC activation. Chelerythrine chloride and HA-100, an isoquinolinesulfonamide derivative, the specific inhibitors of PKC, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), a chelator of intracellular calcium, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), an inhibitor of intracellular calcium release, blocked G-CSF-induced PKC activation in HL-60 cells, and reduced CD11b upregulation in neutrophils, but did not affect ligand-binding or down-modulation of G-CSF-R. Methyl 2,5-dihydroxycinnamate (MDHC), a potent inhibitor of protein tyrosine kinases (PTK), also inhibited PKC activation in response to G-CSF treatment, suggesting that PKC activation may occur downstream of PTK activation. Our results demonstrate the involvement of PKC in G-CSF-R signal transduction, and suggest a common signaling pathway in myeloid cells of granulocytic and monocytic lineages. J. Cell. Biochem. 66:286–296, 1997. © 1997 Wiley-Liss, Inc.     

Inhibitory effect of H-7, H-8 and polymyxin B on liver protein kinase C-induced phosphorylation of endogenous substrates

Inhibitory actions of 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), N-[2-(methylamine)ethyl]-5-isoquinolinesulfonamide [H-8] and polymyxin B on the calcium-activated, phospholipid-dependent protein kinase (protein kinase C) of rat liver were compared. Using a partially purified liver protein kinase C and an exogenous substrate histone-III S, polymyxin B showed maximum inhibition (IC50, 9.5 microM) followed by H-7 (IC50, 25 microM) and H-8 (IC50, 36 microM). These inhibitors also inhibited protein kinase C-induced phosphorylation of endogenous cytosolic and particulate proteins in a dose-dependent manner though polymyxin B was relatively less effective with the particulate fraction. With the aid of protein kinase-C activators and these inhibitors, seven proteins in cytosolic (Mr 170K, 150K, 43K, 34K, 30K, 25K and 19K daltons) and six proteins in particulate (Mr 150K, 43K, 34K, 25K, 19K and 16K daltons) fractions were identified as probable substrates for protein kinase C in liver. The identity of these proteins remains to be determined.     

The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine

Thrombin, 1-oleoyl-2-acetyl-rac-glycerol (OAG), cis- or trans-octadecadienoic acids (linoleic and linolelaidic acid) and the synergistic combination of octadecadienoic acids plus OAG lead to the activation of gel-filtered human platelets, i.e. aggregation via protein kinase C (PKC). Platelet activation by thrombin was only slightly suppressed by polymyxin B, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporine, all being potent inhibitors of PKC in vitro. The OAG-induced aggregation, however, was strongly inhibited by H-7 or staurosporine but not by polymyxin B. In contrast, octadecadienoic acid-induced aggregation was substantially inhibited only by polymyxin B. Synergistic activation by OAG plus octadecadienoic acids was strongly suppressed by all three PKC inhibitors. Our results indicate (1) that the ability of various compounds to inhibit PKC in vitro does not correlate with their inhibitory effects in intact cells and (2) that platelet activation induced by various PKC activators exhibits differential PKC-inhibitor sensitivity.     

Activation of protein kinase-C inhibits vitamin D receptor gene expression.

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of epithelio-mesenchymal transformation of quail embryonic neural cells by inhibition of atypical protein kinase-C

Epithelio-mesenchymal transition, which involves the re-organisation of cell-cell adhesion molecules and the actin cytoskeleton, can be induced in embryonic neural epithelium in vitro by protein kinase-C inhibitors. A non-inhibitory analogue, BIM V, and potent inhibitors of other kinases are not active. This suggests a central role for C-kinases, although the powerful specific C-kinase inhibitors BIM I and Ro 31-8220 show lower than expected activity. Co-inhibition by several kinases is unlikely to account for this, since no potentiation occurs when these are combined with potent inhibitors of other kinases. BIM I and Ro 31-8220 strongly inhibit only conventional calcium-regulated C-kinases; this and the lack of effect of TMB-8, which inhibits calcium release, suggests that novel and/or atypical isoforms are involved. Various potentiators and activators of conventional and novel C-kinases have no obvious effect alone and fail to reduce the effect of staurosporine, suggesting that atypical C-kinases are critical. The presence of C-kinase isoforms in the E2 embryonic neural tissues has been probed on Western blots, revealing immunoreactivity for the atypical isoforms 1 (or 5) and + and the !, %, l and 7 isoforms. Immunofluorecent localisation on sections of embryos has shown the widespread distribution of conventional and novel isoforms but only the atypical isoforms 5 and + are enriched at the apical margins of the neural and other epithelia; they overlap with the cell-cell adhesion molecule N-cadherin and with F-actin. Thus, epithelio-mesenchymal transition in the embryonic neural epithelium in vitro is induced by inhibiting protein kinase activity, probably via an atypical protein kinase-C; atypical protein kinase-C isoforms are present in the tissue at the appropriate developmental stage and subcellular site in cells capable of epithelio-mesenchymal transition.     

Specific inhibition of phorbol ester and DiC8-induced histamine release from human basophils by the protein kinase C inhibitors, H-7 and H-9

The release of histamine and other inflammatory mediators from human basophils is triggered by numerous stimuli, including chemical, physical and receptor-mediated activators. Several mechanisms of cell activation including protein kinase C activation have been proposed to operate in these cells. We used phorbol ester and DiC8 to induce histamine release from human basophils and the protein kinase C inhibitors H-7 and H-9 to inhibit this release. Both DiC8 and TPA induced histamine release were inhibited by H-7 (ID 50 = 37 mcM) and H-9 (IC 50 = 20 mcM). However, anti-IgE, fmlp and A23187-induced histamine release were unaffected. In contrast, the calmodulin antagonists W-7 and perphenazine effectively inhibited histamine release by all five stimuli. Therefore, different biochemical pathways appear to be critical for basophil activation depending on the nature of the stimulus used.     

Stimulation of protein phosphorylations in frog rod outer segments by protein kinase activators. Suppression of light-induced changes in membrane current and cGMP by protein kinase C activators

Addition of protein kinase C activators to electropermeabilized frog rod photoreceptors enhances the phosphorylation of proteins with molecular masses of 54, 24, 19, 17, 12, and 11 kDa. The latter two correspond to components I and II, which are also phosphorylated by cyclic nucleotide-dependent protein kinase. Stimulation of phosphorylation by the protein kinase C activator oleoylacetylglycerol (OAG) is half-maximal at 7.7 microM OAG and is reduced by the protein kinase C inhibitor H-7. In contrast with earlier observations, no effects of calcium, calmodulin, or insulin on protein phosphorylations are observed. We find evidence for only three protein kinases in rod outer segments: a protein kinase C-like activity, cAMP-dependent protein kinase, and rhodopsin kinase. With the exception of components I and II, the substrate proteins for each kinase are distinct. Treatment of intact rods with OAG decreases the amplitude of the photoresponse and dark levels of cGMP up to 40%, as well as depressing the light-stimulated decrease in cGMP levels. These effects are observed between 0.1 and 1 microM OAG. The data suggest that OAG-sensitive reactions may modulate pathways that support the light response.     

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

Metabolic actions of vasopressin,glucagon and adrenalin in the intact rat

Metabolic effects of vasopressin, glucagon and adrenalin were compared, in intact rats, especially in regard to time courses of effects.Hyperglycaemia was transient in response to vasopressin, prolonged following adrenalin, and, surprisingly, was not discernible after glucagon, except in response to a very large dose. Vasopressin decreased and adrenalin increased, the plasma free fatty acid concentration; both hormones decreased the triacylglycerol level. Muscle glycogen concentrations, measured in heart, diaphragm and skeletal muscle, exhibited small changes, with complex time courses, following hormone administration. Vasopressin brought about a rapid but transient activation of hepatic glycogen phosphorylase which resembled that due to adrenalin. The activation by glucagon of phosphorylase was greater and more prolonged, despite the absence of hyperglycaemia. In response to vasopressin, there was an increase in plasma insulin. Incorporation of ¹⁴C from [¹⁴C] glucose into glycogen or fatty acids was not influenced by vasopressin. Taken together, these results may be explained by rapid metabolic action of vasopressin on hepatic glycogenolysis, whereas adrenalin has multiple prolonged actions.     

Calcium-induced aggregation of neuroendocrine protein 7B2in vitro and its modulation by ATP

To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca²⁺ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2_156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.