ucn-1028-c and rottlerin

Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca(2+)-mediated exocytosis. In rat parotid acinar cells, the activation of beta-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The beta-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCdelta inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCdelta, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCdelta, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

Topics: Acetophenones; Amylases; Animals; Benzopyrans; Bucladesine; Cell Membrane; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Isoproterenol; Isoquinolines; Male; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Naphthalenes; Parotid Gland; Peptide Fragments; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Rats; Rats, Sprague-Dawley; Sulfonamides

Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH(3), the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCbeta, or PKCzeta but not by HBDDE or peptide inhibitors for PKCvarepsilon or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCalpha, beta, delta, and zeta by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the gamma, epsilon, eta, mu, theta, or iota PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCalpha is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCbeta, PKCdelta, and PKCzeta in combined diacylglycerol-dependent (PKCbeta and PKCdelta) and -independent (PKCzeta) signaling pathways.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Benzopyrans; Carbazoles; Cell Fusion; Cells, Cultured; Enzyme Inhibitors; Giant Cells, Foreign-Body; Humans; Indoles; Isoenzymes; Isoquinolines; Macrophages; Maleimides; Monocytes; Naphthalenes; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-delta; Signal Transduction

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.

Topics: Acetophenones; Amino Acid Sequence; Antigen-Antibody Complex; Arachidonic Acid; Benzopyrans; Enzyme Activation; Humans; Indoles; Macrophage Activation; Maleimides; Molecular Sequence Data; Monocytes; NADPH Oxidases; Naphthalenes; Oligodeoxyribonucleotides, Antisense; Phosphoproteins; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Superoxides

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Carbazoles; Caspases; Colonic Neoplasms; Drug Synergism; Enzyme Inhibitors; Humans; Indoles; Membrane Glycoproteins; Mitochondria; Naphthalenes; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

1. Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. 2. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. 3. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation.

Topics: Acetophenones; Adenosine Triphosphate; Benzopyrans; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; CD18 Antigens; Cell Aggregation; Cycloheximide; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genistein; Humans; Imidazoles; Indoles; Integrin beta1; Isoenzymes; Maleimides; Mitogen-Activated Protein Kinases; Naphthalenes; Phosphorylation; Phosphotransferases; Protein Kinase C; Protein-Tyrosine Kinases; Pyridines; Staurosporine; Temperature; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine; U937 Cells

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCepsilon isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca(2+)-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCdelta isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs alpha, betaI, betaII, delta, gamma, and lambda failed to enhance either EGF or TRH responses, whereas overexpression of nPKCeta enhanced the EGF response. Neither stable nor transient overexpression of nPKCepsilon produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCdelta construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCdelta, whereas nPKCeta may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca(2+)-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

Topics: Acetophenones; Animals; Benzopyrans; Calcium; Carbazoles; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation; Indoles; Isoenzymes; Maleimides; Naphthalenes; Phorbol Esters; Pituitary Gland; Prolactin; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Rats; Staurosporine; Thyrotropin-Releasing Hormone; Transcription, Genetic

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis.

Topics: Acetophenones; Benzopyrans; Bryostatins; Calcium; Cell Membrane; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Humans; Lactones; Macrolides; Mitogens; Naphthalenes; Protein Isoforms; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate

Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCdelta is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCdelta from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (alpha, betaI, betaII, and epsilon) did not translocate in response to fibroblast growth factors. The PKCdelta subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCdelta adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCdelta isoform. PKC inhibitors selective for the conventional alpha and beta isoforms or dominant negative PKCalpha adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCdelta expression but not by an analogous dominant negative PKCalpha construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCdelta.

Topics: Acetophenones; Adenoviridae; Animals; Benzopyrans; Carbazoles; Culture Media, Serum-Free; Enzyme Activation; Enzyme Inhibitors; Fibroblast Growth Factors; Immunoblotting; Indoles; Isoenzymes; MAP Kinase Signaling System; Naphthalenes; Pituitary Neoplasms; Prolactin; Promoter Regions, Genetic; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Rats; Tumor Cells, Cultured

The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils.

Topics: Acetophenones; Antigens, CD; Benzopyrans; Cells, Cultured; Chemotactic Factors; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Dipeptides; Down-Regulation; Humans; Hydroxamic Acids; L-Selectin; Metalloendopeptidases; N-Formylmethionine Leucyl-Phenylalanine; Naphthalenes; Neutrophil Activation; Neutrophils; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Staurosporine; Tetradecanoylphorbol Acetate

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Topics: Acetophenones; Alkaloids; Benzophenanthridines; Benzopyrans; Bryostatins; Carbazoles; Enzyme Activation; Enzyme Inhibitors; Indoles; Isoenzymes; Lactones; Macrolides; Maleimides; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Recombinant Proteins; Schizosaccharomyces; Sphingosine; Tetradecanoylphorbol Acetate; Transformation, Genetic