ucn-1028-c and herbimycin

Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated.. Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping.. In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH2-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs.. Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

Topics: Animals; Anthracenes; Benzophenanthridines; Cells, Cultured; Chromones; Colon; Flavonoids; Genistein; Imidazoles; Interstitial Cells of Cajal; Intestine, Small; Membrane Potentials; Mice; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Protein-Tyrosine Kinases; Pyridines; Rifabutin

An excessive production of extracellular matrix (ECM) proteins in glomerular mesangial cells is considered to be responsible for the development of mesangial expansion seen in diabetic nephropathy. Mechanical stretch due to glomerular hypertension has been proposed as one of the factors leading to an increase in the production of ECM proteins in mesangial cells, but the precise mechanism of stretch-induced overproduction of ECM proteins has not been elucidated. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) may play a key role in the overproduction of fibronectin (FN) in mesangial cells exposed to mechanical stretch. MAPK, also termed extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), was activated by mechanical stretch in time- and intensity-dependent manners. Stretch-induced activation of ERK was inhibited by herbimycin A, a tyrosine kinase inhibitor, but not by GF109203X or calphostin C, the inhibitors of protein kinase C. Mechanical stretch also enhanced DNA-binding activity of AP-1, and this enhancement was inhibited by PD98059, an inhibitor of MAPK or ERK kinase (MEK). Furthermore, mechanical stretch stimulated the expression of FN mRNA followed by a significant increase in its protein accumulation. PD98059 could prevent stretch-induced increase in the expression of FN mRNA and protein. These results indicate that the activation of ERK may mediate the overproduction of ECM proteins in mesangial cells exposed to mechanical stretch, an in vitro model for glomerular hypertension seen in diabetes.

Topics: Animals; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cell Nucleus; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Flavonoids; Gene Expression Regulation; Glomerular Mesangium; Indoles; JNK Mitogen-Activated Protein Kinases; Kinetics; Lactams, Macrocyclic; Male; Maleimides; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Naphthalenes; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; RNA, Messenger; Stress, Mechanical; Time Factors; Transcription Factor AP-1; Transcription, Genetic

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.

Topics: Alkaloids; alpha Catenin; Benzoquinones; beta Catenin; Binding Sites; Cadherins; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cytoskeletal Proteins; Delta Catenin; DNA Primers; DNA, Complementary; Electrophoresis; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genistein; HT29 Cells; Humans; Kidney; Lactams, Macrocyclic; Membrane Glycoproteins; Mucin-1; Naphthalenes; Phosphoproteins; Quinones; Rifabutin; Staurosporine; Trans-Activators; Transfection

By using tissue miniunits, protein kinase modulators, and topoisomerase inhibitors in short-term incubation (0-90 min) we studied (1) the role of protein phosphorylation in the immediate control of DNA replication in the developing rat cerebral cortex and (2) the mechanism of action for genistein-mediated DNA synthesis inhibition. Genistein decreased the DNA synthesis within less than 30 min. None of the other protein kinase inhibitors examined (herbimycin A, staurosporine, calphostin-C) or the protein phosphatase inhibitor sodium orthovanadate inhibited DNA synthesis and they did not affect the genistein-mediated inhibition. The selective topoisomerase inhibitors camptothecin and etoposide decreased the DNA synthesis to an extent similar to that of genistein and within less than 30 min. In addition, the effects of these substances on topoisomerase I and II were studied. Etoposide and genistein but not herbimycin A, staurosporine, or calphostin-C strongly inhibited the activity of topoisomerase II. Our results (1) strongly suggest that the net rate of DNA replication during the S phase of the cell cycle is independent of protein phosphorylation and (2) indicate that the early inhibitory effect of genistein on DNA synthesis is mediated by topoisomerase II inhibition rather than protein tyrosine kinase inhibition.

Topics: Animals; Anti-Inflammatory Agents; Benzoquinones; Camptothecin; Cerebral Cortex; Dimethyl Sulfoxide; DNA Replication; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Superhelical; Dose-Response Relationship, Drug; Enzyme Inhibitors; Etoposide; Genistein; Lactams, Macrocyclic; Naphthalenes; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Staurosporine; Steroids; Vanadates

Synthesis of plasminogen activator inhibitor type-1 (PAI-1), a major physiological modulator of plasmin generation, is regulated by growth factors and changes in cell shape. To evaluate the specific relationship between PAI-1 gene expression and cytoarchitecture, serum-free cultures of quiescent rat kidney (NRK) cells were exposed to cytochalasin D (CD) at concentrations that disrupt microfilament structure. Treatment with 1-10 microM CD resulted in an increased 1) incidence of rounded cells, 2) relative PAI-1 mRNA content, and 3) fraction of PAI-1 protein-expressing cells. Abrupt increases in each response were evident at a final concentration of 5 microM CD. Maximal levels of induced PAI-1 transcripts (18-fold that of control) occurred 4 hours post-CD addition and declined thereafter but remained elevated (by at least tenfold) for 24 hours. Assessment of the metabolic requirements for CD-induced PAI-1 expression by using the protein synthesis inhibitors puromycin and cycloheximide indicated that PAI-1 transcripts were regulated in a complex manner in response to CD. The predominant mode of induction reflected secondary (protein synthesis-dependent) metabolic processes, although a minor, albeit significant, primary (protein synthesis-independent) pathway was also evident. PAI-1 mRNA levels in NRK cells maintained in serum- and CD-free agarose suspension culture were low or undetectable. Relative abundance of PAI-1 transcripts in suspended cells cultured in the presence of CD, however, closely approximated that of plastic-adherent, CD-treated cells (13-fold over control). NRK cells in suspension culture with or without CD were morphologically identical, remaining spherical and unattached. It appears, therefore, that cell rounding alone is not a sufficient stimulus to induce PAI-1 expression in quiescent NRK cells and that perturbation of the actin skeleton as a consequence of CD treatment is a critical event in the inductive response. A protein tyrosine kinase is likely involved in the CD-mediated signal-transduction cascade, since induced PAI-1 expression can be down-regulated by genistein and herbimycin A but not by calphostin C or tyrphostin B46.

Topics: Animals; Benzoquinones; Cell Line; Cell Size; Cycloheximide; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Gene Expression Regulation; Genistein; Kidney; Kinetics; Lactams, Macrocyclic; Naphthalenes; Nitriles; Nucleic Acid Synthesis Inhibitors; Plasminogen Activator Inhibitor 1; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Puromycin; Quinones; Rats; Rifabutin; RNA, Messenger; Tyrphostins

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.

Topics: Alkaloids; Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzophenanthridines; Benzoquinones; Brain; Carcinogens; Cells, Cultured; Chemotaxis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Lactams, Macrocyclic; Microglia; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP-sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact).

Topics: Activating Transcription Factor 2; Benzoquinones; Blotting, Western; CD2 Antigens; CD3 Complex; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Electrophoresis; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Monocytes; Naphthalenes; Oligonucleotide Probes; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Receptor Cross-Talk; Rifabutin; Signal Transduction; Thionucleotides; Transcription Factors; Transcription, Genetic

Asbestos and the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), increase c-fos and c-jun mRNA levels and AP-1 DNA binding activity in rat pleural mesothelial (RPM) cells, a target cell of asbestos-induced mesotheliomas (N. H. Heintz et al., Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Because protein kinase C (PKC) is the intracellular receptor of phorbol ester tumor promoters and asbestos is a putative tumor promoter in the respiratory tract, we hypothesized that PKC might play a critical role in asbestos-induced cell signaling pathways associated with regulation of proto-oncogenes. Using a panel of PKC antibodies, we identified PKC alpha as the major PKC isozyme in RPM cells. We then pretreated cells with phorbol ester dibutyrate to down-modulate PKC or with calphostin C, a specific PKC inhibitor, to determine if depletion of PKC alpha could block asbestos-induced c-fos/c-jun expression. Quantitation of Northern blots showed that fiber-associated c-fos/c-jun mRNA levels were significantly lower either after PKC alpha down-modulation or pretreatment with calphostin C. In addition, to determine whether tyrosine kinases also were involved in proto-oncogene activation by asbestos, tyrphostin AG82 or herbimycin A was added to RPM cells before exposure to asbestos. These inhibitors decreased crocidolite-induced c-fos but not c-jun levels, suggesting that tyrosine kinases have different regulatory roles in asbestos-induced c-fos versus c-jun signaling pathways. The ability to block induction of asbestos-induced proto-oncogene expression using pharmacological intervention may be important in prevention and treatment of asbestos-induced proliferative diseases including lung cancers, mesothelioma, and pulmonary fibrosis.

Topics: Animals; Asbestos; Benzoquinones; Epithelium; Gene Expression; Isoenzymes; Lactams, Macrocyclic; Naphthalenes; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Quinones; Rats; Rats, Inbred F344; Rifabutin; RNA, Messenger; Time Factors

Recent data suggest involvement of the Janus tyrosine kinase-2 (JAK2) in human GH-induced tyrosine phosphorylation of the GH receptor and the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), leading to activation of the phosphatidylinositol 3-kinase and the acute insulin-like effects in primary rat adipocytes. To investigate the functional role of this kinase, we screened a number of tyrosine kinase inhibitors for their ability to inhibit three rapid effects of GH on primary adipocytes: increased lipogenesis, inhibition of noradrenaline-induced lipolysis, and promotion of JAK2 tyrosine phosphorylation. Only staurosporine was found to inhibit all three effects. The inhibition of lipogenesis and antilipolysis exhibited the same staurosporine dose dependency (IC50, approximately 40 nM) as inhibition of JAK2 and IRS-1 tyrosine phosphorylation as well as binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2. The unidentified cytosolic tyrosine-phosphorylated protein pp95, in contrast, was not affected, suggesting that it is not phosphorylated primarily by JAK2. Protein kinase C does not seem to be directly involved in the insulin-like effects, because the selective protein kinase C inhibitor calphostin C had no effect at levels up to 100 nM above which unspecific cellular effects occurred. Methyl-2,5-dihydroxy cinnamate inhibited GH-induced lipogenesis from [3-3H]glucose and nonstimulated lipogenesis from [2-14C]-pyruvate and [3H]acetate, but was without effect on GH-induced 2-deoxy-D-[1-3H]glucose uptake, JAK2 phosphorylation and antilipolysis, suggesting unspecific effects on mitochondrial metabolism rather than a direct effect on the GH-mediated signal. Tyrphostin 25 and herbimycin A had no effect on any of the parameters studied, except for a slight increase in JAK2 phosphorylation in response to tyrphostin 25. In summary, these data support the role for JAK2 in mediating the insulin-like effects of GH in adipocytes.

Topics: Acetates; Adipocytes; Animals; Benzoquinones; Cells, Cultured; Cinnamates; Deoxyglucose; Enzyme Inhibitors; Epididymis; Human Growth Hormone; Humans; Insulin; Kinetics; Lactams, Macrocyclic; Lipids; Lipolysis; Male; Naphthalenes; Nitriles; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Pyruvic Acid; Quinones; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Rifabutin; Staurosporine; Tyrphostins

Insulin-like growth factor I (IGF-I) stimulates sodium-dependent inorganic phosphate (Pi) transport across the apical plasma membrane of confluent opossum kidney (OK) cells. Previous studies indicated that vanadate, at doses known to inhibit protein tyrosine phosphatases, mimicked the effect of IGF-I and suggested the involvement of tyrosine phosphorylation processes in Pi transport regulation. In this study, protein tyrosine phosphorylation and activation of several cellular signaling pathways were investigated in confluent OK cells in response to IGF-I and vanadate. We report that IGF-I and vanadate induced tyrosine phosphorylation of distinct proteins. Tyrosine phosphorylation of p95 (IGF-I receptor beta-subunit) was rapidly and dose dependently increased in response to IGF-I. Associated with phosphorylation of the receptor, the increase in tyrosine phosphorylation of a protein of 50 kDa was observed. Vanadate did not mimic the effect of IGF-I, but increased phosphorylation of seven major proteins of 170, 140, 100, 83, 70-82, 60, and 35 kDa. Among the different tyrosine kinase inhibitors tested, only staurosporine affected Pi transport up-regulation by IGF-I and vanadate, attenuating the effect of IGF-I and completely blocking the response to vanadate. Staurosporine decreased tyrosine phosphorylation of several constitutively phosphorylated proteins and interfered with the increase in tyrosine phosphorylation induced by vanadate. Phosphorylation of p95 in response to IGF-I was not affected. Staurosporine also markedly decreased constitutive association of the adapter protein Nck with tyrosine-phosphorylated proteins and attenuated increases in phosphotyrosine-associated Nck induced by IGF-I and vanadate. In contrast, signaling to other downstream effectors common to IGF-I and vanadate, such as mitogen-activated protein kinase and phosphatidylinositol-3-kinase, was not affected by staurosporine. In conclusion, our results suggest that although IGF-I and vanadate induce distinct protein tyrosine phosphorylation in OK cells, they activate an overlapping set of signaling molecules, among which Nck appears as an interesting candidate to link activation of tyrosine kinases to the stimulation of Pi transport.

Topics: Adaptor Proteins, Signal Transducing; Animals; Benzoquinones; Cell Line; Enzyme Inhibitors; Epithelium; Genistein; Humans; Insulin; Insulin-Like Growth Factor I; Isoflavones; Kidney; Lactams, Macrocyclic; Naphthalenes; Oncogene Proteins; Opossums; Phosphates; Phosphoproteins; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Signal Transduction; Sodium; Staurosporine; Vanadates

The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.

Topics: Antigen-Presenting Cells; Benzoquinones; Blotting, Western; Calcimycin; Calcium; CD28 Antigens; Cyclosporine; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Naphthalenes; Polycyclic Compounds; Precipitin Tests; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Signal Transduction; Superantigens; T-Lymphocytes

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR-CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the alpha CD3-induced CD3-AK cell response. First, the alpha CD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-L-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR-CD3. The former pathway is primarily PTK-dependent. Activation through TCR-CD3 is a more complex event.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaloids; Animals; Benzoquinones; Female; Genistein; Interleukin-2; Isoflavones; Killer Cells, Lymphokine-Activated; Lactams, Macrocyclic; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Naphthalenes; Perforin; Polycyclic Compounds; Pore Forming Cytotoxic Proteins; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Interleukin-2; Recombinant Proteins; Rifabutin; RNA, Messenger; Signal Transduction; Staurosporine

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces rapid changes in the morphology of the human megakaryoblastic leukemia cell line, MEG-01, as well as changes in adhesion and megakaryocytic differentiation. To investigate the signal transduction pathway of these three phenomena, we studied the effect of herbimycin A, an inhibitor of tyrosine kinase (TK) and the effects of calphostin C, a specific inhibitor protein kinase C (PKC) on TPA treated MEG-01 cells. Both herbimycin A and calphostin C inhibited all three TPA-induced phenomena, suggesting that both pathways are required for these phenomena. Herbimycin A but not calphostin C blocked the tyrosine phosphorylation of cellular proteins. Immunohistochemical staining of PKC using an anti-PKC monoclonal antibody showed that herbimycin A did not interfere with the translocation and subsequent down regulation of PKC induced by TPA, suggesting that the TPA-induced effect on PKC (translocation and probably its activation) is not dependent on TK. Induction of c-fos and c-jun expression by TPA was inhibited by both herbimycin A and calphostin C, suggesting that both PKC and TK pathways are necessary for the induction of the TPA-induced transcription factor AP1, which is a known TPA-inducible early immediate gene product. Taken together, our results show that the tyrosine kinase signal transduction system as well as the PKC pathway is indispensable for the TPA-induced phenomena of morphologic change, cell attachment, early immediate gene expression, and lineage-specific phenotypic expression in the MEG-01 cell line.

Topics: Benzoquinones; Cell Adhesion; Cell Differentiation; Genes, fos; Genes, jun; Humans; Lactams, Macrocyclic; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Naphthalenes; Phosphotyrosine; Polycyclic Compounds; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.

Topics: 3T3 Cells; Animals; Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Cinnamates; Enzyme Inhibitors; Enzyme Stability; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Kinetics; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Naphthalenes; Phosphorylation; Phosphoserine; Phosphotyrosine; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin

The plasma protein fibronectin is an important opsonin in wound repair and host defense. To better understand the process of fibronectin-mediated phagocytosis, we have transfected K562 cells, which endogenously express alpha 5 beta 1, with alpha v beta 3. In these transfectants, antibodies to alpha v beta 3 block phagocytosis of fibronectin-opsonized beads completely, even though half the ingestion occurs through endogenous alpha 5 beta 1 receptors. alpha 5 beta 1-mediated adhesion to fibronectin-coated surfaces is unaffected by alpha v beta 3 ligation. Neither alpha v beta 5 nor alpha M beta 2 ligation affects alpha 5 beta 1 phagocytic function in transfectants expressing these receptors. Pharmacologic data suggest that alpha v beta 3 ligation suppresses the phagocytic competence of high affinity alpha 5 beta 1 receptors through a signal transduction pathway, perhaps involving protein kinase C. In addition to its significance for phagocytosis, alpha v beta 3 regulation of alpha 5 beta 1 function may be significant for its roles in cell migration, metastasis, and angiogenesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzoquinones; Cell Adhesion; Cell Line; Cloning, Molecular; Fibronectins; Flow Cytometry; Genistein; Humans; Integrins; Isoflavones; Isoquinolines; Kinetics; Lactams, Macrocyclic; Leukemia, Erythroblastic, Acute; Naphthalenes; Phagocytosis; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Receptors, Cytoadhesin; Receptors, Fibronectin; Receptors, Vitronectin; Rifabutin; Signal Transduction; Transfection; Tumor Cells, Cultured

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Cells, Cultured; Dogs; Enzyme Activation; Genistein; Inositol 1,4,5-Trisphosphate; Isoflavones; Kidney Medulla; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Data; Naphthalenes; Osmolar Concentration; Phosphorylation; Polycyclic Compounds; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Quinones; Ribosomal Protein S6 Kinases; Rifabutin; Tetradecanoylphorbol Acetate

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein ki

Topics: Benzoquinones; Bone Marrow; Bone Marrow Cells; Carrier Proteins; Cell Division; Cell Line; Ethers, Cyclic; Genistein; Glucose; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Polycyclic Compounds; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate