guanosine-diphosphate and herbimycin

To study the dynamics of Ras-dependent signalling in the course of Herbimycin A induced erythroid differentiation of human erythroleukemia K562 cells.. p21Ras functional activity was analyzed by direct measurement of GTP/GDP ratio in anti-p21Ras immunoprecipitates of K562 cells previously incubated with H3(32)PO4. Dynamics of protein tyrosine phosphorylation was studied using Western blotting. Electrophoretic mobility shift assay was used to monitor Erk2 activation. Phosphotyrosine (pY)-containing proteins bound to recombinant glutathione-S-tranferase (GST)-fused form of adaptor protein Grb2 were identified using GST in vitro binding assay.. It was shown that the relative quantity of GTP associated with Ras protein in non-induced cells varied from 27% to 37% upon 72 h of cell culturing. Herbimycin A caused 15% increase of GTP/GDP ratio at 3rd h. This index decreased during further investigated periods, although it did not reach control values even at 72nd h. Transient rise of Ras-GTP level at 3rd h of incubation in the presence of Herbimycin A correlated with the increase in tyrosine phosphorylation of proteins with apparent molecular weight of 210, 160, 140, 116 and 42 kDa, as well as with the activation of Erk2 and increase of binding of a set of pY-containing proteins with recombinant GST-fusion form of Ras activator, adaptor protein Grb2. Dramatic inhibition of interaction between docking protein Shc and GST-Grb2 was observed at late stages of cell induction (48-72 h) while binding of pY-containing proteins during this period did not differ significantly in control and differentiated cells.. The obtained results suggest that time-dependent changes in Grb2-mediated network of protein-protein interaction events might define implication of Ras-dependent signalling in Herbimycin A-induced erythroid differentiation of K562 cells.

Topics: Adaptor Proteins, Signal Transducing; Benzoquinones; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Erythroid Cells; Glutathione Transferase; GRB2 Adaptor Protein; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Phosphorylation; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; Signal Transduction; Tyrosine

Using human endothelial cells, we define a mechanism that accounts for the induction of interleukin 8 (IL-8) by protein I/IIf, an adhesin from Streptococcus mutans serotype f. We report that protein I/IIf interactions with endothelial cells increased the tyrosine phosphorylation of three cellular components with relative mass of 145,000, 125,000 and 70,000 in endothelial cells. These proteins were identified as phospholipase Cgamma (PLCy), focal adhesion kinase (FAK) and paxillin after immunoprecipitation with monoclonal antibodies (mAbs) and immunoblotting with antiphosphotyrosine mAbs. These results suggested that beta1 integrins could be one of the components implicated in the modulin activity of protein I/IIf. By incubating protein I/IIf with either purified alpha5beta1 integrins or with alpha5beta1 integrins overexpressing CHO cells, we demonstrated that alpha5beta1 integrins act as cell receptors for protein I/IIf. We also showed that protein I/IIf interactions with alpha5beta1 integrins lead to IL-8 secretion. Using specific inhibitors, we demonstrated that protein I/IIf-induced IL-8 release involves mitogen-activated protein kinases (MAPKs), and that PLCgamma and PKC also seem to contribute to protein I/IIf stimulation. However, PI-3K activation is not involved in IL-8 release. Altogether, these results indicate that, after binding to alpha5beta1 integrins, protein I/IIf induces IL-8 release by activating the MAPKs signalling pathways.

Topics: Adhesins, Bacterial; Animals; Apigenin; Bacterial Proteins; Benzoquinones; Cells, Cultured; CHO Cells; Cricetinae; Cytoskeletal Proteins; Endothelium, Vascular; Enzyme Inhibitors; Flavonoids; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genistein; Guanosine Diphosphate; Humans; Immunoblotting; Integrins; Interleukin-8; Isoenzymes; Lactams, Macrocyclic; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Paxillin; Phospholipase C gamma; Phosphoproteins; Phosphorylation; Protein Binding; Protein-Tyrosine Kinases; Quinones; Receptors, Vitronectin; Rifabutin; Streptococcus mutans; Thionucleotides; Type C Phospholipases; Tyrosine

The monoclonal antibody (mAb) R24 is a murine immunoglobulin G3 (IgG3) that reacts with the GD3 disialoganglioside present on melanoma cells as well as a subset of T cells. R24 mAb has induced antitumor responses both alone and in combination with interleukin-2 (IL-2) in clinical trials. We have reported T cell activation via GD3 as measured by the induction of tyrosine phosphorylation. In this study a more detailed analysis of signal transduction after ligation of GD3 was performed in an attempt to understand the mechanism of in vivo therapeutic benefits observed. Analysis of subsequent events indicated that GD3 engagement resulted in phospholipase C(gamma) phosphorylation and calcium flux. When ras-associated events were examined, GD3 signaling resulted in ras activation as determined by GDP/GTP conversion as well as dose-and time-dependent IP3 activation. In addition, the majority of the IP3 activation by GD3 was inhibited by herbimycin A pretreatment. Elucidation of the nature and potential role of this moiety in GD3 signal transduction should be useful. Collectively, these data suggest a novel mechanism of T cell activation via a single, non-protein, surface moiety. This novel form of T cell-mediated activation may permit the delivery and local activation of effector cells at the tumor resulting in site-specific activation of the immune system.

Topics: Benzoquinones; Calcium; Cells, Cultured; Enzyme Inhibitors; Gangliosides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Isoenzymes; Lactams, Macrocyclic; Lymphocyte Activation; Phosphatidylinositols; Phospholipase C gamma; Phosphoproteins; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; Signal Transduction; Staurosporine; T-Lymphocytes; Type C Phospholipases

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Topics: Alkaloids; Amino Acid Sequence; Animals; Base Sequence; Benzoquinones; Cell Cycle Proteins; Cell Line; Ceramides; Chlorocebus aethiops; Diglycerides; DNA Primers; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Kinetics; Lactams, Macrocyclic; Molecular Sequence Data; Muromonab-CD3; Mutagenesis, Site-Directed; Naphthalenes; Phorbol 12,13-Dibutyrate; Point Mutation; Polycyclic Compounds; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Proto-Oncogene Proteins p21(ras); Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Staurosporine; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.

Topics: 3T3 Cells; Adenine Nucleotides; Aluminum Compounds; Animals; Arachidonic Acid; Avian Sarcoma Viruses; Benzoquinones; Cell Line, Transformed; Cholera Toxin; Enzyme Induction; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Kinetics; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Myristic Acid; Myristic Acids; Oncogene Protein pp60(v-src); Phosphatidylcholines; Phospholipase D; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Thionucleotides; Virulence Factors, Bordetella

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Topics: Animals; Benzoquinones; Blotting, Northern; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; DNA Probes; Fusion Proteins, bcr-abl; Gene Expression; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Lactams, Macrocyclic; Mice; Oncogenes; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; RNA; Signal Transduction; Transcription, Genetic

The hematopoietically expressed product of the vav proto-oncogene, Vav, shared homology with guanine nucleotide releasing factors (GRFs) [also called guanosine diphosphate-dissociation stimulators (GDSs)] that activate Ras-related small guanosine triphosphate (GTP)-binding proteins. Human T cell lysates or Vav immunoprecipitates possessed GRF activity that increased after T cell antigen receptor (TCR)-CD3 triggering; an in vitro-translated Vav fragment that contained the putative GRF domain was also active. Vav-associated GRF stimulation after TCR-CD3 ligation paralleled its tyrosine phosphorylation; both were blocked by a protein tyrosine kinase (PTK) inhibitor. Vav also was a substrate for the p56lck PTK. Thus, Vav is a PTK-regulated GRF that may be important in TCR-CD3-initiated signal transduction through the activation of Ras.

Topics: Benzoquinones; Cell Cycle Proteins; Fungal Proteins; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Muromonab-CD3; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Quinones; rap GTP-Binding Proteins; Receptor-CD3 Complex, Antigen, T-Cell; Rifabutin; Signal Transduction; T-Lymphocytes; Tumor Cells, Cultured

Antibody-dependent cellular cytotoxicity is initiated when low affinity Fc receptors (Fc gamma R type III/CD16) on NK cells bind to sensitized (i.e., antibody coated) target cells. Fc gamma R cross-linkage induces the activation of phospholipase C (PLC), which hydrolyses membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. However, the mechanism that couples Fc gamma R stimulation to PLC activation remains unknown. In this study, we investigated whether the Fc gamma R is coupled to PLC via a guanine nucleotide-binding (G) protein or an alternative pathway. Stimulation of electropermeabilized human NK cells with GTP gamma S induced inositol phosphate (IP) release, indicating the presence of a G protein-linked PLC activity in these cells. However, stimulation with both anti-Fc gamma R mAb and GTP gamma S provoked additive rather than synergistic increases in IP formation. Furthermore, exogenous GDP strongly inhibited GTP gamma S-stimulated IP release, but failed to inhibit the response to anti-Fc gamma R mAb stimulation. These results suggested GTP gamma S and anti-Fc gamma R mAb activated PLC through distinct regulatory mechanisms, and that Fc gamma R was not linked to PLC via a G protein. Hence, an alternative transduction mechanism for Fc gamma R-PLC coupling was considered. Antibody-mediated Fc gamma R cross-linkage was shown to rapidly stimulate tyrosine phosphorylation of multiple proteins in NK cells. Pretreatment with the tyrosine kinase inhibitor, herbimycin A, inhibited these phosphorylation events and disrupted the coupling between Fc gamma R ligation and PLC activation. These observations suggest that Fc gamma R in NK cell is coupled to PLC via a G protein-independent, but tyrosine kinase-dependent pathway.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; Benzoquinones; Cell Line; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; In Vitro Techniques; Inositol Phosphates; Killer Cells, Natural; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Quinones; Receptors, Fc; Receptors, IgG; Rifabutin; Signal Transduction; Type C Phospholipases