guanosine-triphosphate 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

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

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Transformed; Enzyme Inhibitors; Genes, ras; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Point Mutation; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Reference Values; Rifabutin; Transformation, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms

In this study, it was shown that the proportion of guanosine triphosphate (GTP)-bound active Ras increased in TPA (12-o-tetradecanoyl phorbol-13-acetate)-induced monocytic differentiation of HL-60 cells. The increase of active Ras was observed at 24 hours after TPA stimulation and attained to threefold (15%) over the proportion in nontreated HL-60 cells. Herbimycin A, an inhibitor of tyrosine kinase, prevented the activation of Ras, as well as the induction of monocytic differentiation. In parallel with the activation of Ras, the proteins with molecular weights of 52, 56, 62, and 190 kD were tyrosine-phosphorylated and formed a complex with GTPase-activating protein (GAP) for Ras. In addition to the 116-kD GAP (type I GAP), the 100-kD GAP (type II GAP) molecule was markedly induced at 24 hours after TPA stimulation of HL-60 cells. These phenomena sustained for a further 24 hours during monocytic differentiation. However, they were not observed during retinoic acid-induced granulocytic differentiation of the cells. The HL-60 transfectants, which expressed a dominant inhibitory Ha-ras Asn17, showed a low level of tyrosine-phosphorylated GAP-associated proteins and did not undergo full differentiation in response to TPA. Taken together, these data indicate that the activation of Ras and GAP complex formation mutually correlate and function downstream of protein-tyrosine kinases in the signaling pathway for monocytic differentiation of HL-60 cells.

Topics: Benzoquinones; Cell Differentiation; Dexamethasone; Enzyme Activation; Granulocytes; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Monocytes; Mutation; Oncogene Protein p21(ras); Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proteins; Quinones; ras GTPase-Activating Proteins; Rifabutin; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured; Tyrosine

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

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.

Topics: Amino Acid Sequence; Benzoquinones; Cell Membrane Permeability; Diglycerides; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Molecular Sequence Data; Phosphatidylinositols; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Receptors, Antigen; Rifabutin; T-Lymphocytes; Transcription, Genetic