A RAS oncogene imparts growth factor independence to myeloid cells that abnormally regulate protein kinase C: a nonautocrine transformation pathway

The factor-dependent cell line FDC-P1 has been utilized as a model of interleukin 3 (IL-3)-dependent myeloid cell proliferation. However, it has been recently observed that active phorbol esters (e.g., phorbol 12-myristate 13-acetate) may entirely replace IL-3 to promote its proliferation. These observations reveal abnormal regulation of protein kinase C (pkC) (absence of downregulation or overexpression). This property allowed a test of the hypothesis that the T24 RAS (codon 12) oncogene acts by constitutive and persistent pkC activation, driving proliferation. FDC-P1 cells were transfected by electroporation with the T24 RAS-containing vector pAL 8, or with a control vector pSVX Zip Neo, and neomycin-resistant clones were selected. Multiple RAS-transfectant clones were categorized for their growth factor requirement and incorporation of the 6.6-kb human mutant H-RAS genome. IL-3-independent clones had incorporated multiple (more than two) copies of the entire 6.6-kb RAS genome. The incorporation of multiple 6.6-kb RAS genomes was correlated with high-level p21 RAS expression. No evidence for autostimulatory growth factor production by clones containing the RAS oncogene was observed. Thus, acquisition of growth factor independence in myeloid cells by abundant expression of a RAS oncogene is linked, in part, to abnormal regulation of pkC, which acts as a collaborating oncogene.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

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.     

Growth hormone induces expression of c-jun and jun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression

Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC. In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.     

Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.

Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.     

Regulation of AP-1 expression and activity in antigen-stimulated mast cells: the role played by protein kinase C and the possible involvement of Fos interacting protein

We have recently observed that protein kinase C (PKC) was involved in the regulation of the accumulation of mRNAs of the AP-1 components in cultured Abelson-transformed murine fetal-liver-derived mast cells stimulated by exocytotic stimuli. Here we analyzed the probable regulatory effect of PKC on the synthesis and DNA-binding activity of AP-1 complexes in immunologic stimulated mast cells. In this study we used the interleukin-3--dependent murine fetal-liver--derived mast cells that were not transformed by the Abelson oncogene. Study of PKC- depleted cells showed PKC dependency of c-fos mRNA accumulation and protein expression in IgE-Ag stimulated cells. In contrast, the c-jun mRNA accumulation was unaffected by PKC depletion, whereas its protein expression was dependent on this enzymatic activity. This suggests the involvement of PKC in the regulation of translation of c-Jun, a level of c-Jun regulation that was not previously described. The amount of AP- 1 DNA-bound complex was also lowered in PKC-depleted cells. Therefore, PKC plays an important regulatory role in different stages of the signal transduction pathway because of IgE-Ag stimulation. Surprisingly, we have observed that although the amount of total synthesized c-Fos began to increase 15 minutes after immunologic stimulation, the amount of c-Fos associated with Juns did not increase, even after 45 minutes. This association was not affected by PKC. Using a Fos-interacting protein (FIP)-cDNA probe, an expression of 2.9 kb mRNA was detected in these cells. Furthermore, immunologic stimulation caused an increase in the amount of a Fos-containing protein complex that bound to an FIP-binding DNA oligonucleotide. Therefore, we propose that this protein complex that contains most of the immunologically induced c-Fos has an important role in IgE-Ag-stimulated signal transduction.     

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.