Growth factor regulation of cell cycle progression and cell fate determination.
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Animals↗
Biomedical subjects
U R Rapp
Publications and source records attributed to U R Rapp.
Cascading towards vascular disorder gene therapy.
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Animals↗
R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.
The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.
3T3 Cells↗
Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.
Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.
Animals↗
Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway.
We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.
3T3 Cells↗
Prostaglandin E suppression of platelet-derived-growth-factor-induced Ito cell mitogenesis occurs independent of raf perinuclear translocation and nuclear proto-oncogene expression.
Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo coincident with the de novo expression of Ito cell PDGF beta receptor messenger RNA. PDGF-induced mitogenesis was studied in cultured rat hepatic Ito cells which resemble the myofibroblast associated with liver injury. Pretreatment with prostaglandin E markedly suppressed the PDGF response in a dose-dependent fashion. The PDGF-induced cascade was studied with or without PGE to determine the level of regulation which induced the observed suppression. PGE caused no apparent diminution in the abundance of the surface PDGF beta receptor nor its subsequent activation and tyrosine phosphorylation following PDGF stimulation. The cytoplasmic 'secondary messengers' mitogen-activated protein kinase pp42-44 and raf kinase, appeared to be comparably induced and therefore unaffected by PGE. Raf perinuclear translocation was also intact and comparable degrees of nuclear egr, fos, and jun expression occurred. Since other studies have suggested that many of these features of the PDGF cascade may be causally and sequentially linked, the data collectively suggests that the dominant PGE mitogenic suppressive effect resides at a raf-MAP parallel pathway or at a nuclear level distal to the induction of these early growth response genes.
Animals↗
Dissociation between activation of Raf-1 kinase and the 42-kDa mitogen-activated protein kinase/90-kDa S6 kinase (MAPK/RSK) cascade in the insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells.
Insulin treatment of untransfected 3T3 L1 cells quickly induced activation of a cytosolic 42-kDa mitogen-activated protein kinase (MAPK) and a 90-kDa S6 kinase (RSK). The activation of these cytosolic kinases was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with inducible ras oncogenes. Furthermore, insulin-induced activation of MAPK and RSK could be blocked by expression of a transfected inducible dominant negative Ras mutant (Asn-17). These results indicate that Ras proteins are obligatory intermediates in the activation of the cytosolic MAPK/RSK cascade by insulin. Insulin treatment of 3T3 L1 cells or expression of transfected ras oncogenes resulted also in hyperphosphorylation of cellular Raf-1. Insulin-induced Raf hyperphosphorylation was inhibited by expression of an inducible dominant negative Ras mutant (Asn-17). We also showed that expression of transfected raf oncogenes induces adipocytic differentiation, as detected by expression of the specific adipocytic marker aP2. In addition, insulin-induced differentiation was significantly blocked by expression of a dominant negative raf mutant. Interestingly, however, the expression of transfected raf oncogenes did not induce MAPK or RSK activation, and the insulin-induced activation of these kinases was not blocked by expression of transfected dominant negative raf mutants. These results are consistent with Raf kinases acting downstream of Ras, but not upstream of MAPK and RSK in insulin-signaling pathways leading to 3T3 L1 differentiation.
3T3 Cells↗
Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium.
Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.
3T3 Cells↗
Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation.
The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.
3T3 Cells↗
The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1).
The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. We have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776 nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors.
3T3 Cells↗
C-raf-1 proto-oncogene expression relates to radiosensitivity rather than radioresistance.
The transfection of several oncogenes, particularly c-raf-1, into mammalian in vitro cell lines has been reported to be associated with increased radioresistance. We have thus investigated (by scanning photodensitometry of western blots) the phenotypic expression of the c-raf-1, c-myc and c-ras protein products in 19 human in vitro cell lines, whose intrinsic cellular sensitivity to 4 MeV photon irradiation has also been determined. High levels of c-raf-1 proto-oncogene product expression did not correlate with increased cellular radioresistance, but rather showed a significant correlation with intrinsic cellular radiosensitivity to photon irradiation for alpha (r = 0.664, P = 0.002), and SF2 (r = -0.655, P = 0.002). There was no significant correlation for the ras family, c-myc or actin. These results conflict with those of previous studies in which transfection of the activated forms of the c-raf-1 oncogene were associated with increased radioresistance, and suggest the possibility that the full length proto-oncogene may influence cellular radiosensitivity in a different manner from that of the activated oncogene.
Blotting, Western↗
The ins and outs of Raf kinases.
Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.
Animals↗
Expression of the raf protooncogene in glial cells of the adult rat cerebral cortex, brain stem and spinal cord.
The raf protooncogenes encode cytoplasmic serine/threonine-specific protein kinases which can be activated from different growth factor receptors by phosphorylation. Our previous immunohistochemical studies proved that raf kinases are present in neurons of the mammalian central nervous system. The present study describes the immunohistochemical localization of raf kinases in glia-like cells of the rat cerebral cortex, spinal cord and brain stem. Small glia-like cells measuring 8-12 microns were observed in the neocortex, the entorhinal and prepiriform allocortical areas and the subcortical white matter. In the hippocampus, the immunoreactive cells were most numerous in the fimbria, the alveus and the molecular layer of the dentate fascia. Ultrastructural studies following preembedding immunohistochemistry revealed that in the cerebral cortex only astrocytes contained raf-protein-like immunoreactivity. Our immunofluorescence studies showed, that the white matter of the spinal cord, the pyramids of the medulla and the basis of the pons contained small glia-like cells, too. No electron microscopic investigations were performed, but the location (white matter tracts) and size (6-12 microns) of these cells suggested their glial nature. On the basis of data from other cell systems we expect that raf kinases participate in growth factor- and cytokine-regulated glial functions of the mammalian central nervous system.
Animals↗
The Raf-1 serine/threonine protein kinase.
Raf-1 belongs to a family of serine/threonine protein kinases which are highly conserved through evolution in multicellular organisms. Raf-1 kinase has gained much attention due to its function as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the cell membrane to changes in the expression of genes involved in the control of cell growth, differentiation and survival. Regulation of Raf-1 activity is complex and involves Ras, as well as several serine/threonine and tyrosine kinases. Through a series of phosphorylation events, extracellular signals are connected through the Raf-1/MAP kinase pathway to activity-regulation of several oncogene-class transcription factors via phosphorylation of specific serine residues. Under ordinary circumstances, the cascade involving Raf-1 eventually leads to changes in gene expression and protein synthesis. Upon constitutive activation of Raf-1 kinase, as a result of genetic changes, a variety of cell types acquire a transformed phenotype.
Animals↗
Transformation by Raf and other oncogenes renders cells differentially sensitive to growth inhibition by a dominant negative c-jun mutant.
In NIH3T3 cells expressing active Raf-1 protein serine/threonine kinase (PSK) c-jun expression is constitutive while c-fos expression is attenuated. This alteration prompted us to determine whether oncogene transformation would render cells differentially sensitive to growth inhibition by a dominant negative mutant of c-jun, TAM 67. Growth inhibition was observed in three types of assays: (1) transfection of TAM 67 into cells stably transformed by a variety of oncogenes, (2) cotransfection of TAM 67 with oncogene expression plasmids into NIH3T3 cells and (3) titration of oncogene-expressing retroviruses on cells stably expressing TAM 67. The results clearly demonstrate that Raf-1 dependent oncogenes, which include receptor protein tyrosine kinases (PTKs)-, intracellular PTKs- and Ras-derived genes share the Raf phenotype of constitutive c-jun expression, attenuated c-fos induction, and high sensitivity to growth suppression by TAM 67. Additionally, the intracellular PSK oncogene, mos and the nuclear oncogenes c-myc, c-fos, and SV40 T antigen were TAM 67-sensitive for transformation. This universal pattern of altered growth regulation in oncogene transformed fibroblast cell lines highlights the potential usefulness of c-jun based inhibitors for control of tumor cell growth.
3T3 Cells↗
v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.
Interleukin-3 (IL-3) is required for the proliferation, survival and differentiation of myeloid progenitors. In the absence of IL-3, murine myeloid 32D.3 cells accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death, or apoptosis. Here we demonstrate that enforced expression of the v-raf oncogene suppresses apoptosis of myeloid 32D.3 cells following the withdrawal of IL-3. Surprisingly, steady state levels of Bcl-2, an oncogene known to suppress apoptosis, were not dependent upon IL-3 in 32D.3 cells and its levels were not augmented in v-raf clones. This suggests that ability of v-raf to suppress apoptosis in the absence of ligand is either Bcl-2 independent or that v-raf kinase promotes Bcl-2 function. v-raf also promoted growth of these cells in the presence of IL-3. v-raf clones proliferated at an increased rate due to a shortened G1 phase and had decreased requirements for IL-3 for growth. Therefore, transformation of myeloid cells by v-raf involves signaling pathways which promote both cell cycle progression and cell survival.
Animals↗
Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase.
The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.
Animals↗
Immunohistochemical detection of raf protein kinase in cerebral cortical areas of adult guinea pigs and rats.
The raf protooncogenes are the cellular counterparts of the v-raf oncogene expressed by a murine sarcoma virus. The raf protooncogenes encode cytoplasmic serine/threonine-specific protein kinases which can be activated from different growth factor receptors by phosphorylation. The mRNAs of raf protooncogenes are found in a large variety of normal adult tissues, including the central nervous system. As concerns the distribution and localization of their protein products (the raf kinases), very few data are to be found in the literature. This is the first detailed description of their light microscopic localization in neocortical and allocortical areas of rodents. Preembedding immunohistochemical studies were performed on vibratome sections from the brains of adult guinea pigs and albino rats. The localizations of two isoenzymes, raf-1 kinase and B-raf kinase, were studied with the help of isoenzyme-specific polyclonal antibodies. Both of the antibodies detected raf protein-like immunoreactivity in many neurons and scattered glial cells of the sensory neocortex, and the cingular, pyriform, perirhinal and entorhinal allocortical areas. Pyramidal and non-pyramidal cells of Ammon's horn, granule cells of the dentate fascia and the large neurons in the hilar region were immunoreactive, too. The findings indicated that B-raf protein kinase and raf-1 kinase are present almost ubiquitously in the neurons of the investigated cortical structures. The intensity of staining obtained with serial dilutions of the antibodies indicated that the cytoplasmic concentration of B-raf kinase is tended to be higher than that of raf-1 kinase. The present findings suggested that the raf kinases are localized in postsynaptic structures, mainly in dendrites and cell bodies. Their cytosolic localization and their ability to undergo intracellular translocation during activation and phosphorylation raise the possibility that they play a pivotal role in the intracellular signaling of neurons.
Amino Acid Sequence↗