Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.

Williams, Nidhi Gupta; Roberts, Thomas M.; Li, Ping

doi:10.1073/pnas.89.7.2922

Cited by 125 publications

(87 citation statements)

References 36 publications

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“…Similarly, the transforming ability of oncogenic ras is dependent on c-Raf-1 (62), and unregulated c-Raf-1 protein kinase activity is itself oncogenic (67). The function of all three protooncogenes is closely interconnected because, although Src and Ras can independently activate c-Raf-1, both are required for full c-Raf-1 activation (43,44,68). The same is true for activation of A-Raf but not B-Raf which can be fully activated by oncogenic Ras alone (44).…”

Section: Discussionmentioning

confidence: 99%

“…B-Raf lacks the tyrosine residues in c-Raf-1 and A-Raf which are phosphorylated by Src, which may explain its lack of sensitivity to activation by Src (44). However, since the activation of c-Raf-1 autokinase activity by v-src is not inhibited by expression of dominant-negative Ha-ras N17 , v-src may at least partially activate c-Raf-1 via a Ras-independent pathway (68).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Oncogenic src, raf, and rasStimulate a Hypertrophic Pattern of Gene Expression and Increase Cell Size in Neonatal Rat Ventricular Myocytes

Fuller

Brown²,

Sugden

1998

Journal of Biological Chemistry

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In response to hormones and growth factors, cultured neonatal ventricular myocytes increase in profile, exhibit myofibrillogenesis, and re-express genes whose expression is normally restricted to the fetal stage of ventricular development. These include atrial natriuretic factor (ANF), ␤-myosin heavy chain (␤-MHC), and skeletal muscle (SkM)-␣-actin. By using luciferase reporter plasmids, we examined whether oncogenes that activate the extracellular signal-regulated kinase cascade (src F527 , Ha-ras V12 , and v-raf) increased expression of "fetal" genes. Transfection of myocytes with src F527 stimulated expression of ANF, SkM-␣-actin, and ␤-MHC by 62-, 6.7-, and 50-fold, respectively, but did not induce DNA synthesis. Stimulation of ANF expression by src was greater than by Ha-ras V12 , which in turn was greater than by v-raf. General gene expression was also increased but to a lesser extent. The response to src F527 was inhibited by dominant-negative Ha-ras N17 . Myocyte area was increased by src F527 , Ha-ras V12 , and v-raf, and although it altered myocyte morphology by causing a pseudopodial appearance, src F527 did not detectably increase myofibrillogenesis either alone or in combination with Ha-ras V12 . A kinase-dead src mutant increased myocyte size to a much lesser extent than src F527 and also did not inhibit ANF-luciferase expression in response to phenylephrine. We conclude that members of the Src family of tyrosine kinases may be important in mediating the transcriptional changes occurring during cardiac myocyte hypertrophy and that Ras and Raf may be downstream effectors.The cardiac myocyte is a terminally differentiated cell that withdraws from cell division at around birth in mammals. In these cells, adaptive hypertrophic growth in response to an increased requirement for contractile power involves increases in cell size, protein content, and myofibrillar organization (reviewed in Ref. 1). There are also changes in gene transcription that distinguish hypertrophy from maturational growth (eutrophy). In response to hypertrophic stimuli, cultured neonatal rat ventricular myocytes initiate a rapid and transient increase in expression of immediate early genes (c-jun, c-fos, and Egr-1), followed by the re-expression of so-called "fetal" genes including atrial natriuretic factor (ANF), 1 ␤-myosin heavy chain (␤-MHC), and skeletal muscle (SkM) ␣-actin. More chronic exposure to hypertrophic agonists also elicits an increase in the expression of constitutively expressed contractile protein genes such as ventricular myosin light chain-2 and cardiac muscle ␣-actin. Many of these changes are seen during hypertrophy of the rat heart in vivo, providing a justification for the use of this model system (reviewed in Ref. 1).A wide variety of agonists induce the hypertrophic phenotype in cultured ventricular myocytes. These include agonists acting through G protein-coupled receptors such as endothelin-1 (2, 3), angiotensin II (4, 5), and ␣ 1 -adrenergic agonists (4, 6, 7). Other hypertrophic stimuli include passive ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Oncogenic src, raf, and rasStimulate a Hypertrophic Pattern of Gene Expression and Increase Cell Size in Neonatal Rat Ventricular Myocytes

Fuller

Brown²,

Sugden

1998

Journal of Biological Chemistry

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“…Raf-1 kinase activation by Ang II was analyzed essentially as described previously (Ohmichi et al, 1992;Williams et al, 1992). Briefly, neuronal cells after Ang II treatment were lysed for 10 min in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 100 M sodium orthovanadate).…”

Section: Measurement Of Ras Raf-1 and Map Kinase Activitymentioning

confidence: 99%

“…Briefly, neuronal cells after Ang II treatment were lysed for 10 min in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 100 M sodium orthovanadate). The lysates were centrifuged at 10,000 ϫ g for 5 min, and supernatants were used for immunoprecipitation with anti-Raf-1 antibody at 4ЊC for 1 hr (Ohmichi et al, 1992;Williams et al, 1992). Immune precipitates were resuspended in 20 l of reaction buffer (20 mM HEPES, pH 7.4, 1 mM NaCl, and 5 mM MgCl 2 ) containing 10 Ci [␥-…”

Section: Measurement Of Ras Raf-1 and Map Kinase Activitymentioning

confidence: 99%

Regulation of Neuromodulatory Actions of Angiotensin II in the Brain Neurons by the Ras-Dependent Mitogen-Activated Protein Kinase Pathway

et al. 1996

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Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamusbrainstem with Ang II resulted in a time-and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT 1 receptor subtype and was associated with the redistribution of AT 1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogenactivated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT 1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuromodulatory actions of Ang II in the brain.

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“…Experiments performed using the Sf9 insect cell system to analyze the activation of Raf-1 have revealed that co-expression of Src, Ras, or PKC with Raf-1 led to its activation (9,10,20,31). To develop a simplified system for the analysis of Raf-1 COOH-terminal modifications, we examined whether treatment of Sf9 cells expressing only RIII-⑀ with H 2 O 2 would lead to a shift in electrophoretic mobility similar to that observed in mammalian cells.…”

Section: Mobility Shift Of a Cooh-terminal Raf-1 Fragment In Mammaliamentioning

confidence: 99%

Sequential Modification of Serines 621 and 624 in the Raf-1 Carboxyl Terminus Produces Alterations in Its Electrophoretic Mobility

Ferrier

Lee

Anderson

et al. 1997

Journal of Biological Chemistry

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The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H 2 O 2 ) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566 -643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.Raf-1, the product of the c-raf-1 protooncogene, is a cytoplasmic serine/threonine protein kinase that is activated in response to various mitogenic signals (1, 2). Activation of Raf-1 results in a cascade of events involving the phosphorylation and subsequent activation of additional proteins, including mitogen-activated protein (MAP) 1 kinase kinase (MEK) and MAP kinase, which ultimately lead to transcriptional activation and mitogenesis (3, 4). Thus the Raf-1 protein kinase plays a central role in cellular signal transduction and is a key player in transmitting signals from the plasma membrane to the nucleus (5, 6).The amino terminus of Raf-1 serves as a regulatory domain, while the catalytic domain resides in the carboxyl terminus, which also may contain regulatory elements (7). Earlier studies showed that the activation of Raf-1 by membrane-associated, protein-tyrosine kinases was dependent on cellular Ras activity (8 -...

show abstract

Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.

Cited by 125 publications

References 36 publications

Oncogenic src, raf, and rasStimulate a Hypertrophic Pattern of Gene Expression and Increase Cell Size in Neonatal Rat Ventricular Myocytes

Oncogenic src, raf, and rasStimulate a Hypertrophic Pattern of Gene Expression and Increase Cell Size in Neonatal Rat Ventricular Myocytes

Regulation of Neuromodulatory Actions of Angiotensin II in the Brain Neurons by the Ras-Dependent Mitogen-Activated Protein Kinase Pathway

Sequential Modification of Serines 621 and 624 in the Raf-1 Carboxyl Terminus Produces Alterations in Its Electrophoretic Mobility

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