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Ulf R Rapp - One of the best experts on this subject based on the ideXlab platform.
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role of melanoma inhibitor of apoptosis ml iap Protein a member of the baculoviral iap repeat bir domain family in the regulation of c Raf kinase and cell migration
Journal of Biological Chemistry, 2012Co-Authors: Tripat Kaur Oberoikhanuja, Ulf R Rapp, Christiaan Karreman, Sarit Larisch, Krishnaraj RajalingamAbstract:Abstract SUMMARY Inhibitors of Apoptosis (IAPs) Proteins are characterized by the presence of evolutionarily conserved Baculoviral Inhibitor of Apoptosis Repeat (BIR) domains, predominantly known for their role in inhibiting caspases and thereby apoptosis. We have previously shown that multi-BIR domain containing IAPs, cIAPs and XIAP can control tumour cell migration by directly regulating the Protein stability of C-Raf kinase. Here, we extend our observations to a single BIR domain containing IAP family member, Melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-Raf and ML-IAP depletion leads to an increase in C-Raf Protein levels, MAPK activation and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-Raf stability and cell migration.
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associations of b and c Raf with cholesterol phosphatidylserine and lipid second messengers preferential binding of Raf to artificial lipid Rafts
Journal of Biological Chemistry, 2002Co-Authors: Mirko Hekman, Heike Hamm, Ana V Villar, Benjamin Bader, Jurgen Kuhlmann, Joachim Nickel, Ulf R RappAbstract:Abstract The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras Proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K D) in the range of 0.5–50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 Proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called Rafts. We demonstrate here that purified Raf Proteins have moderate binding affinity for cholesterol. However, under conditions of lipid Raft formation, Raf association with cholesterol (or Rafts) increased dramatically. Since ceramides also support formation of Rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in Raft regions via association with cholesterol and ceramides and in non-Raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf Protein that is already present at the membrane.
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role of diacylglycerol regulated Protein kinase c isotypes in growth factor activation of the Raf 1 Protein kinase
Molecular and Cellular Biology, 1997Co-Authors: U Smola, Ulf R Rapp, Jorge Moscat, V Wixler, I Eisenmanntappe, M T Diazmeco, U Smola, V Wixler, Geoffrey M. CooperAbstract:The Raf Protein kinases function downstream of Ras guanine nucleotide-binding Proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the «isotype of Protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC« inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC« stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC«also stimulated Raf kinase in baculovirusinfected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKCafunctioned similarly to PKC«in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKCa and PKC« overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerolregulated PKCs function as redundant activators of Raf-1 in vivo.
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role of diacylglycerol regulated Protein kinase c isotypes in growth factor activation of the Raf 1 Protein kinase
Molecular and Cellular Biology, 1997Co-Authors: Hong Cai, Ulf R Rapp, Jorge Moscat, U Smola, V Wixler, I Eisenmanntappe, M T Diazmeco, Geoffrey M. CooperAbstract:The Raf Protein kinases function downstream of Ras guanine nucleotide-binding Proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of Protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.
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95 kilodalton b Raf serine threonine kinase identification of the Protein and its major autophosphorylation site
Molecular and Cellular Biology, 1992Co-Authors: Robert M. Stephens, Ulf R Rapp, G Sithanandam, Terry D. Copeland, David R. Kaplan, Deborah K. MorrisonAbstract:B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf Protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa Protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf Protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these Proteins.
Geoffrey M. Cooper - One of the best experts on this subject based on the ideXlab platform.
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role of diacylglycerol regulated Protein kinase c isotypes in growth factor activation of the Raf 1 Protein kinase
Molecular and Cellular Biology, 1997Co-Authors: Hong Cai, Ulf R Rapp, Jorge Moscat, U Smola, V Wixler, I Eisenmanntappe, M T Diazmeco, Geoffrey M. CooperAbstract:The Raf Protein kinases function downstream of Ras guanine nucleotide-binding Proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of Protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.
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role of diacylglycerol regulated Protein kinase c isotypes in growth factor activation of the Raf 1 Protein kinase
Molecular and Cellular Biology, 1997Co-Authors: U Smola, Ulf R Rapp, Jorge Moscat, V Wixler, I Eisenmanntappe, M T Diazmeco, U Smola, V Wixler, Geoffrey M. CooperAbstract:The Raf Protein kinases function downstream of Ras guanine nucleotide-binding Proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the «isotype of Protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC« inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC« stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC«also stimulated Raf kinase in baculovirusinfected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKCafunctioned similarly to PKC«in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKCa and PKC« overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerolregulated PKCs function as redundant activators of Raf-1 in vivo.
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Ras controls coupling of growth factor receptors and Protein kinase C in the membrane to Raf-1 and B-Raf Protein serine kinases in the cytosol.
Oncogene, 1992Co-Authors: Jakob Troppmair, Joseph T. Bruder, Harald App, Hong Cai, Lauren Liptak, József Szeberényi, Geoffrey M. Cooper, Ulf R RappAbstract:A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf Protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.
Frank Sprenger - One of the best experts on this subject based on the ideXlab platform.
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Raf functions downstream of ras1 in the sevenless signal transduction pathway
Nature, 1992Co-Authors: Barry J Dickson, Frank Sprenger, Deborah Morrison, Ernst HafenAbstract:SPECIFICATION of the R7 cell fate in the developing Drosophila eye requires activation of the Sevenless (Sev) receptor tyrosine kinase, located on the surface of the R7 precursor cell, by its interaction with the Boss Protein, expressed on the surface of the neighbouring R8 cell1–3. Four genes that participate in the intracellular transmission of this signal have so far been identified and molecularly characterized: Rasl, Sos, Gapl and sina (refs 4–8). The Drosophila homologue of the mammalian Raf-1 serine/threonine kinase, which has been implicated in signal transduction pathways activated by many receptor tyrosine kinases (reviewed in refs 9 and 10), is encoded by the Raf locus (also known as l(l)polehole11, DRaf-112 or DRaf13). Here we show that the Drosophila Raf serine/threonine kinase also plays a crucial role in the R7 pathway: the response to Sev activity is dependent on Raf function, and a constitutively activated Raf Protein can induce R7 cell development in the absence of sev function. We also present genetic evidence suggesting that Raf acts downstream of Rasl and upstream of Sina in this signal transduction cascade.
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Raf functions downstream of rasl in the sevenless signal transduction pathway
Nature, 1992Co-Authors: Barry J Dickson, Frank Sprenger, Deborah Morrison, Frank Sprenger, Ernst HafenAbstract:SPECIFICATION of the R7 cell fate in the developing Drosophila eye requires activation of the Sevenless (Sev) receptor tyrosine kinase, located on the surface of the R7 precursor cell, by its interaction with the Boss Protein, expressed on the surface of the neighbouring R8 cell1–3. Four genes that participate in the intracellular transmission of this signal have so far been identified and molecularly characterized: Rasl, Sos, Gapl and sina (refs 4–8). The Drosophila homologue of the mammalian Raf-1 serine/threonine kinase, which has been implicated in signal transduction pathways activated by many receptor tyrosine kinases (reviewed in refs 9 and 10), is encoded by the Raf locus (also known as l(l)polehole11, DRaf-112 or DRaf13). Here we show that the Drosophila Raf serine/threonine kinase also plays a crucial role in the R7 pathway: the response to Sev activity is dependent on Raf function, and a constitutively activated Raf Protein can induce R7 cell development in the absence of sev function. We also present genetic evidence suggesting that Raf acts downstream of Rasl and upstream of Sina in this signal transduction cascade.
Krzysztof Pawlowski - One of the best experts on this subject based on the ideXlab platform.
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the hidden story of heterogeneous b Raf v600e mutation quantitative Protein expression in metastatic melanoma association with clinical outcome and tumor phenotypes
Cancers, 2019Co-Authors: Lazaro Hiram Betancourt, Marcell A Szasz, Magdalena Kuras, Jimmy Rodriguez Murillo, Sugihara Yutaka, Indira Pla, Zsolt Horvath, Krzysztof PawlowskiAbstract:In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRaf V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-Raf V600E Protein, into consideration. As shown by immunohistochemistry, the Protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant Protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target Protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-Raf Protein within metastatic tumors was quantitated for the first time. B-Raf V600E Protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, Protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated Protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and Protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.
Robert Roskoski - One of the best experts on this subject based on the ideXlab platform.
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targeting oncogenic Raf Protein serine threonine kinases in human cancers
Pharmacological Research, 2018Co-Authors: Robert RoskoskiAbstract:The Ras-Raf-MEK-ERK signal transduction cascade is arguably the most important oncogenic pathway in human cancers. Ras-GTP promotes the formation of active homodimers or heterodimers of A-Raf, B-Raf, and C-Raf by an intricate process. These enzymes are Protein-serine/threonine kinases that catalyze the phosphorylation and activation of MEK1 and MEK2 which, in turn, catalyze the phosphorylation and activation of ERK1 and ERK2. The latter catalyze the regulatory phosphorylation of dozens of cytosolic and nuclear Proteins. The X-ray crystal structure of B-Raf-MEK1 depicts a face-to-face dimer with interacting activation segments; B-Raf is in an active conformation and MEK1 is in an inactive conformation. Besides the four traditional components in the Ras-Raf-MEK-ERK signaling module, scaffolding Proteins such as Kinase Suppressor of Ras (KSR1/2) play an important role in this signaling cascade by functioning as a scaffold Protein. RAS mutations occur in about 30% of all human cancers. Moreover, BRafV600E mutations occur in about 8% of all cancers making this the most prevalent oncogenic Protein kinase. VemuRafenib and dabRafenib are B-RafV600E inhibitors that were approved for the treatment of melanomas bearing the V600E mutation. Coupling MEK1/2 inhibitors with B-Raf inhibitors is more effective in treating such melanomas and dual therapy is now the standard of care. VemuRafenib and cobimetanib, dabRafenib and trametinib, and encoRafenib plus binimetinib are the FDA-approved combinations for the treatment of BRafV600E melanomas. Although such mutations occur in other neoplasms including thyroid, colorectal, and non-small cell lung cancers, these agents are not as effective in treating these non-melanoma neoplasms. VemuRafenib and dabRafenib produce the paradoxical activation of the MAP kinase pathway in wild type BRaf cells. The precise mechanism for this activation is unclear, but drug-induced Raf activating side-to-side dimerization appears to be an essential step. Although 63%-76% of all people with advanced melanoma with the BRaf V600E mutation derive clinical benefit from combination therapy, median progression-free survival lasts only about nine months and 90% of patients develop resistance within one year. The various secondary resistance mechanisms include NRAS or KRAS mutations (20%), BRaf splice variants (16%), BRafV600E/K amplifications (13%), MEK1/2 mutations (7%), and non-MAP kinase pathway alterations (11%). VemuRafenib and dabRafenib bind to an inactive form of B-Raf (αC-helixout and DFG-Din) and are classified as type I½ inhibitors. LY3009120 and lifiRafenib, which are in the early drug-development stage, bind to a different inactive form of B-Raf (DFG-Dout) and are classified as type II inhibitors. Besides targeting B-Raf and MEK Protein kinases, immunotherapies that include ipilimumab, pembrolizumab, and nivolumab have been FDA-approved for the treatment of melanomas. Current clinical trials are underway to determine the optimal usage of targeted and immunotherapies.
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Raf Protein serine threonine kinases structure and regulation
Biochemical and Biophysical Research Communications, 2010Co-Authors: Robert RoskoskiAbstract:A-Raf, B-Raf, and C-Raf are a family of three Protein-serine/threonine kinases that participate in the RAS-Raf-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15–30% of all human cancers, and B-Raf mutations occur in 30–60% of melanomas, 30–50% of thyroid cancers, and 5–20% of colorectal cancers. Activation of the Raf kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family Protein-tyrosine kinases and other Protein-serine/threonine kinases. The formation of unique side-to-side Raf dimers is required for full kinase activity. Raf kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-Raf Val600Glu activating mutation. Raf kinase inhibitors lead to the paradoxical increase in Raf kinase activity in cells containing wild-type B-Raf and wild-type or activated mutant RAS. C-Raf plays a key role in this paradoxical increase in downstream MEK-ERK activation.