89,372 research outputs found
Mechanism of regulation of Raf-1 by Ca2+/Calmodulin-dependent kinase II
No full textThe calcium-calmodulin dependent kinase II (CaMKII) is an ubiquitous serine/threonine protein kinase involved in multiple signalings and biological functions. It has been demonstrated that in epithelial and mesenchimal cells CaMKII participates with Ras to Raf-1 activation and that it is necessary for ERK activation by diverse factors. Raf-1 activation is complex. Maximal Raf-1 activation is reached by phosphorylation at Y341 by Src and at S338. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 is not definitively identified.
Aim of my thesis is to go more insight into the molecular mechanisms of CaMKII/Raf-1 interaction and to verify the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To this purpose, I investigated the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors, in COS-7 and NIH3T3 cells. Serum, SrcY527 and RasV12 activated CaMKII. CaMKII was necessary for Raf-1 and ERK activation by all these factors. CaMKII was necessary to the phosphorylation of S338 Raf-1 by serum, fibronectin or oncogenic Ras. Conversely, the inhibition of phosphatidylinositol 3-kinase, which in turn activates Pak3, was ineffective. The direct kinase activity of CaMKII on the serine 338 residue, was demonstrated in vitro by interaction of purified kinases.
These results demonstrate that CaMKII phosphorylates Raf-1 at S338 and partecipates to ERK activation upon different physiologic and pathologic stimuli in the MAPK cascade. This kinase, might have a role in cancers harbouring oncogenic Ras and could represent a new therapeutic target for pharmacological intervention in these tumors
RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development
No full textBackground: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood.
Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation.
Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG
Raf-MEK-ERK signaling pathway is involved in KGA activation in HUVECs.
No full textThe cells were treated with 10ng/ml of TGF-β1 for different indicated periods, from 15 minutes to 90 minutes. (a, c) Representative western blot of p-ERK1/2, T-ERK1/2, p-c-Raf (Ser 259) and T-c-Raf in each group was shown. (b, d, e, f) The histogram are normalized to a GAPDH control and showed the ratio of p-ERK1/2 to T-ERK1/2 and p-c-Raf to T-c-Raf (n≥3). (g, h) HUVECs were pretreated with MEK 1/2 inhibitor (U0126, 10μM) for 30 min. KGA protein expressions were detected by western blot with densitometry analysis (n≥3). *P<0.05 compared to control group. Bars represented means ±SEM.</p
MYC is a metastasis gene for non-small-cell lung cancer.
No full textMetastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development
Raf-1 and B-Raf in vascular development and tumor angiogenesis
No full textAngiogenese wird durch Endothelzellen induziert und ist notwendig für die Entstehung neuer Blutgefäße. Dafür wird die komplexe Koordination von multiplen biologischen Prozessen erfordert: Endothelzell Proliferation, kollektive Zellmigration, Zell-Zell Kommunikation und die Formung eines Lumens. Die korrekte Anordnung eines Vaskulären Netzwerkes ist für einen gesunden Organismus notwendig, da eine Dysfunktion zu einer Vielfalt von Krankheiten führt. Das Verstehen der molekularen Maschinerie welche die Angiogenese kontrolliert wird neue therapeutische Behandlungen für viele menschliche Krankheiten, wie Entzündungskrankheiten oder Krebs ermöglichen. Wir identifizieren Raf-1 als eine essentielle Komponente der molekularen „Sprouting“ Maschinerie. Die Deletion von Raf-1 beeinträchtigt die Endothelzell Kohäsion, das “Sprouting” und die Angiogenese in vivo, was zu einer verminderten Vaskularisierung der Retina (Entwicklungsangiogenese) als auch zu einer reduzierten Tumor Vaskularisierung und Wachstum führt (pathologische Angiogenese). Mechanistisch wird Raf-1 durch das kleine G Protein Rap1 zu VE-Cadherin Komplexen rekrutiert. Dort wird es für die Assoziation des Rho Effektor Rok-α an entstehenden Zell-Zell Kontakten benötigt. Diese Raf-1 vermittelte Feinregulierung von Rok-α ermöglicht die Aktivierung von Myosin an Zell-Zell Kontakten und die zeitgemäße Reifung von Zell-Zell Adhäsionen um die Zell Kohäsion während der „Sprouting“ Angiogenese aufrechtzuerhalten. Im Gegenzug dazu führt die Ablation von B-Raf im Endothelzell/Hämatopoetischem Zellkompartment zu einem vermehrten Wachstum von Tumoren. Wir finden weniger nekrotisches Gewebe dass mit einer geringeren intratumoralen vaskulären Permeabilität korreliert. Die Barriere Funktion von B-Raf KO Endothelzellen wird nicht durch VEGF abgeschwächt, was dafür spricht dass B-Raf für VEGF induzierte Permeabilität benötigt wird. Wir nehmen an dass die Ablation von B-Raf zu einer Blutgefäß Normalisierung führt durch Umkehrung der Hyper-Permeabilität welche typisch für Tumor-assoziierte Gefäße ist. Das würde zu einer erhöhten Funktionalität der Blutgefäße führen und zu einem beschleunigten Tumorwachstum.
Die Ablation von 3 Raf Allelen in Endothelzellen (3AD = B-Raf f/f;Raf-1 f/+ or B-Raf f/+;Raf-1 f/f) führt zum frühen postnatalen Tod der Tiere aufgrund eines Respiratorischen Defektes. Die Lungen der Mäuse sind mit Blut gefüllt und eine Ruptur der pulmonalen Blutgefäße konnte festgestellt werden. In vitro zeigen Endothelzellen von 3AD Mäusen eine reduzierte Resistenz gegenüber angelegtem Scherstress, dass sich durch Desintegration von VE-Cadherin vermittelten Zell-Zell Adhäsionen offenlegt. Die induzierte Deletion von B-Raf und Raf-1 im vaskulären/hämatopoetischen System blockiert fast komplett die Einwanderung neuer Blutgefässe in den Matrigel Plug. Wir schliessen daraus dass die gleichzeitige Inhibition von Raf-1 und B-Raf am effektivsten das Wachstum von Blutgefässen hemmt aber die vaskuläre Homöostase beinträchtigt.Angiogenesis is initiated by endothelial cells and indispensable for the development of new blood vessels. It requires the complex coordination of multiple biological processes: endothelial cell proliferation, collective cell migration, cell-to-cell communication and lumen formation. The correct alignment of the vascular networks is a requirement for a healthy organism since dysfunction leads to a variety of disorders. Understanding the molecular machinery controlling angiogenesis will give rise to novel therapeutic treatments for many human diseases such as inflammatory disorders or cancer. Here we identify Raf-1 as an essential component of the molecular sprouting machinery. Raf-1 dele tion impairs endothelial cell cohesion, sprouting and in vivo angiogenesis, leading to a decreased vascularization of the retina (developmental angiogenesis) and reduced tumor angiogenesis and growth (pathological angiogenesis). Mechanistically, Raf-1 is recruited to VE-Cadherin complexes by the small G Protein Rap1, and is required for the association of the Rho effector Rok-α with nascent adherens junctions (AJ). This Raf-1-mediated fine tuning of Rok-α signaling allows the activation of junctional myosin and the timely maturation of AJ essential for maintaining cell cohesion during sprouting angiogenesis. In contrast, B-Raf ablation in the endothelial/hematopoietic cell compartment increases tumor xenograft growth. We find decreased necrotic areas correlating with lower levels of intratumoral vascular permeability. The barrier function of B-Raf KO endothelial cells is not weakened by VEGF treatment, suggesting that B-Raf is needed to control VEGF-induced vascular permeability. We suggest that B-Raf ablation results in vessel “normalization” by reversing the hyper-permeability typical of tumor-associated vessels; this would increase the functionality of the vasculature and accelerate tumor growth.
Finally, ablation of 3 Raf alleles in endothelial cells (3AD = B-Raf f/f;Raf-1 f/+ or B-Raf f/+;Raf-1 f/f) leads to early postnatal death of the animals because of a respiratory defect. The lungs of these animals are filled with blood and rupture of pulmonary blood vessels could be observed. In vitro, endothelial cells from 3AD mice show reduced resistance to applied shear stress characterized by disintegration of VE-Cadherin mediated junctions. The induced deletion of B-Raf and Raf-1 in the vascular/haematopoietic compartment almost completely abrogates blood vessel invasion in the Matrigel plug assay. We conclude that simultaneous targeting Raf-1 and B-Raf most effectively inhibits blood vessel growth but affects vascular homeostasis
Eintrag von C[hristian] F[riedrich] G[raf] Stolberg[-Wernigerode] und [Johann Wilhelm Ludwig] Gleim / Einschlagblatt mit redaktionellen Anmerkungen
No full textEINTRAG VON C[HRISTIAN] F[RIEDRICH] G[RAF] STOLBERG[-WERNIGERODE] UND [JOHANN WILHELM LUDWIG] GLEIM / EINSCHLAGBLATT MIT REDAKTIONELLEN ANMERKUNGEN
Eintrag von C[hristian] F[riedrich] G[raf] Stolberg[-Wernigerode] und [Johann Wilhelm Ludwig] Gleim / Einschlagblatt mit redaktionellen Anmerkungen ( -
Analysis of B-RAF mutant.
No full text<p><b>A.</b> Sequence alignment, results from 454 pyrosequencing of granuloma cells from patients 1–10 and 16. <b>B.</b> ‘Sanger’ sequencing of patient 16 blood; A/G transition at nucleotide 1795. <b>C.</b> Pedigree of patient 16. Both the patient and his mother carry a <sup>T599A</sup>B-RAF allele, while his father is <sup>wt</sup>B-RAF. <b>D–E.</b> Comparison between <sup>wt</sup>B-RAF 5P_15056 (D, purple), <sup>V600E</sup>B-RAF structure (E, cyan) and the modeled mutant <sup>600DLAT</sup>B-RAF (F, grey). In D, Val600 (yellow) forms a hydrophobic contact with Phe468 (red arrow). In E and F charged residues Asp and Glu (in orange) disrupt the hydrophobic network of interactions, stabilising the active conformation of the P-loop. In F, insertion Asp-Leu-Ala-Thr shifted Val600 and disrupt the hydrophobic cluster. <b>G, H.</b> MEK phosphorylation in 293 T cells. 293 T cells were transiently transfected with with mock or B-RAF mutant expressing vectors (WT, V600E, T599A, 600DLAT, D594A, G596R), and with (H) or without (G) wtC-RAF. Twenty-four hours after transfection, the medium was changed to serum-free DMEM, followed by further 18 hours culture. Total cell lysates were immunoblotted with the indicated antibodies.</p
Expression of Drug Targets in Patients Treated with Sorafenib, Carboplatin and Paclitaxel
No full textIntroduction: Sorafenib, a multitarget kinase inhibitor, targets members of the mitogen-activated protein kinase (MAPK) pathway and VEGFR kinases. Here we assessed the association between expression of sorafenib targets and biomarkers of taxane sensitivity and response to therapy in pre-treatment tumors from patients enrolled in ECOG 2603, a phase III comparing sorafenib, carboplatin and paclitaxel (SCP) to carboplatin, paclitaxel and placebo (CP). Methods: Using a method of automated quantitative analysis (AQUA) of in situ protein expression, we quantified expression of VEGF-R2, VEGF-R1, VEGF-R3, FGF-R1, PDGF-Rβ, c-Kit, B-Raf, C-Raf, MEK1, ERK1/2, STMN1, MAP2, EB1 and Bcl-2 in pretreatment specimens from 263 patients. Results: An association was found between high FGF-R1 and VEGF-R1 and increased progression-free survival (PFS) and overall survival (OS) in our combined cohort (SCP and CP arms). Expression of FGF-R1 and VEGF-R1 was higher in patients who responded to therapy ((CR+PR) vs. (SD+PD+ un-evaluable)). Conclusions: In light of the absence of treatment effect associated with sorafenib, the association found between FGF-R1 and VEGF-R1 expression and OS, PFS and response might reflect a predictive biomarker signature for carboplatin/paclitaxel-based therapy. Seeing that carboplatin and pacitaxel are now widely used for this disease, corroboration in another cohort might enable us to improve the therapeutic ratio of this regimen. © 2013 Jilaveanu et al
Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif.
No full textBRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-Raf(G596R) is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-Raf(G596R) depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation
Analysis of <sup>T599A</sup>B-RAF.
No full text<p>(<b>A, B</b>) Comparison between models of <sup>WT</sup>B-RAF 5P_15056 (A, violet) and <sup>T599A</sup>B-RAF (B, gold). <sup>T599A</sup>B-RAF substitutes a polar uncharged residue with a hydrophobic residue, causing the loss of short-ranged interactions with residues D576 and D594. <b>C</b>. Analysis of MEK and ERK phosphorylation in THP1 cell lines stably transfected with <sup>WT</sup>B-RAF-FLAG and <sup>T599A</sup>B-RAF-FLAG. Experiment was repeated twice with similar results. (<b>D–F</b>) Analysis of MEK and ERK phosphorylation and IL-8 production in U937 cell lines stably transfected with <sup>WT</sup>B-RAF, <sup>T599A</sup>B-RAF, and <sup>D594A</sup>B-RAF. Experiment was repeated twice with similar results.</p
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