Disease relevance of Raf1

• With the use of dominant negative mutants of Ha-Ras and Raf-1, we investigated some of the early signaling events leading to the activation of NF-kappa B by hypoxia [1].
• Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice [2].
• Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload [3].
• Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation [4].
• Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death [5].

High impact information on Raf1

• Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells [6].
• In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes [6].
• Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif [7].
• The Raf-1 serine/threonine kinase is a key component of the MAP kinase cascade, regulating both proliferation and commitment to cell fate [8].
• Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization [8].

Chemical compound and disease context of Raf1

• METHODS AND RESULTS: In an attempt to determine the requirement for the serine/threonine kinase Raf-1 in the pathogenesis of cardiac hypertrophy, we generated transgenic mice with cardiac-specific expression of a dominant negative form of Raf-1 (DN-Raf) [3].
• Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro [9].
• We show that expression of Fas in these lymphomas can be increased not only in the presence of a specific inhibitor (LY294002) of P13 kinase, but also in the presence of specific inhibitor (PD98059) of MEK, downstream target of Raf-1 [10].

Biological context of Raf1

• Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene [11].
• Three mutants: S218D, S222D and S218D/S222D in which we substituted the Raf1/MAPKKK-dependent regulatory phosphorylation sites by aspartic acid residues, displayed increased basal activity when expressed in fibroblasts [12].
• No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus [13].
• These two genes together with Raf1 appear to be members of a large syntenic gene cluster that maps to human chromosome bands 3p25-->p26, mouse chromosome bands 6 C3-->E, and rat chromosome bands 4q41-->q42 [14].
• Similarly, the poor proliferation of Raf-1(-/-) fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect [15].

Anatomical context of Raf1

• Finally, pull-down assays using the Ras-binding domain of Raf1 demonstrated that OSM directly activates K-Ras in fetal hepatocytes [16].
• To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G [17].
• The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation [17].
• Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear [17].
• We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells [18].

Associations of Raf1 with chemical compounds

• We show that the Ras GTPase and Raf1 serine/threonine kinase are required in this pathway [19].
• Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases [19].
• Decrease in K-ras p21 and increase in Raf1 and activated Erk 1 and 2 in murine lung tumors initiated by N-nitrosodimethylamine and promoted by 2,3,7,8-tetrachlorodibenzo-p-dioxin [20].
• Furthermore, Raf-1- deficient fibroblasts are more sensitive than wild- type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor-alpha [15].
• In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk [21].

Physical interactions of Raf1

• Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK [22].
• We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain [23].
• In addition to Raf, we show that v-Src, v-H-Ras and v-Mos activate HIV-LTR expression through the NF-kappa B binding sites and v-H-Ras-induced HIV-LTR expression is mediated by Raf-1 [24].

Enzymatic interactions of Raf1

• Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation [25].

Co-localisations of Raf1

• Phosphorylated ezrin-positive dots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580 [26].

Regulatory relationships of Raf1

• These results indicate that Raf-1 promotes cardiomyocyte survival through a MEK/ERK-independent mechanism [27].
• Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase [28].
• Our data also show by sequential in vitro kinase assays that TNF-alpha enhances KSR phosphorylation of Raf-1 on threonine, enhancing Raf-1 kinase activity toward MAP kinase kinase [29].
• Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation [30].
• Raf-1 regulates Rho signaling and cell migration [31].

Other interactions of Raf1

• Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype [32].
• These data suggest that Tpl-2 activates the MAPK cascade, perhaps through its participation in the assembly of Ras/Raf-1-containing multimolecular complexes [28].
• Since Ras and Raf-1 have been previously shown to work downstream from membrane-associated tyrosine kinases such as Src, we determined if the Src membrane-associated kinase was also activated by low oxygen conditions [1].
• These data suggest that signaling through the Raf-1 pathway is necessary for the optimal expression of p21 in chondrocytes and may play an important role in the control of bone formation [33].
• Raf-1, a direct regulator of MKK1 and MKK2, was activated under these conditions, and a synergistic activation of MKK was observed upon coexpression of Raf-1 and MKP-1 [34].

Analytical, diagnostic and therapeutic context of Raf1

• We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations [32].
• Heretofore, the biomarkers to detect 17-AAG bioactivity (Hsp70, Raf-1, and cyclin-dependent kinase 4) had to be analyzed by Western blot of cellular samples, either from tumor biopsies or peripheral blood leukocytes, a method that is both laborious and invasive [35].
• We have used differential display PCR to search for mRNAs induced by delta Raf-1:ER, an estradiol-dependent form of Raf-1 kinase [36].
• The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells [23].
• Although ligation of both receptors induces Ras and Raf-1 activation, the downstream consequences of these early activation events are not well defined, except for the activation of extracellular signal-regulated kinases (ERK) [37].

References