Alike but Different: RAF Paralogs and Their Signaling Outputs

Desideri, Enrico; Cavallo, Anna Lina; Baccarini, Manuela

doi:10.1016/j.cell.2015.04.045

Cited by 94 publications

(121 citation statements)

References 21 publications

(26 reference statements)

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“…Analysis of p-ERK and p-MEK in colonospheres and tumours showed that this pathway was not inhibited upon RAF1-KD. The levels of p-ERK and p-MEK were either comparable between the samples or increased upon RAF1-KD, consistent with previous studies (figure 5A, B) 22. Moreover, we used Lincscloud database to compare the profiles of differentially regulated genes in RAF1-KD with the profiles generated after the treatment with various MEK-inhibitors (figure 5C).…”

Section: Resultssupporting

confidence: 86%

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer

Borovski

Vellinga

Laoukili

et al. 2016

Gut

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Background and aimColorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target.MethodsColonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence.ResultsKD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture.ConclusionsRAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.

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Section: Resultssupporting

confidence: 86%

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer

Borovski

Vellinga

Laoukili

et al. 2016

Gut

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“…This raises the questions of how does the cell selects between respective RAS proteins and maintains respective effector activation. There are several review articles illustrating the current state of the art regarding the activation mechanism of various effectors [9,11,12,13,21,71,72,73]. HRAS, KRAS and NRAS exhibit remarkable differences beyond their common interaction interfaces for regulators and effectors [74,75,76], especially at their C-terminal hypervariable region (S3 Fig), which has different features, including protein-protein interaction [77,78].…”

Section: Discussionmentioning

confidence: 99%

“…g ., CRAF, PI3Kα, RALGDS, PLCε and RASSF5, and their activation [1,4,5,6,7,8,9,10]. CRAF, a serine/threonine kinase, activates the MEK-ERK axis and controls gene expression and cell proliferation [11]. PI3Kα generates phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) and regulates cell growth, cell survival, cytoskeleton reorganization, and metabolism [12].…”

Section: Introductionmentioning

confidence: 99%

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

Nakhaeizadeh¹,

Amin²,

Nakhaei‐Rad³

et al. 2016

PLoS ONE

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RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

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“…This finding is intriguing, as MEK is considered to be the only established direct substrate of BRAF kinase, especially in the light of prior observations that suggested the effects of vemurafenib on the transcriptome were exclusively MEK dependent (Joseph et al, 2010). CRAF, a paralog of BRAF, has been shown to regulate various other kinases such as ASK1, MST2, PLK1, and ROKa in a MEKindependent manner (Desideri et al, 2015). However, in contrast to the regulation of HMGCL by BRAF V600E , these activities of CRAF were found to be associated with the scaffolding role of CRAF and were independent of its kinase activity.…”

mentioning

confidence: 95%

Addicted to AA (Acetoacetate): A Point of Convergence between Metabolism and BRAF Signaling

Trousil

Zheng

2015

Molecular Cell

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Alike but Different: RAF Paralogs and Their Signaling Outputs

Cited by 94 publications

References 21 publications

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

Addicted to AA (Acetoacetate): A Point of Convergence between Metabolism and BRAF Signaling

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