Raf-1 In Cells Research Articles

Lyso-PAF, a biologically inactive phospholipid, contributes to RAF1 activation

Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor (PAF) to a biologically inactive product Lyso-PAF during inflammatory response. We report that intracellular PLA2G7 is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant NRAS, but not cells expressing BRAF V600E. Mechanistically, PLA2G7 signals through its product Lyso-PAF to contribute to RAF1 activation by mutant NRAS, which is bypassed by BRAF V600E. Intracellular Lyso-PAF promotes p21-activated kinase 2 (PAK2) activation by binding to its catalytic domain and altering ATP kinetics, while PAK2 significantly contributes to S338-phosphorylation of RAF1 in addition to PAK1. Furthermore, the PLA2G7-PAK2 axis is also required for full activation of RAF1 in cells stimulated by epidermal growth factor (EGF) or cancer cells expressing mutant KRAS. Thus, PLA2G7 and Lyso-PAF exhibit intracellular signaling functions as key elements of RAS-RAF1 signaling.

Abstract PL02-02: BRAF and RAS signaling in cancer: From biology to therapeutics.

Abstract The RAS proteins are small G-protein that are activated downstream of cell surface receptors. The RAF proteins are protein kinases that are activated by RAS. RAF proteins then activate the MEK/ERK signalling pathway, which stimulates cell proliferation and drives tumorigenesis. There are three RAS proteins (HRAS, KRAS, NRAS) in humans and the NRAS isoform is mutated in about 25% of melanomas. There are also three RAF proteins in humans (ARAF, BRAF and CRAF) and BRAF is mutated in a further 50% of melanomas. Critically, drugs that target BRAF achieve impressive objective responses in patients with BRAF mutant melanoma, but are ineffective in patients whose melanomas express wild-type BRAF. These data validate mutant BRAF as a therapeutic target in melanoma. An unexpected property of RAF inhibitors is that they drive paradoxical activation of the MEK/ERK pathway in cells that express oncogenic RAS. This is because when they bind to RAF in cells in the presence of oncogenic RAS, these inhibitors drive the formation of CRAF:BRAF heterodimers and CRAF:CRAF homodimers in which one partner is drug-bound and the other is drug-free. Critically, the drug-bound partner acts as a scaffold, or induces a conformation change that hyper-activates the drug-free partner and this partner then drives pathway signalling. Notably, a quarter to a third of patients on BRAF inhibitors develop cutaneous squamous cell carcinomas (cuSCC) and keratoacanthomas. We have used a two-stage skin carcinogenesis model in mice that is driven by oncogenic HRAS to show that while BRAF inhibitors accelerate the development of RAS-driven cuSCC in mice, they do not initiate the formation of these lesions. We found that about 60% of BRAF-inhibitor driven cuSCC in melanoma patients harbour mutations in RAS genes and furthermore show that in mice cuSCC development driven by BRAF inhibitors can be blocked by co-administration of a MEK inhibitor. Thus, we conclude that RAS inhibitors do not act as tumor promoters per se; rather they accelerate the inevitable in susceptible patients and we provide a rational approach to blocking this side effect in BRAF mutant melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL02-02.

Characterization of Ser338 Phosphorylation for Raf-1 Activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.

Pro-apoptotic activity of oncogenic H-Ras for histone deacetylase inhibitor to induce apoptosis of human cancer HT29 cells

To verify the pro-apoptotic activity of oncogenic H-Ras in the increased susceptibility of human cancer cells to histone deacetylase inhibitor (HDACI). The pro-apoptotic activity of oncogenic H-Ras(V12) was verified by its ability to increase susceptibility of human colorectal adenocarcinoma HT29 cells to HDACI for inducing apoptosis and growth inhibition, assayed by various methods. The mode of action of HDACI FR901228 was studied by its ability to modulate protein phosphorylation, acetylation, and expression levels in various signaling pathways, measured by Western blot analysis. Activation of caspase-3, -7, and -8, and serine protease by FR901228 was facilitated by oncogenic H-Ras to induce apoptosis. Expression of H-Ras(V12) changed the intrinsic modulation of Raf in cells responding to FR901228 treatment. Both p21( Cip1 ) and p27( Kip1 ) were induced in FR901228-treated cells arrested in either the G0/G1 or G2/M phase of the cell cycle. Deacetylation of FR901228-induced acetylation of core histones was accelerated by H-Ras(V12) in cells undergoing apoptosis. Expression of H-Ras(V12) increased susceptibility of HT29 cells to HDACI FR901228 and Trichostatin A for inducing apoptosis. The pro-apoptotic activity of H-Ras(V12) responding to HDACI indicates a potential value of this new class of anticancer agents in treating Ras-related human cancers.

Raf-1 and B-Raf promote protein kinase C θ interaction with BAD

PKCθ regulates the proliferation, survival and differentiation of T-cells. Here we show that PKCθ interacts with Raf-1 and B-Raf kinases. Raf-1 enhanced the kinase activity of associated PKCθ, while PKCθ reduced the catalytic activity of associated Raf-1. In contrast, B-Raf binding did not affect PKCθ kinase activity, and PKCθ did not change B-Raf activity. Coexpression of mutationally activated Raf-1 in cells enhanced the phosphorylation of T538 in the PKCθ activation loop. PKCθ and Raf cooperated in terms of binding to BAD, a pro-apoptotic Bcl-2 family protein that is inactivated by phosphorylation. While neither Raf-1 nor B-Raf could phosphorylate BAD, they enhanced the ability of PKCθ to interact with BAD and to phosphorylate BAD in vitro and in vivo, suggesting a new role for Raf proteins in T-cells by targeting PKCθ to interact with and phosphorylate BAD.

Role of Bim in the survival pathway induced by Raf in epithelial cells.

Selective and sustained activation of the Raf/MAP kinase pathway in MCF-10A Delta Raf-ER cells, a spontaneously immortalized human mammary epithelial cell line, was previously shown to protect these cells from suspension-induced cell death, a critical feature of the Ras-transformed phenotype. Although autocrine signalling through the EGF receptor is crucial for the protection induced by Raf in these cells, we report here the existence of an additional, more direct survival mechanism, linking Raf activation to the inhibition of a proapoptotic member of the Bcl-2 family, Bim. While detachment from the matrix results in transcriptional induction of two variants of this BH3-only protein, BimEL and BimL, activation of the Raf/ERK signalling both prevents Bim upregulation specifically and leads to phosphorylation and degradation of the BimEL isoform. This represents an important route to protect epithelial cells from the proapoptotic effect of Bim.

Simian Virus 40 Large Tumor Antigen Modulates the Raf Signaling Pathway

The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.

Contribution of the ERK5/MEK5 Pathway to Ras/Raf Signaling and Growth Control

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.

The MEK1 Proline-rich Insert Is Required for Efficient Activation of the Mitogen-activated Protein Kinases ERK1 and ERK2 in Mammalian Cells

MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.

Activation of Raf as a result of recruitment to the plasma membrane.

The small guanine nucleotide binding protein Ras participates in a growth promoting signal transduction pathway. The mechanism by which interaction of Ras with the protein kinase Raf leads to activation of Raf was studied. Raf was targeted to the plasma membrane by addition of the COOH-terminal localization signals of K-ras. This modified form of Raf (RafCAAX) was activated to the same extent as Raf coexpressed with oncogenic mutant Ras. Plasma membrane localization rather than farnesylation or the presence of the additional COOH-terminal sequence accounted for the activation of RafCAAX. The activation of RafCAAX was completely independent of Ras; it was neither potentiated by oncogenic mutant Ras nor abrogated by dominant negative Ras. Raf, once recruited to the plasma membrane, was not anchored there by Ras; most activated Raf in cells was associated with plasma membrane cytoskeletal elements, not the lipid bilayer. Thus, Ras functions in the activation of Raf by recruiting Raf to the plasma membrane where a separate, Ras-independent, activation of Raf occurs.