B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.

contributed equally to this work Raf-1 protein kinase has been identi®ed as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. 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. Raf-1 ±/± mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. 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. In cells derived from both the raf-1 ±/± and raf-1 FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89-99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras⅐GTP loading by 10 -15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras⅐GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC-and G idependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.Members of the membrane-associated small (21-kDa) G protein Ras family (Ha-Ras, K-Ras, N-Ras) are important in eukaryotic signal transduction (reviewed in Ref. 1). In its GDPligated state, Ras is inactive. By increasing its GTP loading in a regulated manner, Ras acts as a "molecular switch" and transmits signals from a variety of cell surface receptors to downstream effector proteins. The best characterized effector is c-Raf, one of the mitogen-activated protein kinase (MAPK) 1 kinase kinases of the extracellular signal-regulated kinase (ERK) cascade. Ras⅐GTP (but not Ras⅐GDP) has a high affinity for c-Raf, causing it to translocate to the membrane (reviewed in Ref. 2), where, in a process that possibly involves its phosphorylation (3-5), c-Raf becomes fully activated. Several other potential Ras effectors have been identified. These include phosphatidylinositol 3Ј-kinase, other small G proteins, the Ral-GDS family, and other protein kinases (reviewed in Ref. 1). Ras is converted back to its inactive state by its innate GTPase activity, which may be enhanced by GTPase-activating proteins.In the terminally differentiated, cell cycle-arrested ventricular myocyte (in contrast to transformed cell lines and other dividing cells), G protein-coupled receptor (GPCR) agonists such as endothelin-1 (ET-1), bradykinin (BK), and ␣ 1 -adrenergic agonists promote a stronger activation of the ERK cascade members (c-Raf/A-Raf, MAPK kinase 1/2 (MKK1/2), and ERK1/ ERK2) than peptide growth factors (6 -9). This activation is mediated at least in part through the G q /G 11 group of GPCRs (recently also demonstrated in vivo (10)), stimulation of membrane phospho...

The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factorstimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.The Raf-1 serine/threonine-specific protein kinase is the first component of a three-tiered protein kinase cascade that regulates many biological events such as cell growth, differentiation, and apoptosis (for reviews, see references 12, 39, and 44). Raf-1 phosphorylates and activates the dual-specificity mitogen-activated protein kinase (MAPK) kinases MEK1 and MEK2, which in turn activate the MAPKs ERK1 and ERK2. The ERKs phosphorylate and regulate the activity of transcription factors, cytoskeletal proteins, metabolic enzymes, and other protein kinases to modulate cellular responses to extracellular signals. Raf-1 regulation is highly complex. It is cytosolic in unstimulated cells, but following activation of the small G-protein Ras, it translocates to the plasma membrane, where activation takes place (for reviews, see references 23, 38, and 41). Interaction with Ras alone is not sufficient to activate Raf-1 and other membrane-localized events such as oligomerization, interaction with other proteins, and interactions with lipids all appear to play a role.Phosphorylation also plays a key role in Raf-1 activation, and both positive and negative regulatory sites have been mapped (23,38,41). Two sites whose phosphorylation has been shown to be necessary for activation are the serine located at position 338 (S338) and the tyrosine located at position 341 (Y341) (3,15,18,36,40,42). These amino acids are located 10 to 15 amino acids N terminal to the glycine-rich loop of the ATP-binding domain...

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