Mammalian Raf-1 is activated by mutations that restore Raf signaling in Drosophila.

An interaction with the Ras proto-oncogene product is a requirement for Raf-1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras-Raf interaction severely impairs the function of both mammalian (Raf-1) and Drosophila (D-Raf) Raf proteins. In D-Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras-binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf-1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras-Raf-1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf-1, allowing Raf-1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf-1, it also abolishes the ability of wild-type Raf-1 to become activated by Ras. This mutation occurs in the cysteine-rich domain (CRD) of Raf-1 and demonstrates the importance of this region for a productive Ras-Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf-1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf-1 kinase domain.     

The requirement of specific membrane domains for Raf-1 phosphorylation and activation

Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.     

Regulation and role of Raf-1/B-Raf heterodimerization

The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.     

Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.     

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.     

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.     

Raf proteins and cancer: B-Raf is identified as a mutational target

A recent report has shown that activating mutations in the BRAF gene are present in a large percentage of human malignant melanomas and in a proportion of colon cancers. The vast majority of these mutations represent a single nucleotide change of T-A at nucleotide 1796 resulting in a valine to glutamic acid change at residue 599 within the activation segment of B-Raf. This exciting new discovery is the first time that a direct association between any RAF gene and human cancer has been reported. Raf proteins are also indirectly associated with cancer as effectors of activated Ras proteins, oncogenic forms of which are present in approximately one-third of all human cancers. BRAF and RAS mutations are rarely both present in the same cancers but the cancer types with BRAF mutations are similar to those with RAS mutations. This has been taken as evidence that the inappropriate regulation of the downstream ERKs (the p42/p44 MAP kinases) is a major contributing factor in the development of these cancers. Recent studies in mice with targeted mutations of the raf genes have confirmed that B-Raf is a far stronger activator of ERKs than its better studied Raf-1 homologue, even in cell types in which the protein is barely expressed. The explanation for this lies in a number of key differences in the regulation of B-Raf and Raf-1 activity. Constitutive phosphorylation of serine 445 of B-Raf leads to this protein having a higher basal kinase activity than Raf-1. Phosphorylation of threonine 598 and serine 601 within the activation loop of B-Raf at the plasma membrane also regulates its activity. The V599E mutation is thought to mimic these phosphorylations, resulting in a protein with high activity, leading to constitutive ERK activation. B-Raf now provides a critical new target to which drugs for treating malignant melanoma can be developed and, with this in mind, it is now important to gain clear insight into the biochemical properties of this relatively little characterised protein.     

Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.     

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Regulation of Raf through phosphorylation and N terminus-C terminus interaction

Raf kinase is a key component in regulating the MAPK pathway. B-Raf has been reported as an oncogene and is mutated in 60% of human melanomas. The main focus of Raf regulation studies has been on phosphorylation, dephosphorylation, and scaffolding proteins; however, Raf also has its own auto-regulatory domain. Removal of the N-terminal regulatory domain, initially discovered in the viral Raf oncogene (v-Raf), results in a kinase domain with high basal activity independent of Ras activation. In this report, we show that activating phosphorylations are still required for activity of the truncated C-terminal kinase domain (called 22W). The interaction between the N-terminal regulatory domain and the C-terminal kinase domain is disrupted by activated Ras. Mutations in the Ras binding domain, cysteine-rich domain, or S259A do not affect the inhibition of 22W by the N-terminal domain. When phosphomimetic residues are substituted at the activating sites (DDED) in 22W, this results in a higher basal activity that is no longer inhibited by expression of the N-terminal domain, although binding to the N-terminal domain still occurs. Although the interaction between 22W/DDED and the N-terminal domain may be in a different conformation, the interaction is still disrupted by activated Ras. These data demonstrate that N-terminal domain binding to the kinase domain inhibits the activity of the kinase domain. However, this inhibition is relieved when the C-terminal kinase domain is activated by phosphorylation.     

Molecular dynamics simulations of the Ras:Raf and Rap:Raf complexes

The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.     

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.     

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

PKCtheta regulates the proliferation, survival and differentiation of T-cells. Here we show that PKCtheta interacts with Raf-1 and B-Raf kinases. Raf-1 enhanced the kinase activity of associated PKCtheta, while PKCtheta reduced the catalytic activity of associated Raf-1. In contrast, B-Raf binding did not affect PKCtheta kinase activity, and PKCtheta did not change B-Raf activity. Coexpression of mutationally activated Raf-1 in cells enhanced the phosphorylation of T538 in the PKCtheta activation loop. PKCtheta 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 PKCtheta 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 PKCtheta to interact with and phosphorylate BAD.     

B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

SptP,a Salmonella typhimurium type III-secreted protein,inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation

Salmonella has developed ways to modulate host cellular response in order to survive. Although the steps required for such modulation have been incompletely characterized, there is increasing evidence for a role for SptP, a type III secretion protein. In part, the actions of SptP are thought to be mediated through its reported inhibition of the extracellular-regulated kinase (ERK) MAP kinase pathway. In the present studies, a series of transfections were performed in which various constitutively activated components of the MAP kinase pathway were co-transfected with SptP in order to determine the mechanism by which SptP inhibits this MAP kinase activation. SptP was found to inhibit the activation of ERK stimulated by both a constitutively active form of Ras and a partially activated form of Raf-1 containing a phospho-mimetic mutation (Raf Y340D). In contrast, the activation of ERK by constitutively active forms of MAP kinase kinase (MEK) was not inhibited, suggesting that the actions of SptP were mediated by Raf-1. In order to determine how SptP might interfere with activation of Raf, we utilized a membrane-localized form of Raf. Constitutive membrane-localization of Raf (RafCAAX), resulting in partial activation, did not prevent inhibition by SptP. However, introduction of an additional, partially activating (Y340D) phospho-mimetic mutation, to RafCAAX, dramatically reduced the ability of SptP to inhibit Raf action. Comparison of SptP mutants, lacking either GTPase-activating protein (GAP) activity or tyrosine phosphatase activity, further suggested that SptP inhibits both the membrane localization and subsequent phosphorylation required for activation of Raf. Both tyrosine phosphatase activity and GAP activity were responsible for SptP inhibition of Raf(Y340D)-induced ERK activation, but only GAP activity was responsible for inhibition of the membrane localized forms of Raf-1. To assess the biological significance of SptP, we examined tumour necrosis factor (TNF)-alpha induction following Salmonella infection. SptP gene deletion enhanced the capacity of Salmonella to induce TNF-alpha secretion following infection of J774A.1 macrophage cells.     

TC21 causes transformation by Raf-independent signaling pathways.

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.