Ras oncogene at 85D


Table of contents

Ras interaction with RAF (part 2/2)

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. 14-3-3 is shown to bind to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol (Roy, 1998).

A model is proposed that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation. The Ras interaction with Raf's Ras binding domain (RBD) brings the Raf-14-3-3 complex to the membrane and sets in train subsequent activation events: Raf cystine rich doman (CRD)-Ras interactions then act in concert with CRD-phosphatidylserine interactions and lead to (1) partial uncovering and activation of the Raf kinase domain, (2) displacement of 14-3-3 from the Raf N terminus, and (3) more favorable presentation of Raf Y340 and Y341 for phosphorylation. At some early point in the activation process, 14-3-3 is also displaced from the Raf-1 C terminus; displacement of 14-3-3 allows for dephosphorylation of S259 and S621. Successful completion of all these events is required for full Raf-1 activation. A continuing interaction between activated Raf-1 and 14-3-3 is not required to maintain the activity of Raf-1 at the plasma membrane, because such an interaction cannot be demonstrated in vivo. Following rephosphorylation of S621 and/or S259, 14-3-3 rebinds to inactive Raf-1 and sequesters it to the cytosol. This model explains why 14-3-3 functions as a negative regulator in some assays (because it must be displaced from Raf-1 for activation and may be involved in removing Raf-1 from the plasma membrane) but appears to be essential for Ras-to-Raf-1 signaling in genetic assays (because it is permissive for Ras-dependent membrane recruitment and activation) (Roy, 1998).

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. However, the exact mechanism of their action is still largely unknown. A requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation has been demonstrated in this study. Expression of dominant negative forms of 14-3-3 results in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. However, phosphorylation of Raf-1 is insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogates kinase activity, whereas addition of 14-3-3 restores it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions (Thorson, 1998).

cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to three-fold increase in overall phosphorylation of Raf on serine/threonine residues; treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity has been uncertain. Active Raf is shown to be almost completely deactivated in vitro as a consequence of the displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by the addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. It is proposed that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo (Tzivion, 1998).

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation. p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338. The origin of the signal for Pak-mediated Raf-1 activation has been investigated by examining the roles of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acts synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding are also readily activated by RasV12. This indirect activation of Raf-1 by Ras is blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85delta, a mutant of the PI 3-kinase regulatory subunit, inhibits the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also block both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1 (Sun, 2000).

Although Raf-1 is a critical Ras effector target, the issue of how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, the identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) is unexpected and suggests a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. Mutations of Ras that impair Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminish binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, it is suggested that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131) (Drugan, 1995).

The direct physical association between Ras and the Raf-1 kinase promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras. To clarify the role of the Raf cysteine-rich domain in Ras-Raf binding, the ability of two distinct Raf fragments to interact with Ras were compared using both in vitro Ras binding and in vivo Ras inhibition assays. Both Raf sequences 2-140 and 139-186 (designated Raf-Cys) show preferential binding to active, GTP-bound Ras in vitro. Raf-Cys antagonizes oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements and focus-forming activity in NIH 3T3 cells and insulin-induced germinal vesicle breakdown in Xenopus laevis oocytes in vivo. This inhibitory activity suggests that Raf-Cys can interact with Ras in vivo. Taken together, these results suggest that Ras interaction with two distinct domains of Raf-1 may be important in Ras-mediated activation of Raf kinase activity (Brtva, 1995).

Raf-1 interacts directly with the second messenger ceramide. TNF-mediated cellular activation encompasses the rapid activation of phospholipases, such as PC-phospholipase C, phospholipase A2, and spingomyelinases, the latter producing the novel second messenger ceramide. Several intracellular targets of ceramide have been described; their roles in TNF signaling are not yet fully understood. Protein kinase C zeta is a ceramide-responsive kinase that can function as a molecular switch between pleiotropic signals of TNF, and is an important negative regulator of apoptotic signals induced by UV irradiation. This study also describes a ceramide-activated phosphatase.

TNF induces a ceramide-activated protein kinase (CAPK) located at the plasma membrane, which directly phosphorylates and activates Raf-1 in intact myelomonocytic cells. It is currently unknown whether Raf-1 activation is a general consequence of TNF stimulation; also unknown is its specific role in TNF signal pathways. Raf-1 is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. Mechanisms of Raf-1 regulation by TNF and its messenger ceramide have been investigated in cell-free assays and in insect and mammalian cell lines. In vitro, ceramide specifically binds to the purified catalytic domain of Raf-1 and enhances association with activated Ras proteins, but does not affect the kinase activity of Raf-1. Cell-permeable ceramides induce a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, is followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase is observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfer with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulate v-Src-induced Raf-1 kinase activity. TNF also induces the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action (Müller, 1998).

To dissect the mechanism of Raf activation, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type, (His)6-, and FLAG-Raf-1 are activated in a Ras- and ATP-dependent manner by transformed membranes; not activated however, are Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS). Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) are activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induces de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, is not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, is stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhances the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. These findings suggest a model for activation of Raf-1, wherein (1) Raf-1 associates with Ras-GTP, (2) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (3) Raf-1 activity is further increased by a membrane cofactor (Dent, 1995).

The structure of the Ras-binding domain of human c-Raf-1 (residues 55-132) has been determined in solution by nuclear magnetic resonance (NMR) spectroscopy. The fold of Raf55-132 consists of a five-stranded beta-sheet, a 12-residue alpha-helix, and an additional one-turn helix. It is similar to those of ubiquitin and the IgG-binding domain of protein G, although the three proteins share very little sequence identity. The Ras-binding site is contained within a spatially contiguous patch comprised of the N-terminal beta-hairpin and the C-terminal end of the alpha-helix (Emerson, 1995).

Agents that increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), it has been shown that cGMP analogs inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. The cGMP analogs inhibit epidermal growth factor (Egf)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by Egf was not impaired in G-kinase-expressing cells treated with cGMP analogs. Activation of G-kinase inhibits c-Raf kinase activation and G-kinase phosphorylates c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 is insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protects cells from inhibition of Egf-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase is not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induces MAP kinase phosphatase 1 expression, but this occurs later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (1) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (2) by inducing MAP kinase phosphatase 1 expression (Suhasini, 1998).

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 has been thought to be Ras independent. The extracellular signal-regulated kinases (ERKs) are mitogen-activated protein kinases (MAPKs) that are activated by PKC. ERKs appear to mediate the effects of PKC on differentiation, secretion, proliferation, and hypertrophy. A study was undertaken to explore the role of Ras in transducing signals from PKC to the ERKs. Stimulation of PKC in COS cells leads to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras block activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC is not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases (Marais, 1998).

The pathway involving the signaling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the catalytic domain of Raf-1 regulates Raf-1's activation in response to Ras, Src and epidermal growth factor. The p21-activated protein kinase Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42. These results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. Since guanine-nucleotide-exchange activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism might exist through which one Ras effector pathway can be influenced by another (King, 1998).

Inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and Raf is the critical target of PI 3-kinase regulation. To investigate how PI 3-kinase regulates Raf, sites on Raf1 required for regulation by PI 3-kinase were examined and the mechanisms involved in this regulation were explored. Serine 338 (Ser338), which 1s critical for fibronectin stimulation of Raf1, is phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) is independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, is also dependent on PI 3-kinase activity, and expression of a kinase-inactive Pak-1 mutant blocks phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins (Chaudhary, 2000).

The Raf family of serine/threonine protein kinases couples growth factor receptor stimulation to mitogen activated protein kinase activation, but the regulation of members in this family remains poorly understood. Using phospho-specific antisera, it has been shown 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 that prevent phosphorylation at either site block Raf-1 activation: 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 activity, 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 (Mason, 1999).

Cyclic adenosine monophosphate (cAMP) produces tissue-specific effects involving growth, differentiation, and gene expression. cAMP can activate the transcription factor Elk-1 and induce neuronal differentiation of PC12 cells via its activation of the MAP kinase cascade. These cell type-specific actions of cAMP require the expression of the serine/threonine kinase B-Raf and activation of the small G protein Rap1. Rap1, activated by mutation or by the cAMP-dependent protein kinase PKA, is a selective activator of B-Raf and an inhibitor of Raf-1. In PC12 cells, Rap1 and B-Raf are localized to the cell membrane and cytosol, respectively. 8-CPT stimulates the association of B-Raf with Rap1 within membranes. This action is specific for both cAMP and Rap1; no association of B-Raf with Rap1 as detected within membranes following treatment with EGF or in untreated cells, nor is B-Raf detected in immunoprecipitates using Ras antibody Y13-238. The dependence on GTP of this interaction was examined in COS-7 cells transfected with B-Raf and histidine-tagged Rap1b (His-Rap) or His-RapV12. B-Raf and its kinase activity are detected in eluates. The small amount of B-Raf associating with His-Rap1 is increased in cells cotransfected with PKA. The highest level of B-Raf is detected in cells cotransfected with His-RapV12. Only eluates from B-Raf-transfected cells contain B-Raf activity, as measured by immune complex assay using B-Raf antisera. B-Raf activity associated with His-Rap1 is greatly stimulated by PKA, to a level similar to that associated with His-RapV12. The expression of equal amounts of His-Rap was confirmed by immunoblotting with Rap1 antisera. These data suggest that the association of activated B-Raf protein with Rap1 is increased upon GTP loading, stimulated by PKA or by a V12 mutation. Therefore, in B-Raf-expressing cells, the activation of Rap1 provides a mechanism for tissue-specific regulation of cell growth and differentiation via MAP kinase (Vossler, 1998).

The oncogenes RAS and RAF were first identified as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells was examined. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) leads to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, the induction of the cyclin-dependent kinase (CDK) inhibitors p21(Cip1) and p16(Ink4a) occurs. Ablation of p53 and p21(Cip1) expression by use of the E6 oncoprotein of HPV16 demonstrates that expression of these proteins is not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence are elicited in IMR-90 cells by the ectopic expression of p16(Ink4a) alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevents Raf from inducing p16(Ink4a) and also prevents Raf-induced senescence. It is concluded that the kinase cascade initiated by Raf can regulate the expression of p16(Ink4a) and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active (Zhu 1998).

Several protein kinases [e.g. pp60(src), v-Raf] exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, the binding of p50(cdc37) to hsp90 and to Raf was examined. p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and intact TPR proteins compete for p50(cdc37) binding to hsp90, but a protein fragment containing a TPR domain does not compete. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient for binding. It is proposed that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols (Silverstein, 1998).

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. Cdc37 is a protein kinase targeting subunit of heat shock protein 90. In this study, the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling was studied biochemically. Coexpression of wild-type p50(cdc37) with Raf-1 results in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiates v-Src-mediated Raf-1 activation. p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant that is unable to recruit Hsp90 into the Raf-1 complex inhibits Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevents both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells is also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular (Grammatikakis. 1999).

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogate the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells (Ramana, 2000).

Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Activated and inhibitory mutants of Trk effectors were transfected into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling the contributions of individual signaling mediators to be observed. While Ras is both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induce distinct morphologies. Activated Raf-1 causes axon lengthening comparable to NGF, while active Akt increases axon caliber and branching. These results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons (Markus, 2002).

RAF interaction with downstream effectors

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1, indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. However, MEK1 can synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism that allows fast signal amplification and could prolong activation of Raf-1 (Zimmermann, 1997).

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. B-Raf has been shown in this study to be activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocks the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. These results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor [Son of sevenless (SOS)] nor the cAMP-responsive Rap-1 exchange factor (Epac), participates in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP (Busca, 2000).

The Raf-1 kinase is regulated by phosphorylation, and Ser259 has been identified as an inhibitory phosphorylation site. The dephosphorylation of Ser259 is an essential part of the Raf-1 activation process. The fraction of Raf-1 that is phosphorylated on Ser259 is refractory to mitogenic stimulation. Mutating Ser259 elevates kinase activity because of enhanced binding to Ras and constitutive membrane recruitment. This facilitates the phosphorylation of an activating site, Ser338. The mutation of Ser259 also increases the functional coupling to MEK, augmenting the efficiency of MEK activation. These results suggest that Ser259 regulates the coupling of Raf-1 to upstream activators as well as to its downstream substrate MEK, thus determining the pool of Raf-1 that is competent for signaling. These results also suggest a new model for Raf-1 activation where the release of repression through Ser259 dephosphorylation is the pivotal step (Dhillon, 2002).

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism (Wan, 2004).

To define the role of the Raf serine/threonine kinases in nervous system development, B-Raf and C-Raf, two of the three known mammalian Raf homologs, were conditionally targeted using a mouse line expressing Cre recombinase driven by a nestin promoter. Targeting of B-Raf, but not C-Raf, markedly attenuated baseline phosphorylation of Erk in neural tissues and led to growth retardation. Conditional elimination of B-Raf in dorsal root ganglion (DRG) neurons did not interfere with survival, but instead caused marked eduction in expression of the glial cell line-derived neurotrophic factor receptor Ret at postnatal stages, associated with a profound reduction in levels of transcription factor CBF-β. Elimination of both alleles of Braf, which encodes B-Raf, and one allele of Raf1, which encodes C-Raf, affected DRG neuron maturation as well as proprioceptive axon projection toward the ventral horn in the spinal cord. Finally, conditional elimination of all Braf and Raf1 alleles strongly reduced neurotrophin-dependent axon growth in vitro as well as cutaneous axon terminal arborization in vivo. It is concluded that Raf function is crucial for several aspects of DRG neuron development, including differentiation and axon growth (Zhong, 2007).

Evolutionary homologs: Table of contents

Ras85D: Biological Overview | Regulation | Protein Interactions | Effects of Mutation | Ras as Oncogene | References

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