Ras oncogene at 85D


Table of contents

Ras interaction with RAF (part 1/2)

In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes, including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, a genetic screen was conducted for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling (Rocheleau, 2002).

The Rap proteins are members of the Ras superfamily of low molecular weight monomeric GTP-binding proteins. Rap1A was identified originally based on its homology with the Drosophilia Ras-related gene (Dras3) and independently from its ability to induce a flat phenotype in v-Ki-Ras-transformed fibroblasts. However, the physiological role of Rap1 in regulating intracellular signaling events appears to be enigmatic and can be either positive or negative depending on the particular cell context. Nevertheless, in multiple cell types, several studies have demonstrated that Rap1 can function as a suppressor of Ras-mediated downstream signaling. For example, both the Ras- and T-antigen-dependent transformation can be reversed by Rap1 expression. Several studies have demonstrated that both Rap and Ras can bind the same regulators (p120RasGap) and effectors (RalGDS, Raf1 and B-Raf) in a GTP-dependent manner. Rap1 activation (formation of the GTP-bound state) occurs through interaction with the Rap1-specific 140 kDa guanylnucleotide exchange factor C3G. In addition, increased expression of C3G can also suppress the v-Ki-Ras-transformed phenotype in a manner similar to increased expression of Rap1. Crk represents another family of small adaptor proteins that are composed of SH2 and SH3 domains. In particular, CrkII is composed of a single N-terminal SH2 domain and two tandem SH3 domains. Under basal conditions, C3G is associated with CrkII through interactions of the central SH3 domain of CrkII with several proline-rich regions in C3G. Similar to the function of Grb2, the N-terminal SH2 domain can then direct the association of CrkII with several tyrosine-phosphorylated proteins, including the epidermal growth factor (EGF) and nerve growth factor (NGF) receptors, IRS1/2, Cbl, paxillin and pp130Cas. Whether targeting of the CrkII-C3G complex to these tyrosine-phosphorylated proteins is responsible for the positive and/or negative signaling properties of Rap1 remains to be determined. In any case, since Rap1 can function as a suppressor of Ras downstream signaling, there must be a cellular mechanism that rapidly inhibits Rap1 function to allow for Ras activation of downstream signals. Recently, several studies have suggested that the activation of Ras can be limited by a negative feedback loop which results in the serine/threonine phosphorylation of SOS and dissociation of the Grb2-SOS complex. It was therefore hypothesized that a more proximal signaling pathway leading to the dissociation of CrkII from C3G may also exist to inactivate Rap1 prior to the activation of the Ras/Raf/MEK/ERK pathway. Insulin stimulation results in a rapid dissociation of the CrkII-C3G complex, inactivation of Rap1 and decreased association of Rap1 with the Raf1 serine kinase. In addition, the uncoupling of the CrkII-C3G complex occurs in a Ras-independent manner but is directly dependent on the phosphorylation of CrkII at Tyr221 (Okada, 1998 and references).

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor results in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 is associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there is an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, a rapid insulin-stimulated dissociation of the CrkII-C3G complex is observed that occurs in a Ras-independent manner. The dissociation of the CrkII-C3G has been recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII is also dose dependent and demonstrates that insulin reduces the affinity of CrkII for C3G without any effect on CrkII protein levels. The reduction in CrkII binding affinity is reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade. In this model, activation of the insulin receptor induces the tyrosine phosphorylation of Shc and thereby generates the formation of the Shc-Grb2-SOS ternary complex necessary for Ras activation. In parallel, the insulin receptor tyrosine phosphorylates CrkII, inducing the dissociation of the CrkII-C3G complex and the inactivation of Rap1. Inactivation of Rap1 allows Raf1 to associate with Ras, and its subsequent activation results in the stimulation of the ERK kinase module (Okada, 1998).

Ras recruits Raf to the plasma membrane for activation by a combination of tyrosine phosphorylation and other as yet undefined mechanism(s). The Raf zinc finger is not required for plasma membrane recruitment of Raf by Ras but is essential for full activation of Raf at the plasma membrane. Membrane targeting cannot compensate for the absence of the zinc finger. One facet of the zinc finger activation defect is revealed using a constitutively activated Raf mutant. Targeting Raf Y340D,Y341D to the plasma membrane increases activity incrementally, but full activation requires coexpression with activated Ras. This sensitivity to regulation by Ras at the plasma membrane is abrogated by mutations in the Raf zinc finger but is unaffected by mutation of the minimal Ras binding domain. These data show for the first time that Ras has two separate roles in Raf activation: recruitment of Raf to the plasma membrane through an interaction with the minimal Ras binding domain and activation of membrane-localized Raf via a mechanism that requires the Raf zinc finger (Roy, 1997).

The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. A ras variant, ras12V,37G, has been identified that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V,37G. Cell fractionation localizes both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhances the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment (Mineo, 1997).

The GTP-bound form of Ras is capable of interacting directly with RasGAP, neurofibromin, and the Raf kinases. Although believed to be endowed with some signaling capacity, RasGAP and neurofibromin act primarily to negatively regulate Ras. The Raf kinases are considered the primary targets of Ras signaling. Activation of the Raf kinases is the first step in a cascade of multiple protein kinases, including Mek, Erk1, and Erk2. Using mutagenesis, peptide epitope scanning, and in vitro reconstitution of protein interactions, specific sites of association between the Ras-GTP and c-Raf-1 proteins have been identified. The interaction between these contact points is essential for the plasma membrane localization of Raf, which ultimately leads to kinase activation. The formation of this protein complex is negatively regulated by protein kinase A (PKA) through phosphorylation of the c-Raf-1 N-terminus. Phosphorylation of c-Raf-1 serine 43 is believed to cause an N-terminal cap structure to cover the Ras docking site (Marshall, 1995a).

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. A consensus Ras binding sequence shared among a subset of Ras effectors has now been identified. Peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, the demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents (Clark, 1996).

The activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupts interaction with Ras and prevents Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1. Further analysis of this mutant demonstrates that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. These findings have also shown that phosphorylation events play a role in regulating Raf-1. Sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1 have been identified. Some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (Morrison, 1995b).

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. Autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, it is proposed that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation (Cutler, 1998).

Raf can interact with a number of proteins, including Ras, Src and 14-3-3. The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. Raf can be activated by Ras and Src and the activation of Ras by Src requires tyrosine phosphorylation of Raf. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. A system has been established to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras is the only component in these membranes required for activation, since purified recombinant farnesylated K-Ras.GTP (but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP) is able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurs under conditions in which phosphorylation is prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras is substantially inhibited. The activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1; in vitro activation of c-Raf-1 by Src requires the presence of ATP. Therefore it is proposed that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms (Stokoe, 1997).

The binding of Raf-1 to Ras results in translocation of the kinase to the plasma membrane and facilitates its activation by an unknown mechanism. While an N-terminal fragment consisting of amino acid residues 2 through 130 of Raf-1 is able to bind Ras, residues 131-147 were found to be critically important for conferring high affinity binding to Ras. Surprisingly, a second domain between residues 52-64 is an essential element for Raf-Ras interaction, although it does not appear to form an independent binding site for Ras (Ghosh, 1994).

The signaling pathway comprising Raf, MEK (mitogen-activated protein kinase, or ERK kinase), and ERK (extracellular signal-regulated kinase) lies downstream of the small guanine nucleotide binding protein Ras and mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, differentiation, and senescence, depending on the duration and strength of the external stimulus and on cell type. Another pathway that lies downstream of Ras includes phosphatidylinositol (PI) 3-kinase and Akt (or protein kinase B) and also regulates these cellular responses, acting either synergistically with or in opposition to the Raf pathway. Coordination of the two pathways in a single cellular response may depend on cell type or the stage of differentiation. Akt interacts with Raf and phosphorylates this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibits activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt. These results demonstrate that Akt antagonizes Raf activity by direct phosphorylation of Ser259. This modification creates a binding site for 14-3-3 protein, a negative regulator of Raf. Similarly, phosphorylation of BAD or the forkhead transcription factor FKHRL1 by Akt also promotes binding of 14-3-3 protein. In all three instances, phosphorylation by Akt inactivates the function of its substrate. Cross talk between the Raf-MEK-ERK and the PI 3-kinase-Akt pathways, mediated by direct interaction of Akt with and its phosphorylation of Raf, may switch the biological response from growth arrest to proliferation, as shown for MCF-7 cells, and may also modulate senescence or differentiation as shown for myoblast differentiation, depending on the cellular system (Zimmermann, 1999).

Cellular proliferation and differentiation of cells in response to extracellular signals, are both controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Described here are two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signaling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibits growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection reveals that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associates with Ras but does not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibits the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signaling (Wakioka, 2001).

Spred-1 is tyrosine phosphorylated in response to stem cell factor (SCF), platelet-derived growth factor (PDGF) and EGF, and efficient phosphorylation of Spred-1 requires the KBD region. Using immunofluorescence microscopy, endogenous Spred-2 was found to be localized to the plasma membrane. Membrane localization of Spred was confirmed by exogenously expressed Spred fused to enhanced green fluorescent protein (EGFP). The C-terminal SPR domain is essential for plasma membrane localization, since a deletion mutant lacking SPR domain (GFP-C) is localized in the cytoplasm (Wakioka, 2001).

The molecular mechanism by which Spred suppresses the Ras-MAP kinase pathway was investigated. Since one of the nuclear targets of MAP kinase is Elk-1, a transcription factor of the Ets family, EGF-induced activation of MAP kinase can be monitored by measuring the rate of Elk-1-dependent transcription. In 293 cells, forced expression of Spred-1 or -2 dose-dependently suppresses EGF-dependent Elk-1 activation. The negative effect of Spred-1 and -2 is comparable to that of Ras GTPase activating protein (rasGAP) and N17-Ras, and Spred-1 and -2 are more potent inhibitors than is murine Sprouty-4 or the Raf kinase inhibitor protein 1. Both EVH-1 and SPR domains are necessary for the suppression of Elk-1 activation. Replacement of the EVH-1 domain of Spred-1 with that of Wiskott-Aldrich syndrome protein (WASP) abolishes the inhibitory activity of Spred-1, suggesting that the EVH-1 domain of Spred-1 may interact with a specific target required for suppression of the MAP kinase pathway. In contrast, the KBD region is not essential but required for efficient suppression of the MAP kinase pathway (Wakioka, 2001).

Ras directly interacts with and activates Raf. Raf phosphorylates and activates MEK, which in turn phosphorylates and activates MAP kinases. Spred inhibits activation of Elk-1 induced by active Ras (V12-Ras), but not that induced by active MEK or active Raf (N-Raf). Therefore, the target of Spred is probably located between Ras and Raf. To test this hypothesis, the effect of Spred-1 on EGF-induced Ras and Raf activation was examined. Interestingly, Spred sustains Ras activation, whereas it inhibits Raf activation, as measured by autophosphorylation and by in vitro kinase assay. Furthermore, like rasGAP, Spred inhibits the phosphorylation of Raf on Ser 338, which is required for Raf activation, but not on Ser 259, which is not. Thus, Spred inhibits MAP kinase activity by suppressing Raf activation (Wakioka, 2001).

continued: Ras interaction with RAF part 2/2

Evolutionary homologs: Table of contents

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

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