An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ.

The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures. Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF). Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation. PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane. The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP. In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation. Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras.     

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.     

Ras signalling on the endoplasmic reticulum and the Golgi

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.     

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.     

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.     

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.     

A permissive function of phosphoinositide 3-kinase in Ras activation mediated by inhibition of GTPase-activating proteins

The activation status of the guanosine triphosphate (GTP)-binding protein Ras is dictated by the relative intensities of two opposing reactions: the formation of active Ras-GTP complexes, promoted by guanine-nucleotide exchange factors (GEFs), and their conversion to inactive Ras-GDP as a result of the deactivating action of GTPase-activating proteins (GAPs). The relevance of phosphoinositide 3-kinase (PI 3-kinase) to these processes is still unclear. We have investigated the regulation of Ras activation by PI 3-kinase in the myelomonocytic U937 cell line. These cells exhibited basal levels of Ras-GTP, which were suppressed by two PI 3-kinase inhibitors and a dominant-negative PI 3-kinase. In addition, PI 3-kinase inhibition aborted Ras activation by all stimuli tested, including foetal calf serum (FCS) and phorbol 12-myristate 13-acetate (TPA). Significantly, TPA does not activate PI 3-kinase in U937 cells, indicating that PI 3-kinase has a permissive rather than an intermediary role in Ras activation. Investigation of the mechanism of PI 3-kinase action revealed that inhibition of PI 3-kinase does not affect nucleotide exchange on Ras but abrogates Ras-GTP accumulation through an increase in GAP activity. These findings establish blockage of GAP action as the mechanism underlying a permissive function of PI 3-kinase in Ras activation.     

Role of Ras in ethanol modulation of angiotensin II activated p42/p44 MAP kinase in rat hepatocytes

Angiotensin II plays a role in both liver cell proliferation and liver injury but the effects of ethanol on angiotensin II signaling in liver are not clearly understood. We have investigated the role of Ras in ethanol modulation of p42/p44 mitogen-activated protein kinase (MAPK) stimulated by angiotensin II (Ang II) in primary cultures of rat hepatocytes. Hepatocytes were incubated with ethanol (100 mM) for 24 h, then stimulated with Ang II (100 nM). The level of p42/p44 MAPK phosphorylation was measured by Western blot analysis and Ras activation was assessed by specific binding of Ras-GTP (activated form) to a GST-RBD fusion protein containing Ras-binding domain (RBD) of Raf-1. Ethanol potentiated p42/p44 MAPK activation by Ang II, whereas ethanol alone did not significantly affect phosphorylation of p42/p44 MAPK. Ang II increased Ras activity by about 2 fold. Ethanol exposure increased Ang II stimulated Ras activity by an additional about 2 fold. Ethanol alone elicited a small increase in basal Ras activity. Pretreatment with manumycin A (10 microM), a Ras farnesylation inhibitor, partially blocked both Ang II-activated and ethanol-potentiated MAPK activities. These data provided the first evidence that ethanol potentiation of Ang II stimulated p42/p44 MAPK is mediated, in part, by Ras in hepatocytes.     

PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.     

Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes

Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-kappaB- and Erk-dependent pathways. In addition, NT-stimulated NF-kappaB activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-kappaB, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-kappaB and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.     

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, H?hn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.     

Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.     

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.     

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.     

Characterization of a Peptide that Specifically Blocks the Ras Binding Domain of p75

The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid_1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.     

Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.     

Input from Ras is required for maximal PI(3)K signalling in Drosophila

Class I phosphoinositide 3-kinases (PI(3)Ks) are activated through associated adaptor molecules in response to G protein-coupled and tyrosine kinase receptor signalling. They contain Ras-binding domains (RBDs) and can also be activated through direct association with active GTP-bound Ras. The ability of Ras to activate PI(3)K has been established in vitro and by overexpression analysis, but its relevance for normal PI(3)K function in vivo is unknown. The Drosophila class I PI(3)K, Dp110, is activated by nutrient-responsive insulin signalling and modulates growth, oogenesis and metabolism. To investigate the importance of Ras-mediated PI(3)K activation for normal PI(3)K function, we replaced Dp110 with Dp110(RBD), which is unable to bind to Ras but otherwise biochemically normal. We found that Ras-mediated Dp110 regulation is dispensable for viability. However, egg production, which requires large amounts of growth, is dramatically lowered in Dp110(RBD) flies. Furthermore, insulin cannot maximally activate PI(3)K signalling in Dp110(RBD) imaginal discs and Dp110(RBD) flies are small. Thus, Dp110 integrates inputs from its phosphotyrosine-binding adaptor and Ras to achieve maximal PI(3)K signalling in specific biological situations.     

Increased cell growth due to a new lipase-GEF (Phospholipase D2) fastly acting on Ras

We report the novel finding that Phospholipase D2 (PLD2), through its PX and PH domains, binds specifically to Ras and catalyzes the GDP/GTP exchange (i.e., is a GEF), with potency comparable to Ras-GRF-1, a known Ras-GEF. Cells overexpressing PLD2-GEF inactive mutants (F129Y and R172C/L173A) fail to stimulate cell proliferation compared to the wild type-expressing cells. The GEF effect on Ras follows a faster kinetics than other GTPase substrates (such as Rac2 or Rac1) and is a better substrate, too. The GEF action is due to PLD2 (protein) itself, independent of the lipase product PA. PA can still have a fine-tuning regulatory effect on Ras-GTP depending upon its cellular concentration. Rapidly growing human breast cancer cells MDA-MB 231 (but not the slow growing MCF7 counterpart) have high levels of endogenous PLD2-GEF which correlates with high Ras activation. The PLD2-“GEF” activity is even higher than the classical “lipase” activity and is abrogated with GEF single point mutants, particularly F129Y, and concomitantly with a slow rate of cell growth. This can be crucial to cancer biology in that not only Ras mutations explain abnormal growth, but the existence of a new GEF for Ras: a GEF molecule that happens to be a phospholipase.     

Calcium and calmodulin are essential for Ras-GRF1-mediated activation of the Ras pathway by lysophosphatidic acid

The exchange factor Ras-GRF1, also called CDC25Mm, couples calcium signaling and G-protein-coupled receptors to Ras and downstream effectors. Here we show that when expressed in different cell lines Ras-GRF1 strongly enhances the level of active Ras (Ras-GTP) and the activity of mitogen-activated protein kinases (MAPK). Moreover, in NIH 3T3 fibroblasts it potentiates the effect of lysophosphatidic acid (LPA) on Ras protein and MAPK activity. Calmodulin and cytosolic free calcium are essential for Ras and MAPK activation induced by LPA and mediated by Ras-GRF1, as shown by the finding that BAPTA-AM, an intracellular calcium chelator, and calmodulin inhibitors completely abolished this effect. This report demonstrates the relevance of calmodulin in addition to calcium for the response of Ras-GRF1 to LPA.     

Nonconventional trafficking of Ras associated with Ras signal organization

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.