Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.     

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.     

Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.The Ras, Raf, MEK, and extracellular signal-regulated kinase (ERK) proteins are core components of one of the major signaling cascades regulating normal cell proliferation—the Ras pathway. Not surprising, deregulation of Ras pathway signaling is a major contributor to human cancer and has recently been linked with several developmental disorders, such as Noonan''s, LEOPARD, and cardiofaciocutaneous (CFC) syndromes (28). Given its importance to both normal and disease states, much effort has been directed toward elucidating the mechanisms that modulate Ras pathway signaling. Of all the pathway components, regulation of the Raf proteins has proved to be the most complex, involving inter- and intramolecular interactions, a change in subcellular localization, and phosphorylation and dephosphorylation events (6, 32).In mammalian cells, there are three Raf family members: A-Raf, B-Raf, and C-Raf (12). In their inactive state, all Raf proteins are found in the cytosol, with the N-terminal regulatory domain acting as an autoinhibitor of the C-terminal kinase domain (4, 5, 13). 14-3-3 dimers bind to phosphorylation sites present in both the N- and C-terminal regions and stabilize the autoinhibited state (22). To activate the Raf proteins, autoinhibition mediated by the N terminus must be relieved and the kinase domain must adopt the active catalytic conformation (6, 31, 32). Under normal signaling conditions, Ras activation helps mediate these events by recruiting the Raf proteins to the plasma membrane, which induces the release of 14-3-3 from the N-terminal binding site and facilitates phosphorylation of the Raf kinase domain (19). For the C-Raf and A-Raf proteins, phosphorylation occurs in two regions of the kinase domain, the negative-charge regulatory region (N-region) and the activation segment (4). In contrast, the N-region of B-Raf exhibits a constitutive negative charge due to increased basal phosphorylation of an activating serine site and the presence of two aspartic acid residues (18); thus, only phosphorylation of the activation segment is required. Phosphorylation of the activation segment serves both to destabilize the “inactive” catalytic conformation maintained by hydrophobic interactions between the glycine-rich loop and the activation segment and to stabilize the “active” catalytic conformation, whereas the negative charge of the N-region helps to disrupt the autoinhibitory activity of the N-terminal domain (5, 30, 31).Because the N-region of B-Raf exhibits a constitutive negative charge, B-Raf possesses higher basal kinase activity than other family members and is more susceptible to mutational activation (9, 11, 17). In particular, B-Raf is a major contributor to human cancer: somatic mutations in the B-Raf gene are detected in ∼50% of malignant melanomas and many colorectal, ovarian, and papillary thyroid carcinomas (7). Of the oncogenic mutations identified in B-Raf, the vast majority cluster to the two regions of the kinase domain responsible for maintaining the inactive catalytic conformation—the glycine-rich loop and the activation segment (31). Based on enzymatic activity, the oncogenic B-Raf proteins have been divided into three groups: those with high activity (130- to 700-fold more active than wild-type [WT] B-Raf), those with intermediate activity (64- to 1.3-fold more active), and surprisingly, those with impaired catalytic activity (0.8 to 0.3 of WT B-Raf activity) (31). Further analysis has revealed that all oncogenic B-Raf proteins heterodimerize constitutively with C-Raf and activate C-Raf in a Ras-independent manner that requires an intact C-Raf activation segment as well as the binding of 14-3-3 to the C-terminal pS621 binding site on C-Raf (11). Importantly, for the oncogenic B-Raf proteins with impaired kinase activity, the binding and activation of C-Raf are required for ERK activation in vivo (31). Interestingly, heterodimerization of B-Raf and C-Raf also occurs under normal signaling conditions; however, in this case, heterodimerization is Ras dependent and occurs at the plasma membrane following mitogen stimulation (11, 27).Once activated, either by upstream signaling or by mutational events, all Raf proteins are capable of initiating the phosphorylation cascade that results in the sequential activation of MEK and ERK. ERK then phosphorylates targets in both the cytoplasm and the nucleus that are required for cell proliferation. Strikingly, the Raf proteins themselves are also substrates of activated ERK. In regard to C-Raf, ERK-dependent feedback phosphorylation has been shown to instigate a regulatory cycle whereby phosphorylation of the feedback sites down-modulates C-Raf signaling, after which the hyperphosphorylated C-Raf protein is dephosphorylated and returned to a signaling-competent state through dephosphorylation events involving protein phosphatase 2A (PP2A) and the Pin1 prolyl-isomerase (8). For B-Raf, two ERK-dependent feedback sites, S750 and T753, have been identified, and phosphorylation of these sites has been reported to have a negative regulatory effect (3).In this study, we have further investigated the impact of feedback phosphorylation and heterodimerization on B-Raf signaling. Here we find that both normal and oncogenic B-Raf proteins are phosphorylated on four S/TP sites (S151, T401, S750, and T753) by activated ERK. Through mutational analysis, we find that phosphorylation of B-Raf at S151 inhibits binding to activated Ras, whereas phosphorylation of each of the feedback sites contributes to the disruption of B-Raf/C-Raf heterodimers. Moreover, we find that events influencing B-Raf/C-Raf heterodimerization, such as feedback phosphorylation and 14-3-3 binding, can alter the signaling activity of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity as well as that of B-Raf and C-Raf proteins containing mutations identified in CFC and Noonan''s syndromes, respectively.     

Diacylglycerol Kinase �� Augments C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase η (DGKη) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKη1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKη serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKη-silenced HeLa cells, this study revealed that DGKη activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKη enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKη physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKη-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKη acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.The Ras/Raf/MEK³/ERK signaling pathway is critical for the transduction of the extracellular signals to the nucleus, regulating diverse physiological processes such as cell proliferation, differentiation, and survival (1, 2). The binding of extracellular ligands, such as growth factors and cytokines, to cell surface receptors activates Ras. The Raf serine/threonine kinase transmits signals from activated Ras to the downstream protein kinases, MEK1 and MEK2, subsequently leading to activation of ERK1 and ERK2.In mammals, the Raf kinase consists of three isoforms, A-Raf, B-Raf, and C-Raf (Raf-1). It is clinically known that both B-Raf and C-Raf mutations are associated with human cancers (3–5). Knock-out mouse studies demonstrated that each individual Raf isoform has distinct functions, although the three Raf isoforms have high homology in the amino acid sequence (6). The mechanisms underlying C-Raf activation are complicated and thus are not completely understood (3). In response to extracellular signals, C-Raf is initially recruited from cytosol to the plasma membrane and undergo conformational changes by binding directly to the active Ras (7). In addition, other modifications and factors are required for the sufficient activation of C-Raf. For example, dephosphorylation of Ser-259 and phosphorylation of Ser-338, Tyr-341, Thr-491, and Ser-494 are critical for the activation of C-Raf (8–11). Feedback phosphorylation of C-Raf by ERK was also reported to be important for the modulation of C-Raf activity (12, 13). C-Raf activity is regulated by the interaction with 14-3-3 protein (14). Moreover, the heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf, has been reported to play an essential role in the activation of the MEK-ERK signaling pathway (15–17). Although B-Raf and C-Raf are the central regulatory components in the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade involved in a variety of pathophysiological events, the activation mechanisms of C-Raf by B-Raf are still unclear.Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol to generate phosphatidic acid. DGK has been recently recognized as an emerging key regulator in a wide range of cell signaling systems (18–20). To date, 10 mammalian DGK isozymes have been identified. They characteristically contain two or three protein kinase C-like C1 domains and a catalytic region and are subdivided into five subtypes according to their structural features (18–20). Their structural variety and distinct expression patterns in tissues allow us to presume that each DGK isozyme has its own biological functions. Indeed, recent studies have revealed that individual DGK isozymes play distinct roles in cell functions through interactions with unique partner proteins such as protein kinase C (21, 22), Ras guanyl nucleotide-releasing protein (23, 24), phosphatidylinositol-4-phosphate 5-kinase (25), chimerins (26, 27), AP-2 (28), and PSD-95 (29).DGKη belongs to the type II DGKs containing a pleckstrin homology domain at the N terminus and the separated catalytic region (19, 30). Two alternative splicing products of DGKη have been identified as DGKη1 and -η2 (31). DGKη2 possesses a sterile α-motif (SAM) domain at the C terminus, whereas DGKη1 does not. This study demonstrated that the expression levels of DGKη1 and -η2 were regulated differently by glucocorticoid, and that they were translocated from the cytoplasm to endosomes in response to stress stimuli as osmotic shock and oxidative stress (31). However, the physiological roles of DGKη remain unknown.This study showed that siRNA-dependent knockdown of DGKη inhibits cell proliferation of the HeLa cells. In addition, DGKη is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade activated by epidermal growth factor (EGF). Intriguingly, DGKη regulates recruitment of B-Raf and C-Raf from cytosol to membranes and their heterodimerization. Moreover, this study demonstrated that DGKη activates C-Raf but not B-Raf in an EGF-dependent manner. The data show DGKη as a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.     

Interactions of Ras proteins with the plasma membrane and their roles in signaling

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.     

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.     

Ras proteins control mitochondrial biogenesis and function in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The evolutionarily conserved Ras proteins function as a point of convergence for different signaling pathways in eukaryotes and have been implicated in both aging and cancer development. In Saccharomyces cerevisiae the plasma membrane proteins Ras1 and Ras2 are sensing the nutritional status of the environments, e.g., the abundance and quality of available carbon sources. The cAMP-protein kinase A pathway is the most explored signaling pathway controlled by Ras proteins; it affects a large number of genes, some of which are important to defend the cell against oxidative stress. In addition, recent analysis has shown that the Ras system of yeast is involved in the development of mitochondria and in regulating their activity. As a sensor of environmental status and an effector of mitochondrial activity, Ras serves as a Rosetta stone of cellular energy transduction. This review summarizes the physical and functional involvement of Ras proteins and Ras-dependent signaling pathways in mitochondrial function in S. cerevisiae. Since mitochondria produce harmful reactive oxygen species as an inevitable byproduct and are partly under control of Ras, illuminating these regulatory interactions may improve our understanding of both cancer and aging.     

The role of collagen structure in mitogen stimulation of ERK,cyclin D1 expression,and G1-S progression in rat hepatocytes

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.     

Structural determinants of Ras-Raf interaction analyzed in live cells

The minimum structure of the Raf-1 serine/threonine kinase that recognizes active Ras was used to create a green fluorescent fusion protein (GFP) for monitoring Ras activation in live cells. In spite of its ability to bind activated Ras in vitro, the Ras binding domain (RBD) of Raf-1 (Raf-1[51-131]GFP) failed to detect Ras in Ras-transformed NIH 3T3 fibroblasts and required the addition of the cysteine-rich domain (CRD) (Raf-1[51-220]GFP) to show clear localization to plasma membrane ruffles. In normal NIH 3T3 cells, (Raf-1[51-220]GFP) showed minimal membrane localization that was enhanced after stimulation with platelet-derived growth factor or phorbol-12-myristate-13-acetate. Mutations within either the RBD (R89L) or CRD (C168S) disrupted the membrane localization of (Raf-1[51-220]GFP), suggesting that both domains contribute to the recruitment of the fusion protein to Ras at the plasma membrane. The abilities of the various constructs to localize to the plasma membrane closely correlated with their inhibitory effects on mitogen-activated protein kinase kinase1 and mitogen-activated protein kinase activation. Membrane localization of full-length Raf-1-GFP was less prominent than that of (Raf-1[51-220]GFP) in spite of its strong binding to RasV12 and potent activation of mitogen-activated protein kinase. These finding indicate that both RBD and CRD are necessary to recruit Raf-1 to active Ras at the plasma membrane, and that these domains are not fully exposed in the Raf-1 molecule. Visualization of activated Ras in live cells will help to better understand the dynamics of Ras activation under various physiological and pathological conditions.     

Localization of Ras signaling complex in budding yeast

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins. Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.     

Epstein-Barr virus latent membrane protein 2A mediates transformation through constitutive activation of the Ras/PI3-K/Akt Pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.     

Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras.

Two dominant inhibitory Ras mutant proteins were analyzed by microinjection. One, [Asn-17]Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras. The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP. RAST also had a mutation that blocked its localization to the plasma membrane. In NIH 3T3 cells [Asn-17]Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras. In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras. These conclusions were based on two separate types of analysis. The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras. Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein. Whereas RAST was active in soluble form. [Asn-17]Ras required membrane localization for activity. Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on [Asn-17]Ras. These results are consistent with the possibility that [Asn-17]Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells. The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.     

Coating cells with cationic silica-magnetite nanocomposites for rapid purification of integral plasma membrane proteins

This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica–magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell‐lysis‐buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10‐fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10⁷ Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN‐based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.