High density lipoprotein-induced signaling of the MAPK pathway involves scavenger receptor type BI-mediated activation of Ras

High density lipoprotein (HDL) stimulates multiple signaling pathways. HDL-induced activation of the mitogen-activated protein kinase (MAPK) pathway can be mediated by protein kinase C (PKC) and/or pertussis toxin-sensitive G-proteins. Although HDL-induced activation of MAPK involves Raf-1, Mek, and Erk1/2, the upstream contribution of p21(ras) (Ras) on the activation of Raf-1 and MAPK remains elusive. Here we examine the effect of HDL on Ras activity and demonstrate that HDL induces PKC-independent activation of Ras that is completely blocked by pertussis toxin, thus implicating heterotrimeric G-proteins. In addition, the HDL-induced activation of Ras is inhibited by a neutralizing antibody against scavenger receptor type BI. We conclude that the binding of HDL to scavenger receptor type BI activates Ras in a PKC-independent manner with subsequent induction of the MAPK signaling cascade.     

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

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

Growth factor-independent proliferation of erythroid cells infected with Friend spleen focus-forming virus is protein kinase C dependent but does not require Ras-GTP

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.     

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.     

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2 ± 13.7% decrease for annexin A1 RNA interference; 78.7 ± 16.3% decrease for anti-Annexin A1 antibody blocking; 11.2 ± 6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.     

Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity

nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.     

Calmodulin modulates H-Ras mediated Raf-1 activation

We have previously demonstrated that, in COS-1 cells, inhibition of calmodulin increases Ras-GTP levels although it decreases Raf-1 activity and consequently MAPK. The present study analyzes the role of calmodulin in the regulation of Raf-1. First we show, using FRET microscopy, that inhibition of Raf-1 was not a consequence of a decreased interaction between H-Ras and Raf-1. Besides, the analysis of the phosphorylation state of Raf-1 showed that calmodulin, through downstream PI3K, is essential to ensure the Ser338-Raf-1 phosphorylation, critical for Raf-1 activation. We also show that the expression of a dominant negative mutant of PI3K impairs the calmodulin-mediated Raf-1 activation; in addition, both calmodulin and PI3K inhibitors decrease phospho-Ser338 and Raf-1 activity from upstream active H-Ras (H-RasG12V) and this effect is dependent on endocytosis. Importantly, in H-Ras depleted COS-1 cells, calmodulin does not modulate MAPK activation. Altogether, the results suggest that calmodulin regulation of MAPK in COS-1 cells relies upon H-Ras control of Raf-1 activity and involves PI3K.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts

We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins beta1, alpha4, alpha5, alpha6 and the activation epitope of beta1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras(V12), H-Ras(V12)T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in beta1-dependent manner, the expression of H-Ras(V12) or H-Ras(V12)T35S, but not H-Ras(V12)Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, whereas those with H-Ras(V12)Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1. (5) Most of the cells having H-Ras(V12), H-Ras(V12)T35S, or Raf-1 became annexin-V(high)/propidium iodide (PI)(high or low) and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)(high)/PI(low) after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces beta(1)-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.     

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.     

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.     

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.     

Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells

We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how 1alpha,25(OH)(2)D(3) up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with 1alpha,25(OH)(2)D(3) (1 nm) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D(3) stimulation of Raf-1. 1alpha,25(OH)(2)D(3) rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D(3) activates Ras in myoblasts. The protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,25(OH)(2)D(3)-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,25(OH)(2)D(3) could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D(3) exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a 1alpha,25(OH)(2)D(3) target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.     

Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.     

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes

Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.     

Activation of Raf-1 is defective in annexin 6 overexpressing Chinese hamster ovary cells.

Annexin 6 is a Ca2+-dependent phospholipid-binding protein involved in membrane trafficking. In this study we demonstrate the association of Raf-1 with recombinant rat annexin 6. Raf-annexin 6 interaction was shown to be independent of cell activation by epidermal growth factor (EGF) or phorbol esters (12-O-tetradecanoyl-phorbol-13-acetate (TPA)). A stable Chinese hamster ovary (CHO)-anx6 cell line overexpressing annexin 6 was established to examine the function of annexin 6. In these cells, no increase of Ras-GTP levels, induced by EGF or TPA, was detected. In addition, the activity of Raf was completely inhibited, whereas the mitogen-activated protein kinase-P was unaffected.     

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.