hSef co-localizes and interacts with Ras in the inhibition of Ras/MAPK signaling pathway

To study the mechanism of the inhibitory effects of Sef (similar expression to fgf genes) on Ras/mitogen-activated protein kinase (MAPK) signaling pathway, we observed cellular localization of this protein. Immunofluorescent staining results show that Sef locates in the vesicles of the cytoplasm without bFGF treatment but co-localizes with Ras on the plasma membrane (PM) in response to bFGF stimulation. The coimmunoprecipitation assay demonstrates that Sef interacts with Ras or RasG12V, respectively. We observed that Sef inhibited FGF induced, but not RasG12V mediated, signal transduction. We propose that Sef interacted with Ras in the inhibition of Ras/MAPK signaling pathway.     

Characterization of heterotrimeric G protein complexes in rice plasma membrane

Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells. Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit. When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.     

The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation

The Ca(2+)-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, is involved in the inactivation of mitogen-activated protein kinase pathway. However, a precise role of CAPRI in immune responses is still unknown. Here we showed that overexpression of CAPRI suppresses antigen-induced degranulation and cytokine production in mast cells (RBL cells). Antigen elicited the translocation of CAPRI to the plasma membrane from the cytoplasm, which was concomitant with the increase in the intracellular Ca(2+) concentration. The nuclear import of extracellular signal-regulated kinase 2 (ERK2) occurred after the re-localization of CAPRI to the cytoplasm in the mast cells, suggesting that the early phase of ERK2 activation is eliminated. A mutant of GAP-related domain, CAPRI(R472S), showed a feeble translocation to the plasma membrane but did not affect the degranulation, ERK2 activation, and cytokine production. The results suggested that the translocation of CAPRI to the plasma membranes regulates crucially cellular responses in mast cells.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.     

Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling.

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).     

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.     

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.     

Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C

We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.     

Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.     

Phospholipase C-beta 2 interacts with mitogen-activated protein kinase kinase 3

Phospholipase C (PLC)-beta enzymes (isoenzymes beta 1-beta 4) are activated by G protein subunits, leading to the generation of intracellular messengers which mobilize calcium and activate protein kinase C. It has recently been recognized that these enzymes interact with and are regulated by proteins other than G proteins. Using the yeast two-hybrid technique to screen a leukocyte library we identified mitogen-activated protein kinase kinase 3 (MKK3) as a partner of PLC-beta 2. The interaction was confirmed by co-immunoprecipitation assays which indicated that MKK3 interacts with PLC-beta 2, but not with other PLC-betas. PLC-beta 2 interacted weakly with MKK6, which is related to MKK3, but not with the other MKK3 tested. The region of PLC-beta 2 involved in the interaction with MKK3 was mapped to the C-terminus of PLC-beta 2. p38MAPK also co-immunoprecipitated with PLC-beta 2. The data suggest that PLC-beta 2 serves an unappreciated role assembling components of the p38MAPK signaling module.     

The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes

PTEN is a tumor suppressor with sequence homology to protein-tyrosine phosphatases and the cytoskeleton protein tensin. PTEN is capable of dephosphorylating phosphatidylinositol 3,4, 5-trisphosphate in vitro and down-regulating its levels in insulin-stimulated 293 cells. To study the role of PTEN in insulin signaling, we overexpressed PTEN in 3T3-L1 adipocytes approximately 30-fold above uninfected or control virus (green fluorescent protein)-infected cells, using an adenovirus gene transfer system. PTEN overexpression inhibited insulin-induced 2-deoxy-glucose uptake by 36%, GLUT4 translocation by 35%, and membrane ruffling by 50%, all of which are phosphatidylinositol 3-kinase-dependent processes, compared with uninfected cells or cells infected with control virus. Microinjection of an anti-PTEN antibody increased basal and insulin stimulated GLUT4 translocation, suggesting that inhibition of endogenous PTEN function led to an increase in intracellular phosphatidylinositol 3,4,5-trisphosphate levels, which stimulates GLUT4 translocation. Further, insulin-induced phosphorylation of downstream targets Akt and p70S6 kinase were also inhibited significantly by overexpression of PTEN, whereas tyrosine phosphorylation of the insulin receptor and IRS-1 or the phosphorylation of mitogen-activated protein kinase were not affected, suggesting that the Ras/mitogen-activated protein kinase pathway remains fully functional. Thus, we conclude that PTEN may regulate phosphatidylinositol 3-kinase-dependent insulin signaling pathways in 3T3-L1 adipocytes.     

Modulation of the inward rectifier potassium channel IRK1 by the Ras signaling pathway.

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.     

Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma.     

A function for phosphoinositide 3-kinase beta lipid products in coupling beta gamma to Ras activation in response to lysophosphatidic acid

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.     

Plugging PKA into ERK scaffolds

Cancers often arise in part through derangements in protein kinase signaling. A striking example of this is the finding that approximately 30% of human tumors have mutations in Ras or B-Raf, leading to aberrant ERK kinase activation. Kinase signaling networks are often organized by scaffolding and anchoring proteins that help shape the dynamics of signal processing. AKAP-Lbc associates with the ERK scaffold protein KSR-1 to organize a growth factor and cAMP responsive signaling network. AKAP-Lbc also directs PKA phosphorylation of KSR-1 on a critical residue to ensure maximal signaling efficiency.     

Trafficking of cGMP-dependent protein kinase II via interaction with Rab11

cGMP-dependent protein kinase II (cGK-II) is implicated in several physiological functions including intestinal secretion, bone growth, and learning and memory, but the detailed mechanisms are still unclear. To identify proteins that are involved in cGMP/cGK-II signaling, we performed yeast two-hybrid screening and identified Rab11b as a cGK-II-interacting protein that regulates the slow-recycling pathway. Interestingly, cGK-II interacted with the GDP-bound form of Rab11b (Rab11b S25N), but not the GTP-bound form, in mammalian cells. Immunofluorescence staining revealed that Rab11b S25N promoted the translocation of cGK-II from the plasma membrane to the cytoplasm and that the localization of cGK-II extensively overlapped with Rab11b. Furthermore, treatment with a membrane-permeable cGMP analog caused the rapid retranslocation of cGK-II and Rab11b S25N to the membrane. These data indicate that Rab11b is necessary for the trafficking of cGK-II and that the cGMP/cGK-II signaling pathway is closely related to Rab11b recycling pathway.     

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.     

Agonist-dependent traffic of raft-associated Ras and Raf-1 is required for activation of the mitogen-activated protein kinase cascade

Stimulation of HIRcB fibroblasts with insulin leads to accumulation of active components of the mitogen-activated protein kinase cascade in endocytic compartments. However, the factors that regulate the mobilization of these components through the endocytic pathway and the relevance of this event to cellular signaling remain unclear. Here we report that Ras proteins are associated with lipid rafts in resting HIRcB fibroblasts. Ras is rapidly internalized into the endocytic compartment following stimulation with insulin. The redistribution of Ras is independent of its activation. Attachment of the C-terminal 20 amino acids of Ha-Ras to green fluorescent protein was sufficient to target this construct to the same loci as the endogenous Ras protein, indicating that Ras distribution is a consequence of the association of its lipid modified C terminus with membranes. Depletion of plasma membrane cholesterol delocalized Ras and blocked insulin-dependent Ras traffic. Cholesterol depletion also blocked insulin-dependent phosphorylation of MEK and mitogen-activated protein kinase (MAPK) but had no effects on the translocation and activation of Raf-1. A second inhibitor of endocytosis, cytochalasin D, also blocked insulin-dependent MAPK phosphorylation. Taken together, these results suggest that mobilization of active Raf-1 through the endocytic compartment is required for completion of the MAPK cascade.