The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation*

Kendall D. CareyDagger , Robert T. Watson§, Jeffrey E. Pessin§, and Philip J. S. StorkDagger

From the Dagger  Vollum Institute, Department of Cell and Developmental Biology, L474 Oregon Health Sciences University, Portland, Oregon 97201 and the § Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

Received for publication, July 12, 2002, and in revised form, November 8, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mitogen-activated protein (MAP)1 kinase family regulates diverse physiological processes including cell growth, differentiation, and death. Activation of one of these MAP kinases (the extracellular signal-regulated kinase or ERK) is initiated by the recruitment of the MAP kinase kinase kinase Raf-1 to the small G protein Ras, a resident plasma membrane protein. The Ras family consists of three members (Ha-Ras, Ki-Ras, and N-Ras) (1) that display overlapping but distinct patterns of expression and function (2). Ras is tethered to the membrane via a carboxyl CAAX motif containing a cysteine followed by two aliphatic amino acids (A) and a carboxyl-terminal amino acid (X) that directs the attachment of a farnesyl moiety (3, 4). In addition to farnesylation, a second signal assists in correct membrane targeting. For Ha-Ras and N-Ras, this second signal is a palmitoyl moiety that is introduced on a neighboring cysteine. In Ki-Ras, this second site is a polybasic domain. The requirement of Ras membrane localization for Raf-1 activation has been confirmed by mutating the terminal cysteine in a constitutively active Ras mutant, RasV12, resulting in a mutant that cannot activate Raf-1 (5).

Membrane regions rich in cholesterol and sphingolipids, termed "rafts" or detergent-insoluble glycolipid-enriched complexes have been proposed to participate in signaling events by organizing additional molecules, such as c-Src, G protein subunits, and phospholipases, into discrete membrane domains (6). Recent attention has focused on the role of these specialized microdomains in Ras signaling (7-10). A number of groups have shown that both Ras isoforms Ki-Ras (11) and Ha-Ras are targeted to rafts (7-9, 12-14). Others have suggested that Ha-Ras, but not Ki-Ras, can be targeted to raft microdomains (10, 15). Targeting of Ras isoforms to specific membrane domains may be determined by the characteristics of the lipid modifications on Ras, as well as other sequences found within the hypervariable region (hvr) (10). Localization of Ras isoforms to distinct membrane microdomains may influence selectivity of signaling among the Ras isoforms (2). For example, Ki-Ras is thought to couple well to Raf-1 but unlike Ha-Ras, couples poorly to phosphatidylinositol 3-kinase (16, 17). Differences between Ha-Ras and Ki-Ras in their promotion of cell survival have also been noted (18), suggesting that distinct localization of Ras isoforms dictate signaling pathways, as recently proposed (19). Ras isoforms have been reported to display distinct dependences on caveolin in their coupling to Raf-1 (9, 15).

Raf-1 recruitment to the membrane can be achieved independently of Ras by the addition of Ras carboxyl-terminal sequences to the carboxyl terminus of Raf-1. The addition of twenty amino acids from the carboxyl terminus of Ki-Ras onto Raf-1 (Raf-KiCAAX) is sufficient to redirect Raf-1 to the membrane where it is constitutively active (20, 21). Maximal activity of this chimera, however, requires additional phosphorylation events (22, 23), consistent with the requirement of specific kinases for full activation for wild type Raf-1 (24-26). In particular, two phosphorylations on Ser-338 and Tyr-341 have been shown to be required for full activity (27). Recently, two additional sites within the kinase activation loop have also been shown to be required (28). Phosphorylation of serine 338 may be mediated by the serine/threonine kinase PAK (p21-associated protein) (29) and phosphorylation of tyrosine 341 can be carried out by Src family tyrosine kinases (23, 27, 30). The participation of specific membrane microdomains in these modifications is not known.

In this study, we examined the membrane requirements for the post-translational modification of Raf-1. In addition, we took advantage of chimeric Raf-1 molecules that are targeted to specific membrane domains to determine the specificity of these domains for raft localization, phosphorylation, and constitutive activation of Raf-1. Understanding the molecular basis for Raf-1 activation by Ras may also help explain the actions of the related small G protein Rap1, which recruits Raf-1, but unlike Ras, cannot activate it. In the present study we address whether membrane localization also plays a role in Rap1-mediated inhibition of Raf-1 activation by Ras. We found that Raf-1 phosphorylation was intimately linked to proper membrane targeting and that the ability of Ras and Rap1 to support Raf-1 phosphorylation dictated the biochemical actions of both Ras and Rap1 on Raf-1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Cell Culture, Transfections, and Stimulations-- COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected using LipofectAMINE 2000 according to the manufacturer's recommendations. Unless otherwise noted, cells were transfected with a total of 10 µg of plasmid DNA, with pcDNA3.1 (vector) used to adjust DNA amounts where necessary. After 24 h, transfected cells were switched to low serum-containing medium and incubated for a further 12 h. Cells were stimulated with 50 ng/ml epidermal growth factor (EGF) for the indicated times. For cholesterol depletion experiments, serum-starved cells were preincubated for 1 h with 2% methyl beta -cyclodextrin (CD, Sigma) in Dulbecco's modified Eagle's medium prior to stimulation with EGF.

Plasmids-- The cDNA encoding Ha-Ras and Ha-RasV12 were tagged at their amino termini with a FLAG epitope. The cDNA encoding Ki-Ras and Ki-RasV12 were tagged at their amino termini with a Myc epitope. The cDNA encoding N-Ras and N-RasV12 were tagged at their amino termini with a hemagglutinin (HA) epitope and were purchased from the Guthrie Institute, Sayre, PA. All other cDNAs were tagged at the amino termini with a 2× FLAG epitope (Kodak) by PCR and introduced into the BamH1 and XbaI sites of pcDNA3.1 vector (Invitrogen), unless otherwise indicated. Raf-1 and B-Raf mutations were introduced by PCR using the QuickChange site-directed mutagenesis kit (Stratagene). The minimal membrane targeting domain of Ha-Ras(-hvr) (31) was added to the 3'-end of FLAGRaf-1 by PCR using a primer with Raf-1 sequences along with an in-frame XhoI site (adding amino acids Leu and Glu) and the last nine amino acids of Ha-Ras (CMSCKCVLS), a stop codon, and an XbaI site. This construct is designated Raf-HaCAAX(-hvr). Raf-Ha-RasCAAX(+hvr), containing both the minimal membrane targeting domain and the hypervariable domain, was constructed using a primer with an in-frame XhoI site, the carboxyl-terminal 27 amino acids of Ha-Ras (IRQHKLRKLNPPDESGPGCMSCKCVLS), a stop codon, and an XbaI site by PCR. Raf-Rap1CAAX was constructed using the same strategy with the carboxyl-terminal 24 amino acids of Rap1b (LVRQINRKTPVPGKARKKSSCQLL) introduced into the XhoI-XbaI site of Raf-Ha-RasCAAX(+hvr). B-Raf-Ha-RasCAAX, B-Raf-Rap1CAAX chimeras were constructed using a similar strategy. RapE63-Ha-RasCAAX(+hvr) was constructed by adding an in-frame XhoI site by PCR into RapE63 at base pair 480 and cloning into the EcoR1-XhoI sites of FLAGRaf-Ha-RasCAAX, thereby replacing the Rap membrane targeting motif with the Ha-Ras hypervariable domain and CAAX motif. The full-length coding sequences of human Ha-Ras, bovine Rap1b, RapE63-Ha-RasCAAX, Raf-Rap1CAAX, and Raf(Y341D)-Rap1CAAX were introduced in-frame into the pEGFP-C1 cloning vector (Clontech). Wild type (CAV/WT) and mutant caveolin-3 (CAV/DGV) plasmids were constructed as per Watson et al. (32).

Western Blotting and Immunoblotting-- COS-7 cells were stimulated and lysates prepared as described. Protein concentrations were determined by using the Bio-Rad protein assay dye reagent according to manufacturer's recommendations. Equal amounts of lysate were immunoprecipitated with either FLAG M2 antibody coupled to agarose (Sigma) or anti-Myc antibody (9E10) coupled to agarose (Santa Cruz Biotechnology) where indicated and examined by Western blot as previously described (33). Samples were separated by SDS-PAGE and transferred to PVDF membrane. Expression of FLAG and Myc-tagged proteins was detected using monoclonal FLAG M2 antibody or monoclonal anti-Myc 9E10 antibody. Rabbit polyclonal anti-phospho-MEK1/2 antibody (Cell Signaling Technology) was used to detect activated GST-MEK1 (Upstate Biotechnology). Polyclonal anti-phospho-ERK1/2 (Cell Signaling Technology) was used to detect activated MycERK2 (pMycERK2). Phosphorylation of Raf-1 at Ser-338 or at Tyr-341 was detected using anti-phospho-Raf-1 Ser-338 or -phosphotyrosine (pTyr, 4G10) (Upstate Biotechnology). All experiments were repeated at least three times, and representative blots are shown. The expression of endogenous N-Ras, Ki-Ras, and Ha-Ras was examined using subtype-specific Ras antisera from Santa Cruz Biotechnology.

Cell Fractionation-- COS-7 cells were washed twice in phosphate-buffered saline (PBS) before scraping into 0.5 ml of hypotonic lysis buffer (10 mM Tris, pH 7.5, 5 mM Mg2Cl, 25 mM NaF, 25 mM beta -glycerophosphate, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). After 10 min., cells were homogenized at 4 °C by 50 strokes in a tight fitting Dounce homogenizer. Nuclei and unbroken cells were pelleted by centrifugation at 1500 × g. Supernatants were then centrifuged at 100,000 × g in a Beckman TLA 45 rotor at 4 °C for 30 min. The supernatant was collected and designated the cytosolic fraction (S100), and the pellet was resuspended in 250 µl of hypotonic lysis buffer and designated the membrane (P100) fraction.

Sucrose Gradients-- Membrane microdomains were isolated based on their buoyant density using isopycnic equilibrium sucrose density gradient centrifugation. A non-detergent method for lipid raft isolation was used based on the method of Smart et al. (34) but modified for equilibrium centrifugation, and sucrose was used instead of Optiprep. Briefly, transfected COS-7 cells (2 × 106 cells) were rinsed twice in PBS and scraped into 0.5 ml of MES buffer (25 mM MES, pH 6.5, 10 mM NaCl, 5 mM Mg2Cl, 10 µg/ml aprotinin, 10 mg/ml leupeptin, 25 mM NaF, 25 mM beta -glycerophosphate, 5 mM sodium orthovanadate). Cells were homogenized at 4 °C by 30 passes through a 23-gauge syringe and sonicated on ice for 30 s at setting 2, 30 s at setting 3, and 30 s at setting 4 (Sonic Dismembrater, Fisher). The lysate was mixed with 0.5 ml of 90% sucrose in MES buffer and placed at the bottom of a 2.4-ml Beckman ultracentrifuge tube. The gradient was constructed by overlaying the 45% sucrose/lysate mixture with 1.2 ml of 35% sucrose, 1 ml of 30% sucrose, 1 ml of 25% sucrose, and ultimately 1 ml of 5% sucrose. The tubes were centrifuged at 4 °C in a Beckman SW 55 rotor for 16 h at 48,000 rpm. A visible band 3-4 mm from the top was observed after centrifugation and corresponded to the lipid raft/caveolae fraction. Twelve 0.43-ml fractions were collected from the top of the gradient. From each fraction, 40 µl was removed for refractometry and protein determination using the Bradford method (BioRad). The remainder of each fraction was diluted with 1 ml of MES buffer to dilute out the sucrose, and membranes and proteins were pelleted at 100,000 × g for 45 min in a TLA 45 rotor. Pellets were resuspended in Laemmli buffer, separated by SDS-PAGE, and transferred to a PVDF membrane for analysis by immunoblotting. Only the first 10 fractions are shown for each gradient. Similar results were obtained using the sodium carbonate method of raft preparation (10).

Immunofluorescence-- COS-7 cells were grown on glass coverslips and transfected with 100 ng of FLAGRaf cDNAs using LipofectAMINE 2000. Cells were fixed in PBS containing 4% formaldehyde, permeablized in PBS containing 0.1% Triton X-100, and blocked with PBS, 5% horse serum. Localization of transfected proteins was detected with a 1:2000 dilution of FLAG M2 antibody (Sigma) in 5% horse serum, 0.01% Tween followed by a 1:10,000 dilution of an anti-mouse-fluorescein isothiocyanate conjugate in 5% horse serum, 0.01% Tween. Cells were visualized using a Zeiss Axioplan 2 microscope. To examine the expression patterns of the Myc-tagged wild type caveolin (CAV/WT) or DGV mutant (CAV/DGV) constructs, cells were transfected with 5 µg of plasmid cDNAs as described above. The expressed caveolin constructs were detected by mouse anti-Myc monoclonal antibody (Santa Cruz Biotechnology) at 1:100 dilution, followed by Texas-Red anti-mouse secondary antibody at 1:100 (Jackson Immunoresearch Laboratories). Endogenous caveolin was detected by rabbit anti-caveolin polyclonal antibody (Transduction Laboratories) at 1:200 dilution, followed by Alexa-488 anti-rabbit secondary antibody at 1:100 dilution (Molecular Probes). Cells were imaged with a Zeiss 510 scanning laser confocal microscope.

Raf-1 Kinase Assays-- COS-7 cells were transfected, lysed, and lysates prepared as described (28). FLAGRaf proteins were immunoprecipitated from 500 µg of cell lysate with 30 µl of FLAG M2 agarose at 4 °C for 6 h. Immune complexes were washed twice with lysis buffer and once with kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM beta -glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 5 µg/ml aprotinin). Pellets were resuspended in 40 µl of kinase assay buffer with 1.5 mM Mg2Cl and 7.5 µM ATP along with 0.4 µg of GST-MEK1 (Upstate Biotechnology), and the reaction was incubated for 30 min at 30 °C. The kinase reaction was terminated by adding 45 µl of 2× Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE, and transferred to PVDF membrane. Raf-1 activity was evaluated by immunoblotting with anti-phospho-MEK1/2 antibody.

Phospho-MycERK2 Assay-- For MycERK2 assays, treated and untreated cells were lysed in ERK assay buffer and activation of MycErk2 was detected as described previously (33).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Raf-1 Is Relocalized to Raft Microdomains upon EGF Stimulation-- Activation of Raf-1 by Ras-dependent signals induced by growth factors is associated with a redistribution of Raf-1 from the cytoplasm to the cell membrane, where it associates with Ras (35). Recent studies suggest that Ras may be localized to specific cholesterol-rich membrane microdomains called rafts (11), and upon Ras activation, Raf-1 may also be recruited to rafts (12).

Proteins that localize to cholesterol-rich microdomains (rafts) can be detected within low density fractions of sucrose density gradients (10, 14, 36). The method is illustrated in Fig. 1A. The density gradient achieved following equilibrium centrifugation is shown in Fig. 1B with the corresponding protein concentrations. One of these raft proteins, caveolin-1, was used to identify the density of these cholesterol-rich raft domains (Fig. 1C, top panel) (6). Using this technique, we show that endogenous Raf-1 was excluded from raft microdomains in untreated cells (Fig. 1C, second panel), and EGF treatment induced the redistribution of endogenous Raf-1 into raft domains (Fig. 1C, third panel). A significant fraction of Raf-1 protein was also detected at higher densities within the gradient.


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Fig. 1.   EGF stimulation relocalizes Rap-1 to raft microdomains. A, schematic illustration of the sucrose density gradient assay. Isopycnic centrigugation is used to achieve an equiibrium gradient. The lipid-associated proteins migrate to buoyant densities, where they can be detected within the top fractions of the gradient. The localization of Raf-1 in EGF-treated cells is shown as an example. B, sucrose density gradient profiles of COS-7 cells. Cells were lysed and proteins prepared for sucrose equilibrium density gradient centrifugation as described under "Experimental Procedures." The top twelve 0.43-ml fractions are shown (open squares). The protein content of each fraction is shown as black circles. C, growth factor stimulation recruits Raf-1 to lipid rafts. Cells were treated with or without EGF as indicated, and proteins separated into the indicated fractions by equilibrium centrifugation on a sucrose density gradient (fraction 1 is top). Fractions 1-10 were pelleted and proteins separated by SDS-PAGE. The position of caveolin-1 within the gradient is shown in the upper panel using caveolin-1 antisera. The presence of endogenous Raf-1 within fractions (1-10) of each gradient is shown in the middle and lower panels, using antisera to Raf-1. D, sucrose density gradients of protein fractions containing Raf-1 and Raf mutants. Cells were transfected with wild type FLAG-tagged Raf-1 (Raf-1 WT), treated with or without EGF or CD, as indicated, and proteins separated into the indicated fractions by sucrose density gradients. The presence of FLAGRaf-1 constructs within fractions 1-10 of each gradient is shown in the lower two panels, using FLAG antibody. E, activated Ras proteins recruit Raf-1 into raft microdomains. COS-7 cells were transfected with FLAGRaf-1 and either vector, Ha-RasV12 (top panel), Ki-RasV12 (second panel), N-RasV12 (third panel), or vector (bottom panel) as indicated, and proteins were separated into the indicated fractions by sucrose density centrifugation. The presence of FLAGRaf-1 within fractions (1-10) of each gradient is shown using the FLAG antibody.

Similar results were seen using transfected cDNAs encoding wild type Raf-1 tagged with the FLAG epitope. We show that wild type Raf-1 was excluded from raft microdomains in untreated cells (Fig. 1D, top panel), and EGF treatment induced the redistribution of wild type Raf-1 into raft domains (Fig. 1D, middle panel). Raft domains can be disrupted by the cholesterol-depleting agent CD (11). In the presence of CD, EGF recruitment of Raf-1 to low density fractions was inhibited (Fig. 1D, bottom panel), confirming that these low density fractions represented cholesterol-rich membrane microdomains. This provides strong evidence that the buoyant fractions containing Raf-1 represented cholesterol-rich membrane microdomains consistent with rafts. Raf-1 could also be redistributed to rafts in cells transfected with constitutively active mutants of Ha-Ras, Ki-Ras, and N-Ras (Fig. 1E).

The localization of Ras proteins to rafts is not completely understood, and some controversies remain (37) with some groups showing that Ha-Ras, but not Ki-Ras, requires raft localization for full activity (9, 15). COS-7 cells express detectable levels of Ki-Ras and N-Ras (Fig. 2A; middle and right panels, untr.). The expression of Ha-Ras was not detected (Fig. 2A; left panel, untr.). As a control, cells were transfected with each Ras isoform and lysates probed with isoform-specific antisera. Endogenous Ki-Ras and N-Ras were localized to lipid rafts in resting cells (Fig. 2B). Similar results were seen in cells transfected with wild type as well as constitutively active (V12) Ha-Ras, Ki-Ras, and N-Ras mutants, although transfected N-Ras wild type appeared in higher density fractions as well (Fig. 2, C and D).


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Fig. 2.   Ras isoforms are localized to raft domains. A, Western blot of Ras isoforms expressed in COS-7 cells. Cells were left untransfected or transfected with FLAG-HaRas, Myc-KiRas, or HA-NRas. Proteins were assayed by Western blot for endogenous and transfected Ras proteins, using isoform-specific Ras antibodies directed against Ha-Ras, Ki-Ras, and N-Ras as indicated. B, sucrose density gradients of membrane fractions from unstimulated COS-7 cells. The presence of endogenous Ki-Ras (top panel) and N-Ras (bottom panel) are shown using Ki-Ras or N-Ras antisera, respectively. C, sucrose density gradients of membrane fractions containing transfected Ras proteins. Cells were transfected with WT, FLAG-HaRas, Myc-KiRas, or HA-NRas as indicated, and proteins were separated into the indicated fractions by sucrose density gradients The presence of Ha-Ras (top panel), Ki-Ras (middle panel), and N-Ras (lower panel) within fractions 1-10 of each gradient were analyzed using FLAG, Myc, and HA antibodies respectively. D, sucrose density gradients of membrane fractions containing transfected Ras proteins. Cells were transfected with constitutively active FLAG-HaRasV12, Myc-KiRasV12, or HA-NRasV12, as indicated. The presence of Ha-RasV12 (top panel), Ki-RasV12 (middle panel), and N-RasV12 (lower panel) within fractions 1-10 of each gradient were analyzed using FLAG, Myc, and HA antibodies respectively.

Raf-1 Activation and Ser-338 Phosphorylation Require Intact Raft Microdomains-- It has long been appreciated that Raf-1 recruitment to Ras is insufficient by itself to trigger Raf-1 activation and that additional post-translational modifications are required (27). The two best studied modifications are serine phosphorylations on serine 338 and tyrosine 341. Fig. 3A demonstrates this requirement. Constitutively active Ras (RasV12) activated wild type Raf-1 (Raf-1 WT), as measured by in vitro Raf-1 assay. However, Raf-1 that was mutated at either Ser-338 to alanine (RafS338A) or Tyr-341 to alanine (RafY341A) could no longer be activated by Ha-RasV12 (Fig. 3A).


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Fig. 3.   Ser-338 phosphorylation and activation requires intact raft domains. A, Raf-1 kinase assays. HaRasV12 and either FLAG-tagged Raf-1, RafS338, RafY341A, or pcDNA3 vector was transfected into COS-7 cells. Equivalent amounts of Raf protein were immunoprecipitated using the FLAG antibody and assayed for the ability to phosphorylate MEK (pMEK) in vitro (top panel). The levels of FLAGRaf (middle panel) and MycRasV12 (lower panel) are shown. B, cells were transfected with FLAG Raf-1 WT and treated with EGF and/or CD as indicated. Lysates were subjected to FLAG immunoprecipitation and assayed for p338 (upper panel) and the ability to phosphorylate MEK (pMEK) in vitro (middle panel). The levels of FLAGRaf-1 expression are shown in the lower panel, using FLAG antibody. C, cells were transfected with FLAGRaf-1WT or FLAGRafY341A and treated with EGF and/or CD as indicated. Lysates were subjected to FLAG immunoprecipitation and assayed for tyrosine phosphorylation with phospho-Tyr antibody (pTyr) (upper panel). The levels of FLAGRaf-1 expression are shown in the lower panel, using FLAG antibody.

Raft microdomains were required for phosphorylation on Ser-338, since CD inhibited EGF-induced phosphorylation of FLAGRaf-1 on Ser-338 (p338) and Raf-1 activation (Fig. 3B). These data suggest that targeting to raft domains was required for full Raf-1 activation and phosphorylation at Ser-338. In contrast, tyrosine phosphorylation of FLAGRaf-1 was not affected by CD (Fig. 3C, upper panel, lanes 1-4). The absence of phosphorylation of Tyr-341 in FLAGRafY341A is provided as a negative control (Fig. 3C, upper panel, lanes 5-8).

Recent studies have shown that Ras proteins are localized to specialized raft domains called caveolae (11). These microdomains are enriched for caveolin, and a requirement for caveolin can be assessed using interfering mutants such as CAV/DGV, a truncated form of caveolin-3 (9, 10, 32). In COS-7 cells, expression of CAV/DGV was detected within cytoplasmic vesicles, as previously reported (9, 10) and interferes with the localization of caveolin-1 (Fig. 4A). CAV/DGV did not interfere with the ability of either EGF (Fig. 4B) or constitutively active Ras mutants (Fig. 4C) to activate Raf-1 or stimulate Ser-338 phosphorylation.


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Fig. 4.   Interfering mutants of caveolin do not disrupt Ras function. A, epifluorescent micrographs of CAV/WT and CAV/DGV. COS-7 cells were transfected with Myc-CAV/WT (WT; panels 2, 4, and 6) or Myc-CAV3/DGV (DGV; panels 1, 3, 5, and 7). The expression of Myc-CAV/DGV is shown in panels 1 and 2 (anti-Myc; red). The expression of endogenous caveolin-1 is shown in panels 3 and 4 (anti-caveolin; green). Merges of the red and green images are shown in panels 5 and 6. Panel 7 shows a magnification of the boxed area in panel 5. Bar is 10 µM. The zoom field is 10 × 10 µm. B, cells were transfected with FLAGRaf-1 and either vector (-) or Myc-CAV/DGV (+), and treated with EGF or left untreated, as indicated. Lysates were subjected to FLAG immunoprecipitation and assayed for p338 (top panel) and the ability to phosphorylate MEK (pMEK) in vitro (second panel). The levels of FLAGRaf-1 expression are shown in the third panel, using FLAG antibody. The levels of Myc-CAV/DGV expression are shown in the fourth panel, using Myc antibody. C, cells were transfected with FLAGRaf-1 along with Ha-RasV12 (Ha), Ki-RasV12 (Ki), or N-RasV12 (N), and vector (V), with CAV/DGV; (+) or without CAV/DGV; (-). Lysates were subjected to FLAG immunoprecipitation and assayed for p338 (top panel) and the ability to phosphorylate MEK (pMEK) in vitro (second panel). The levels of FLAGRaf-1 expression are shown in the third panel, using FLAG antibody. The levels of Myc-CAV/DGV expression are shown in the fourth panel, using Myc antibody.

Tyr-341 Is Required for Targeting to Raft Microdomains and Phosphorylation of Ser-338-- We next examined the requirement of Ser-338 and Tyr-341 in Raf-1 localization. EGF was able to direct RafS338A into rafts (Fig. 5A, upper panel), but was not able to direct RafY341A into rafts (Fig. 5A, lower panel). Neither Raf mutant RafS338A nor RafY341A entered rafts in the absence of EGF stimulation (data not shown). This suggests that phosphorylation of Tyr-341 participates in localization of Raf-1 to lipid rafts, while Ser-338 phosphorylation occurs once proper localization has been achieved. In Fig. 5B, we show that the tyrosine at residue 341 was essential for EGF and Ha-RasV12 to phosphorylate Ser-338 (Fig. 5B, upper panel) and activate Raf-1 (Fig. 5B, middle panel). Replacing tyrosine with aspartate in the mutant Raf Y341D did not affect EGF actions (Fig. 5B).


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Fig. 5.   The requirement for Tyr-341 can be overcome by targeting Raf-1 to raft domains. A, sucrose density gradients of membrane fractions containing Raf-1 mutants. Cells were transfected with FLAG-tagged RafY341A, and RafS338A, treated with EGF as indicated, and proteins separated into the indicated fractions by sucrose density gradients, as described in the legend to Fig. 1. The presence of FLAGRaf constructs within each gradient is shown in both panels, using FLAG antibody. B, phosphorylation and activation of Raf-1 mutants by EGF. FLAG-tagged Raf-1, RafY341A, or RafY341D were transfected into COS-7 cells, and cells were either treated with EGF or left untreated, as indicated. Lysates were subjected to FLAG immunoprecipitation and assayed for p338 (upper panel) and the ability to phosphorylate MEK (pMEK) in vitro (middle panel). The levels of FLAGRaf proteins are shown in the lower panel, using FLAG antibody. C, immunofluorescence of Raf-Ras chimeras. COS-7 cells were transfected with FLAG-tagged Raf-1 WT, Raf-HaCAAX(+hvr), and Raf-HaCAAX(-hvr), as indicated. Cells were prepared for epifluorescent microscopy as described under "Experimental Procedures," and representative cells are shown. D, sucrose density gradients of Raf-Ras chimeras. Cells were transfected with FLAG-tagged Raf-HaCAAX(+hvr), Raf Y341AHaCAAX(+hvr), and Raf S338AHaCAAX(+hvr), and left untreated. Proteins were separated as in Fig. 1 and the presence of the chimera within each fraction is shown, using FLAG antibody. E, lack of requirement of Tyr-341 for Ser-338 phosphorylation in targeted chimeras. Cells were transfected with Raf-1 WT, Raf-HaCAAX(+hvr), RafS338A-HaCAAX(+hvr), or RafY341AHaCAAX(+hvr), and treated with or without EGF, as indicated, and assayed for p338 (upper panel), or Raf-1 kinase activity (pMEK, middle panel). The levels of FLAG-containing proteins are shown in the lower panel.

The Requirement of Tyr-341 Phosphorylation for Raf-1 Activation Can Be Overcome by Targeting to Raft Domains-- Raf-1 can be constitutively targeted to the membrane following the attachment of a Kirsten Ras carboxyl-terminal domain (Raf-KiCAAX) (20, 21). Here, we examined the localization of a related chimera created by fusing the Raf-1 protein to the carboxyl 27 amino acids of Ha-Ras including both the CAAX domain and the hvr; Raf-HaCAAX(+hvr) (16). As expected, wild type Raf-1 was located within the cytoplasm of resting cells (Fig. 5C, left panel), and the chimera Raf-HaCAAX(+hvr) was present on the plasma membrane (Fig. 5C, middle panel). The chimera Raf-HaCAAX(-hvr) that lacked hvr sequences was also present on the plasma membrane (Fig. 5C, right panel).

Raf-HaCAAX(+hvr) is constitutively localized within raft domains (Fig. 5D, upper panel). Unlike Raf Y341A, the introduction of Y341A into Raf-HaCAAX(+hvr) [RafY341A-HaCAAX(+hvr)] did not prevent raft localization (Fig. 5D, middle panel), constitutive phosphorylation of Ser-338 or activation of Raf-1 (Fig. 5E). These data suggest that phosphorylation of Tyr-341 is required for raft localization, Ser-338 phosphorylation, and activation of wild type Raf-1 but that Tyr-341 phosphorylation is not required if Raf-1 is constitutively targeted to rafts. In contrast, mutating Ser-338 to alanine, in the chimera RafS338A-HaCAAX(+hvr), completely abolished activation of Raf-1 (Fig. 5E), without affecting raft localization (Fig. 5D, lower panel), demonstrating the requirement of phospho-Ser-338 for kinase activation, but not raft localization.

In addition to the CAAX domain, Ha-Ras contains a hypervariable region that influences specific membrane localization (10). Here we examined chimeras either containing the Ha-Ras hypervariable region (+hvr) or lacking these sequences (-hvr). Like Raf-HaCAAX(+hvr), Raf-HaCAAX(-hvr) was present within the particulate (P100) fraction (Fig. 6A) and was detected on the plasma membrane (Fig. 5C, right panel). Unlike Raf-HaCAAX(+hvr), Raf-HaCAAX(-hvr) was excluded from low density gradient fractions (Fig. 6B, upper panel), suggesting that despite its membrane localization, the chimera was targeted differently than Raf-HaCAAX(+hvr). Raf-HaCAAX(+hvr) gradients were included as a control (Fig. 6B, lower panel).


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Fig. 6.   Ectopic targeting of Raf-1 to raft domains is required for constitutive Ser-338 phosphorylation, activation of Raf-1, and activation of ERKs. A, localization of Raf-Ras chimeras. Cells were transfected with FLAG-tagged Raf-1 WT and the Raf-Ras chimeras as indicated and fractionated into S100 and P100 fractions, and FLAG-containing proteins detected by Western blot. B, sucrose density gradients of Raf-HaCAAX(-hvr). Cells were transfected with FLAG-tagged Raf-HaCAAX(-hvr), and left untreated. Proteins were separated as in the legend to Fig. 1, and the presence of the chimera within each fraction is shown (upper panel), using FLAG antibody. Gradients of lysates expressing Raf-HaCAAX(+hvr) are shown as a control (lower panel). C, raft-targeted chimeras show constitutive activity. Cells were transfected with MycERK2 and individual Raf-1 chimeras. Cells transfected with wild type Raf-1 were also treated with EGF as indicated. FLAG-containing proteins were recovered by immunoprecipitation and examined for Ser-338 phosphorylation (p338, first panel), and phosphorylation of MEK in vitro, as in Fig. 1 (pMEK, second panel). The position of FLAGRaf proteins is shown in the third panel (FlagRaf). In the lower two panels, the lysates were subjected to Myc immunoprecipitation and the recovered MycERK2 examined for phosphorylation (pMycERK2) or total MycERK levels (MycERK2).

Only Raf-HaCAAX(+hvr) (lane 4), but not Raf-HaCAAX(-hvr) (lane 3) displayed constitutive phosphorylation of Ser-338 (Fig. 6C, p338, first panel). Raf-HaCAAX(+hvr), but not Raf-HaCAAX(-hvr), was active in Raf-1 kinase assays in vitro (Fig. 3C, pMEK, second panel) and ERK activation assays in vivo (Fig. 6C, pMycERK2, fourth panel). As a control, EGF stimulation of wild type Raf-1 was included, which resulted in phosphorylation of Ser-338 and activation of both Raf-1 and ERK (lanes 1 and 2). High levels of activation of Raf-HaCAAX(+hvr), but not Raf-HaCAAX(-hvr), were also seen in coupled in vitro kinase assays (data not shown). Therefore, both Ser-338 phosphorylation and biochemical activity of these chimeras paralleled raft localization.

Y341D Restored Raft Localization and Activity to Raf-HaCAAX(-hvr)-- For Raf-HaCAAX(-hvr), membrane targeting was not sufficient to trigger Ser-338 phosphorylation and Raf-1 activity. Since Tyr-341 phosphorylation potentiates the localization of Raf-1 within rafts, which may be required for subsequent Ser-338 phosphorylation, we tested whether the Y341D mutation could restore Ser-338 phosphorylation in the HaCAAX(-hvr) chimera. Indeed, this mutant, RafY341D-HaCAAX(-hvr), but not RafS338D-HaCAAX(-hvr), was capable of entering raft domains (Fig. 7A). Moreover, RafY341D-HaCAAX(-hvr) was phosphorylated on Ser-338 and showed constitutive activity to levels similar to those seen with Raf-HaCAAX(+hvr) (Fig. 7B). Furthermore, the introduction of Y341D into Raf-HaCAAX(+hvr) did not significantly increase Raf-1 activity (Fig. 7, B and C). These data suggest that one of the functions of Tyr-341 is to localize Raf-1 to specific membrane microdomains permitting efficient phosphorylation of Ser-338 and coupling to downstream effectors.


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Fig. 7.   Mutation of Y341D restores phosphorylation of Ser-338, activation, and raft localization of inactive Raf/Ras(-hvr) chimeras. A, raft localization of RafY341DHaCAAX(-hvr). Cells were transfected with RafY341DHaCAAX(-hvr) and proteins fractionated as described in the legend to Fig. 1. RafS338D-HaCAAX(-hvr) is shown as a control (lower panel). The presence of FLAG-containing proteins within each fraction is shown. B, Ser-338 phosphorylation and Raf-1 activity assays. Cells were transfected with either FLAG-tagged Raf-HaCAAX(-hvr), RafY341DHaCAAX(-hvr), Raf-HaCAAX(+hvr), or RafY341DHaCAAX(+hvr) and immunoprecipitated using FLAG antibody and assayed for Ser-338 phosphorylation (upper panel) and the ability to phosphorylate MEK (pMEK) in vitro (middle panel). The levels of FLAGRaf-1 expression are shown in the lower panel, using FLAG antibody. C, Raf-1 assays from three independent experiments, as in Fig. 4. The data are shown as fold activation above that seen for Raf-1 WT, with S.E.

Rap1 Is Unable to Activate Raf-1 Because it Cannot Induce Tyr-341 Phosphorylation-- The ability of small G proteins to recruit Raf-1 to the membrane is not sufficient for full activation of Raf-1 (22). For example, Rap1 is a small G protein within the Ras family that can associate with Raf-1 but cannot activate it (38). Unlike Ras, which is located at the plasma membrane, Rap1 is located in vesicular membranes (39-41). This localization is directed by carboxyl-terminal sequences of Rap1 that contain a distinct CAAX motif that regulate the attachment of geranyl modifications that direct Rap1 to vesicular membranes (42, 43). This could be shown using GFP fusions to the RapE63 protein (GFP-Rap), which, unlike GFP alone (Fig. 8A, a), was localized to perinuclear vesicles within the cytoplasm (Fig. 8A, b). In contrast, GFP-Ha-RasV12 was detected at the plasma membrane, consistent with recent reports (19) (Fig. 8A, c). The chimera GFP-RapE63-Ha-RasCAAX was also present on the plasma membrane, confirming that the carboxyl-terminal sequences of Ha-Ras could redirect ectopic proteins (Fig. 8A, d).


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Fig. 8.   The inability of Rap1 to activate Raf-1 is due to the inability of Rap1 to induce Ser-338 phosphorylation. A, GFP epifluorescence. Cells were transfected with GFP (a), GFP-RapE63 (b), GFP-HaRasV12 (c), and GFP-RapE63-HaRas-CAAX (d). The locations of the transfected proteins were examined by epifluoresent microscopy, and representative cells are shown. B, Rap1 supports neither Raf-1 activation nor Ser-338 phosphorylation. Cells were transfected with Raf-1 WT along with pcDNA3, RapE63, RapE63-HaCAAX(+hvr) or HaRasV12 and examined for Ser-338 phosphorylation (p338) and Raf-1 activity (pMEK). C, Ser-338 phosphorylation and activation of RafY341D by Rap1. Cells were transfected with RafY341D along with either pcDNA3, RapE63, RapE63-HaCAAX(+hvr) or HaRasV12 and examined for Ser-338 phosphorylation (p338) and kinase activity of Raf-1 (pMEK). D, Raf-Rap1 chimeras are not constitutively active. Cells were transfected with either FLAG Raf-1 wild type (WT), Raf-Rap1CAAX, RafY341D-Rap1CAAX, or Raf-HaCAAX(+hvr). Cells were treated with EGF (+) or left untreated (-) and lysates immunoprecipitated using FLAG antibody and assayed for both Ser-338 phosphorylation (upper panel) and Raf-1 activity in vitro (middle panel). The levels of FLAGRaf expression are shown in the lower panel, using FLAG antibody. E, GFP epifluorescence. Cells were transfected with GFP-Raf-Rap1CAAX (a) or GFP-RafY341-Rap1CAAX (b). The locations of the transfected proteins were examined by epifluoresent microscopy, and representative cells are shown.

Activated Rap1 does not activate Raf1 (44). Rap1's inability to permit Ser-338 phosphorylation and activation of Raf-1 could be partially overcome by swapping the Rap1 CAAX domain with that of Ras (RapE63/Ha-RasCAAX) (Fig. 8B). Although RapE63 could not activate wild type Raf-1, it could activate RafY341D, as measured by Ser-338 phosphorylation and kinase activation (Fig. 8C). These data suggest that the inability of RapE63 to activate Raf-1 was due to the inability of Raf-1 to be correctly phosphorylated when recruited by Rap1, since Rap1 was capable of supporting Ser-338 phosphorylation in the Y341D mutation. These data also suggest that the inability of Rap1 to activate Raf-1 is not just a consequence of the interaction between Rap1 and Raf-1, as has been proposed (44) but may also be dictated by the localization of Rap1. However, Ha-RasV12 was better than RapE63-HaCAAX(+hvr) in activating both Raf-1 and RafY341D (Fig. 8, B and C), suggesting that sequences within Ras distinct from the carboxyl-terminal membrane-targeting domain are critical for maximal activation of Raf-1.

To examine the effect of relocalizing Raf-1 to Rap1-containing membranes, we generated chimeras of Raf-1 fused to the Rap1 carboxyl-terminal CAAX motif (Raf-Rap1CAAX) (Fig. 8D). Raf-Rap1CAAX was not constitutively active and could not be activated (Fig. 8D, middle panel) or phosphorylated on Ser-338 by EGF (Fig. 8D, upper panel). These data suggest that Raf-1 needs to be targeted to specific membranes in order to be activated and that Raf-1 targeting to Rap1-specific membrane domains does not support Ser-338 phosphorylation or activation. The inability of Rap1 to direct the proper phosphorylation of Raf-1/Rap chimeras could be overcome by introducing negative charges into Raf-1 at Tyr-341. Mutation of Tyr-341 to aspartate to generate RafY341D-RapCAAX increased the basal levels of both phosphorylation of Ser-338 and Raf-1 activation compared with Raf-Rap1CAAX (Fig. 8D), which were not further increased by EGF. In Fig. 8E, we show the subcellular localization of GFP-fusion proteins, GFP-Raf-Rap1CAAX (Fig. 8E, a), GFP-RafY341-Rap1CAAX (Fig. 8E, b). Both chimeras are largely localized to perinuclear regions, with little or no staining detected at the cell surface.

Rap1 Activation of B-Raf Requires Aspartic Acid at Residues Asp-447/Asp-448-- The Y341D mutation in Raf-1 resembles the naturally occurring sequence within the Raf isoform, B-Raf. B-Raf lacks tyrosines at the site corresponding to Tyr-341 in Raf-1 (448 in B-Raf). Instead, it contains aspartic acids at residues 447 and 448, that appear to mimic phosphorylation at these sites (27). Moreover, B-Raf was phosphorylated at the serine corresponding to Ser-338 in Raf-1 (Ser-445 in B-Raf) in resting cells (Fig. 9A, lane 1, upper panel), and remained phosphorylated at this residue in the presence of the raft-disrupting agent, CD (data not shown).


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Fig. 9.   The ability of B-Raf to be activated by Rap1 requires aspartic acid residues at positions Asp-447/Asp-448 in B-Raf. A, p445 phosphorylation and B-Raf activity assays. Cells were transfected with FLAGB-Raf wild type (B-Raf WT) or FLAGB-RafYY (B-RafYY) along with either pcDNA3, HaRasV12, or RapE63, as indicated. Lysates were subjected to FLAG immunoprecipitation and assayed for both p338 (upper panel) and Raf-1 activity in vitro (pMEK, middle panel). The levels of FLAGB-Raf expression are shown in the lower panel, using FLAG antibody. B, ERK activation by B-Raf requires targeting to either Rap1- or Ras-containing membranes. Cells were transfected with MycERK2 and either FLAG B-Raf WT, B-Raf-HaCAAX(+hvr), or B-Raf-Rap1CAAX and immunoprecipitated using FLAG antibody and assayed for Ser-445 phosphorylation (p445, upper panel). In the middle panel (pMycERK2), the total lysates were assayed for phosphorylation of MycERK2. The levels of FLAGB-Raf expression are shown in the lower panel, using FLAG antibody.

Unlike Raf-1, B-Raf can be activated by the small G protein Rap1 (44). Because of this, Rap1 can activate MEK and ERK in B-Raf-expressing cells (45-47). The ability of Rap1 to activate B-Raf is shown in Fig. 9A (lanes 1-3). Both constitutively active mutants of Ras (RasV12) and Rap1 (RapE63) could activate wild type B-Raf (B-RafWT) (Fig. 9A, lanes 2 and 3). In contrast to B-RafWT, expression of a mutant B-Raf in which the aspartic acid residues were mutated to the corresponding tyrosines residues in Raf-1 (B-RafYY) showed no basal phosphorylation on Ser-445 (Fig. 9A, lane 4). B-RafYY was no longer activated by constitutively active Rap1 (RapE63), as measured by both Ser-445 phosphorylation and kinase activity (lane 6). Ha-RasV12 stimulated Ser-445 phosphorylation and activity of the B-RafYY mutant (lane 5). When Rap1-CAAX sequences were coupled to B-Raf, the resulting chimera, B-Raf-Rap1CAAX, was phosphorylated on p445 and activated ERKs to a similar degree as B-Raf-HaCAAX(+hvr) (Fig. 9B). Although wild type B-Raf was constitutively phosphorylated on Ser-445 and displayed detectable constitutive kinase activity against MEK in vitro (Fig. 9A, lane 1), it could not activate ERKs unless it was targeted to Ras or Rap1 (Fig. 9B), reflecting the requirement of specific membrane targeting for the B-Raf activation of MEK/ERK in vivo. Therefore, we propose that B-Raf's ability to mimic phosphorylation at residues 447 and 448 is critical for its ability to be activated by Rap1.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multiple Ras Isoforms Localize Raf-1 to Raft Microdomains-- Both endogenous Ki-Ras and N-Ras were readily detected in COS-7 cells and localized to raft domains. In contrast, Ha-Ras was not detected in COS-7 cells, using isoform-specific antisera. This is consistent with the results of Kranenburg et al. (11), who also detected little or no Ha-Ras in COS-7 cells. In that study, Ki-Ras, largely, and N-Ras, partially, colocalized with caveolin. Colocalization with caveolin did not appear essential for activation as EGF activated N-Ras but not Ki-Ras in COS-7 cells (11). We show that the cholesterol-depleting agent CD blocked EGF activation of Raf-1 kinase activity and Ser-338 phosphorylation, consistent with CD's ability to block EGF activation of MEK/ERK in these cells (11). Although, it has been shown that CD potentiated activation of Ki-Ras (but not N-Ras), CD completely blocked the coupling of Ras to Raf-1/MEK/ERK (11).

Ser-338 Phosphorylation of Raf-1 Requires Targeting to Raft Microdomains-- Recent studies suggest that the ability of small G proteins to regulate signaling cascades is dictated not only by the specificity of effector utilization, but also by their subcellular localization (2). Differences in the localization of specific Ras isoforms within rafts has been reported by some (2) but not others (11). Paradoxically, disruption of rafts by CD could completely inhibit coupling to downstream effectors, while actually increasing the GTP loading of selected Ras isoforms (11). This may reflect the need for selected Ras isoforms to shuttle in and out of the raft (10, 48). In this study, we focused our attention on the requirement of raft localization not on Ras activation but activation of the proximal downstream effector Raf-1. We show that one of the functions of raft localization is that it permits phosphorylation of Raf-1 on Ser-338.

Localization of Raf-1 to rafts appears to be required for full activation of ERKs (12). Using the cholesterol-depleting agent CD, this group and others (11, 13) have shown that disruption of raft microdomains interferes with signaling of Raf-1 to ERKs. Raf-1 activation also requires phosphorylation at serine 338. This activating phosphorylation occurs within the plasma membrane for Raf-1, subsequent to Raf-1 recruitment to Ras (27). Using CD to disrupt rafts, we show that intact rafts are required for proper phosphorylation at 338. Therefore, the requirement of raft localization for full Raf-1 activity is coupled to Ser-338 phosphorylation, extending previous studies showing that the membrane-localized Ser-338 kinase was required for Raf-1 activation by oncogenic Ras (29). A candidate kinase, PAK, has been proposed (49-51); however, its role has been challenged (52).

CAV/DRG Does Not Disrupt Ras Activation of Raf-1 in COS-7 Cells-- A number of studies have demonstrated that at least two types of rafts exist; those that contain caveolin and those that do not (6, 11, 32). Caveolins are integral components of caveolae, 50-100-nm vesicular invaginations of the plasma membrane involved in vesicular trafficking and cell signaling (53). The role of caveolin in ERK signaling has received much recent attention, and both positive and negative affects on ERKs have been reported (9, 11, 54-57). Ras isoforms appear heterogeneous in their ability to couple to caveolins and to localize to caveolin-containing membranes. For example, in one study, Ki-Ras largely colocalized with caveolin in COS-7 cells, whereas N-Ras only partially colocalized with caveolin (11). In BHK cells, Ki-Ras was largely excluded from caveolin-containing membrane fractions (10).

The caveolin-3 mutant DGV (CAV/DGV) acts as an interfering mutant of caveolin function (10, 32). In BHK cells, this mutant inhibits Raf-1 activation by Ha-RasV12 but not by Ki-RasV12 (N-Ras was not examined) (9, 15). In these cells, CAV/DGV expression increased the buoyant density of Ras-containing membranes. The effect of this shift was reflected in the redistribution of caveolin and to a lesser extent of Ha-Ras, with no redistribution of Ki-Ras detected (9). This suggests that Ki-Ras, and to a lesser extent H-Ras, remains localized to low density raft domains even in the presence of CAV/DGV. This is likely true for N-Ras as well, since N-Ras only partially colocalizes with caveolin-containing membranes.

We have shown that disrupting caveolin function by overexpressing CAV3/DGV did not block the activation of Raf-1 by EGF, Ha-RasV12, Ki-RasV12, or N-RasV12 in these cells. Our finding that CAV3/DGV did not block EGF activation of Raf-1 supports the results of Kranenburg et al. (11), and may reflect the prominent role of endogenous Ki-Ras and N-Ras in EGF action in COS-7 cells. However, the inability of CAV/DGV to block the action of Ha-RasV12 on Raf-1 appears to conflict with the results of Roy et al. (9). This may reflect differences in the membrane compositions of the BHK cells (9, 10) and the COS-7 cells used in this study. For example, Ki-Ras, which this group and others (11) identified in raft microdomains in COS-7 cells, has been localized to non-raft domains in BHK cells (10).

In this study we compared the ability of distinct CAAX motifs to potentiate the phosphorylation and activation of a variety of chimeric Raf-1/CAAX proteins whose carboxyl-terminal domains were derived from Ha-Ras or Rap1. We show that a Raf-1 chimera that included the complete carboxyl-terminal membrane targeting domains from Ha-Ras was localized to rafts, showed both constitutive activity and phosphorylation of Ser-338, and activated ERKs. Chimeras containing only the minimal membrane-targeting motif [Raf-HaCAAX(-hvr)], however, had no basal activity. We suggest that the lack of activity of this chimera was a direct consequence of its inability to be phosphorylated on serine 338. The ability of Y341D to restore the raft localization and Ser-338 phosphorylation of Raf-1-HaCAAX(-hvr) and kinase activity argues that localization to specific raft microdomains may be necessary and sufficient for Ser-338 phosphorylation and activation of Raf-1. Recent studies have proposed that hvr sequences help shuttle Ha-Ras out of the rafts in a GTP-dependent fashion (10) and cooperate in effector utilization (16). Differences in the localization of mutant Ha-Ras proteins and Raf/Ras chimeras may be due to the influence of sequences in Ras mutants that are absent from the Raf/Ras chimeras. It is also possible that activated Ras shuttles Raf-1 into the raft where it is phosphorylated on Ser-338 and subsequently exits the raft, as suggested by recent studies (10, 48).

Phosphorylation of Tyr-341 Is Required for Proper Raft Localization and Subsequent Phosphorylation of Ser-338-- Upon EGF stimulation, RafS338A was localized to a raft microdomain. Moreover, RafS338A-HaCAAX(+hvr) was constitutively localized to a raft domain. These data demonstrate that Ser-338 phosphorylation was not required for raft localization, but likely occurs subsequently. This is consistent with a model that Raf-1 activation by Ha-Ras requires post-translational modifications, including Ser-338 phosphorylation, which occurs within specialized microdomains.

We show here that Tyr-341 phosphorylation of Raf-1 was a prerequisite for Ser-338 phosphorylation, consistent with previous results (27). Marais and co-workers (27) also showed that tyrosine phosphorylation by Src enhanced Ser-338 phosphorylation of Raf-1. The data presented here suggest that one of the consequences of Tyr-341 phosphorylation may be the repositioning of Raf-1 near potential Ser-338 kinases. The requirement of Tyr-341 in Raf-1 activation and Ser-338 phosphorylation, however, could be overcome by membrane targeting, suggesting that one of the functions of Tyr-341 phosphorylation is to facilitate proper membrane localization. Indeed, RafY341A mutants were unable to enter rafts upon EGF stimulation, unless linked to ectopic raft-targeting domains.

One explanation for the increased phosphorylation on Ser-338 seen in Y341D mutants is that this reflects the strong cooperativity between the phosphorylations of these sites (50). However, in studies examining the ability of PAK to phosphorylate Raf-1 in vitro, this was not the case (58). Another explanation is that Y341D mutants relocalize Raf-1 to sites of Ser-338 phosphorylation. Phosphorylation of Tyr-341 has been proposed to function in concert with pS338 to provide a negatively charged surface on the Raf-1 protein (27). We suggest that one additional function of phosphorylation of Tyr-341 that is distinct from that of Ser-338, is to target Raf-1 to specific membrane sites that participate in subsequent phosphorylations.

Rap1 Association with Raf-1 Is Not Sufficient for the Phosphorylation of Tyr-341-- The inability of some small G proteins to activate Raf-1 despite recruiting Raf-1 to the membrane also suggests that recruitment to the membrane is not sufficient for Raf-1 activation. One small G protein that binds Raf-1, without activating it, is Rap1 (44). Chimeric Ras/Rap1 proteins that replace membrane-targeting domains of Ras with those of Rap1 are growth inhibitory (42), but this inhibition can be relieved by constitutively active Raf-1, suggesting that the inhibitory effects of this chimera were due to impaired Raf-1 activation.

One proposed function for Ras is the displacement of the 14-3-3 protein from its binding site on residue 259 within Raf-1 (35). The inability to displace 14-3-3 from Raf-1 may explain the inability of selected G proteins to activate Raf-1 (59). However, for Rap1, such a model has been ruled out (59). This suggests that other mechanisms account for the inability of Rap1 to activate Raf-1. Studies have demonstrated that activation of endogenous Rap1 limits Ras activation of Raf-1 (33, 38, 44). It has been proposed that Rap1 interferes with Ras by trapping the Ras/Raf-1 complex in an inactive conformation (60, 61). However, recent studies have demonstrated that Ras and Rap1 occupy distinct subcellular regions (39, 41, 43), even following Rap1 activation (40).

In part because of its distinct location, Rap1 has been proposed to inhibit Ras activation of Raf-1 by sequestering Raf-1 from Ras. This is consistent with studies showing a loss of Ras/Raf-1 association (and a parallel increase in Rap1/Raf-1 association) upon Rap1 activation (38). Data presented here suggest a possible explanation for the inability of Rap1 recruitment of Raf-1 to activate Raf-1-the inability of Rap1 to support Raf-1 phosphorylations. First, Raf-1 chimeras that were targeted to Rap1-containing membranes via Rap1CAAX motifs were neither activated nor phosphorylated on Ser-338. Second, retargeting Rap1 by swapping in Ha-RasCAAX sequences allowed Rap1 to activate Raf-1 and to phosphorylate Ser-388. Mutation of Raf-1 to mimic Tyr-341 phosphorylation (Y341D) resulted in a Raf-1 protein that could be activated and phosphorylated on Ser-338 following Rap1 activation, suggesting that the phosphorylation on Tyr-341 can partially overcome Rap1's inability to activate Raf-1. This may be due to the lack of specific Tyr-341 kinases within Rap1 domains. Therefore, we propose that Rap1 prevents Raf-1 activation by positioning it away from tyrosine kinases that are required for Tyr-341 phosphorylation. One of the functions of Tyr-341 phosphorylation might be to provide a regulatable interaction with proteins or lipids to participate in proper targeting of Ras/Raf-1 (62, 63).

The Lack of Dependence of B-Raf on Tyrosine Phosphorylation Accounts for Its Activation by Rap1-- The Y341D mutation in Raf-1 resembles the naturally occurring sequences within the Raf isoform, B-Raf. B-Raf lacks a tyrosine at the site corresponding to Tyr-341 in Raf-1 (448 in B-Raf). Like Raf-1Y341D, B-Raf is constitutively phosphorylated on the nearby serine (Ser-338 in Raf-1, Ser-445 in B-Raf). Although B-Raf was constitutively active in in vitro kinase assays, we show that membrane recruitment was required to permit B-Raf to activate MEK and ERKs in vivo. Moreover, targeting of B-Raf chimeras via either Rap1-CAAX or Ras-CAAX was sufficient. The ability of B-Raf-Rap1 chimeras to activate ERKs confirms that the requirement for membrane localization for B-Raf activation by small G proteins is less stringent than that of Raf-1. The mutant of B-Raf in which Asp-447/Asp-448 was replaced by tyrosines (B-RafYY) behaved like Raf-1; it was no longer activated by Rap1, but retained the ability to be activated by Ras. The unique specificity of Rap1 for B-Raf activation, but not Raf-1 activation, can be largely explained by the distinct requirements of each kinase for specific membrane targeting for phosphorylation and activation. Future studies examining the ability of Rap1 to support additional critical phosphorylations, including Thr-491 and Ser-494 in Raf-1 (Thr-598 and Ser-601 in B-Raf) (28, 64) may be informative as well.

It has been proposed that sequences within the cysteine-rich domain (CRD) of Raf-1 and B-Raf dictated the contrasting actions of Rap1 on each Raf isoform (44). However, the ability of Rap1 to activate RafY341D, as well as the ability of Rap/Ras chimeras to activate wild type Raf-1, both argue strongly that the interactions between Rap1 and Raf-1 are not the only determinants of Raf-1 inhibition. It should be noted that Rap1/Ras chimeras were not as effective as Ras in activating/phosphorylating Raf-1, suggesting that Ras also provides an activation function that is distinct from localization (22, 59, 65). Furthermore, the lack of activation of B-RafYY by Rap1 suggests that interactions between the B-Raf CRD and Rap1 are also not sufficient to promote activation, although they may be important (44). We propose that the carboxyl-terminal domain of Rap1 provides specificity to Rap1 signaling in addition to that provided through the interaction between Rap1's effector loop and the Raf CRDs.

In conclusion, we show that Raf-1 phosphorylation at Ser-338 requires membrane targeting of Raf-1 to specific raft microdomains. We propose that tyrosine phosphorylation of Tyr-341 potentiates Ser-338 phosphorylation by facilitating proper membrane localization. This two-step mechanism is outlined in Fig. 10 and may explain the contrasting actions of Ras and Rap1 on Raf isoforms.


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Fig. 10.   A model of sequential phosphorylation of Raf-1 for full activation. EGF induces the recruitment of Raf-1 to plasma membrane-bound Ras. Following this association of Ras with Raf-1 two sequential modifications occur. The first modification is the phosphorylation of Tyr-341 by membrane-bound Tyr-341 kinases, whose activities are induced by EGF and/or Ras activation. This phosphorylation may relocalize the Ras/Raf-1 complex within specialized plasma membrane microdomains where a second phosphorylation on Ser-338 can occur that renders the Raf-1 molecule competent in phosphorylating downstream effectors like MEK/ERK. Rap1 signaling is depicted in the lower portion of the figure. Rap1 activation does not lead to phosphorylation of Raf-1 or activation of MEK/ERK, although Raf-1 is recruited to Rap1-containing membranes. In contrast, B-Raf is constitutively phosphorylated on Ser-445 (Ser-338 equivalent site), and the adjacent tyrosines in Raf-1 are replaced with aspartate residues (Y447D/Y448D), and therefore do not need to be phosphorylated upon recruitment to Rap1. In this case, Rap1 is capable of coupling B-Raf to MEK/ERK signaling. We suggest that the lack of Tyr-341 activity within Rap1 domains is the limiting step in Rap1's inability to activate Raf-1. Gray and white circles represent Tyr-341 kinases and Ser-338 kinases, respectively.


    ACKNOWLEDGEMENTS

We thank Brian Hunter for scientific expertise and technical assistance. We are grateful for the help of Brian Hunter, Tara Dillon, and Snigdha Mishra for critical reading of the manuscript, and Chris Fenner for secretarial assistance.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health Grant RO1 CA72971 (to P. J. S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Vollum Institute, L-474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-5494; Fax: 503-494-4976; E-mail: stork@ohsu.edu.

Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M207014200

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein; PVDF, polyvinylidene difluoride; HA, hemagglutinin; p338, phosphorylation of FLAGRaf-1 on Ser-338; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pMEK, phosphorylated MEK; WT, wild type; CD, methyl beta -cyclodextrin; hvr, hypervariable domain; CRD, cysteine-rich domain; EGF, epidermal growth factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; CAV, caveolin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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