Activation of H-ras61L-Specific Signaling Pathways Does Not Require Posttranslational Processing of H-ras

Experimental Cell Research 257, 89 –100 (2000) doi:10.1006/excr.2000.4874, available online at http://www.idealibrary.com on

Activation of H-ras 61L-Specific Signaling Pathways Does Not Require Posttranslational Processing of H-ras Kristen C. Hart, Scott C. Robertson, and Daniel J. Donoghue* ,1 Department of Chemistry and Biochemistry and *Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0367

ample, targeting of H-ras to the plasma membrane involves farnesylation of Cys186, cleavage of the last three amino acids (187–189), methylation of the exposed C-terminal Cys residue, and palmitoylation of Cys181 and Cys184 (reviewed in [4]). Mutation or overexpression of ras protein is implicated in approximately 30% of all human tumors. K-ras is mutated in 80 –90% of pancreatic tumors and 30 – 60% of lung and colon carcinomas. N-ras mutations are implicated in a significant portion of human leukemias and cancers of the liver, skin, and thyroid. H-ras protein is often mutated in skin, prostate, thyroid, or small intestine cancers (reviewed in [5, 6]). Posttranslational modifications of ras proteins are critical for their proper plasma membrane localization. Therefore, a significant amount of research has been devoted to the development of therapeutic agents that inhibit these processing steps. These drugs include farnesyltransferase inhibitors (FTI), CAAX peptide and chemical mimetics, farnesyl pyrophosphate analogues, and various natural products, many of which are designed to prevent the initial farnesyl modification of ras proteins, especially H-ras (reviewed in [7–10]). While it is clear that posttranslational modifications are necessary for targeting this family of proteins to appropriate membranes, it is less certain whether these modifications are important for activation of signaling pathways. For example, the ras-related protein RhoB regulates actin stress fiber formation and is localized to internal vesicles and the nuclear membrane via geranylgeranyl and farnesyl lipid moieties [11, 12]. While a nonprenylated RhoB can no longer transform cells, it is still able to activate transcription through the serum response element (SRE), present in the promoter of c-fos and other genes [13]. This suggests that at least some signaling functions of small GTPases are independent of their posttranslational modifications and/or localization. In the case of H-ras, posttranslational modifications may be required for activation of c-Raf-1 and B-Raf [14, 15]. Also, recent work suggests that H-ras farnesylation is important for in vitro activation of p110␥, but not p110␣, PI 3-kinase activity [16]. It has been shown that for K-ras4B, C-terminal

We have previously demonstrated that H-ras 61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras 61L-induced activation of the Raf/ MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras 61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras 61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras 61L. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications. © 2000 Academic Press Key Words: H-ras; MAPK cascade; posttranslational modifications; protein localization; signal transduction.

INTRODUCTION

The ras family of small GTP-binding proteins regulate a number of processes, including proliferation, differentiation, cytoskeletal structure, and intracellular trafficking [1–3]. This family includes H-ras, K-ras4A and -4B, and N-ras, which are localized to the inner surface of the plasma membrane by various posttranslational modifications and amino acid motifs. For ex1

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processing is required for activation of MAPK in a cell-free system [17]. Several other studies also point to a role for the lipid modifications of small GTP-binding proteins in mediating protein–protein interactions [18, 19]. Mutations which abolish the lipid modification sites also result in mislocalization of ras to the cytosol or other intracellular membranes [20, 21]; thus, it has been difficult to distinguish the role of posttranslational modifications in activation of ras effectors from their crucial role in localizing ras to the plasma membrane. In an approach first used by Lacal et al. (1988) [22], and later by Buss et al. (1989) [23], H-ras was targeted to the plasma membrane using an N-terminal myristylation signal, demonstrating that myristylation can substitute for C-terminal lipid modifications and restore ras 61L transforming activity. Subsequently, our laboratory demonstrated that a transmembrane anchor domain could be used to target H-ras derivatives to the plasma membrane with retention of significant transforming potential, even in the absence of full posttranslational processing [24]. It has become clear that the combined activation of several signal transduction pathways is required for ras-mediated transformation [25–29]. To clarify the signaling role of H-ras posttranslational modifications, we examined the ability of transmembrane domainanchored H-ras 61L derivatives containing the “3S” C-terminal mutation, which abolishes all posttranslational processing [30, 31], to activate the MAPK cascade, the Rac/Rho pathway, and the PI 3-kinase pathway and induce neurite outgrowth in PC12 cells and the expression of the immediate-early gene c-fos. Our results demonstrate that while the posttranslational modifications of H-ras may enhance interactions with (or activation of) downstream effectors, they are not required for H-ras 61L signaling. MATERIALS AND METHODS Plasmid constructs. Several of the H-ras constructs utilized in this work were described previously [24], although their nomenclature has been changed to allow their names to more closely reflect their function; the clones TM(3)-ras 61L-3S, TM(22)-ras 61L-SAAX, and TM(22)-ras WT-SAAX are identical to the clones previously described [24] as E1(QI)-3-61L-3S, E1(QI)-22-61L-SAAX, and E1(QI)-22-WTSAAX, respectively. To generate the TM(3)-ras WT-3S derivative, the HindIII–FspI fragment of pKH079 (TM(3)-ras 61L-3S) was swapped into the HindIII– FspI vector fragment from pKH074 (TM(3)-ras WT-3S) [24]. To generate the new “3S” derivatives with a 22-amino-acid linker region, the HindIII–FspI fragments from plasmids pKH088 (TM(22)-ras WTSAAX) and pKH089 (TM(22)-ras 61L-SAAX) were swapped into the HindIII–FspI vector fragment from either pKH074 (for TM(22)ras WT-3S) or pKH079 (for TM(22)-ras 61L-3S). Mutations in all constructs were verified by dideoxy nucleotide sequencing. c-Raf-1 in pcDNAI/Amp was a kind gift of Dr. M. Urich. pFL700 plasmid, encoding the luciferase gene under control of the c-fos promoter (⫺700 nt), was a gift from Dr. Q. Hu. GST-p42 MAPK expression plasmid pEBG-ERK2 was a generous gift from Dr. I. Sanchez. GST-

MAPK and GST-MEK bacterial expression vectors were generously provided by Dr. C. J. Marshall. Focus assays. Focus-forming assays using NIH3T3 cells were performed as previously described [24]. Subcellular fractionation. 293T cells were transfected by calcium phosphate precipitation, as previously described [24], and then starved overnight in 0.2% FBS. The following day, cells were collected, washed with PBS, and resuspended in HB-EDTA (10 mM Tris, pH 7.4, 2 mM EDTA) plus inhibitors (1 mM sodium vanadate, 1 mM PMSF, 1 mM sodium fluoride, 2.25 ␮g/ml aprotinin, 1.0 ␮g/ml leupeptin) on ice for 15 min and then lysed by homogenization in a tight-fitting Dounce homogenizer (50 strokes). Nuclei were removed by centrifugation for 5 min at 2500 rpm. NaCl was added to the supernatant to a final concentration of 0.15 M, and the lysates were centrifuged at 100,000g for 30 min at 4°C. The supernatant was collected as the cytoplasmic fraction (C), while the pellet or membrane fraction (M) was washed twice with HB-EDTA and transferred to an Eppendorf tube. Both fractions were microcentrifuged for 5 min and then the supernatant of C fractions was transferred to new tubes, and the supernatant of M fractions discarded. To C fractions, 10⫻ TNE (500 mM Tris, pH 8.0, 10% NP-40, 20 mM EDTA) was diluted 1/10 and 10% SDS was diluted 1/100. M fractions were resuspended in 1⫻ TNE plus 0.1% SDS, 10⫻ HB-EDTA was added to 1⫻, and NaCl added to a final concentration of 0.15 M. Both sets of fractions were vortexed very well, microcentrifuged for 5 min at 4°C, and quantitated using Bio-Rad protein assay reagent. Equivalent amounts of protein for each fraction were loaded and separated by SDS–PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis as described below. Indirect immunofluorescence. NIH3T3 cells were plated on glass coverslips, transfected, and processed for immunofluorescence as previously described [24]. All antibodies were diluted in 3% BSA/PBS, and all incubations performed at room temperature. Coverslips were mounted on glass slides with 90% glycerol in 0.1 M Tris, pH 8.5, plus phenylenediamine to prevent fading and photographed using a Nikon Microphot-FXA, fitted with a cooled CCD camera (Hamamatsu C5810). To determine the localization of ras derivatives, rat mAb Y13-238 (Ab-2, Oncogene Science) was used (1:500) and detected with rhodamine-conjugated goat anti-rat secondary. For colocalization studies of ras derivatives with transfected c-Raf-1, cells were starved overnight before fixation, then incubated with a mixture of rat mAb Y13-238 ras antiserum (1:500) and rabbit polyclonal C-12 Raf antiserum (1:500, Santa Cruz), and detected with a mixture of rhodamine-conjugated goat anti-rat (ICN) and fluorescein-conjugated goat anti-rabbit (Boehringer-Mannheim) secondary antibodies. Images of cells for Fig. 3 were overlaid using Adobe Photoshop 4.0. For ruffling experiments, REF-52 cells were transfected with ras derivatives as described for transformation assays [24] and then starved overnight before fixation. Immunofluorescence was performed as described for localization of ras derivatives, except that fluorescein-conjugated phalloidin (1:1000, Sigma) was included in the secondary antibody. For MAPK translocation experiments, NIH3T3 cells were cotransfected with pEBG-ERK2 and ras derivatives and starved overnight before fixing. In this case, coverslips were incubated with a mixture of primary antisera, including Y13-238 ras antibody (1:500) and rabbit polyclonal GST antibody Ab-1, (1:200, Oncogene Science). The secondary antibody mixture contained Texas red-conjugated goat anti-rat (ICN) and fluorescein-conjugated goat anti-rabbit sera (Boehringer-Mannheim). For neurite outgrowth experiments, PC12 rat pheochromocytoma cells were maintained on collagen-coated plates (Biocoat, Becton–Dickinson) in RPMI 1640 plus 10% heatinactivated horse serum and 5% fetal bovine serum, plus antibiotics. Cells were plated onto polyethylenimine-coated glass coverslips and transfected using Effectene (Qiagen). Immunofluorescence reactions were performed as described above for localization of H-ras derivatives, 3– 4 days posttransfection. Immune complex/in vitro kinase assays. To examine Raf activation, COS-1 cells were transfected as previously described [24] with

SIGNALING BY H-ras 61L DERIVATIVES 10 ␮g of indicated ras derivatives. Cells were starved overnight, harvested in 500 ␮l 1⫻ PBS, centrifuged for 15 min at 4°C, then lysed in 1 ml of RFR buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 50 mM ␤-glycerophosphate, 1 mM PMSF, 1 ␮g/ml pepstatin A, 1 ␮g/ml leupeptin, 2.25 ␮g/ml aprotinin, and 1 mM sodium vanadate). Ten microliters of Raf antibody (C-12, 100 ␮g/ml; Santa Cruz) was added, and the lysates were incubated at 4°C with rotation for 2 h or overnight. Protein A–Sepharose was added, and the mixture was incubated for an additional 1 h. Immune complexes were washed three times with RFR buffer, once with NP-40 lysis buffer (20 mM Tris, pH 7.5, 137 mM NaCl, 1% NP-40, 1 mM sodium vanadate, 5 mM EDTA, 10% glycerol, 10 ␮g/ml aprotinin), and once with buffer D [32]. One-half of the immunoprecipitate was used in the kinase assay, which was performed as previously described [32]. A portion of the remaining immunoprecipitate was resolved by SDS–PAGE and examined by immunoblotting with Raf antibody to confirm equal amounts of c-Raf-1 in each reaction. Lysates were also blotted for ras to confirm equivalent expression of each construct, and immunofluorescence was also used to verify ras protein expression. This assay was repeated three times with similar results. For PI 3-kinase assays, 293T cells were transfected with H-ras derivatives, starved overnight, washed twice in buffer A (137 mM NaCl, 20 mM Tris, pH 7.5, 1 mM MgCl 2, 1 mM CaCl 2, 100 ␮M sodium vanadate), and then lysed in lysis buffer (buffer A plus 1% NP-40, 10% glycerol). Equivalent amounts of lysate were immunoprecipitated with anti-v-H-ras sera (Ab-2, Oncogene Science) and collected with Protein G-PLUS Agarose (Santa Cruz Biotechnology). Beads were washed thrice with PBS/1% NP-40, twice with 0.1 M Tris, pH 7.5/0.5 M LiCl, and twice with TNE (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA), all with 100 ␮M sodium vanadate. Twenty micrograms per reaction of PI (Avanti Polar Lipids) in CHCl 3 was dried, resuspended in lipid sonicating solution (10 mM Hepes, pH 7.5, 1 mM EGTA), and sonicated for 5 min in a water bath sonicator. Twenty microliters of immunoprecipitated sample was combined with 10 ␮l lipid solution, 10 ␮l ATP solution [20 ␮Ci [ 32P]ATP (3000 Ci/mM), 40 ␮M ATP, 10 mM MgCl 2, 20 mM Hepes, pH 7.0], and 60 ␮l TNE, mixed, and incubated at room temperature for 10 min. The reaction was stopped by adding 20 ␮l 8 N HCl and 160 ␮l 1:1 CHCl 3/MeOH, then vortexed, centrifuged for 30 s, and the bottom layer saved. Equal amounts of sample were spotted onto TLC plates (silica gel 60, Merck) coated with 10% potassium oxalate, and chromatography was performed in the following solvent system: CHCl 3/MeOH/H 2O/NH 4OH 60:47:11.3:2. TLC plates were air-dried and exposed to film. Immunoblotting. 293T cells were transfected as described above, starved overnight, then harvested in 1 ml RFR buffer, collected into Eppendorf tubes, lysed by vortexing, and centrifuged for 15 min at 4°C. Equivalent amounts of clarified lysates were analyzed by SDS– PAGE, transferred to nitrocellulose, and blotted using PhosphoMAPK antisera (NEB), ERK1/ERK2 antisera (Zymed) or ras antisera (Ab-2, Oncogene Science). For c-Raf-1 immunoblots, C-12 Raf polyclonal antiserum (Santa Cruz) was used. Immunoreactive proteins were visualized using HRP-conjugated secondary antibodies and enhanced chemiluminescence (Amersham). Reporter assays. NIH3T3 cells were transfected with 2 ␮g of pFL700 fos-luciferase reporter plasmid and 8 ␮g of ras plasmids as described above. After transfection (18 –20 h), plates were rinsed once with TS and starved for approximately 48 h in DME plus 0.2% bovine calf serum. Cells were rinsed twice in cold PBS, lysed in 250 ␮l of Reporter Lysis Buffer (Promega), and subjected to luciferase assays according to manufacturer’s instructions.

RESULTS

Description of ras Derivatives To examine the role of posttranslational modifications in the signaling activity of H-ras, transmembrane

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FIG. 1. Diagram of transmembrane domain-anchored H-ras derivatives. The TM-ras constructs are targeted to the plasma membrane using a transmembrane domain anchor as described previously [24]. The black boxed region represents a linker region of either 3 amino acids (designated “TM(3)”) or 22 amino acids (designated “TM(22)”) in length which separates the transmembrane domain from the H-ras domain. Derivatives were made of both wild-type H-ras (designated “ras WT”) and an oncogenic version, which contains the point mutation Gln61 3 Leu (designated “ras 61L”). All derivatives contain the “3S” mutation, in which Cys181, Cys184, and Cys186 of H-ras are mutated to Ser. Note that the entire H-ras portion of the fusion proteins is always localized in the cytoplasm of the cell.

domain-anchored derivatives were constructed from ras WT or ras 61L, an oncogenic mutant of H-ras (Fig. 1). These constructs are similar to those previously described [24]; however, for convenience note that the constructs previously referred to as E1(QI) derivatives, which denoted the origin of the transmembrane domain, are now referred to as TM-ras derivatives instead. The derivatives described here contain mutations that change the three C-terminal Cys residues (Cys181, Cys184, and Cys186) to Ser (designated 3S). These mutations abolish the normal posttranslational processing of H-ras by preventing the crucial first step, farnesylation of Cys186 [30, 31], and also remove the upstream palmitoylation sites. In order to target the TM-ras constructs shown in Fig. 1 to the plasma membrane, we exploited the first transmembrane domain of the E1 protein of infectious bronchitis virus (IBV) as an integral membrane protein anchor. The E1 protein is normally localized to the cis-Golgi membranes, which is specified by the sequence of the first of three transmembrane domain regions in E1. When a point mutation (Gln37 3 Ile) is introduced, this transmembrane domain effectively targets proteins to the plasma membrane [33, 34]. The TM-ras derivatives are oriented with the N-terminus of the ras-derived domain near the inner surface of the plasma membrane, in contrast

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to H-ras, which is normally attached to the plasma membrane via farnesyl and palmityl modifications at the C-terminus. However, the crystal structure of H-ras predicts that the N-terminus and C-terminus of H-ras are normally in close proximity [35], so the difference in orientation between normal H-ras and the TM-ras fusion proteins should not be a factor. While the N-terminus of the TM-ras targeting sequence is actually extracellular, the entire ras portion of the fusion proteins is always in the cytosol (Fig. 1). As previously reported, TM-ras derivatives of ras 61L were able to transform cells even when C-terminal processing is blocked by mutation, provided that a linker of sufficient length is introduced between the membrane anchor and the ras protein [24]. In this study, a subset of TM-ras WT-3S or TM-ras 61L-3S derivatives containing either the inactive 3-amino-acid linker (previously described [24]) or the active 22-amino-acid linker (new for this study) were used (designated TM(3) or TM(22), respectively). To verify the transforming activity, NIH3T3 cells were transfected with the ras constructs and subjected to a focus forming assay. The plasma membrane-targeted derivative of ras 61L, TM(22)-ras 61L-3S, exhibited focus forming activity in transformation assays, which averaged approximately 60% of lipidated ras 61L activity (data not shown). All other transmembrane domain-anchored derivatives used in this study were inactive in transformation assays, even when they carried the activating 61L mutation. TM-ras Derivatives Are Targeted to the Plasma Membrane To confirm that the transmembrane domain-anchored derivatives of H-ras are indeed targeted to the membrane, subcellular fractionation experiments were performed. 293T cells transfected with a subset of the constructs were separated into C and M fractions, and the localization of H-ras protein in these fractions was examined by immunoblotting. As evidenced by Fig. 2A, ras 61L protein is found almost exclusively in the membrane fraction of cell lysates, as are the two TM-ras derivatives, TM(3)-ras WT-3S and TM(22)-ras 61L-3S. In contrast, ras 61L-3S, a cytosolic and inactive derivative of H-ras, is localized primarily in the cytoplasm. The localization of H-ras derivatives was also examined by immunofluorescence microscopy. The staining pattern for TM-ras derivatives of ras WT or ras 61L is similar to that seen for lipidated, processed ras WT and ras 61L (data not shown; see also Figs. 3, 6, 8, and 10). The TM-ras fusion proteins are processed through the ER/Golgi apparatus, so some immature protein is detectable within the secretory pathway of positive cells, in addition to the mature protein that has reached the plasma membrane. In contrast, cellular H-ras is cytosolic until modified by processing enzymes, when it

FIG. 2. Subcellular localization of H-ras derivatives and endogenous c-Raf-1. NIH3T3 cells transfected with various H-ras derivatives were separated into cytosolic (C) and membrane (M) fractions and analyzed by SDS–PAGE and immunoblotting. (A) Immunoblot of transfected H-ras proteins; (B) immunoblot of endogenous c-Raf-1.

then associates with the plasma membrane. Considered together, the biochemical fractionation and immunofluorescence experiments described here confirm that the TM-ras derivatives localize appropriately to the plasma membrane. Recruitment of Raf to Membranes One of the critical effectors of ras is the serine/threonine kinase c-Raf-1, whose activation involves recruitment to the plasma membrane by GTP-bound ras [36, 37]. It has been previously demonstrated that c-Raf-1 is associated with the plasma membrane of cells transformed by oncogenic ras [38]. To determine whether TM-ras derivatives lacking C-terminal lipid modifications retain the ability to recruit Raf, doublelabel immunofluorescence was performed on cells cotransfected with ras 61L derivatives and human c-Raf-1. To verify that the two proteins were indeed colocalized, images of coexpressing cells were superimposed to produce the overlay images in Fig. 3. c-Raf-1 was cytoplasmic in unstimulated or serum-starved cells (Fig. 3A), displaying no obvious plasma membrane staining or colocalization with Golgi elements, which were detected by staining with fluorescein-conjugated lectin (Fig. 3B). Cells expressing ras 61L caused a relocalization of co-expressed c-Raf-1 to the plasma membrane (Fig. 3C and 3D). When Figs. 3C and 3D were overlaid, this colocalization was even more evident (Fig. 3E). The integral membrane derivative of ras, TM(22)ras 61L-3S, was also able to recruit c-Raf-1 to the plasma membrane (Figs. 3F and 3G). These images, representative of several independent experiments, indicate that the TM-ras derivative retains the ability to recruit c-Raf-1 to the plasma membrane. To verify the immunofluorescence results, biochemical fractionation experiments were also performed. In

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FIG. 3. Colocalization of TM(22)-ras 61L-3S derivative with c-Raf-1. NIH3T3 cells were cotransfected with the membrane-targeted TM(22)ras 61L-3S clone and c-Raf-1 and subjected to immunofluorescence. (A and B) c-Raf-1 is normally localized in the cytoplasm of unstimulated cells, in a pattern distinct from intracellular membranes such as the Golgi apparatus; red staining, anti-Raf; green staining, Lens culinaris lectin. (C–E) ras 61L recruits c-Raf-1 to the plasma membrane; red staining, anti-ras; green staining, anti-Raf; yellow staining, resulting overlay of C and D. (F–H) TM(22)-ras 61L-3S is also able to recruit c-Raf-1 to the plasma membrane. The staining patterns for TM(22)-ras61L-3S are virtually identical to those seen for ras 61L; red staining, anti-ras; green staining, anti-Raf; yellow staining, resulting overlay of F and G.

cells transfected with various H-ras derivatives, there was a clear and significant recruitment of endogenous c-Raf-1 protein to the membrane fraction of cells expressing the active derivatives ras 61L and TM(22)ras 61L-3S (Fig. 2B). Expression of TM(3)-ras WT-3S, or the cytosolic mutant ras 61L-3S, did not cause a relocalization of c-Raf-1 to the membrane fraction (Fig. 2B). These results are consistent with the immunofluorescence data shown in Fig. 3 and indicate that cellular c-Raf-1 protein can be recruited to the plasma membrane by TM(22)-ras 61L-3S, as is true for ras 61L. The nontransforming derivative of ras, TM(3)ras 61L-3S, was correctly localized to the plasma membrane but was unable to recruit c-Raf-1 to the plasma membrane (data not shown). In addition, ras WT and its integral membrane derivatives did not cause a relocalization of c-Raf-1 to the plasma membrane (data not shown), indicating that c-Raf-1 recruitment was specific to the activated form of H-ras. Moreover, since the H-ras derivative TM(22)-ras 61L-3S contains C-terminal mutations that prevent normal lipid modifications, we conclude that posttranslational modifications at the

C-terminus of H-ras were not required for plasma membrane recruitment of c-Raf-1. Activation of Raf Once recruited to the plasma membrane by ras, Raf becomes activated by a complex series of modifications, which may involve both tyrosine and serine phosphorylations [39 – 41], regulatory interactions with 14-3-3 proteins [42, 43], and interaction with lipids and second messengers [44 – 46]. To determine if Raf protein recruited to the plasma membrane by TM(22)-ras 61L-3S was indeed activated, in vitro kinase assays were performed. Endogenous c-Raf-1 was immunoprecipitated from serum-starved cells expressing H-ras derivatives and then incubated with MEK and MAPK in a coupled assay. MAPK activity was determined using [␥- 32P]ATP and myelin basic protein as a substrate and represents a measure of the activity of the immunoprecipitated c-Raf-1 protein. The extent of c-Raf-1 kinase activation above mock-transfected cells is shown in Fig. 4 and was normalized to the amount of c-Raf-1 in

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FIG. 4. Kinase activation of c-Raf-1 in cells expressing TM-ras derivatives. Kinase activity of c-Raf-1 immunoprecipitated from starved COS-1 cells transfected with the indicated H-ras derivatives was assayed in a coupled in vitro kinase assay, using MBP as a final substrate. A portion of the immune precipitate was subjected to immunoblotting to confirm the presence of c-Raf-1 in each sample. The graph presents in vitro kinase activity normalized to Raf protein levels in the samples as fold activation over that of mock-transfected cells. This assay was performed three times with similar results.

each sample. The transforming derivative of ras 61L (TM(22)-ras 61L-3S) stimulated c-Raf-1 kinase activity approximately fivefold over that seen in mock-transfected cells (Fig. 4). This was approximately 50% of the activity observed in ras 61L-transfected cells (Fig. 4). Ras protein expression was also examined by immunoblotting of whole cell lysates and found to be equivalent in each sample (data not shown). These results suggest that, once recruited to membranes by ras, c-Raf-1 activation does not require normal C-terminal lipid modifications or other processing events of H-ras.

Mock-transfected cells, or cells expressing ras 61L-3S, a cytoplasmic and inactive derivative, did not exhibit any detectable phosphorylated MAPK forms (Fig. 5A). Similar levels of MAPK expression were observed for all samples examined (Fig. 5B), as determined by antiERK1/2 immunoblot analysis. The expression level of each ras derivative was also examined by immunoblotting using ras-specific antisera (Fig. 5C). In this particular experiment, ras 61L was expressed at a significantly lower level than TM(22)-ras 61L-3S. However, it was still enough to activate the MAPK cascade. Once activated, MAPK translocates from the cytoplasm to the nucleus [52], where it phosphorylates transcription factors, such as Elk-1 and Sap-1a [53]. To examine changes in the localization of MAPK, doublelabel immunofluorescence was performed on cells cotransfected with ras derivatives and a GST-ERK2 (p42 MAPK) construct. GST-p42 MAPK was localized within the cytoplasm of serum-starved cells (Fig. 6B). The transforming derivative TM(22)-ras 61L-3S stimulated translocation of GST-p42 MAPK to the nucleus (Figs. 6I and 6J), as did ras 61L (Figs. 6E and 6F). The nontransforming derivative TM(3)-ras 61L-3S did not relocalize MAPK to the nucleus, as reflected by the cytoplasmic staining of GST-p42 MAPK (Figs. 6G and 6H). ras WT did not cause translocation of GST-p42 MAPK to the nucleus (Figs. 6C and 6D). TM-ras derivatives of ras WT were also inactive in this assay (data not shown). The recruitment and activation of Raf, in addition to the robust translocation of MAPK to the nucleus by the nonprocessed TM(22)-ras 61L-3S derivative, demonstrates that posttranslational modifications are not necessary for H-ras-mediated activation of the MAPK cascade.

Activation of MAPK Cascade by ras Derivatives Raf activation leads to phosphorylation and activation of MEK [47], the upstream activator of MAPK. Activation of the two mammalian forms of MAPK (p42 and p44) involves phosphorylation of both threonine and tyrosine residues by the dual-specificity kinase activity of MEK [48 –51]. To determine whether the c-Raf-1 kinase activity observed in the in vitro assays above correlated with an in vivo activation of the MAPK pathway, 293T cells were transfected with several H-ras derivatives, and cell lysates were analyzed for the presence of phosphorylated forms of MAPK. Cells expressing either ras 61L or the plasma membranelocalized derivative TM(22)-ras 61L-3S contained activated MAPK, as determined by immunoblotting using an antibody specific for the phosphorylated forms of p42 MAPK and p44 MAPK (Fig. 5A), indicating that the MAPK pathway was indeed activated in these cells.

FIG. 5. Activation of MAPK in response to TM(22)-ras 61L-3S and ras 61L. 293T cell lysates expressing derivatives of H-ras were analyzed for phosphorylated forms of MAPK by SDS–PAGE and immunoblotting. (A) Anti-phospho-MAPK blot; (B) anti-MAPK (ERK1/2) blot; (C) anti-H-ras blot. Arrows to the right indicate the positions of phosphorylated (pp) and nonphosphorylated (p) forms of MAPK in A and B and processed and unprocessed forms of ras 61L in C. The bracket in C indicates the position of the TM(22)-ras 61L-3S protein.

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of reporter gene activity was induced by ras 61L when compared to mock-transfected cells (Fig. 7). Approximately 70-fold induction of luciferase activity was generated by expression of TM(22)-ras 61L-3S, which is unable to undergo posttranslational modifications (Fig. 7). The other derivatives of ras WT or ras 61L tested did not exhibit any significant activation of the reporter plasmid (Fig. 7). Ras protein levels were found to be approximately equivalent, as examined by immunoblotting of lysates and by indirect immunofluorescence (data not shown). These results suggest that only plasma membrane localization of H-ras, not C-terminal processing events such as lipidation and methylation, is necessary for induction of immediate-early gene expression. Activation of Membrane Ruffling and PI 3-Kinase Another pathway involved in transformation by ras is the Rac/Rho pathway, which controls cytoskeletal actin organization, cell morphology, membrane ruffling, and stress fiber formation [2, 3]. To examine whether these ras-mediated cytoskeletal changes require the presence of posttranslational modifications, REF-52 cells transfected with H-ras derivatives were starved overnight and then stained with fluoresceinconjugated phalloidin, an actin-binding mushroom toxin. In addition, the same cells were also stained with ras-specific antibodies and examined by doublelabel immunofluorescence. As shown in Fig. 8, mocktransfected cells exhibited an organized, regular actin filament network and no membrane ruffles (Figs. 8A

FIG. 6. Translocation of p42MAPK into the nucleus in response to TM-ras derivatives. NIH3T3 cells cotransfected with expression plasmids encoding H-ras constructs and GST-p42MAPK were starved and then fixed and processed for double-label immunofluorescence. (Left column) Anti-ras staining; (right column) anti-GST staining. (A and B) Cells expressing GST-p42 MAPK alone; (C and D) coexpression of ras WT and GST-p42 MAPK; (E and F) coexpression of ras 61L and GST-p42 MAPK; (G and H) coexpression of TM(3)-ras 61L-3S and GST-p42 MAPK; (I and J) coexpression of TM(22)-ras 61L-3S and GST-p42 MAPK.

Transcriptional Activation by ras 61L Derivatives Immediate-early genes, such as fos and jun, are induced by a number of growth factors, and fos expression is known to be stimulated by H-ras [54]. To examine whether immediate-early genes were expressed in response to ras derivatives lacking C-terminal modifications, luciferase assays were performed using a reporter plasmid consisting of 700 bases of the c-fos promoter driving expression of a luciferase gene. NIH3T3 cells were transiently cotransfected with H-ras derivatives and the fos reporter plasmid, starved, lysed, and assayed for luciferase activity. A 240-fold stimulation

FIG. 7. Induction of c-fos promoter-driven gene expression by TM-ras derivatives. NIH3T3 cells were cotransfected with various H-ras derivatives and a fos-luciferase reporter plasmid, starved, and then subjected to luciferase assay. Results presented are fold induction of the reporter gene compared to that of mock-transfected cells and were repeated three times with similar results. Error bars represent the standard deviation derived from triplicate readings of each sample.

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lipid modification at the C-terminus of H-ras are also not necessary for activation of PI 3-kinase. To determine this more directly, we performed an in vitro PI 3-kinase assay using H-ras immunoprecipitates from ras-transfected cells. We detected an increase in H-rasassociated PI 3-kinase activity only in cells expressing ras 61L or TM(22)-ras 61L-3S, when compared to mocktransfected cells (Fig. 9A). Protein expression levels were verified by Western blotting of cell lysates (Fig. 9B). These results show that PI 3-kinase activation by H-ras is at least partially lipid-independent. Induction of Neurite Outgrowth in PC12 Cells Activated H-ras is known to be a potent inducer of neurite outgrowth in PC12 cells [57], an effect commonly used to assess the ability of a protein to stimulate differentiation pathways. Sustained activation of the MAPK pathway is thought to be a crucial event for this response [58, 59]. While TM(22)-ras 61L-3S can activate the MAPK pathway, as discussed above, we also wished to determine whether this derivative would induce the sustained activation required for initiation of this differentiation pathway. PC12 cells were transfected with several H-ras derivatives and analyzed by immunofluorescence microscopy for neuron-like processes in the transfected cells. Figure 10 shows representative cells from these experiments. Formation of neurites was evident as early as 48 h posttransfection FIG. 8. Membrane ruffling induced by H-ras derivatives. REF-52 cells were transfected with indicated H-ras derivatives, starved, and then subjected to immunofluorescence. (Left column) Anti-ras staining; (right column) fluorescein-conjugated phalloidin staining of actin. (A and B) Mock-transfected cells; (C and D) ras 61L; (E and F) TM(3)-ras 61L-3S; (G and H) TM(22)-ras 61L-3S. Arrows in D and H indicate areas of ruffling and/or lamellipodia.

and 8B). Expression of ras 61L led to changes in the cytoskeleton, including membrane ruffling and the appearance of focal complexes (arrows, Figs. 8C and 8D). The transforming derivative TM(22)-ras 61L-3S invoked similar cytoskeletal changes in these cells (Figs. 8G and 8H). The TM(3)-ras 61L-3S derivative did not result in any cytoskeletal changes (Figs. 8E and 8F). Expression of ras WT or TM(22)-ras WT-3S had no effect on the cytoskeleton (data not shown). These results demonstrate that changes in the cytoskeletal organization, mediated through the Rac/Rho pathway, can be stimulated by H-ras 61L in the absence of C-terminal lipid modifications. Cytoskeletal changes induced by H-ras also involve activation of the PI 3-kinase pathway [29], which leads to activation of the Akt and S6 kinases and provides yet another way for ras to regulate cellular responses to growth factor stimulation [55, 56]. The membrane ruffling induced by TM(22)-ras 61L-3S suggests that the

FIG. 9. H-ras-associated PI 3-kinase activity in cells expressing various H-ras derivatives. (A) 293T cells were transfected with the indicated constructs, immunoprecipitated with v-H-ras antisera, and subjected to an in vitro kinase assay using phosphatidylinositol (PI) as a substrate. The arrows indicate the positions of the origin and PIP product. (B) Cell lysates were also subjected to Western blotting with the same antisera, to confirm protein expression.

SIGNALING BY H-ras 61L DERIVATIVES

FIG. 10. TM(22)-ras 61L-3S induces potent neurite outgrowth in PC12 cells. PC12 cells transfected with various H-ras derivatives were subjected to immunofluorescence analysis to examine neurite outgrowth. (A) Mock-transfected cells; (B) ras 61L-3S; (C) TM(3)-ras WT3S; (D) TM(22)-ras 61L-3S; (E) H-ras 61L.

in cells expressing either TM(22)-ras 61L-3S or ras 61L (Figs. 10D and 10E). Nearly all of the cells positive for these ras proteins exhibited neurite outgrowth, and some cells were very flattened in appearance, with large lamellipodia. In contrast, cells expressing TM(3)ras WT-3S or ras 61L-3S remained very rounded and refractile, similar to the mock-transfected cells (Figs. 10A–10C). These results indicate that lipid modifications of H-ras are also not required for sustained activation of the MAPK pathway and induction of neurite outgrowth in this assay. DISCUSSION

The processing events that contribute to ras protein lipidation and membrane binding are well understood (reviewed in [4]); however, several questions remain concerning their function. Several elegant studies have demonstrated that C-terminal lipid modifications of H-ras are important for efficient membrane association and transformation [21–23]. Prevention of these lipid modifications, however, also results in loss of plasma membrane association, rendering it difficult to determine the role that the lipid moieties themselves play in signaling mediated by H-ras. Derivatives of H-ras which contain an N-terminal myristylation signal derived from either viral gag or src proteins [22, 23] demonstrated that alternative membrane localization signals could restore the transforming activity of a nonprenylated H-ras mutant, H-ras186S, which lacks the farnesylation site at Cys186. However, subsequent work demonstrated that myristylated H-ras186S was able to undergo palmitoylation, despite a lack of farnesylation [21, 60]. The ras 61L constructs described here

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extend these earlier studies in two significant ways: (1) by efficiently targeting a ras 61L derivative to the plasma membrane using a transmembrane domain anchor instead of a lipid anchor and (2) by eliminating posttranslational processing of H-ras by using the “3S” mutation described previously [30, 31]. Using these transmembrane domain-anchored ras derivatives, we demonstrate that a ras 61L derivative, TM(22)-ras 61L-3S, containing the 3S mutation which abrogates posttranslational processing and lipidation, can stimulate activation of the MAPK pathway, the Rac/Rho and PI 3-kinase pathways, induction of c-fos gene expression, and neurite outgrowth in PC12 cells. Taken together, these results demonstrate that posttranslational modifications of ras, which include farnesylation, cleavage, methylation, and palmitoylation, are not absolutely required for ras activity and primarily serve as plasma membrane anchor motifs. One of the major pathways through which ras triggers downstream signaling events is the MAPK pathway, which is stimulated by the membrane recruitment and subsequent activation of Raf by ras. Plasma membrane-localized derivatives of Raf containing the C-terminal membrane targeting sequences of K-ras were found to be activated [36, 37], suggesting that the primary role of ras is to bring Raf to the membrane, facilitating activation by other mechanisms. Our results clearly demonstrate that recruitment of Raf by H-ras does not depend on the presence of lipid modifications, but only on the localization of ras to the plasma membrane. However, there is evidence that, at least in vitro, farnesylation of H-ras significantly increases its ability to activate c-Raf-1 and B-Raf [14, 15]. Also, recent studies suggest that the Raf zinc finger domain contributes to optimal binding of ras, by interacting with a noneffector loop epitope present only on prenylated ras [61, 62]. Thus, H-ras lipid modifications, while not absolutely required, may facilitate or enhance activation of the Raf/MAPK pathway in vivo. In virtually all of the assays performed, TM(22)ras 61L-3S was a less potent activator of signaling pathways than lipidated ras 61L. For example, in the Raf kinase assay in Fig. 4, the TM(22)-ras 61L-3S derivative is only 42% as active as ras 61L. Similarly, induction of fos gene expression by TM(22)-ras 61L-3S, as shown in Fig. 7, measures ⬃30% of that seen with ras 61L. The other assays are less quantitative in nature. While our results confirm that lipid modifications are not required for ras 61L activity, they equally suggest that lipid modifications enhance the ability of ras 61L to activate or interact with its downstream effectors. Whether this is through direct interaction of farnesyl or palmityl lipid moieties with other proteins or due to a specific conformation mediated by C-terminal modification is not clear at this time. We have demonstrated that the posttranslational modifications which serve to anchor H-ras to the

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plasma membrane are not required for the activation of several downstream pathways. However, lipid modification of proteins may serve alternative functions in addition to appropriate membrane targeting. For example, prenylation of a novel plant calmodulin, CaM53, directs its localization to either the plasma membrane or the nucleus, allowing the protein to coordinate different functions in response to metabolic changes [63]. In mammalian cells, detergent-resistant membrane rafts (DRMs) function in the formation of caveolae and plasma membrane invaginations, the latter playing a role in signal transduction, endocytosis, and cholesterol trafficking. Recent experiments demonstrated that DRMs preferentially incorporate acylated proteins, those with palmitoylation and/or myristylation, and contain very few prenylated proteins, such as Rab5 and Rap1 [64]. Recently, Choy et al. [65] examined the endomembrane trafficking of N-ras, K-ras, and H-ras using GFP-tagged derivatives. They found that the CAAX motif was necessary and sufficient to target ras proteins to peri-Golgi vesicles, where they underwent methylation, one of the initial posttranslational steps in ras protein processing. The four ras homologues (H-ras, N-ras, and Ki-ras4A and -4B) differ significantly in their abilities to activate Raf, stimulate cell migration, and induce transformation [66]. Since the proteins are identical in their Switch I and II regions, differences in activity may reflect the variety in C-terminal sequences and posttranslational processing. These differences may mediate alternative plasma membrane localization and/or association with guanine nucleotide exchange factors or other effector proteins. Thus, the C-terminal region of ras proteins can, in some cases, confer targeting information to highly specific regions of the plasma membrane. Although our results demonstrate that H-ras does not require reversible membrane association in order to function, the potential reversibility of lipid modifications [67] also renders this a versatile method for regulating protein function and localization in vivo. An important factor in regulating the activity of transmembrane domain-anchored H-ras derivatives involves the length of the linker region between the transmembrane domain and the H-ras domain. For example, derivatives containing a linker region only 3 amino acids in length are completely inactive [24; this study]. This likely reflects the inability of the ras fusion protein to interact with key substrates, due to its close proximity to the inner surface of the plasma membrane. Longer linker regions, such as the 11-aminoacid linker used previously [24] and the 22-amino-acid linker used in the present study, would more closely mimic the flexible C-terminus of H-ras; this C-terminal domain is ␣-helical in nature [68, 69] and, in conjunction with normal lipid modifications, may provide for a natural linker or spacer. We cannot exclude the possibility that the orientation of N-terminally anchored ras

differs from that of native H-ras in a manner that affects its function. However, the crystal structure of H-ras predicts that the N-terminus is normally in close proximity to the C-terminal portion [35], suggesting that a membrane anchor at either end of the protein would result in a similar orientation with respect to the membrane. Since ras proteins play such a significant role in the progression of human tumors, it is crucial to understand the many factors contributing to their activity. It is clear from this and many other studies that localization to the plasma membrane is an absolute requirement for ras protein function. Therefore, many groups have labored to develop farnesyltransferase and geranylgeranyltransferase enzyme inhibitors which could abrogate plasma membrane association of ras proteins and revert the transformed phenotype of cells. Understanding the role of lipid modifications in all aspects of ras signaling is necessary for the development of more effective cancer treatment strategies. Transmembrane domain-anchored derivatives of lipidated proteins provides a versatile method for investigating the functions of posttranslational modifications of other membrane-associated proteins. All of our experiments have been conducted with H-ras derivatives, but similar studies could also be performed to examine N-ras or K-ras for the importance of posttranslational modifications and C-terminal poly-basic motifs. Compared to myristylation, a transmembrane anchor provides stable membrane association, allowing examination of the importance of reversible membrane association for protein function. The transmembrane domain anchor can also be adapted to target proteins to other specific cellular membranes. For example, the wild-type E1 transmembrane domain sequence can very efficiently target proteins to the early Golgi membranes [33, 34]. This could be useful in studying the function of Rab1, which is localized to the ER and Golgi membranes [70]. Resident Golgi enzymes called glycosyltransferases have well-characterized transmembrane domain anchor motifs, which could be used to target proteins to all regions of the secretory pathway (reviewed in [71]). The merging of these two fields of study—lipid processing of proteins and membrane anchoring motifs— could provide significant advances in the understanding of posttranslational lipid modifications. We thank Jim Feramisco for valuable suggestions concerning ruffling experiments and microscopy and Laura Castrejon for editorial assistance. We also thank the following people for reagents provided: Jan Buss, Qianjin Hu, Chris Marshall, Irma Sanchez, George Smith III, and Marc Urich. We thank Darren Kamikura for assistance with cellular fractionation protocols. For helpful advice on Raf kinase assays, we thank both Deborah Morrison and Chris Marshall. This work was funded by Grant RPG-95-080-03-CSM from the American Cancer Society and Grant 5 RO1 DE12581 from the National Institutes of Health.

SIGNALING BY H-ras 61L DERIVATIVES

require palmitoylation or a polybasic domain for plasma membrane localization. Mol. Cell. Biol. 14, 4722– 4730.

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