RI-Mediated Signaling Ε Proteins in Fc Geranylgeranylated And

MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in Fc εRI-Mediated Signaling This information is current as of September 26, 2021. Timothy E. Graham, Janet R. Pfeiffer, Rebecca J. Lee, Donna F. Kusewitt, A. Marina Martinez, Terry Foutz, Bridget S. Wilson and Janet M. Oliver J Immunol 1998; 161:6733-6744; ; http://www.jimmunol.org/content/161/12/6733 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in Fc⑀RI-Mediated Signaling

Timothy E. Graham,* Janet R. Pfeiffer,* Rebecca J. Lee,2* Donna F. Kusewitt,† A. Marina Martinez,* Terry Foutz,* Bridget S. Wilson,* and Janet M. Oliver3*

Cross-linking the high affinity IgE receptor Fc⑀RI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2؉ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated ⑀ proteins in the Fc RI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the Downloaded from farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates Fc⑀RI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to Fc⑀RI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase C␥-mediated inositol (1,4,5) trisphosphate production, sustained http://www.jimmunol.org/ -Ca2؉ influx, and Ca2؉-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insen sitive) proteins in Fc⑀RI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2؉ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2؉ release from intracellular stores and secretion. We identified no clear role for Ras in Fc⑀RI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin. The Journal of Immunology, 1998, 161: 6733–6744.

n rat basophilic leukemia cells (RBL-2H3), cross-linking the others. Protein-tyrosine phosphorylation in turn activates a signaling by guest on September 26, 2021 ⑀ high affinity receptor for IgE (Fc RI) activates the receptor- cascade leading to inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) produc- I associated tyrosine kinases, Lyn and Syk, as well as Bruton’s tion (10, 11), Ca2ϩ mobilization (12, 13), Ras activation (14), and the tyrosine kinase (1–3), and causes the tyrosine phosphorylation of activation of the ERK and JNK MAP kinases (8, 15–17). These bio- multiple substrates, including immunoreceptor tyrosine-based ac- chemical and ionic responses lead to functional responses, including ␤ ␥ ␣␤␥ tivation motifs in the and subunits of the heterotrimeric ( 2) secretion, actin polymerization, membrane ruffling, the assembly of Fc⑀RI itself (4); two phospholipase C␥ (PLC␥)4 isoforms, PLC␥1 actin plaques implicated in cell spreading, and increased cytokine pro- and PLC␥2 (5); the guanine nucleotide exchange factor Vav (6); duction (reviewed in Ref. 18). phosphatidylinositol 3-kinase (7, 8); the adaptor protein Grb2 (9); and Previously, we used lovastatin to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the pathway to isoprenoid and cholesterol biosynthesis (19, 20). ϩ Departments of *Pathology and †Cell Biology and Physiology and Cancer Research This treatment inhibits Ag-stimulated 45Ca2 influx, Ins(1,4,5) and Treatment Center, University of New Mexico Health Sciences Center, Albuquer- P production, secretion, ruffling, and spreading in RBL-2H3 que, NM 87131 3 cells. The signaling pathway was restored by the addition of Received for publication June 15, 1998. Accepted for publication August 28, 1998. mevalonate, which is a precursor of the farnesyl and gera- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance nylgeranyl pyrophosphate substrates of protein prenyltrans- with 18 U.S.C. Section 1734 solely to indicate this fact. ferases. Neither dolichol nor cholesterol, downstream of 1 This work was supported in part by National Institutes of Health Grants GM50562 isoprenoid synthesis and metabolism in the cholesterol biosyn- (to B.S.W.) and GM49814 and HL56384 (to J.M.O.). T.E.G. was supported in part by thetic pathway, restored signaling responses. These studies American Cancer Society Institutional Research Grant 192 and by a Howard Hughes Medical Institute Medical Student Research Training Fellowship. identified critical roles for isoprenylated proteins in the cou- ⑀ 2 Present address: Department of Molecular and Cellular Biology, University of Cal- pling of Fc RI cross-linking to biochemical and functional re- ifornia, Berkeley, CA 94720. sponses. However, they could not determine whether the active 3 Address correspondence and reprint requests to Dr. Janet M. Oliver, Department of species belonged to the relatively limited group of proteins with Pathology, Cell Pathology Division, University of New Mexico Health Sciences Cen- C-terminal CAAX motifs that are modified by farnesylation ter, Suite 201, 2325 Camino de Salud, Albuquerque, NM 87131. E-mail address: [email protected] (mostly Ras isoforms but also Rap2, RhoB, the ␥ subunit of trans- 4 ␥ ␥ ducin, type 1 Ins(1,4,5)P 5-phosphatase, and others) (21–23) or to the Abbreviations used in this paper: PLC , phospholipase C- ; Ins(1,4,5)P3, inositol- 3 1,4,5-trisphosphate; ERK, extracellular signal-regulated kinase; MAPK, mitogen-ac- larger group of proteins with C-terminal CAAX, CC, or CXC motifs tivated protein kinase; MEK, MAPK/ERK kinase; GST, glutathione S-transferase; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; MBP, myelin basic protein; that are modified by geranylgeranylation (most of the Rho, Rab, and JNK, c-jun NH2 kinase. Ral family members; reviewed in Ref. 24).

Here, we use the benzodiazepine-based farnesyltransferase in- For Southern blot analysis of genomic ras, DNA was isolated from hibitor BZA-5B described by James et al. (25) to analyze the con- supernatants remaining after mRNA isolation by the MicroFastTrack kit as tributions of farnesylated proteins to Fc⑀RI signaling and, by com- follows: the EDTA concentration of the supernatant was brought to 20 mM, and proteinase K was added to a final concentration of 0.5 mg/ml; the parison with lovastatin, to identify roles for geranylgeranylated mixture was incubated overnight at 45°C with shaking; and DNA was then proteins in the signaling pathway. BZA-5B is a CAAX peptido- extracted with phenol/chloroform and precipitated with ethanol. DNA was mimetic that competes for the farnesyltransferase linking a C15 also isolated from the liver of a Wistar rat (Harlan Sprague-Dawley, Indi- farnesyl group to a cysteine within carboxyl-terminal CAAX se- anapolis, IN). DNA samples (3 ␮g each) were digested to completion with the restriction endonucleases BamHI, EcoRI, and HindIII (Life Technolo- quences on specific proteins. Like lovastatin, BZA-5B inhibits the gies), size fractionated in 0.8% agarose, and transferred to GeneScreen- farnesylation and Ag-induced activation of most Ras species (26, Plus. DNA blots were probed with radiolabeled v-H-ras, v-K-ras, and 27). Unlike lovastatin, BZA-5B and other CAAX peptidomimetics N-ras probes as described above. have little or no inhibitory activity toward type I geranylgeranyl- To detect alternatively spliced forms of K-ras, mRNA prepared from ϫ 6 transferase, which catalyzes the posttranslational geranylgeranyla- adherent RBL-2H3 cells (20 10 /cells) was reverse transcribed to cDNA using the RiboClone System (Invitrogen, San Diego, CA). This cDNA, as tion of proteins, especially Rho and Ral family members, the well as cDNA from Wistar rat brain (a gift of Dr. K. K. Caldwell, Uni- CAAX box of which usually ends in a C-terminal leucine residue. versity of New Mexico), was denatured and amplified by PCR using a They are also inactive against type II geranylgeranyltransferase, single sense primer from exon 1 (5Ј-TGTGGTAGTTGGAGCTGGTGG- which acts on the Rab proteins that have C-terminal CC or CXC 3Ј) and antisense primers reflecting either the exon 4A alternative splice form (5Ј-AATTTTCACACAGCCAGGAGT-3Ј) or the exon 4B alternative sequences (reviewed in Refs. 28 and 29). splice form (5Ј-GTACACCTTGTCCTTGACTTC-3Ј). As a positive con- Our results show that lovastatin and BZA-5B prevent both the trol, glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was amplified farnesylation and Ag-induced activation of the Ras isoforms found from the same cDNA using commercial primers (Clontech Laboratories, Downloaded from in RBL-2H3 cells. Neither inhibitor substantially affects Fc⑀RI- Palo Alto, CA). Amplified products were size fractionated by electrophore- mediated tyrosine kinase activation, the earliest event in the Fc⑀RI- sis in a 1% agarose formaldehyde gel containing ethidium bromide (0.5 ␮g/ml). Sizes of DNA bands were estimated based on the mobilities of coupled signaling pathway, or the activation of MEK and ERK1/ DNA standards (Life Technologies). ERK2, which are downstream of Ras in many cell types. Both To detect transforming mutations in H-ras species expressed in RBL- inhibitors block cell division. However, a series of lovastatin-sen- 2H3 cells, cDNA was prepared from RBL-2H3 mRNA, and exons 1 and 2

sitive responses to Fc⑀RI cross-linking were either unaffected or of the H-ras gene were amplified by PCR using the primers of Wang et al. http://www.jimmunol.org/ (32). The amplified material was cloned into the Bluescript vector (Strat- enhanced by BZA-5B. Analysis of these similarities and differ- agene) and used to transform Escherichia coli SURE cells (Stratagene); ences suggests Ras-independent pathways to MEK and ERK ac- colonies were screened using a radiolabeled v-H-ras probe (Oncor). Nu- tivation in RBL-2H3 cells and both predicted and novel roles for cleotide sequence was determined using a Sequenase version 2.0 sequenc- farnesylated and geranylgeranylated proteins in Fc⑀RI-coupled ing kit (United States Biochemical, Cleveland, OH) with the vector-spe- signaling. cific primers recommended by the manufacturer. Exons 1 and 2 of the K-ras gene were similarly amplified using previously described primers (33–35) and sequenced. Materials and Methods Reagents Ras prenylation by guest on September 26, 2021 The farnesyltransferase inhibitor BZA-5B and its inactive analogue RBL-2H3 cells were incubated for 2 h with BZA-5B or carrier. The me- BZA-7B were generous gifts of Dr. James Marsters (Genentech, San Fran- dium was then supplemented with 10 ␮M lovastatin to inhibit the endog- cisco, CA). Stock solutions (100 mM) of BZA-5B and BZA-7B were pre- enous production of mevalonic acid, plus 10 ␮Ci/ml [3H]mevalonolactone pared in DMSO and stored at Ϫ20°C. For use, BZA-5B and BZA-7B were (40–60 Ci/mmol; American Radiolabeled Chemical, St. Louis, MO), and diluted into normal saline containing 100 mM reduced glutathione (used to incubation continued for 20 h. Cells were lysed in 1 ml of lysis buffer A (10 maintain BZA-5B in its reduced form) as carrier and then further diluted in mM Tris, pH 7.2; 50 mM NaCl; 20 mM sodium pyrophosphate; 50 mM culture medium to final concentrations of 1 mM reduced glutathione and NaF; 2 mM iodoacetamide; 5 ␮M ZnCl; 0.5% Triton X-100; 100 ␮M ␮ 0.1% DMSO. Lovastatin was solubilized as described in Kita et al. (30). NaVO4; 0.1% BSA; 10 g/ml aprotinin and leupeptin; and 1 mM PMSF) Mouse monoclonal anti-DNP IgE was prepared as described in Liu et al. and Ras proteins immunoprecipitated from the clarified lysates with the (31). The MEK inhibitor PD98058 was from BioMol (Plymouth anti-Ras mAb Y13-259 prebound to protein A-Sepharose (Pharmacia, Pis- Meeting, PA). cataway, NJ). Beads were washed with buffer A. Immunoprecipitated proteins were separated by SDS-PAGE on 25-cm 12–15% gradient gels, and Cell culture, drug treatment, and activation the gels were incubated with a fluorographic enhancer (Amersham, Arlington Heights, IL) and exposed for 20 days to X-OMAT MR film RBL-2H3 cells were grown as adherent monolayers on tissue culture flasks Ϫ in MEM (Life Technologies, Grand Island, NY) supplemented with 15% at 70°C for fluororadiography. Alternatively, the incorporation of [3H]mevalonolactone product into total immunoprecipitated protein was FCS, penicillin-streptomycin, and L-glutamine. For microscopy, cell mono- ␮ layers were cultured on 13-mm glass coverslips. For secretion assays, cell determined by boiling the beads for 5 min in 500 l of 1 N HCl and monolayers were grown in 24-well tissue culture plates. Unless otherwise measuring radioactivity from Ras-bound isoprene groups by liquid scintil- noted, incubations were for 24 h with 10 ␮M lovastatin or for 72 h with 10 lation counting. or 100 ␮M BZA-5B; control cells were incubated with carrier alone. In most experiments, IgE receptors were primed by the addition of 1 ␮g/ml Ras activation anti-DNP-IgE during the last 12–14 h of incubation. After excess IgE was washed away, cells were activated with 0.01–1.0 ␮g/ml DNP-BSA (Mo- Drug-treated RBL-2H3 cells were transferred to inorganic phosphate-free lecular Probes, Eugene, OR). medium, with 10% dialyzed FCS plus IgE and drugs and/or carrier for 2 h, followed by a 2-h incubation in inorganic phosphate-free medium supple- 32 Characterization of Ras isoforms in RBL-2H3 cells mented with 333 ␮Ci/ml [ P]orthophosphate (Amersham). Cells were activated for 2 min with 1 ␮g/ml DNP-BSA and washed with ice-cold PBS,

For Northern blot detection of ras expression, mRNA was prepared from and 1 ml of lysis buffer B (50 mM Tris, pH 7.4; 10 mM MgCl2; 500 mM ϳ50 ϫ 106 RBL-2H3 cells using the MicroFastTrack mRNA isolation kit NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; and 0.05% sodium (Invitrogen, San Diego, CA). mRNA (1.75 ␮g) was size fractionated by dodecyl sulfate) was added. Ras proteins were immunoprecipitated using electrophoresis in 1.2% agarose formaldehyde gels and transferred to mAb Y13-259 and washed with modiﬁed buffer B (0.1% Triton X-100 and GeneScreenPlus (NEN-Dupont Research Products, Boston, MA). v-H-ras, 0.005% SDS) followed by 5 mM Tris-phosphate (pH 7.4). Radiolabeled v-K-ras, and N-ras human cDNA probes (Oncor, Gaithersburg, MD) were guanine nucleotides were eluted from the immunoprecipitated proteins radiolabeled by random priming (Prime-It II kit, Stratagene, La Jolla, CA). with 5 mM Tris-phosphate, 2 mM EDTA, and 2 mM DTT, pH 7.0. Carrier Hybridizations followed the membrane manufacturer’s instructions. Blots nucleotides were added to the eluate and the mixtures separated by one- were exposed to X-OMAT AR ﬁlm (Eastman Kodak, Rochester, NY) at dimensional TLC on polyethyleneimine-cellulose plates (36). Guanine nu- Ϫ70°C using an intensifying screen. cleotides (GMP, GDP, and GTP) on dried plates were localized under UV The Journal of Immunology 6735

light. Dried plates were exposed to phosphor screens (Molecular Dynam- Cell proliferation and apoptosis analyses ics, Sunnyvale, CA) for 3–4 days and scanned, and radioactivity in GTP and GDP spots was quantified using ImageQuant software (Molecular Dy- Effects of prenylation inhibitors on cell growth were determined by [3H]thymidine incorporation assays. Cells were harvested at 24-h intervals namics). Data were expressed as the percentage total Ras in the GTP-bound 5 form using the following equation: % GTP-Ras ϭ cpm in GTP/cpm in and counted, and 10 cells were incubated in suspension for1hat37°C ␮ 3 (GTP ϩ 1.5 GDP). with 3.5 Ci of nonmethylated 6-[ H]thymidine (Amersham) in a total of 0.35 ml. The cells were collected by centrifugation and washed, and the Protein-tyrosine phosphorylation final pellet was solubilized in 0.25 ml 10% SDS plus 0.25 ml 1 N NaOH. Radioactivity in aliquots of the solubilized material was determined by ␮ 32 RBL-2H3 cells were radiolabeled with 100–200 Ci/ml [ P]orthophos- liquid scintillation counting. Cell viability was determined in the same phate, activated with DNP-BSA, and lysed as described for Ras activation. ␮ cell preparations by addition of trypan blue to a separate sample of each Clarified lysates were incubated overnight with 1 g of affinity-purified, culture and observation of dye exclusion in a light microscope. Apo- polyclonal anti-phosphotyrosine Ab (generated by J. Potter and G. Deanin, ptosis was measured by staining cells in Krishan buffer (0.1% sodium University of New Mexico, as described in Ref. 37) precoupled to protein citrate, pH 7.4, containing 0.3% Nonidet P-40, 0.005% propidium io- A-Sepharose beads (Pharmacia). Phosphoproteins were eluted from the dide, and 0.02 mg/ml ribonuclease A). Fluorescence was measured us- beads with 1 mM phenylphosphate in the presence of 0.01% OVA and the ing a Becton Dickinson FACSscan flow cytometer with CellFIT soft- protease inhibitors described above. Phosphoproteins were separated by ware. BD Modfit software was used to identify uniform sub-G0/G1 10% SDS-PAGE and detected by autoradiography. peaks representing apoptotic cells. Raf-1, MEK, and ERK activation Results RBL-2H3 cells were lysed with 1 ml of ice-cold lysis buffer C (25 mM HEPES, pH 7.5; 150 mM NaCl; 0.5% Triton X-100; 0.5% Brij-96; 0.1 mM RBL-2H3 cells express nononcogenic H-ras,K-rasA, and K-rasB,

EGTA; 1 mM NaVO3; and protease inhibitors). Kinases were immunopre- but not N-ras Downloaded from cipitated from clariﬁed lysates using kinase-speciﬁc polyclonal Abs from Santa Cruz Biotechnology (Santa Cruz, CA; C-12 for Raf-1, C-18 for Fig. 1A shows the results of Northern blot analyses to detect ras MEK; C-4 for ERK1/ERK2), all prebound to protein A-Sepharose beads. species in RBL-2H3 mRNA preparations. A single band with ap- Beads were washed three times in ice-cold buffer C, twice more in the same parent molecular mass of ϳ1.5 kDa was detected when mRNA buffer but containing 0.05% Triton X-100 and 0.05% Brij-96, and once in blots were hybridized with a radiolabeled H-ras-directed probe. A ␮ 25 mM HEPES, pH 7.5. Kinase reactions were initiated by adding 40 l K-ras-directed probe revealed a single band with apparent molec- per sample of reaction buffer (30 mM Tris-Cl, pH 7.5; 15 mM MgCl2; and

ϳ http://www.jimmunol.org/ 0.1 mM EGTA) containing 3 ␮g per sample kinase-dead MEK (glutathione ular mass of 1.8 kDa. No bands were detected with a N-ras- S-transferase (GST)-MEK; K97A; Upstate Biotechnology, Lake Placid, directed probe (data not shown). NY) for Raf-1, kinase-dead ERK (GST-ERK; K91A; a kind gift of Dr. In Fig. 1B, PCR amplification of cDNA was used to detect K- Alan Saltiel, Parke-Davis, Ann Arbor, MI) for MEK, myelin basic protein ras splice variants in mRNA from RBL-2H3 cells and Wistar rat (MBP; Sigma, St. Louis, MO) for ERK, and 10 ␮Ci per sample [␥-32P]ATP (Redivue; Amersham). After 20 min at 30°C, reactions were brain. RBL-2H3 cells contain both K-ras4a and K-ras4b, whereas terminated by the addition of 10 ␮lof8ϫ Laemmli buffer and boiling for rat brain contains only K-ras4b. Fig. 1C shows Southern blot anal- 10 min. Proteins were separated by 12% SDS-PAGE and phosphoproteins ysis of H- and K-ras genomic DNA from RBL-2H3 cells, derived visualized by autoradiography. Data were quantified using a Phosphor- originally from a Wistar rat mastocytoma, and Wistar rat liver. Imager (Molecular Dynamics). Restriction fragment maps generated from genomic DNA from by guest on September 26, 2021 Ins(1,4,5)P3 assays both sources are identical, suggesting that no major mutation or Levels of Ins(1,4,5) P in TCA extracts of activated cells were determined rearrangement of H- and K-ras occurred in the cell line. Exons 1 3 and 2 of oncogenic ras genes typically encode proteins with amino using the Ins(1,4,5) P3-specific radioreceptor assay of Challiss et al. (38) with modifications described in Ref. 8. acid substitutions at residues 12, 13, 59, or 61 (41, 42). These Microscopy mutant Ras proteins are constitutively maintained in the GTP- bound (active) state. Direct nucleotide sequencing of these con- RBL-2H3 cell monolayers were fixed in 2% glutaraldehyde and dehydrated served sites established that neither H-ras nor K-ras has trans- and carbon coated as previously described (39). Surface topography was observed with a Hitachi S800 scanning electron microscope. forming mutations in RBL-2H3 cells (data not shown). IL-4 production BZA-5B inhibits Ras farnesylation in RBL-2H3 cells RBL-2H3 cells (ϳ50 ϫ 106 cells/condition) were incubated for 2 h without Newly prenylated Ras species were detected from the incorpora- ␮ or with 1 g/ml DNP-BSA and then lysed, and mRNA was isolated as tion of [3H]mevalonolactone into Y13-259-precipitable proteins in above using the FastTrack mRNA isolation kit. cDNA was prepared by ␮ reverse transcription, denatured, and amplified for 35 cycles (1 min at the presence of 10 M lovastatin, which inhibits the de novo pro- 94°C, 1 min at 53°C, and 2 min at 72°C) in the presence of PCR primers duction of isoprene precursors. mAb Y13-259 immunoprecipitated for IL-4 (sense, 5Ј-TTTAGGCTTTCCAGGAAGT-3Ј; antisense, 5Ј-GAG a doublet of newly synthesized, prenylated Ras from [3H]meval- ATCATCAACACTTTGAAC-3Ј) or G3PDH (control). Amplified prod- onolactone-labeled control cells (Fig. 1D); separate experiments ucts were size fractionated by 1% agarose gel electrophoresis in the pres- ␮ (not shown) using isoform-specific Abs established that the upper ence of 0.5 g/ml ethidium bromide. Sizes of DNA bands were estimated ␮ based on the mobilities of DNA standards (Life Technologies); the pre- band is H-Ras, and the lower band is K-Ras. BZA-5B (100 M) dicted product for IL-4 is ϳ300 bp. blocked incorporation of [3H]mevalonolactone products into this Degranulation doublet of Ras proteins by greater than 70% (Fig. 1D; compare lanes 1 and 2). Y13-259 Western blots of replicate Y13-259 im- RBL-2H3 cell monolayers in 24-well plates (ϳ2 ϫ 105 cells per well) were 3 mune complexes (not shown) detected very similar levels of H- loaded overnight with [ H]serotonin (400 nCi/ml; NEN-Dupont). Secretion and K-Ras protein in control and BZA-5B-treated cells. Thus, the was measured from the release of this preloaded mediator as described 3 (40). Results are reported as percentage of total [3H]serotonin released in reduced signal from [ H]mevalonolactone labeling is due to re- 20 min by duplicate samples and are corrected for spontaneous release duced protein prenylation, not to reduced Ras protein concentra- from unstimulated cells in the same set. tions. Indeed, BZA-5B-treated cells probably overexpress Ras pro- Single-cell Ca2ϩ measurements teins. This was indicated by the presence in anti-Ras immunoblots

2ϩ of BZA-5B-treated cells of an additional higher molecular mass [Ca ]i mobilization was measured by ratio imaging microscopy of fura- 2-loaded cells as previously described (13). Each experiment provided band that was completely nonprenylated but showed cross-reac- time-resolved analyses of Ca2ϩ levels for 6–10 individual cells within a tivity with anti-ubiquitin Abs. This band, representing perhaps single ﬁeld of view. 10% of the total signal in anti-Ras blots, presumably represents 6736 Fc⑀RI SIGNALING PATHWAYS AND PRENYLATION INHIBITORS Downloaded from http://www.jimmunol.org/

FIGURE 1. Analysis of ras isoform expression and farnesylation in RBL-2H3 cells. A, Identification of major ras isoforms by Northern blotting. H-ras- and K-ras-directed probes identified single bands. No bands were observed with the N-ras-directed probe (not shown). B, RBL-2H3 cells express both K-rasA and K-rasB mRNA. Rat brain (control) and RBL-2H3 cell cDNA preparations were amplified using primers specific for regions of K-ras exon 4a (lanes 1 and 2) and K-ras exon 4b (lanes 4 and 5). G3PDH was used as a positive control (lanes 7 and 8) and primers only as negative controls (lanes by guest on September 26, 2021 3, 6, and 9). C, Analysis of genomic H- and K-ras DNA by Southern blotting. No H- or K-ras RFLP was identified between Wistar rat liver genomic DNA and RBL-2H3 genomic DNA. D, BZA-5B inhibits Ras farnesylation. Ras proteins were immunoprecipitated from lysates of control (lane 1) and 100 ␮M BZA-5B-treated (lane 2)[3H]mevalonolactone-labeled cells, separated by SDS-PAGE, and imaged by fluorography. Units shown beneath each lane are relative band densities estimated by photodensitometry. newly synthesized Ras that was marked for degradation. As an of immune complexes prepared with the Raf-1-specific Ab, C-12, independent measurement, we used liquid scintillation counting to to phosphorylate kinase-dead GST-MEK. The results in Fig. 3A quantify the effect of BZA-5B on [3H]mevalonolactone incorpo- (top) show that Fc⑀RI-induced Raf-1 activity peaked within 5 min ration into Ras immunoprecipitates. These experiments confirmed and remained elevated for at least 10 min. Raf-1 activation was that 100 ␮M BZA-5B inhibits the prenylation of Ras species by abolished in cells exposed for 24 h to 10 ␮M lovastatin. In con- more than 70%. In contrast, 1 ␮M and 10 ␮M concentrations of trast, 72-h incubation with 100 ␮M BZA-5B resulted in only a BZA-5B had no measurable effect on Ras prenylation (data not modest reduction in Raf-1 activation. shown). Ag-mediated Ras activation is inhibited by lovastatin and ⑀ BZA-5B Fc RI-induced MEK and ERK1/ERK2 activation is not inhibited by lovastatin or BZA-5B Ag-induced Ras activation was determined from the increase in the percentage of Ras in the GTP-bound (active) form in Y13-259 MEK and ERK activities toward kinase-dead GST-ERK and MBP, immunoprecipitates prepared from cell lysates (Fig. 2). In control respectively, were similarly measured in immune complex kinase ⑀ cells, incubation with Ag caused an increase in GTP-Ras to ap- assays. Fc RI cross-linking activates MEK (Fig. 3A, middle) and proximately twice resting levels within 2 min. Cells treated for ERK1/ERK-2 (Fig. 3A, bottom). Activation was maximal within 5 72 h with 100 ␮M BZA-5B or for 24 h with 10 ␮M lovastatin min after cross-linking and persisted for at least 10 min. The rate responded to Fc⑀RI cross-linking with either no change or a small and extent of MEK and ERK1/ERK2 activation were very similar decrease in the percentage of Ras in the GTP-bound form. in control, BZA-5B-treated, and lovastatin-treated cells. RBL-2H3 cells were also treated with the MEK inhibitor Ag-stimulated Raf-1 activation is reduced by BZA-5B and PD98059 (43). In contrast with the prenylation inhibitors, 1-h abolished by lovastatin treatment with 100 ␮M PD98059 inhibited the Fc⑀RI-induced ac- In many cells, activated Ras couples directly to the serine/threo- tivation of MEK (Fig. 3B, top) and of ERK1/ERK2 (Fig. 3B, nine kinase Raf-1. Raf-1 activity was determined from the ability bottom). The Journal of Immunology 6737

FIGURE 2. Inhibition of Ras activation by prenylation inhibitors. [32P]Orthophosphate-labeled control cells and BZA-5B-treated (100 ␮M for 72 h) and lovastatin-treated (10 ␮M for 24 h) cells were incubated with or without Ag (1.0 ␮g/ml DNP-BSA) for 2 min. Ras proteins were immunoprecipitated from cell lysates, guanine nucleotides were eluted, and radioactivity incorporated into GDP and GTP was measured. Data, expressed Downloaded from as the percentage of total Ras in the GTP-bound form, are the average of duplicate samples and are representative of two separate experiments.

Lovastatin and BZA-5B have little effect on Ag-induced protein- tyrosine kinase activation http://www.jimmunol.org/ Previously, Shakarjian et al. (44) reported a small inhibition of Ag-induced protein-tyrosine phosphorylation in lovastatin-treated cells. Fig. 4 shows the results of similar studies in our laboratory; FIGURE 4. Ag-stimulated tyrosine phosphorylation is not significantly arrows indicate bands identified in previous studies (2, 40) as the affected by BZA-5B or lovastatin. [32P]Orthophosphate-labeled cells were Fc⑀RI-associated tyrosine kinases Lyn and Syk, which are impli- incubated for 2 min without or with 1 ␮g/ml DNP-BSA, and then antiphos- cated in signal initiation and signal propagation, respectively (40). photyrosine-reactive proteins were isolated and analyzed by SDS-PAGE. Arrows indicate phosphoproteins previously identified as Lyn and Syk. In control cells (lanes 1 and 2), Fc⑀RI cross-linking caused the tyrosine phos-

phorylation of multiple proteins. Basal and Ag-stimulated protein phos- by guest on September 26, 2021 phorylation was unaffected by BZA-5B treatment (lanes 3, 4, and 6). Ag- induced protein phosphorylation was modestly reduced by lovastatin (lane 5).

Lovastatin pretreatment had no effect on the overall pattern of protein-tyrosine phosphorylation in Ag-stimulated cells (Fig. 4; compare lanes 2 and 5). Incubation with BZA-5B also had no effect on the pattern of basal and Ag-induced protein-tyrosine phosphorylation (Fig. 4; compare lanes 1 and 2 with lanes 3, 4, and 6). Lovastatin, but not BZA-5B, modestly reduced the extent of Ag-induced phosphorylation. These results reveal no substantial role for protein prenylation in the processes of tyrosine kinase activation that initiate the Fc⑀RI-coupled signaling cascade.

BZA-5B does not inhibit Ag-induced Ins(1,4,5)P3 synthesis, membrane/cytoskeletal responses, or IL-4 production A series of lovastatin-sensitive responses to Fc⑀RI cross-linking were unaffected by the farnesyl-speciﬁc inhibitor BZA-5B. Assays

of Ag-induced Ag-activated Ins(1,4,5)P3 synthesis are shown in FIGURE 3. Effects of inhibitors on Ag-stimulated Raf-1, MEK, and Fig. 5. As previously reported, lovastatin inhibited Ag-stimulated ERK1/ERK2 activation. IgE-primed RBL-2H3 cells were activated for the Ins(1,4,5)P production (20). Ag-induced Ins(1,4,5)P production indicated times with 0.1 ␮g/ml DNP-BSA and then lysed, and kinases were 3 3 was the same in control cells and in cells treated with 100 ␮M immunoprecipitated from the clariﬁed lysates. Kinase activity was determined from the in vitro phosphorylation of kinase-dead GST-MEK (Raf- BZA-5B. 1), kinase-dead ERK (MEK-1), and MBP (ERK1/ERK2). In A, cells were Assays of Ag-induced membrane rufﬂing and spreading are pretreated with prenylation inhibitors. In B, cells were pretreated for 1 h shown in Fig. 6. Unstimulated RBL-2H3 cells adhered to glass or with MEK inhibitor. Data in each case are representative of three separate plastic surfaces and showed a modest spreading response (Fig. experiments. 6A). Cross-linking the Fc⑀RI transformed the dorsal surface from 6738 Fc⑀RI SIGNALING PATHWAYS AND PRENYLATION INHIBITORS

FIGURE 7. Ag-stimulated production of IL-4 mRNA is inhibited by lovastatin, but not by BZA-5B. mRNA was isolated from RBL-2H3 cells, and RT-PCR ampliﬁcation of IL-4 message (top set of bands) was performed with G3PDH as a positive control (bottom set of bands). A 2-h stimulation of control cells by 1 ␮g/ml DNP-BSA or 1.25 ␮M ionomycin induced IL-4 production (lanes 2 and 5, respectively). IL-4 mRNA was also present in Ag-stimulated, BZA-5B-treated cells (lane 3). Lovastatin FIGURE 5. Ag-stimulated Ins(1,4,5)P production is inhibited by lova- 3 inhibited IL-4 mRNA production induced by Ag (lane 4) but not by iono- Downloaded from statin but not by BZA-5B. Ins(1,4,5)P3 levels were measured in acid ex- mycin (lane 6). Results are typical of three experiments. tracts of control and drug-treated Ag-activated (1 ␮g/ml DNP-BSA) cells. Results are representative of three experiments, each performed in duplicate. Error bars show the ranges of the duplicates. duction of IL-4 mRNA in RBL-2H3 cells. IL-4 production induced by the Ca2ϩ ionophore ionomycin was not inhibited by lovastatin.

BZA-5B potentiates Ag-induced secretion and Ca2ϩ store a microvillous to a lamellar topography and caused a dramatic http://www.jimmunol.org/ increase in cell spreading and adhesion (Fig. 6B). As reported pre- release viously (20), cells treated for 24 h with 10 ␮M lovastatin became Remarkably, two lovastatin-sensitive responses to Fc⑀RI cross- rounded and poorly adherent (Fig. 6C) and no longer showed ruf- linking, secretion, and the release of Ca2ϩ from stores, were en- ﬂing and spreading responses to Ag (Fig. 6D). In contrast, 72-h hanced in BZA-5B-treated cells. Secretion data are shown in Fig. incubation with 100 ␮M BZA-5B did not alter the morphology of 8. As reported previously, 24-h incubation of RBL-2H3 cells with resting cells (Fig. 6E). Furthermore, BZA-5B-treated cells re- 10 ␮M lovastatin blocked the Ag-induced release of [3H]serotonin sponded to Ag by vigorous rufﬂing and spreading (Fig. 6F). (20). Treatment for 1 h with 1 ␮Mor10␮M concentrations of the The production of cytokines, including IL-4, is a late (2–4 h) MEK inhibitor PD98059 also inhibited Fc⑀RI-induced secretion. response to Fc⑀RI cross-linking that depends on Ca2ϩ mobiliza- In contrast, 72 h incubation of RBL-2H3 cells with BZA-5B in- by guest on September 26, 2021 tion, signaling to the nucleus, and transcriptional activation (re- creased the Ag-stimulated secretion of [3H]serotonin. Secretion viewed in Ref. 45). The results in Fig. 7 show that Ag-induced was also induced in RBL-2H3 cells by ionomycin. Like Ag-in- IL-4 message production was the same in control and BZA-5B duced secretion, ionomycin-induced secretion was inhibited by lo- (100 ␮M)-treated cells. Lovastatin inhibited the Ag-induced pro- vastatin and PD98059 and enhanced by BZA-5B.

FIGURE 6. Different effects of lovastatin and BZA-5B on Ag-stimulated ruffling and spreading. IgE- primed cell monolayers were incubated for 10 min without (A, C, and E) or with (B, D, and F)1␮g/ml DNP-BSA and then processed for scanning electron microscopy. Lov- astatin-treated cells (C) are rounded in comparison with control (A) and BZA-5B-treated (E) cells. Fc⑀RI cross-linking induces flattening and ruffling responses in control (B) and BZA-5B-treated (F) cells. Lovastat- in-treated cells (D) show incomplete flattening and ruffling following Fc⑀RI cross-linking. Results are typical fields of cells from one of three replicate experiments. Bar ϭ 1 ␮m. The Journal of Immunology 6739

FIGURE 8. Secretion is impaired in cells treated with lovastatin or PD98059 but potentiated in BZA-5B-treated cells. [3H]Serotonin release was measured during a 20-min incubation of IgE-primed cells with Ag or 2ϩ ionomycin; suboptimal concentrations of stimuli were used to maximize FIGURE 9. Ca mobilization responses of control cells and BZA- and Ϯ lovastatin-treated RBL-2H3 cells. Fura2-labeled, IgE-primed cells were the enhancing effect of BZA. Data represent the average SD of four Downloaded from replicate experiments, each performed in duplicate. Data were corrected for imaged on a microscope stage, and ﬂuorescence intensities at 360 and 380 spontaneous degranulation (ϳ4% of total [3H]serotonin in 20 min). Note nm were measured using a charge-coupled device camera interfaced to a 2ϩ that during these experiments, we observed that BZA-5B (10–100 ␮M), computer. Ca concentrations were calculated from the ratios of these but not lovastatin, increased the overnight incorporation of [3H]serotonin measurements using corrections and algorithms described in Reference 13. ␮ into granules by nearly twofold (not shown). Electron microscopy revealed Ag (0.1 g/ml DNP-BSA) was added at the arrow. Each panel represents 2ϩ no accompanying increase in granule density or size in BZA-5B-treated the Ca response of a single cell from a typical experiment. A total of 20–30 cells in three or four separate experiments were observed for each cells. Because secretion data are expressed as percentage of total http://www.jimmunol.org/ [3H]serotonin, the potentiation of secretion is independent of BZA-5B- condition. induced increases in mediator uptake.

ϩ The Ca2ϩ mobilization response induced by Ag in a typical ulated in the continual presence of extracellular Ca2 . Results are IgE-primed control cell is illustrated in Fig. 9A. Before stimula- given in Table I. In control cells, the average lag time from Ag ϩ tion, cells maintained a low basal concentration of free Ca2ϩ. Ad- addition to the release of intracellular Ca2 stores was 81 s. The ϩ dition of 0.1 ␮g/ml DNP-BSA resulted, after a characteristic delay, magnitude of the Ca2 response was 59.8 nM ⅐ s. The average lag 2ϩ 2ϩ by guest on September 26, 2021 in an abrupt elevation in [Ca ]i that was maintained for at least 10 time from Ag addition to a Ca response in lovastatin-treated min. Previous work showed that the Ca2ϩ spike resulted from cells was 58 s, slightly (but not significantly) less than the 81-s lag Ca2ϩ release from intracellular stores and that the maintained el- time of control cells. However, lovastatin reduced the magnitude ϩ evation required Ca2ϩ influx (12, 13). of the Ca2 response by almost 50%. The average lag times to ϩ Lovastatin-treated cells also released Ca2ϩ stores in response to Ca2 response in cells treated with 10 and 100 ␮M BZA-5B were Ag (Fig. 9B). Consistent with previous evidence that lovastatin 47 s and 40 s, respectively, significantly less than the 81-s average ϩ inhibits Ag-induced [45Ca2ϩ] uptake, the maintained component lag time to Ca2 response in Ag-stimulated control cells. In BZA- ϩ of the Ca2ϩ mobilization response was significantly smaller in 5B-treated cells, the maintained Ca2 elevation was nonoscillatory lovastatin-treated cells than in control cells. Furthermore, in ap- and either was of comparable magnitude with that of control cells proximately one-third of cells (17 of 49) the Ca2ϩ response con- (cells treated with 10 ␮M BZA-5B) or was modestly reduced in sisted of fairly regular oscillations, interpreted as cycles of stores comparison with that of control cells (cells treated with 100 ␮M release and uptake, superimposed on a slowly rising baseline (as in BZA-5B). Fig. 9B). Comparable oscillations were not observed in any of 44 Ag-stimulated control cells. Effects of lovastatin and BZA-5B on RBL-2H3 cell proliferation Fig. 9, C and D, shows that the typical Ag-stimulated Ca2ϩ and survival responses of cells treated with 10 and 100 ␮M BZA-5B is non- Incubation of RBL-2H3 cells with BZA-5B slowed but did not oscillatory and appears very similar to that of control cells. How- abolish the incorporation of [3H]thymidine into DNA (Fig. 10A). ever, 16% (23 of 140 cells) of cells treated with BZA-5B, includ- The reduced rate of thymidine incorporation was apparent within ing the one illustrated in Fig. 9D, showed Ca2ϩ oscillations prior 24 h of BZA-5B addition and was maximal by 48 h. Growth in- to Fc⑀RI cross-linking. Spontaneous oscillations were not ob- hibition was seen at 10 ␮M BZA-5B, at which most newly syn- served in any of 62 control cells. Because spontaneous oscillations thesized Ras protein could still be farnesylated (data not shown). in some cells were observed in the absence of [Ca2ϩ] (data not Even at the highest dose of drug (100 ␮M), BZA-5B-treated cells shown), they apparently result from the release and reuptake of remained viable beyond 96 h of treatment, as assessed by trypan intracellular Ca2ϩ stores. The observations suggest that a farnesy- blue exclusion (not shown). In contrast, lovastatin impaired pro- lated protein acts in both resting and Ag-stimulated cells to sup- liferation within 24 h (Fig. 10A), and cell viability, assessed by press Ca2ϩ stores release. trypan blue exclusion, was reduced within 48 h. Protocols described in Lee and Oliver (13) were used to measure Apoptosis was measured by flow cytometry of propidium io- lag time, defined as the time from Ag addition to a Ca2ϩ spike in dide-treated cells. When RBL-2H3 cells were cultured in the pres- cells stimulated in the absence of extracellular Ca2ϩ, and response ence of carrier alone, 100% of cells were distributed within the 2ϩ magnitude, defined as the area under the [Ca ]i curve integrated G0/G1, S, and G2/M phases of the cell cycle (Fig. 10B, panel 1). for 4 min following the onset of the Ca2ϩ response in cells stim- Twenty-four hours after heat shock (90 min at 43°C), all of the 6740 Fc⑀RI SIGNALING PATHWAYS AND PRENYLATION INHIBITORS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 10. Effects of lovastatin and BZA-5B on RBL-2H3 cell proliferation and survival. A,[3H]Thymidine incorporation assays showing that both inhibitors block cell proliferation. Results are the average of duplicate experiments. B, Apoptosis assays. Cells were incubated with the DNA-speciﬁc dye and propidium iodide, and DNA fragmentation was analyzed by ﬂow cytometry. Panel 1, Control cells; panel 2, cells in apoptosis induced by heat shock; panel 3, cells treated for 72 h with BZA-5B, showing no apoptotic cells; panel 4, cells treated for 24 h with lovastatin, showing little apoptosis; panel 5, most cells treated with lovastatin for 48 h are in apoptosis. Results are typical of three replicate experiments.

cells were apoptotic, as indicated by a broad peak of fragmented pathway. These results indicated that lovastatin inactivates isopre- DNA (Fig. 10B, panel 2). No apoptotic cells were detected after nylated proteins required for Fc⑀RI-stimulated signaling, presum- 72 h of BZA-5B treatment (Fig. 10B, panel 3). There was also very ably by preventing their association with membranes. Here, we little apoptosis in cultures exposed to lovastatin for 24 h (Fig. 10B, used the farnesyl-speciﬁc inhibitor BZA-5B to analyze the contri- panel 4). Results in Fig. 10B, panel 5, show that 48 h of lovastatin butions of farnesylated proteins to Fc⑀RI signaling and, by com- treatment leads to apoptosis. parison with lovastatin, to identify roles for geranylgeranylated proteins in the signaling pathway. We focused attention on Ras Discussion isoforms because these are the principal farnesylated species im- We demonstrated previously that Fc⑀RI-stimulated signaling re- plicated in receptor-mediated signaling pathways. A role for Ras in sponses are inhibited by lovastatin, a cholesterol biosynthetic Fc⑀RI-coupled signaling was suggested previously by evidence pathway inhibitor that blocks the synthesis of the farnesyl and that Fc⑀RI cross-linking stimulates the tyrosine phosphorylation of geranylgeranyl pyrophosphates needed to isoprenylate a set of pro- Vav, a GDP-GTP exchange factor with activity toward Ras and teins recognizable by their C-terminal CAAX, CXC, or CC motifs other GTPases (6, 46), as well as by reports that Fc⑀RI cross- (19, 20, 24). Signaling responses could be restored by adding me- linking stimulates assembly of the Ras-stimulatory Shc-Grb2-SoS valonic acid, which is a direct precursor of these isoprenoid de- complex (9, 14). rivatives, but not by dolichol and cholesterol, which are down- We began by identifying the ras isoforms in RBL-2H3 mast stream of isoprenoid metabolism in the cholesterol biosynthetic cells and demonstrating their activation by Fc⑀RI cross-linking. The Journal of Immunology 6741

Table I. Ag-stimulated Ca2ϩ responses in lovastatin- and BZA-5B-treated RBL-2H3 cells a

Average Lag Average Integral Treatment n Time (SD) pnϫ 103 (SD ϫ 103) p

Control 29 81 s (34) 25 59.8 nM ⅐ s (17.0) Lovastatin 21 58 s (32) Ͻ 0.05 28 33.9 nM ⅐ s (8.5) Ͻ 10Ϫ6 10 ␮M BZA-5B 30 47 s (23) Ͻ 10Ϫ5 26 51.0 nM ⅐ s (15.3) NS 100 ␮M BZA-5B 28 40 s (26) Ͻ 106 23 39.8 nM ⅐ s (14/7) Ͻ 10Ϫ4 a 2ϩ 2ϩ ␮ Average lag times to Ca response and [Ca ]i integrals in cells pretreated for 72 h with GSH (control), 10 or 100 M BZA-5B, or for 24 h with 10 ␮M lovastatin. Stimulation in each case was with 10 ng/ml DNP-BSA. The number of cells included in each average (n) and the standard deviation (SD) are indicated. The average lag time in lovastatin-treated cells includes individual cell responses from two experiments; all other averages include all cells observed in three experiments. The 2ϩ 2ϩ 2ϩ average lag times from Ag addition to Ca response were measured in cells stimulated in the absence of Cao . [Ca ]i 2ϩ 2ϩ integrals were calculated for cells stimulated in the presence of Cao . [Ca ]i was integrated over 240 s following the initial 2ϩ 2ϩ Ag-stimulated increase in [Ca ]i. Normal distributions of both lag times and [Ca ]i integrals are assumed. Values of p give the signiﬁcance of each condition compared to control using the Student’s t test. NS is not signiﬁcant at p Ͻ 0.05.

Four principal Ras species, H-ras,N-ras,K-rasA, and K-rasB, that Ag-stimulated Raf-1 activation occurs in RBL-2H3 cells by a have been described in animal cells (41). RBL-2H3 cells express pathway that, instead of requiring Ras, requires the activation of Downloaded from all of these isoforms except for N-ras. Ras proteins are not am- members of the Rho family of geranylgeranylated GTPases (52, plified or expressed in oncogenic forms in RBL-2H3 cells. Ras- 53) and so is more sensitive to inhibition by lovastatin than by GTP levels increase in Ag-stimulated cells, confirming that Fc⑀RI BZA-5B. Unexpectedly, neither BZA-5B nor lovastatin has any cross-linking activates Ras. Importantly, BZA-5B and lovastatin effect on MEK1 or ERK1/ERK2 activation. These data clearly abolish the Ag-induced increase in Ras-GTP levels, indicating that establish the presence of Raf-1-independent pathways to MEK ac- both drugs inhibit Ras activation. Although no Ras activation tivation in RBL-2H3 cells. In contrast with lovastatin and BZA- http://www.jimmunol.org/ could be measured, BZA-5B caused only a 70% inhibition of Ras 5B, the MEK inhibitor PD98058 prevented Ag-induced ERK1/ prenylation. Others have shown that the K-Ras4B isoform (47, 27), ERK2 activation. These results locate MEK conventionally as well as N-ras and K-Ras4A (17), can serve as an in vitro sub- upstream of the ERK MAP kinases in RBL-2H3 cells. strate for geranylgeranyl transferase-1. Thus, it is possible that Why do mast cells use apparently Ras- and Raf-1-independent some of the residual prenylated Ras reflects a pool of BZA-5B- pathways to Fc⑀RI-mediated ERK1/ERK2 activation when they resistant geranylgeranylated K-Ras. Because Ag-induced Ras ac- clearly contain the elements of the classical Grb2/SoS/Ras/Raf/ tivation measured by assays of total Ras-GTP levels is completely MEK pathway defined in fibroblasts? One explanation is that he- inhibited in BZA-5B-treated cells, it is likely that this residual

matopoietic cells may use individual variations on the general sig- by guest on September 26, 2021 ⑀ prenylated Ras is not activated by Fc RI cross-linking. We recog- naling sequences established in fibroblasts. Consistent with this, nize, however, that our assay may fail to detect an increase in GTP preliminary evidence that BZA-5B reduces both basal and Ag- bound to a minor isoform of Ras. stimulated JNK activity toward its substrate, GST-c-Jun, in RBL- The Fc⑀RI-coupled signaling cascade is initiated by the activa- 2H3 cells raises the possibility that mast cell Ras might activate a tion of two receptor-associated tyrosine kinases, Lyn and Syk, non-ERK member of the MAP kinase family (T.E.G. and B.S.W., which in turn phosphorylate multiple substrates (reviewed in Ref. unpublished results). Another explanation is that Ag-stimulated 48). We found that BZA-5B does not alter the Ag-stimulated ty- mast cells may activate MAP kinases by pathways that are inde- rosine phosphorylation of RBL-2H3 proteins, including the Fc⑀RI- pendent of Ras-related GTPases. In other cells, protein kinase C associated kinases Lyn and Syk, and we confirmed previous evi- isozymes appear to link certain receptors directly to Raf and MEK dence (44) that Ag-stimulated protein tyrosine phosphorylation is isoforms, bypassing Ras (53). Importantly for our studies, James et also not substantially inhibited by lovastatin. Thus, it is unlikely that prenylation inhibitors target the earliest events in the Fc⑀RI al. (26) reported that BZA-5B blocks epidermal growth factor- signaling cascade. stimulated ERK activation in H-Ras-transformed Rat-1 fibroblasts, In many cell systems, receptor-mediated tyrosine kinase activa- but not in untransformed cells. One explanation is that the un- tion results in Grb2/SoS-mediated Ras activation that couples di- transformed cells contained a small (undetectable in biochemical rectly to the serine/threonine kinase, Raf-1, resulting in Raf-1 re- assays) amount of BZA-5B-resistant Ras. An alternative ex- cruitment to the plasma membrane and activation. Activated Raf-1 planation is that Rat-1 cells lacking oncogenic Ras, like RBL-2H3 in turn phosphorylates the dual-specificity kinases MEK-1 and cells, favor a Ras-independent pathway to receptor-mediated ERK MEK-2, which finally phosphorylate and activate the MAP kinases activation. ERK-1 and ERK-2, which are implicated in transcriptional acti- We showed previously that lovastatin inhibits a series of Fc⑀RI- vation (reviewed in Refs. 49–51). We confirmed that Raf-1 shows mediated responses, including Ag-stimulated Ins(1,4,5)P3 produc- 2ϩ increased activity after Fc⑀RI cross-linking. We also showed in- tion, a measure of PLC␥ activation; Ca influx, attributed in large 2ϩ 2ϩ creased MEK and ERK1/ERK2 activities in Ag-treated cells. Nev- part to the coupling of Ca stores release to capacitative Ca ertheless, we failed to obtain support for the classical (Ras 3 Raf entry (13); and a series of functional responses including secretion, 3 MEK 3 ERK) pathway of ERK1/ERK2 activation in RBL- ruffling, spreading, and IL-4 production. In the present study, 2H3 cells. First, only lovastatin, and not BZA-5B, strongly inhibits BZA-5B failed to inhibit any of these signaling responses. These Ag-stimulated Raf-1 activation. The persistent Raf-1 activation in data implicate geranylgeranylated (lovastatin-sensitive, BZA-5B- BZA-5B-treated cells could be mediated by a small amount of insensitive) proteins acting downstream of Ag-stimulated protein- BZA-5B-resistant geranylgeranylated K-RasB, which was not de- tyrosine phosphorylation in the regulation of a diverse array of tected in our assay for Ras-GTP levels. Alternatively, it is possible signaling responses. 6742 Fc⑀RI SIGNALING PATHWAYS AND PRENYLATION INHIBITORS

The inhibition of ruffling, spreading, and actin plaque assembly potentiation) and its sensitivity to unusually low concentrations of by lovastatin, which inhibits all protein prenylation, but not by farnesylation inhibitors. There is precedent for inhibition of farne- BZA-5B, which spares protein geranylation, was predictable based sylation of at least one non-Ras protein, nuclear lamin B, at on evidence from Hall that different members of the geranylgera- BZA-5B concentrations too low to inhibit Ras prenylation (63). nylated Rho family of GTPases control the formation of filopodia We speculate that the target protein may be the type I Ins(1,4,5)P3 (CDC42), ruffles (Rac) and adhesion and spreading (Rho) (re- 5-phosphatase. This enzyme associates with membranes via its far- viewed in Ref. 54). Indeed, Guillemot and colleagues (55) recently nesylated C-terminal CAAX motif (22, 64). Its substrates are phos- reported that expressing dominant negative mutant forms of phatidylinositol 4,5-bisphosphate and Ins(1,4,5)P3. In particular, it CDC42 in RBL-2H3 cells decreases Fc⑀RI-induced adhesion and 2ϩ converts the Ca -mobilizing metabolite, Ins(1,4,5)P3, to the in- actin plaque assembly, while expressing dominant negative Rac1 active metabolite, Ins(1,4)P2. In its nonfarnesylated form, type I abolishes ruffling. The selective inhibition by lovastatin of secre- Ins(1,4,5)P 5-phosphatase would be expected to be soluble rather tion was also predictable based on evidence from Prepens and 3 than membrane associated, reducing its access to Ins(1,4,5)P3 in- colleagues (56) that Clostridium difficile toxin B, which targets the teracting with its receptors at the endoplasmic reticulum mem- geranylgeranylated GTPases RhoA and CDC42, blocks Ag-in- ϩ brane and so increasing Ins(1,4,5)P -mediated Ca2 store release duced secretion from RBL-2H3 cells. Other geranylgeranylated 3 and secretion. GTPases, including Rab3B and Rab3D cloned from RBL-2H3 In course of these studies, we observed that the MEK inhib- cells (57) and primary rat mast cells (58), have also been impli- itor PD98059 potently inhibits secretion. This result supports cated in vesicular trafficking and secretion (59).

previous evidence that ERK activation is required for Ag-stim- Downloaded from Our data also localize geranylgeranylated proteins to sites in the Fc⑀RI-coupled signaling cascade that have not previously been ulated secretion in RBL-2H3 cells (16). Nevertheless, since described. First, we found that lovastatin, but not BZA-5B, inhibits MEK and ERK activation occur normally in lovastatin-treated cells, the inhibition of secretion by lovastatin is not explained Ag-stimulated Ins(1,4,5)P3 synthesis. This result implicates a geranylgeranylated protein in the control of Ag-induced PLC␥ ac- by this pathway. tivation. We cannot as yet deﬁne the protein. However, recent Ras activation plays a pivotal role in the pathways coupling studies have shown that the recruitment and activation of PLC␥ growth factor receptors to signal transduction pathways, at least in http://www.jimmunol.org/ isoforms by Fc⑀RI cross-linking in RBL-2H3 cells is a complex ﬁbroblast cell lines. It was therefore surprising that our studies process requiring not only tyrosine phosphorylation but also inter- failed to reveal any clear role for Ras in Fc⑀RI-mediated signaling actions with at least one other enzyme, phosphatidylinositol 3-ki- in RBL-2H3 cells. We considered the possibility that the principal nase (5, 8). Other investigators have shown that Rac and CDC42 role of Ras is in mast cell growth regulation rather than acute associate with phosphatidylinositol 3-kinase (60, 61). Thus, it is aspects of Fc⑀RI signaling. Consistent with this hypothesis, both possible that a geranylgeranylated GTPase of the Rho family is lovastatin and BZA-5B blocked DNA synthesis within 24 h. involved in the regulation of PLC␥ activation by inositol BZA-5B concentrations that abolish Ras activation did not de- phospholipids. crease cell viability or induce apoptosis, at least over a 96-h period.

ϩ by guest on September 26, 2021 Second, we found that Ag-stimulated Ca2 influx is inhibited by In contrast, lovastatin impaired cell viability and induced apoptosis lovastatin but not by BZA-5B. One explanation is simply that the within 48 h, presumably reflecting important roles for geranylgera- reduced Ins(1,4,5)P3 levels in lovastatin-treated cells are sufficient nylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in the ϩ to support the initial Ca2 stores release response but too low to pathways that protect against apoptosis. Relatively low concentra- maintain the stores in an empty state, as needed for capacitative tions of BZA-5B that only modestly reduced Ras prenylation also 2ϩ influx. However, we showed previously that vigorous Ca stores arrested cell growth. One explanation is that a non-Ras-farnesy- release and influx can be supported at low Ag concentrations that lated protein with greater sensitivity to BZA-5B is essential for induce less Ins(1,4,5)P3 synthesis than occurs in lovastatin-treated RBL-2H3 cell proliferation. Alternatively, if a Ras pathway con- cells (13). Alternatively, previous studies by Wilson et al. (11) and tributes to mast cell proliferation, even a small amount of nonfar- Fasolato et al. (62) have implicated GTP-binding proteins in the 2ϩ 2ϩ nesylated Ras may block cell proliferation by trapping Raf or an- coupling of empty Ca stores to capacitative Ca influx. The other effector molecule in an inactive cytoplasmic complex. inhibition of Ca2ϩ influx by lovastatin but not by BZA-5B now Previously, Lerner et al. (65) invoked the induction of signal cur- suggests that the putative coupling protein belongs to the family of tailing Ras-Raf complexes to explain how another farnesyl-spe- geranylgeranylated GTPases. Because lovastatin-treated cells syn- cific inhibitor, FTI-277, may block the growth of Ras-transformed thesize IL-4 mRNA in response to ionomycin but not to Ag, we cells at concentrations 100-fold lower than those required to abol- speculate that the Ag-induced production of IL-4 depends largely ϩ ish Ras farnesylation. on this lovastatin-sensitive Ca2 influx pathway. Secretion also ϩ Previous investigators have shown that in general BZA-5B requires Ca2 influx (10, 12). However, lovastatin inhibits ionomycin-induced secretion, indicating roles for additional gera- and other farnesylation inhibitors do not significantly inhibit the nylgeranylated proteins downstream of Ca2ϩ influx in the pathway proliferation of non-ras-transformed tumor cells and tissue cul- linking Fc⑀RI cross-linking to degranulation. ture lines (25, 26, 66). However, a screen by Sepp-Lorenzino et Most interestingly, BZA-5B enhances Ag-induced secretion and al. (67) of a large panel of cell lines lacking Ras mutations for reduces the lag time from Ag addition to Ca2ϩ stores release. The sensitivity to the farnesyltransferase inhibitor FTI L-744832 re- BZA-5B-induced potentiation of secretion and of Ca2ϩ stores revealed that all seven of the hematopoietic cell lines in the panel lease was observed at BZA-5B concentrations too low to affect Ras were sensitive. This suggests that the proliferation of hemato- farnesylation and activation. We suggest, therefore, that these poietic system cells expressing nononcogenic Ras isoforms may changes reflect the inactivation by BZA-5B of a non-Ras- be unusually sensitive to farnesyltransferase inhibitors. Never- farnesylated protein that is normally involved in the suppression of theless, BZA-5B-treated RBL-2H3 cells remain viable for ex- Ca2ϩ stores release and secretion. Properties of this protein that tended periods. Thus, growth arrest of hematopoietic cells may can be inferred from our data include its relatively slow turnover not compromise the use of farnesyltransferase inhibitors in can- rate (since 72-h incubation with BZA-5B is required to observe cer therapy. The Journal of Immunology 6743

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