Suppression of Integrin Activation by Activated Ras or Raf Does Not Correlate with Bulk Activation of ERK MAP Kinase

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Suppression of Integrin Activation by Activated Ras or Raf Does Not Correlate with Bulk Activation of ERK MAP Kinase
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  Molecular Biology of the CellVol. 13, 2256–2265, July 2002 Suppression of Integrin Activation by Activated Ras orRaf Does Not Correlate with Bulk Activation of ERKMAP Kinase Paul E. Hughes,* †‡ Beat Oertli,* ‡ Malene Hansen, § Fan-Li Chou,*Berthe M. Willumsen, § and Mark H. Ginsberg*  *The Division of Vascular Biology, Department of Cell Biology. The Scripps Research Institute,La Jolla, California 92037; and  § Department of Molecular Cell Biology, Institute of Molecular Biology,Copenhagen University, Oester Farimagsgade 2A, DK-1353, Copenhagen K, Denmark Submitted October 5, 2001; Revised February 7, 2001; Accepted April 1, 2002Monitoring Editor: Mary C. Beckerle The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrinfamily of cell adhesion receptors. The Ras family of small GTP-binding proteins and theirdownstream effectors are key players in regulating integrin activation. H-Ras can suppressintegrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, aclosely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrinactivation. To gain insight into the regulation of integrin activation by Ras GTPases, we created aseries of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required forfull suppressive activity. Furthermore, the suppressive chimeras were weak activators of theERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may beindependent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating thatthe ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulkERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrinactivation is a Raf kinase induced regulatory event that can be mediated independently of bulkactivation of the ERK MAP-kinase pathway. INTRODUCTION Interactions of integrin cell adhesion receptors with theirextracellular ligands are important for cell migration,growth, and survival (Schwartz  et al. , 1995; Schwartz, 1997;Clark  et al. , 1998). A characteristic feature of many integrinsis their ability to alter their affinity for ligands in response tointracellular signals, a process termed “activation”(Hughesand Pfaff, 1998).Presently, the signal transduction cascades controlling in-tegrin activation are incompletely understood (Hughes andPfaff, 1998). However, several observations suggest thatmembers of the Ras family of small GTP-binding proteinsand their downstream effectors are critically involved in theregulation of integrin activation (Zhang  et al. , 1996; Hughes et al. , 1997; Reedquist  et al. , 2000). Activated H-Ras cansuppress the activation of certain   1 and   3 integrins infibroblasts via its effector serine/threonine kinase Raf-1(Hughes  et al. , 1997). This activity of H-Ras is implicated inthe control of cell morphology, cell movement, and assem- bly of the extracellular matrix (Hughes  et al. , 1997; Brenner  etal. , 2000). The suppressive activity of H-Ras does not requireprotein synthesis or mRNA transcription, and furthermore,suppression can be reversed by MAP kinase phosphatase 1(Hughes  et al. , 1997). Thus, suppression appears to be me-diated by a MAP kinase and correlates with activation of theERK1/2 MAP kinase pathway.In contrast, to H-Ras other closely related small GTP- binding proteins, such as R-Ras and Rap1, have the oppositeactivity, promoting rather than suppressing integrin activa-tion (Zhang  et al. , 1996; Osada  et al. , 1999; Caron  et al. , 2000;Reedquist  et al. , 2000; Shimizu, 2000). In CHO cells, activatedR-Ras can antagonize the Ras/Raf suppressor pathway, andin fibroblasts, myeloid cells and bone marrow–derived mastcells activated R-Ras stimulates integrin activation and inte-grin-dependent adhesion (Zhang  et al. , 1996; Osada  et al. ,1999; Sethi  et al. , 1999; Kinashi  et al. , 2000). Activation of PI3-kinase is involved in R-Ras stimulation of integrins inhematopoietic cells, but in fibroblasts, the critical effectors Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01–10–0480. Article and publication date are at–10–0480.  Corresponding author. E-mail address: ‡ Both authors contributed equally to this work. † Present address: SUGEN, 230 East Grand Avenue, South SanFrancisco, CA 94116.2256 © 2002 by The American Society for Cell Biology  are as yet unidenti fi ed (Osada  et al. , 1999; Kinashi  et al. , 2000;Oertli  et al. , 2000). Thus, H-Ras and R-Ras have opposingeffects on integrin activation in  fi  broblasts.The exact mechanisms by which H-Ras and R-Ras exerttheir opposing effects on integrin function are uncertain.Indeed, both of these small GTPases have remarkably sim-ilar effector domains, and interact with many of the samedownstream targets (Bos, 1998; Campbell  et al. , 1998; Re-uther and Der, 2000). To gain insight into this question, wemapped the regions of H-Ras responsible for suppression of integrin activation by creating a series of H-Ras/R-Ras chi-meras. We found that a 35-amino acid stretch of H-Rasencompassing residues 149 – 175 is required for full suppres-sive activity. Signi fi cantly, certain suppressive chimeras hadlittle effect on the activation of ERK1/2 MAP kinases. Fur-thermore, blockade of ERK1/2 activation by either a phar-macological inhibitor of MEK kinase or by coexpression of MAP kinase phosphatase 3 (MKP3) did not alter the abilityof H-Ras to suppress integrin activation. Thus, activation of the bulk of ERK1/2 is not required for integrin suppression.In addition, ERK activation per se is insuf  fi cient for integrinsuppression because an activated variant of MEK1 was un-able to suppress integrin activation. These results raise thepossibilities that Raf-1 can activate ERK1/2- and MEK1/2-independent pathways to suppress integrins. Alternatively,our data do not eliminate the possibility that a small pool of ERK1/2, acting at a discrete subcellular location, mediatesintegrin suppression. MATERIALS AND METHODS  Antibodies and Reagents The activation-dependent anti-  IIb  3  mAb, PAC1, and activatingantibody, anti-LIBS6, have previously been described (Shattil  et al. ,1985; Frelinger  et al. , 1990). The anti-Tac antibody 7G7B6 was ob-tained from the American Type Culture Collection (ATCC, Rock-ville, MD) and was biotinylated with biotin- N  -hydroxysuccinimide(Sigma, St. Louis, MO). The   IIb  3  speci fi c peptidomimetic inhibitorRo43 – 5054 was a generous gift of Dr. Beat Steiner (Hoffmann-LaRoche, Basel, Switzerland). The MEK kinase inhibitor U0126 wasobtained from Promega (Madison, WI) and used according to themanufacturer ’ s instructions. 4-Hydroxy tamoxifen (4  OHT) was ob-tained from Sigma (St. Louis, MO) and used at a  fi nal concentrationof 300 nM cDNA Constructs, Cell Lines, and Transfection The mammalian expression vectors encoding H-Ras(G12V),R-Ras(G38V), and HA-ERK2 have been described previously(Hughes  et al. , 1997; Sethi  et al. , 1999). The H-Ras/R-Ras chimeraswere constructed using splice overlap PCR mutagenesis with pSG5-R-Ras(G38V) and pcDR-H-Ras(G12V) as templates. The ampli fi edDNA was ligated into the  Eco RI site of pcDNA3.1 (Invitrogen, SanDiego, CA). All chimeras and mutant constructs were veri fi ed byDNA sequencing before further analysis. The mammalian expres-sion vectors pMCL- MEK1(  N3, S222D) and pSG5-MKP3 weregenerous gifts of Dr. N. Ahn (Howard Hughes Medical Institute,University of Colorado, Boulder, CO) and Dr. Steven Keyse (ICRFMolecular Pharmacology Unit, Ninewells Hospital, Dundee, UnitedKingdom), respectively. The plasmid pDCR-H-Ras(G12V) was a giftfrom Dr. M.H. Wigler (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY) and pSG5-R-Ras(G38V) was generously provided byDr. Julian Downward, (Signal Transduction Laboratory, ICRF, Lon-don, United Kingdom). PMX-Raf-1:ER vector was obtained fromMartin McMahon (University of California, San Francisco, CA).PMX-Raf-1:ER contains a mutated form of the mouse estradiolreceptor-binding domain that is sensitive to 4  OHT, but insensitiveto 17-  -estradiol and Phenol Red in the cell culture medium (Danie-lian  et al. , 1993). The PMX-RAF-1:ER vector express Raf-1:ER andeGFP (enhanced green  fl uorescent protein) from a single bicistronicmRNA with the translation of the 5   coding region for eGFP pro-moted by the presence of an internal ribosomal entry site (IRES)from encephalomyocarditis virus. The pGEX expression vector en-coding the central cell-binding domain of   fi  bronectin as a GST-fusion protein has been described previously (Ramos and DeSi-mone, 1996). GST-fusion proteins were produced as described(Ramos and DeSimone, 1996). Chinese Hamster Ovary (CHO)-K1cells were obtained from the ATCC (American Type Culture Col-lection). The generation of CHO   -py cells has been describedpreviously (Baker  et al. , 1997). These cells stably express the poly-oma large T antigen and bear a recombinant chimeric integrin thathastheextracellularandtransmembranedomainsofintegrin  IIb  3 joined to the cytoplasmic domains of integrin   6A  1A(  IIb  6A  3  1). All cells were cultured in DMEM (BioWhittaker,Walkersville, MD) containing 10% FCS, 1% nonessential aminoacids, 2 mM glutamine (Sigma), 100 U/ml penicillin, and 100  g/mlstreptomycin. Raf-1:ER cells were generated by cotransfecting CHOcells with PMX-RAF-1:ER and a G418 resistance vector. After selec-tion with G418, GFP-expressing cells were isolated by FACS.  Flow Cytometry PAC1 binding was measured by two-color  fl ow-cytometry as de-scribed previously (O ’ Toole  et al. , 1994; Hughes  et al. , 1996). Brie fl y,48 h after transfection cells were harvested by a brief trypsinizationand washed in DMEM/1% BSA. Cells, 5  10 5 , were incubated with0.1% PAC1 ascites in the presence of the competitive inhibitorRo43 – 5054 at 1   M or anti-LIBS6 ascites. After 30-min incubation atroom temperature cells were washed with cold DMEM/1% BSAand incubated with the biotinylated anti-Tac antibody 7G7B6 for 30min on ice. After washing, cells were incubated with 10% FITC-conjugated goat anti-mouse IgM (TAGO) and 4% phycoerythrin-streptavidin (Molecular Probes Inc., Eugene, OR) for another 30 minon ice. Cells were washed in ice-cold PBS and resuspended in PBS.Then cells were analyzed on a FACScan (Becton Dickinson, Moun-tain View, CA)  fl ow cytometer as described (Hughes  et al. , 1997),and the collected data were analyzed using CellQuest software(Becton Dickinson). To obtain numerical estimates of integrin acti-vation, we calculated an activation index (AI), de fi ned as 100  (F o  F r )/(F o LIBS6  F r ), where F o  is the median  fl uorescence intensity(MFI) of PAC1 binding; F r  is the MFI of PAC1 binding in thepresence of competitive inhibitor (Ro43 – 5054, 1   M); and F o LIBS6 isthe MFI of PAC1 binding in the presence of 2   M anti-LIBS6(Hughes  et al. , 1996). The percentage inhibition was calculated as100(AI o  AI)/AI o , where AI o  is the activation index in the absenceof the cotransfected test cDNA and AI is the activation index in itspresence.FN 9 – 11 binding was assayed by two-color  fl ow cytometry. Cellswere harvested by a brief trypsinization, followed by neutralizationwith the addition of complete media. Cells were then pelleted by a brief centrifugation, washed, and resuspended in Tyrode ’ s buffer.The harvested cells were then aliquoted into three pools containingeither Tyrode ’ s buffer alone, Tyrode ’ s buffer plus 5 mM EDTA, andTyrode ’ s buffer plus the activating anti-  1 mAb 9EG7 (10   g/ml;PharMingen, San Diego, CA). The cells were then incubated for 15min at room temperature. After the addition of biotinylated GST FN9 – 11 the cells were incubated at room temperature for an additional15 min. After washing in ice cold Tyrode ’ s, the cells were incubatedon ice for 30 min with 4% phycoerythrin-streptavidin (MolecularProbes Inc.). The cells were then washed in ice cold Tyrode ’ s andanalyzed on a FACScan (Becton Dickinson)  fl ow cytometer. Thecollected data were analyzed using CellQuest software (BectonDickinson).Integrin Suppression and ERK ActivationVol. 13, July 2002 2257   Measurement of ERK Phosphorylation CHO cells were transfected using Lipofectamine (Life Technologies,Rockville, MD) as described (Hughes  et al. , 1996). Transfectionswere performed in duplicate to allow for parallel analysis of bothERK phosphorylation and PAC1 binding by  fl ow cytometry. Twen-ty-four hours after transfection the cells were washed and placed inmedium containing 0.5% fetal calf serum. Forty-eight hours aftertransfection cells were washed and lysed in a buffer containing amixture of protease and phosphatase inhibitors (Hughes  et al. , 1997).Phosphorylated ERK was detected by fractionating 20   g of wholecell lysate on a 4 – 20% SDS-polyacrylamide gels, transferring tonitrocellulose membranes, and immunoblotting with an mAb thatrecognizesonlythephosphorylatedformsofERK1andERK2(SantaCruz Biotechnology, Santa Cruz, CA). To determine the totalamount of ERK present in each of the lysates, the blots were thenstripped and immunoblotted with either polyclonal antibodies rec-ognizing ERK1 and ERK2 (Santa Cruz Biotechnology) or the mAb12CA5 to detect HA-ERK2. RESULTS Residues 148–171 of H-Ras Are Required for Suppression of Integrin Activation Despite their sequence similarity, the small GTP bindingprotein R-Ras and H-Ras have opposing effects on integrinactivation in CHO cells (Figure 1, A and B; Sethi  et al. , 1999).H-Ras suppresses integrin activation as assessed by reduc-tion in the binding of the activation-speci fi c mAb, PAC1 toCHO cells expressing the active chimeric integrin,  IIb  6A  3  1. In contrast, activated R-Ras does not sup-press, but instead reverses Ras-initiated suppression (Figure1B) through the activation of an as yet unidenti fi ed effector.To map the region(s) of H-Ras responsible for suppressingintegrin activation, we generated a series of chimeric pro-teins composed of portions of the C terminus of H-Ras fused Figure 1.  H-Ras and R-Ras are ho-mologous small GTP-binding pro-teins with opposing effects on inte-grin activation. (A) Sequencealignment of H-Ras and R-Ras. as-terisk, shared amino acids; arrows,sites where exchanges were made togenerate each chimera. Shaded box,the amino acid residues responsiblefor effector binding; open box, theresidues comprising the C-terminalprenylation motif. Prenylation dif-fers between these two proteins; H-Ras is farnesylated, whereas R-Rasis predicted to be geranylgerany-lated. (B) The effects of activated H-Ras and R-Ras on integrin activationin CHO cells.   -py cells, which ex-press recombinant chimeric integrin  IIb  6A  3  1A, were transientlytransfected with an expression vec-tor encoding the transfection re-porter Tac-  5 alone and Tac-  5 plusH-Ras(G12V). In a separate transfec-tion, Tac-  5 plus H-Ras(G12V) wascotransfected with a plasmid encod-ing R-Ras(G38V). The cells were har-vested and stained for Tac expression(ordinate) to identify transfected cellsandPAC1binding(abscissa)toassesstheactivationstateoftherecombinant  IIb  6A  3  1A. In the H-Ras(G12V)-transfected cells there is a leftwardshiftofthedotplotintheupperquad-rants as a result of an inhibition of PAC1 binding. This shift is com-pletely reversed by the cotransfectionof activated R-Ras(G38V). In theempty vector control transfectionthere was no suppression of PAC1 binding in the Tac-  5-expressing(transfected) cells.P.E. Hughes  et al. Molecular Biology of the Cell2258  to the N-terminus of R-Ras, and reciprocal chimeras com-posed of portions of the C terminus of R-Ras fused to theN-terminus of H-Ras (Figures 2A and 3A). All chimerascontained either an activating H-Ras(G12V) or R-Ras(G38V)mutations to ensure they were in a GTP-bound state andthus able to engage downstream effectors.AnalysisofbothsetsofH-Ras/R-Raschimeraspinpointedresidues 148 – 171 of H-Ras as critical sequences for suppres-sion of integrin activation. Of the H-Ras C-terminal chime-ras, all those containing the H-Ras C-terminal 42 residuessuppressed PAC1 binding (Figure 2). In contrast, a chimeracontaining the last 15 residues of H-Ras, R-Ras(203)H-Ras(175 – 189), failed to suppress PAC1 binding. R-Ras inwhich the C-terminal prenylation sequence was replacedwith that of H-Ras (R-RasCVLS) also did not suppress.Indeed, both R-Ras(203)H-Ras(175 – 189) and R-RasCVLS ap-peared to increase activation slightly (Figure 2B), suggestingthat they retained R-Ras function. This hypothesis was con- fi rmed by the  fi nding that they could reverse the suppres-sive effects of H-Ras (our unpublished results). Thus, anal-ysis of chimeras composed of varying portions of the Cterminus of H-Ras indicates that sequences C-terminal of Lys 147 are necessary for suppressive activity. Furthermore,the C terminus of H-Ras beginning with Asp 175 is not suf- fi cient to convey suppressive activity to R-Ras.Chimeras composed of portions of the C terminus of R-RasfusedtotheN-terminusofH-Rasalsoindicatedtheimportanceof residues 148 – 171 of H-Ras. Chimeras containing H-Ras se-quences N-terminal of Leu 171 , H-Ras(171)R-Ras(199 – 218),H-Ras(174)R-Ras(204 – 218), and H-Ras(CVLL), all suppressedPAC1 binding (Figure 3B). In contrast, those chimeras contain-ing H-Ras sequences N-terminal of Lys 147 (H-Ras(147)R-Ras(175 – 218), H-Ras(59)R-Ras(86 – 218), lacked suppressive ac-tivity.Furthermore,bothoftheseconstructsseemedtoincreaseactivation slightly (Figure 3B) signifying that they retainedR-Ras function. Indeed, these chimeras could reverse the sup-pressive activity of H-Ras (our unpublished results). Thus, theanalysis of both the H-Ras and R-Ras C-terminal chimeras Figure 2.  Analysis of H-Ras C-terminal chimeras indicates thatresidues 148 – 171 of H-Ras are critical for the suppression of integrinactivation. (A) A schematic representation of the H-Ras C-terminalchimeras. Open box, H-Ras sequences;  fi lled box, the R-Ras portionof each chimera. (  )   -py cells were transiently transfected induplicate with either a control expression vector or vectors encod-ing either H-Ras(G12V) or the indicated chimera. After 48 h, inte-grin activation was measure by PAC1 binding. Depicted is the meanpercent inhibition of integrin activation relative to that of the emptyvector  SEM of three independent determinations. Figure 3.  Analysis of R-Ras C-terminal chimeras indicates theimportance of H-Ras residues 148 – 171 for the suppression of inte-grin activation. (A) A schematic representation of the R-Ras C-terminal chimeras. Open box, H-Ras sequences,  fi lled box, the R-Rasportion of each chimera. (  )   -py cells were transiently transfectedin duplicate with either a control expression vector or vectors en-coding either H-Ras(G12V) or the indicated chimera. After 48 h,integrin activation was determined by PAC1 binding. Depicted isthe mean percent inhibition of integrin activation relative to that of the empty vector  SEM of three independent determinations.Integrin Suppression and ERK ActivationVol. 13, July 2002 2259  pinpointed residues 148 – 171 of H-Ras as those critical forsuppression of integrin activation. Suppression of Integrin Activation Does Not Correlate with ERK Activation Suppression of integrin activation involves activation of aMAP kinase pathway and appeared to be due to the activa-tion of the ERK1/2 MAP kinases (Hughes  et al. , 1997). There-fore, we examined capacity of the chimeras to activate ERKas assessed by reactivity with a phosphorylation-speci fi cantibody. To our surprise, many of the suppressive chimerasactivated ERK poorly (Figure 4). R-Ras(85)H-Ras(60 – 189)and R-Ras(174)H-Ras(148 – 189) were potent suppressors of PAC1 binding (Figure 2B), yet they had little effect on ERKphosphorylation. All chimeras were expressed to the samelevels and in agreement with the data presented in Figure 4;none of them detectably stimulated ERK kinase activity (ourunpublished observations). These data suggest that H-Ras – initiated suppression of integrin activation could occur in-dependently of bulk ERK activation.To further test the idea that suppression could be inde-pendent of ERK activation, we blocked ERK activation andexamined the ability of H-Ras(G12V) to suppress integrinactivation. First, we tested the effect of coexpressing MAPkinase phosphatase 3 (MKP3), a phosphatase that speci fi -cally binds and dephosphorylates ERK1 and ERK2 (Muda  etal. , 1996; Keyse, 2000). Second, we used a MEK kinase in-hibitor, U0126 (Favata  et al. , 1998), to block H-Ras – inducedMEK activation, and thus, the activation of its downstreamtarget kinases, ERK1 and ERK2. The ability of H-Ras(G12V)to suppress PAC1 binding was largely unaffected by theaddition of U0126 or by coexpression of MKP3 (Figure 5A).Nevertheless, both of these treatments inhibited the bulk of ERK1/2 activation (Figure 5B). Thus, bulk ERK1/2 activa-tion can be blocked without reducing the ability of H-Ras tosuppress integrin activation. Direct Activation of ERK1/2 Is Insufficient toSuppress Integrin Activation The previous experiments suggested that ERK activationwas not necessary to for Ras to suppress integrin activation.To assess whether ERK activation was suf  fi cient for suppres-sion, we activated ERK by transfecting CHO cells withMEK1(  N3, S222D), a constitutively activated variant of MEK1 (Mansour  et al. , 1994). Unlike H-Ras(G12V), the activevariant of MEK1 failed to suppress integrin activation (Fig-ure 6A), whereas it strongly activated ERK1/2 (Figure 6B).Thus, the activation of ERK by this activated MEK1 variantwas insuf  fi cient to suppress integrin activation. Suppression of Activation of Integrin   5  1 by Activated Raf-1 Is Independent of Bulk ERK  Activation We have previously demonstrated that activated variants of Raf-1 can suppress integrin activation. In addition, analysesof H-Ras effector loop mutants suggest that only those ca-pable of coupling ef  fi ciently to Raf-1 are effective suppres-sors of integrin activation (Hughes  et al. , 1997; Sethi  et al. ,1999; our unpublished results). These data suggest that thesuppression of integrin activation by H-Ras is via a Raf-1 – dependent pathway. The data presented in this article takeour understanding of Ras/Raf-mediated suppression fur-ther by suggesting that suppression is independent of bulkERK activation. However, one of the limitations of this datais that we have only analyzed the suppression of chimeric  IIb  3 integrins. Therefore, to determine if this pathwaycould suppress the function of native integrins, we exam-ined if Raf-1 could suppress the activation of integrin   5  1.When activated, integrin   5  1 binds soluble fragments of  fi  bronectin containing the cell binding domain with highaf  fi nity. The soluble fragment of   fi  bronectin used in theseexperiments was a fusion protein, composed of glutathioneS-transferase (GST) and the 9, 10, and 11 type III repeats of  fi  bronectin, (FN 9 – 11) that make up the RGD-containingcentral cell binding domain of   fi  bronectin (Ramos and DeSi-mone, 1996). We measured FN 9 – 11 binding to endogenousintegrin   5  1 in a CHO cell line stably expressing Raf-1:ER,a conditionally active form of Raf-1. Raf-1:ER is a fusion of amodi fi ed form of the hormone-binding domain of the mouseestrogen receptor and the kinase domain of Raf-1 (Samuelsand McMahon, 1994; Chen  et al. , 1999). Raf-1 activity israpidly induced after the addition of 4  OHT to the culturemedium at a  fi nal concentration of 300 nM (Chen  et al. , 1999).In the absence of 4  OHT, the Raf-ER cells bound FN 9 – 11(Figure 7A). Binding was through integrin   5  1 as it wasinhibited by an anti-  5  1 antibody, PB1 (our unpublishedresults). FN 9 – 11 binding was inhibited by Raf-1 activationafter the addition of 300 nM 4  OHT (Figure 7B). However, binding could be reconstituted by addition of the exogenous Figure 4.  The suppressive chimeras R-Ras(85)H-Ras(60 – 189) andR-Ras(174)H-Ras(148 – 189) have little effect on ERK phosphoryla-tion.   -py cells were transiently transfected with an expressionvector encoding HA-ERK2 alone or in combination with vectorsencoding H-Ras(G12V), R-Ras(G38V), R-Ras(85)H-Ras(60 – 189), orR-Ras(174)H-Ras(148 – 189). Twenty-four hours after transfection thecells were placed in medium containing 0.5% FCS, and 48 h aftertransfection the cells were lysed and 30   g of cell lysate was re-solved on 4 – 20% SDS gel and transferred to a nitrocellulose mem- brane. The blots were probed with an mAb-speci fi c for phosphor-ylated ERK (top panel), then stripped, and reprobed with an mAb(12CA5) to verify equal expression of HA-ERK2 (bottom panel).P.E. Hughes  et al. Molecular Biology of the Cell2260
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