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Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3932|Page 1 of 5Video ArticleAffinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant RhoProtein(GST-RhoA(G17A)from Epithelial Cell LysatesFaiza Waheed1,2,Pamela Speight1,2,Qinghong Dan1,2,Rafael Garcia-Mata3,Katalin Szaszi1,21Keenan Research Centre,Li Ka Shing Knowledge Institute,St.Michaels Hospital2Department of Surgery,University of Toronto3Department of Cell and Developmental Biology,University of North Carolina at Chapel HillCorrespondence to:Katalin Szaszi at szasziksmh.caURL:http:/ Biology,Issue 61,Rho Family Small GTPases,Guanine-nucleotide exchange factor(GEFs),Affinity Precipitation Assay,expression of proteins in E.Coli,Purification of GST-tagged Protein,microbead assayDate Published:3/31/2012Citation:Waheed,F.,Speight,P.,Dan,Q.,Garcia-Mata,R.,Szaszi,K.Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant RhoProtein(GST-RhoA(G17A)from Epithelial Cell Lysates.J.Vis.Exp.(61),e3932,doi:10.3791/3932(2012).AbstractProteins of the Rho family of small GTPases are central regulators of the cytoskeleton,and control a large variety of cellular processes,includingcell migration,gene expression,cell cycle progression and cell adhesion 1.Rho proteins are molecular switches that are active in GTP-boundand inactive in GDP-bound state.Their activation is mediated by a family of Guanine-nucleotide Exchange Factor(GEF)proteins.Rho-GEFsconstitute a large family,with overlapping specificities 2.Although a lot of progress has been made in identifying the GEFs activated by specificsignals,there are still many questions remaining regarding the pathway-specific regulation of these proteins.The number of Rho-GEFs exceeds70,and each cell expresses more than one GEF protein.In addition,many of these proteins activate not only Rho,but other members of thefamily,contributing further to the complexity of the regulatory networks.Importantly,exploring how GEFs are regulated requires a method tofollow the active pool of individual GEFs in cells activated by different stimuli.Here we provide a step-by-step protocol for a method used toassess and quantify the available active Rho-specific GEFs using an affinity precipitation assay.This assay was developed a few years ago inthe Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7.The assay takes advantage of a nucleotide free mutant RhoA,with ahigh affinity for active GEFs.The mutation(G17A)renders the protein unable to bind GDP or GTP and this state mimics the intermediate statethat is bound to the GEF.A GST-tagged version of this mutant protein is expressed and purified from E.coli,bound to glutathione sepharosebeads and used to precipitate active GEFs from lysates of untreated and stimulated cells.As most GEFs are activated via posttranslationalmodifications or release from inhibitory bindings,their active state is preserved in cell lysates,and they can be detected by this assay8.Capturedproteins can be probed for known GEFs by detection with specific antibodies using Western blotting,or analyzed by Mass Spectrometry toidentify unknown GEFs activated by certain stimuli.Video LinkThe video component of this article can be found at http:/ of E.coli with the pGEX-RhoA(G17A)Construct1.Prepare LB-Agar by dissolving 2.5 g LB and 1.5 g Agar in 100 ml dH2O.Autoclave and cool to an estimated 50-55 C,which as a rule ofthumb,is when the flask can be held comfortably.2.Prepare Ampicillin(Amp)stock by dissolving 50 mg/ml in dH2O.Syringe filter and freeze unused aliquots.Add 100 l of Amp stock(finalconcentration 50 g/ml)to the LB-Agar from 1.1.Swirl to mix and pour into 10 cm bacterial dishes(15-20 ml/dish).Allow it to solidify(15-30min.)and store unused plates inverted at 4 C for 2-3 weeks.3.To transform E.Coli,quickly thaw an aliquot of DH5 competent cells in an ice bath.Add 1 l of pGEXRhoA(G17A)DNA diluted to 25-50 ng/l.Flick the tube to mix and incubate on ice for 30 minutes.Heat shock at 42 C for 45 seconds and place back on ice for 2 minutes.Add 900l SOC medium and grow for one hour at 37 C with shaking.4.Spread 50-100 l of the transformed bacteria on an LB-Agar-Amp plate using a bent sterile Pasteur pipette.Incubate the plate right side up ina 37 C incubator for 5 minutes and then invert and grow overnight.5.A single colony will be picked from the plate for preparation of the GST-tagged protein(step 2.1).For future use,wrap and store platesinverted at 4 C for about 3 weeks.In addition,bacterial stocks can be prepared for more prolonged storage by growing individual colonies in2 ml sterile LB-Amp overnight at 37 C with shaking.Mix an aliquot with sterile 80%glycerol in a 1:1 ratio and freeze at-80 C.Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3932|Page 2 of 52.Preparation of GST-RhoA(G17A)Beads1.Prepare LB by adding 25 g LB to 1 L dH20 and autoclaving.When cool,add 50 l Amp from stock to 50 ml LB(50 g/ml final concentration).Inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 C with agitation.When at full density(OD600 1.0)dilute with 450 ml LB-Amp and grow for an additional 30 minutes at 37 C.2.Prepare a 100 mM stock solution of Isopropyl B-D-thiogalactopyranoside(IPTG)by dissolving 0.238 g in 10 ml dH2O.Store in aliquots at-20 C.Induce bacteria to produce Rho protein by adding 500 l 100 mM IPTG to 500 ml culture(a final concentration of 100 M).Reducetemperature to 22-24 C and grow for 16 h hours.3.Spin culture at 3600 g for 10 minutes at 4 C.If needed,the 500 ml culture can be divided into 50 ml tubes for centrifugation.Freeze pellet(s)for at least 1 hour(or preferably overnight)at-80 C.4.Prepare 200 ml lysis buffer containing 20 mM HEPES(0.95 g)/pH 7.5;150 mM NaCl(1.75 g);5 mM MgCl2(0.203 g);1%TX-100(2 ml).Prepare stock solutions of 1M DTT(1.542 g in 10 ml dH2O)and 100 mM PMSF(0.174 g/10 ml EtOH).To prepare lysis buffer+,supplement10 ml with 1mM DTT(10 l of stock)and 1 mM PMSF(100 l of stock)and one Complete Mini Protease Inhibitor tablet.5.Working on ice,add 10 ml lysis buffer+to the pellets from step 2.3.Resuspend thoroughly by gentle vortexing and pipetting.Avoid foaming.Sonicate on ice for 1 minute at setting 4 with 50%pulse.Spin the sonicated lysate at 15,000-20,000 g for 15 minutes at 4 C,and remove theclarified sonicate(supernatant)to a sterile capped 15 ml tube.6.Prepare the Glutathione Sepharose by gently mixing the original tube containing a 75%slurry and transfer 335 l into a 15 ml tube.Use awide bore tip to pipette beads.Add 10 ml cold PBS,and spin 500 g for 5 minutes at 4 C.Discard the supernatant,add 1 ml lysis buffer+tothe beads and spin as for previous wash.Discard the supernatant and add lysis buffer+to make a 50%slurry.7.Add 250 l of equilibrated bead slurry to the supernatant from step 2.5.Rotate at 4 C for 45 minutes.8.Prepare 500 ml HBS containing 20 mM HEPES(2.38 g)/pH 7.5 and 150 mM NaCl(4.38 g)in dH2O.Prepare stock solutions of 1M MgCl2(0.952 g in 10 ml dH2O)and 1M DTT(1.542 g in 10 ml dH2O).To prepare HBS+,supplement 100 ml just before use with 5 mM MgCl2(50 lfrom stock)and 1 mM DTT(100 l from stock).9.Spin the beads from step 2.7 at 500 g for 5 minutes at 4 C.Discard the supernatant and wash beads 2x with 10 ml lysis buffer+,and 2xwith 10 ml HBS+.After the final wash,make a 50%slurry by resuspending the beads in HBS+supplemented with BD BaculoGold proteaseinhibitor(20 l of 50 x BD BaculoGold/ml).10.Dilute 10 l of the final beads preparation with 2x Laemmli sample buffer containing-mercaptoethanol.Make Bovine Serum Albumin(BSA)standards.Use a 2 mg/ml stock(0.02 g of BSA in 10 ml dH2O).Mix 10 l of stock with 10 l Laemmli(20 g final);5 l of stock with 5 l ofdH2O and 10 l Laemmli(10 g final);and 2.5 l stock with 7.5 l dH2O and 10 l Laemmli(5 g final).Boil all samples(5 min).Spin thebead sample and run supernatant with BSA standards and molecular weight markers on a 10%SDS-polyacrylamide gel.11.Prepare the Comassie Blue stain(0.1 g in 10 ml Acetic Acid,40 ml Methanol and 50 ml dH2O)and the destain solution(500 ml dH2O,400ml methanol and 100 ml acetic acid).Store at room temperature.Stain the gel for 20-30 minutes,remove the dye(it can be reused multipletimes)and rinse with destain solution twice.Continue to destain with gentle shaking for several hours until bands are clearly visible.12.Estimate the concentration of GST-RhoA(G17A)coupled to the beads using the BSA standards as a reference(Fig 2).Aliquot an equalvolume of beads containing 10-15 g protein into 1.5 ml micro centrifuge tubes.Store beads at 4 C to use within a day.Freeze at-80 C inHBS+/glycerol in a 3:1 ratio to use within a few days.3.GEF Pulldown Assay with Nucleotide-free RhoA(G17A)Beads1.Grow cells in 10 cm dishes to confluence.Serum deprive for at least 3 hours and treat as required.2.Prepare lysis buffer+as in step 2.4.Prepare enough lysis buffer for 700 l/dish plus some extra amount to allow for pipetting errors.Add theprotease inhibitors just before use.3.Working on ice,remove culture medium from the dishes and wash with ice-cold HBS.Remove all the HBS and add 700 l lysis buffer+toeach dish.Swirl plates to cover all areas,scrape and collect lysates into numbered 1.5 ml tubes.Spin at 15,000 g for 1 min at 4 C.Thesupernatant will be used for the assay.4.If your cell number is equivalent in all dishes being tested,you can omit doing a protein assay,and move to step 3.5.Otherwise measure theprotein concentration of each supernatant using Bio-Rad quick protein assay and equalize the supernatant for volume and concentration.Theamount of total protein depends on the cell types used(typically 1-1.5 mg protein for LLC-PK1 cells).5.Remove 30 l of each supernatant and mix with 30 l 2x reducing Laemmli sample buffer,boil and set aside for step 3.7.Add remainingsupernatants to aliquots of the GST-RhoA(G17A)beads from step 2.12.Rotate for 45 minutes at 4 C.6.Spin beads at 6800 g for 10 seconds at 4 C.Discard the supernatant and wash the beads 3x with lysis buffer,spinning in the same waybetween washes.Completely remove the final wash using a 1 cc syringe fitted with a 30 G needle and add 20 l 2x reducing Laemmli samplebuffer.Boil for 5 min.Spin to pellet beads and either run the supernatant immediately(preferable)or store it at-80 C for later analysis.7.Run 20 l total cell lysates and all of the precipitated protein samples on the appropriate percentage SDS-polyacrylamide gel for the size ofGEF you are studying.Detect your GEF of choice by Western blotting using a specific antibody.4.Representative ResultsPart 1 and 2 of the protocol describes preparation of GST-RhoA(G17A)coupled to GSH-sepharose beads and its testing by SDS-PAGE(seeoutline of protocol on Fig 1).A typical Coomassie stained gel is shown on Fig 2.The sample with the eluted protein should contain a single bandat approximately 50 kDa(Fig 2,lane 6).The concentration of the protein can be estimated using the BSA reference samples.In the exampleon Fig 2,the concentration of RhoA(G17A)is estimated to be 15 g/10 l.Thus,aliquots of 10 l/tube were prepared.The typical yield in ourhand is 15-20 g protein from 10 ml bacterial lysis.Part 3 of the protocol describes the affinity precipitation assay(see overview on Fig 1).Asuccessful GEF assay detecting activation of the exchange factor GEF-H1 is shown on Fig 3.The RhoA(G17A)protein captured some GEF-H1from the control(untreated)cell lysates,suggesting that GEF-H1 has basal activity.The amount precipitated however increases in cells treatedwith the inflammatory cytokine Tumor Necrosis Factor-(TNF-),consistent with the notion that TNF-activates GEF-H1 5,7.Importantly,theJournal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3932|Page 3 of 5total cell lysates show similar amounts of GEF-H1 in the control and the treated sample,suggesting that the treatment did not alter GEF-H1levels and the input used in the assay is equal.Figure 1.Overview of the protocol.Figure 2.Representative result of the bead preparation protocol.A Coomassie stained gel with successful GST-RhoA(G17A)bead preparationis shown.Bead sample and BSA protein standards were separated by SDS-PAGE using a 10%acrylamide gel.To test the beads,10 l of thefinal bead slurry containing GST-RhoA(G17A)is diluted 1:1 with reducing Laemmli sample buffer and boiled for 5 minutes.The beads were spunbriefly and the supernatant loaded on the gel.The following samples were loaded:Lane 1:molecular weight marker(MW)(FroggaBio BLUeyeprestained protein ladder);Lanes 2-4:5,10 and 20 g Bovine Serum Albumin(BSA);Lane 5 is empty;Lane 6:10 l of the freshly preparedRhoA(G17A)beads.After separation is completed,the gel is stained using Coomassie Blue and subsequently destained to reveal proteins.Themolecular weight of the GST-RhoA(G17A)protein is roughly 50 kDa and runs around the level of the 48 kDa marker.The concentration of theGST-Rho protein in this particular sample is estimated to be around 15 g/10 l slurry.Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3932|Page 4 of 5 Figure 3.Representative GEF activation assay showing TNF-induced activation of GEF-H1.Confluent LLC-PK1 cells were treated with 10ng/ml TNF-for 5 minutes.Following treatment the cells were lysed and active GEFs were captured using GST-RhoA(G17A)bound beads.Thepresence of GEF-H1 in the precipitated proteins(top blot)and total cell lysate samples(bottom blot)was detected using Western blotting withan anti-GEF-H1 antibody(Cell Signaling).Please note the increased amount of precipitated GEF-H1 in TNF-treated versus non-treated cellsrelative to the equivalent inputs,indicating a successful result.Figure 4.A bad result.Although the GEF pulldown assay shown here resulted in some GEF-H1 captured by the beads,the amountsprecipitated from control and TNF-treated cells are the same.Thus TNF-,a know activator of GEF-H1 in this case did not induce activation.Inthis particular experiment subsequent troubleshooting suggested that the TNF-used was not fresh enough and probably degraded.DiscussionThe method presented here is the only available non-radioactive activation assay for GEFs that can follow the active pool of GEFs in cells.The assay is similar to the precipitation assays used for following activation of small GTPases as well as GEFs against Rac and Cdc42.Thoseassays use different GST-tagged proteins and have slight differences from the one described here,however the basic steps are the same.Thus,this protocol can easily be adapted for other small GTPase and GEF activation assays.The presented GEF assay was recently modified for application for nuclear fractions 9,10.With further modifications,testing of GEF activation inother subcellular compartments might also be possible.We use the presented method to study activation of GEFs in epithelial cell lines5,6,7.With some op