small gtpases and their regulators, part b

DAVID LAMBETH 27, Department of BiD- chemistry, Emory University MediCal School, Atlanta, Georgia 30322 PAUL LANG 35, INSERM CJF 93-O1, Facultd de Pharmacie-Universit~ Paris-Sud, 922

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P r e f a c e Rho-related GTP-binding proteins constitute a functionally distinct group in the small GTPase superfamily Like Ras, they control intracellular signal transduction pathways, and it is now firmly established that Rho- related GTPases regulate the organization of the actin cytoskeleton of all eukaryotic cells Accordingly, this family of GTPases controls cell adhesion, cell movement, and cytokinesis

This volume describes a wide range of experimental approaches that have been used to study the function of Rho-related GTPases both in vitro

and in vivo The availability of recombinant proteins has been of enormous benefit in characterizing the biochemical and biological activities of the GTPases and of the proteins with which they interact The first part of this volume deals with expression systems used both in Escherichia coli and in insect cells The driving force for the enormous interest now being taken

in the Rho family of GTPases stems from their demonstrated biological roles, particularly as regulators of adhesion and movement Thus many of the cellular assays that have been used to establish these effects are included

in this volume The ultimate test for any cellular activity attributed to a GTPase is the ability to reconstitute that activity in vitro To date, this has been achieved only for Rac-dependent activation of phagocytic NADPH oxidase, and several chapters are devoted to this topic

Although the area has already generated an enormous amount of gen- eral interest, the functional analysis of small GTPases is still in its infancy There are many more surprises to come as the biochemical details of the pathways controlled by small GTPases are elucidated The prize is a molecular explanation of many aspects of contemporary cell biology

We are extremely grateful to all the contributors who have taken the time to commit their expertise to paper, and are confident that their efforts will be greatly appreciated by the scientific community Dr Hall thanks the Cancer Research Campaign (UK), the Wellcome Trust, and the Medical Research Council (UK) for providing the funds and environment that have allowed him to work in this very exciting area

ALAN HALL

W E BALCH CHANNING J DER

xiii

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C o n t r i b u t o r s to V o l u m e 2 5 6 Article numbers arc in parentheses following the names of contributors

Affiliations listed are currenl

ARIE ABO (5, 29), Onyx Pharmaceuticals,

Richmond, California 94806

PETER ADAMSON (19), Vascular Biology Re-

search Centre, Kings College London, Lon-

don, W8 7AH, United Kingdom

DANIEL E H AFAR (15), Department of Mi-

crobiology and Molecular Genetics, Univer-

sity of California-Los Angeles, Los Angeles,

California 90024

SOHAIL AHMED (14), Department of Neuro-

chemistry, Institute of Neurology, London

WC1N 1PJ, United Kingdom, and Institute

of Molecular and Cell Biology, National

University of Singapore, Singapore 0511

KLAUS AKTORIES (21), Institute of Pharma-

cology and Toxicology, Albert-Ludwigs

University, D-79104 Freiburg, Germany

PONTUS ASPENSTROM (25), Department of

Zoological Cell Biology, Arrhenius Labo-

ratories E5, The Wenner-Gren Institute,

Stockholm University, S106-91, Sweden

DAVID BALTIMORE (17), Massachusetts Insti-

tute of Technology, Cambridge, Massachu-

setts 02139

PATR1C1A BEROEZ-AULLO (32), Laboratoire

de Biologie Mol(culaire et S4quencage,

Universit~ Bordeaux II, 33076 Bordeaux,

France

JACQUES BERTOGLIO (35), INSERM CJF 93-

01, Facultd de Pharmacie-Universit~ Paris-

Sud, 92296 Chatenay Malabry Cedex,

France

GARY M BOKOCH (4, 28), Departments of

Immunology and Cell Biology, The Scripps

Research Institute, La Jolla, California

92037

PATR1CE BOQUET (32), Unit~ des Toxines Mi-

crobiennes, Institut Pasteur, 75724 Paris,

France

EDWARD P BOWMAN (27), Department of

Biochemistry, Emory University Medical School, Atlanta, Georgia 30322

RICHARD A CERIONE (2, 9, 12), Department

of Pharmacology, Cornell University, Ith- aca, New York 14853

PIERA CICCHETTI (17), Institute for Genetics, University of Cologne, Cologne D-50674, Germany

DAGMAR DIEKMANN (23), CRC Oncogene and Signal Transduction Group, MRC Lab- oratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom

SIMON T DILLON (20), Department of Micro- biology and Molecular Biology, Tufts Uni- versity School qfl Medicine, Boston, Massa- chusetts 02111

OLIVIER DORSEUIL (39), Institut Cochin de G4n4tique Mol&ulaire, 1NSERM UnitO

257, 75014 Paris, France

ALESSANDRA EVA (38), Laboratory of Cellu- lar and Molecular Biology, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892

LARRY a FEIG (20), Department of Biochem- istry, Tufts University, School of Medicine, Boston, Massachusetts 02111

PHILIPPE FORT (18), Institute of Molecular Ge- netics, University Montipellier, F 340.33 Montpellier, France

ROSEMARY FOSTER (13), MGM Cancer Cen- ter and Department of Medicine, Harvard Medical School, Charlestown, Massachu- setts 02129

GERARD GACON (39), Institut Cochin de G~n- (ique Mol~culaire, 1NSERM Unit4 257,

75014 Paris, France

MURIELLE GIRY (32), Unit~ des Toxines Mi- crobiennes, Institut Pasteur, 75724 Paris, France

IX

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X CONTRIBUTORS TO VOLUME 256

ALAN HALL (1, 8, 23), MRC Laboratory for

Molecular Cell Biology and Department of

Biochemistry, University College London,

London WC1E 6BT, England

CHRISTINE HALL (14), Institute of Neurology,

London WC1N 1P J, United Kingdom

JOHN F HANCOCK (10), Onyx Pharmaceuti-

cals, Richmond, California 94806

MATTHEW J HART (9), Department of Phar-

macology, Ithaca, New York 14853

DOUGLAS I JOHNSON (30), Department of Mi-

crobiology and Molecular Genetics, Univer-

sity of Vermont, Burlington, Vermont05405

INGO JUST (21), Institute of Pharmacology and

Toxicology, Albert-Ludwigs University,

D-79104 Freiburg, Germany

ULLA G KNAUS (4), Department oflmmunol-

DAy, The Scripps Research Institute, La

Jolla, California 92037

ROBERT KOZMA (14), Institute of Neurology,

London WC1N IPJ, United Kingdom, and

Institute of Molecular and Cell Biology, Na-

tional University of Singapore, Singapore

0511

J DAVID LAMBETH (27), Department of BiD-

chemistry, Emory University MediCal

School, Atlanta, Georgia 30322

PAUL LANG (35), INSERM CJF 93-O1, Facultd

de Pharmacie-Universit~ Paris-Sud, 92296

Chatenay Malabry Cedex, France

GI~RALD LECA (39), INSERM Unit~131,

Association Chlude Bernard, Institute

d'Hematologie-HOpital Saint-Louis, Paris,

France

EMMAUEL LEMICHEZ (32), Unit~ des Toxines

Microbiennes, Institut Pasteur, 75724

Paris, France

DAVID LEONARD (2,12), Department of Phar-

macology, Cornell University, Ithaca, New

York 14853

THOMAS LEUNG (16, 24), Institute of Molecu-

lar and Cell Biology, National University of

Singapore, Singapore 0511

Louis LIM (14, 16, 24), Institute of Neurology,

London WC1N 1PJ, United Kindgom, and

Institute of Molecular and Cell Biology, Na-

tional University of Singapore, Singapore

TORU MIKI (11), Laboratory of Cellular and Molecular Biology, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892

PETER J MILLER (30), Department of Micro- biology and Molecular Genetics, University

of Vermont, Burlington, Vermont 05405

TAKAKAZU MIZUNO (3), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka

565, Japan

NARITO MORII (22), Department of Pharma- cology, Kyoto University Faculty of Medi- cine, Kyoto 606, Japan

HIROYUKI NAKANISHI (3), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka

565, Japan

SHUH NARUMIYA (22, 31), Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto University, Kyoto 606, Japan

MICHAEL F OLSON (25), CRC Oncogene and Signal Transduction Group, MRC Labora- tory for Molecular Cell Biology, University College London, London WCIE 6BT, United Kingdom

Huort PATERSON (19), Section of Cell and Molecular Biology, Chester Beatty Labora- tories, Institute of Cancer Research, London SW3 6B J, United Kingdom

MARK R PHILIPS (7), Departments of Medi- cine and Cell Biology, New York University School of Medicine, New York, New York 10016

MICHAEL H PILLINGER (7), Department of Medicine, New York University School of Medicine, New York, New York 10016

MICHEL R POPOFF (32), Unit~ des Toxines Microbiennes, Institut Pasteur, 75724 Paris, France

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CONTRIBUTORS TO VOLUME 256 xi EMILIO PORFIRI (10), Onyx Pharmaceuticals,

Richmond, California 94806

JAMES POSADA (30), Department of Microbi-

ology and Molecular Genetics, University

of Vermont, Burlington, Vermont 05405

MARK T QUINN (28), Veterinary Molecular

Biology, Department of Microbiology,

Montana State University, Bozeman, Mon-

tana 59717

ANNE J RIDLEY (33, 3,4), Ludwig Institute

for Cancer Research, London WCIP 8BT,

United Kingdom

SUSAN E RITTENHOUSE (26), Jefferson Can-

cer Institute and Cardeza Foundation for

Hematologic Research, Philadelphia, Penn-

sylvania 19107

DAVID ROBERTSON (19), Haddow Labora-

tories, Institute of Cancer Research, Sutton,

Surrey, SM2 5NG, United Kingdom

TAKUYA SASAKI (6, 37), Department of Mo-

lecular Biology and Biochemistry, Osaka

University Medical School, Suita, Osaka

565, Japan

ANTHONY W SEGAL (29), Division of Molec-

ular Medicine, University College London,

London WCIE 6J J, United Kingdom

ANNETTE J SELF (1, 8), MRC Laboratory for

Molecular Cell Biology, University College

London, London WC1E 6BT, United

Kingdom

JEFFREY SETTLEMAN (13), MGH Cancer Cen-

ter and Department of Medicine, Harvard

Medical School, Charlestown, Massachu-

setts 02129

MARIE-JOSE STASlA (36), Laboratoire d'En-

zymologie, Centre Hospitalier Universitaire

de Grenoble, Grenoble, France

YOSHIMI TAKAI (3, 6, 37), Department of Mo-

lecular Biology and Biochemistry, Osaka

University Medical School, Osaka 565, Ja-

pan, and Department of Cell Physiology, National Institute for Physiological Sci- ences, Okagaki 444, Japan

KENJI TAKAISHI (37), Department of Molecu- lar Biology and Biochemistry, Osaka Uni- versity Medical School Suita 565, Japan

KAZUMA TANAKA (6), Department of Molec- ular Biology and Biochemistry, Osaka Uni- versity Medical School Suita, Osaka 565, Japan

TOMOKO TOMINAGA (31), Department of Cel- lular and Molecular Physiology, National Institute for Physiological Sciences, Oka- zaki 444, Japan

DAVID J UHLINGER (27), Department of Biochemistry, Emory University Medical School Atlanta, Georgia 30322

A1ME VASQUEZ (39), 1NSERM Unit 131, As- sociation Claude Bernard Research Center,

92140 Clamart, France

PIERRE V VIGNAIS (36), Laboratoire de Bio- chimie, Departement de Biologie Molecu- laire et Structurale, CEA CEN-Grenoble, F-38054 Grenoble, France

SYLVIE VINCENT (18), Institute of Molecular Genetics, University Montipellier, F 34033 Montpellier, France

OWEN N WITrE (15), Molecular Biology In- stitute and Howard Hughes Medical Insti- tute, University of California-Los Angeles, Los Angeles, California 90024

DANIELA ZANGR1LLI (38), Laboratory of Cel- lular and Molecular Biology, National Can- cer Institute, National Institutes of Health, Bethesda, Maryland 20892

Y1 ZHENG (2, 9), Department of Pharma- cology, Cornell University, Ithaca, New York 14853

MICHAEL ZIMAN (30), Department of Molecu- lar and Cell Biology, University of Califor- nia, Berkeley, Berkeley, California 94720

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[ 1 ] Rho/Rac/G25K FROM E coli 3

[ 1] P u r i f i c a t i o n o f R e c o m b i n a n t R h o / R a c / G 2 5 K

f r o m E s c h e r i c h i a coli

By ANNETTE J SELF a n d A L A N H A L L Introduction

The purification of Ras-related GTP-binding proteins from recombinant sources has proved to be invaluable for studying their biochemical properties and biological effects The simplest expression systems have made use

not posttranslationally modified Yeast and baculovirus-Sf9 (Spodaptera

they are eukaryotic hosts, the GTPases expressed are at least partially modified 1'2 A wide range of expression levels has been reported for Ras- related proteins in E coli; in the case of Ras, yields of 7.5 mg/liter of culture have been obtained, 3 whereas others such as Rap1, for example, have proved much more difficult to make in a stable form Members of the Rho family have been relatively difficult to express in E coli in large amounts; as described below, we obtain yields of around 0.1-1 rag/liter The mammalian Rho subfamily consists of RhoA, B, and C, Racl and

2, G25K/CDC42, RhoG, and TC10 4-9 These proteins are 30% identical to Ras in amino acid sequence and 55% identical to each other, and their overall three-dimensional structure is expected to be very similar to that

of Ras 1° RhoA, B, and C are 85% identical to each other, with almost all

x S G Clark, J P McGrath, and A D Levinson, Mol Cell Biol 5, 2726 (1985)

2 M J Page, A Hall, S Rhodes, R H Skinner, V Murphy, M Sydenham, and P N Lowe,

J Biol Chem 264, 19147 (1989)

3 A M De Vos, L Tong, M V Milburn, P M Matias, J Jancarik, S Noguchi, S Nishimura,

K Mitra, E Ohtsuka, and S Kim, Science 239, 888 (1988)

4 p Madaule and R Axel, Cell 41, 31 (1985)

5 j Didsbury, R F Weber, G M Bocock, T Evans, and R Synderman, J Biol Chem 264,

9 S Vincent, P Jeanteur, and P Fort, MoL Cell Biol 12, 3138 (1992)

10 E F Pai, W Kabsch, U Krengal, K C Holmes, J John, and A Wittinghofer, Nature 341,

209 (1989)

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4 EXPRESSION AND PURIFICATION [ l]

of the divergence being at the carboxy-terminal end of the proteins; R a c l and 2 are 92% identical to each other with 15 amino acids different; and

G 2 5 K and C D C 4 2 H s are the closest related isoforms with only 9 amino acid differences b e t w e e n them All R h o family m e m b e r s contain a C- terminal C A A X box motif (A = aliphatic amino acid; X = L for R h o and Rac; X = F for C D C 4 2 / G 2 5 K ) , and all are posttranslationally modified in vivo by the addition of a C 20 geranylgeranyl isoprenoid, u 13 Interestingly,

R h o B also a p p e a r s to be a substrate for the f a r n e s y l t r a n s f e r a s e J 4

Like all small G T P a s e s , the R h o - r e l a t e d proteins are regulated by guanine nucleotide exchange factors ( G E F s ) and GTPase-activating proteins ( G A P s ) , and the characterization of these regulatory proteins has relied

on a source of r e c o m b i n a n t protein All G A P s and m o s t G E F s are active

in vitro on E c o l i - p r o d u c e d , nonmodified R h o - r e l a t e d GTPases E coli-

p r o d u c e d r e c o m b i n a n t proteins are also very useful for studying the biological function of the R h o subfamily by microinjection because the G T P a s e s

b e c o m e posttranslationally modified and functionally active after in-

j e c t i o n J 5

T o characterize the function of R h o - r e l a t e d proteins, we have purified

R h o A , R a c l , and G 2 5 K f r o m E coli using the glutathione S-transferase ( G S T ) gene fusion vector p G E X - 2 T (Pharmacia L K B Biotechnology, Inc.) 16 As described in the following section, the yields of these proteins

f r o m this vector are not as high as have b e e n r e p o r t e d for other proteins expressed using this system, but purification is e x t r e m e l y rapid and the final p r e p a r a t i o n s are of high purity

C o n s t r u c t i o n of V e c t o r s

c D N A s g e n e r a t e d by the p o l y m e r a s e chain reaction ( P C R ) and encoding h u m a n R h o A , R a c l , and G 2 5 K were fused to the carboxy-terminal end of the S c h i s t o s o m a ] a p o n i c u m glutathione S-transferase gene by cloning into the B a m H I / E c o R I sites of p G E X - 2 T (see Fig 1) Expression of the fusion protein is u n d e r the control of the tac p r o m o t e r , and the nucleotide sequences across the fusion junctions are shown in Fig lb A f t e r cleavage 11M Katayama, M Kawata, Y Yoshida, H Horiuchi, T Yamamoto, Y Matsuura, and

Y Takai, J Cell Biol 266, 12639 (1991)

12 B T Kinsella, R A Erdman, and W A Maltese, J Biol Chem 15, 9786 (1991)

13 H Yamane, C C Farnsworth, H Xiec, T Evans, W N Howald, M H Gelb, J A Glomset,

S Clarke, and B K K Fung, Proc Natl Acad Sci U.S.A 88, 286 (1991)

a4 p Adamson, C J Marshall, A Hall, and P A Tilbrook, J Biol Chem 267, 20033 (1992) 15H F Paterson, A J Self, M D Garrett, I Just, K Aktories, and A Hall,

J Cell Biol 111, 1001 (1990)

16 D B Smith and K S Johnson, Gene 67, 31 (1988)

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[1] Rho/Rac/G25K FROM E coli 5

ILeu Val Pro Arg~Gly serlpro GIy lie His Arg Asp

GST CTG G-I-F CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG

BamHl [ _ _ ] EcoRl Stop codons

Smal GST CTG G-I-r CCG CGT GGA TCC CCG GCT rhoA

GST CTG GTT CCG CGT GGA TCC CCG CAG., racl

GST CTG GTT CCG CGT GGS TCC CCG CAG., GZSK

codon

2 FIG 1 Structure of the glutathione S-transferase vector pGEX-2T (a) Schematic represen- tation of pGEX-2T (b) Nucleotide sequence of pGEX-2T and of pGEX-2T containing RhoA, Racl, and G25K cDNAs across the fusion junction

with thrombin it is predicted that the GTPases will each have Gly-Ser-Pro fused to the second codon of the native sequence The p G E X - 2 T vectors

containing R h o A , R a c l , and G25K were each introduced into the E coli

strain JM101 and stored as glycerol stocks at - 7 0 °

P u r i f i c a t i o n of Wild-Type RhoA, Rac 1, a n d G 2 5 K

Growth and Purification

One hundred milliliters of L-broth containing 50/~g/ml ampicillin is

inoculated with E coli containing the expression plasmids taken from the

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6 EXPRESSION AND PURIFICATION [ l ] glycerol stock After overnight incubation at 37 °, the culture is diluted 1 : 10 into fresh, prewarmed (37 °) L-broth/ampicillin and is incubated for 1 hr in two 2-liter flasks in a bacterial shaker at 37 ° To induce fusion protein expression, isopropyl-/3-D-thiogalactopyranoside (IPTG) is added to 0.1

mM (0.5 ml of a 0.1 M stock made in water and stored at -20°), and the culture is incubated with shaking for a further 3 hr After induction, the cells are collected in l-liter buckets by centrifugation at 4000 rpm for 10 min at 4 ° and then resuspended (on ice) in 3 ml of cold lysis buffer [50

mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF)]

We have noted that many purification procedures for GST fusion proteins use buffers containing phosphate, a chelator of magnesium ions 16 In low magnesium concentrations, Rho-related GTPases rapidly lose their bound guanine nucleotide (see [9] in this volume) and are unstable It is therefore important that phosphate buffers or other chelators of magnesium such as EDTA are not used in the purification procedure and that there

is an excess of free magnesium in all buffers used

Resuspended bacteria are lysed by sonication on ice (three times at 1 min each) We use a small probe on an MSE Soniprep 150 sonicator at an amplitude of 14 tzm, and the bacterial suspension is kept cool at all times

As lysis occurs the suspension turns from a light creamy color to a muddy brown and becomes somewhat more viscous The sonicate is centrifuged

at 10,000 rpm for 10 min at 4 °, and the supernatant (4 ml) is carefully transferred to a 5-ml bijou tube (Sterillin) Some 30-50% of GST-RhoA,

G S T - R a c l , and GST-G25K produced by this expression system in JM101

is found in the pellet after centrifugation of the sonicate

Glutathione-agarose beads (Sigma G4510) or glutathione-Sepharose 4B beads (Pharmacia) are prewashed with several volumes of lysis buffer and kept as a 1 : 1 suspension One milliliter of this suspension is added to the supernatant and is incubated for 30 min on a rotating wheel at 4 ° The beads are pelleted in a benchtop centrifuge at 4000 rpm for 1 min, and the supernatant is removed and discarded The beads are then washed with 5

ml of cold lysis buffer (without DTT and PMSF) five times to remove unbound proteins Recovery of bound protein can be achieved in one of two ways

a Recovery o f Fusion Protein The GST fusion protein can be eluted from the beads by competition with free glutathione An equal volume (0.5 ml) of freshly prepared release buffer [50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgC12, 1 mM DTT + 5 mM reduced glutathione (Sigma G4251) (final pH 7.5)], is added to the washed beads and incubated for 2 min at 4 ° on a rotating wheel The beads are pelleted and the supernatant

Trang 9

[11 Rho/Rac/G25K FROM E coli 7

is removed The procedure is repeated, and the two supernatants are pooled (1 ml) and dialyzed overnight (see later)

are transferred to a 1.5-ml microcentrifuge tube and resuspended in 0.5 ml

of thrombin digestion buffer (50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 2.5

mM CaCI2,5 mM MgCI2, 1 mM DTT) containing 5 units of bovine thrombin (Sigma T6634) The suspension is incubated at 4 ° on a rotating wheel overnight After thrombin digestion, the beads are pelleted in a microcentrifuge (1 min), and the supernatant is removed Sometimes after thrombin digestion, the cleaved protein remains partly associated with the beads so

we routinely incubate the beads with another 0.5 ml of high salt/DTY buffer (50 mM Tris-HC1, 7.6, 150 mM NaC1, 5 mM MgC12, 1 mM DTT) for 2 rain at 4 ° After centrifugation the two supernatants are pooled (1 ml) The efficiency of thrombin cleavage of G S T - R h o A and G S T - R a c l approaches 100%, but GST-G25K is more resistant and usually only 50% is cleaved

by an overnight incubation with thrombin

Thrombin can be removed by adding 10 ~1 of a suspension of p-amino- benzamidine-agarose beads (Sigma) to the supernatant and incubating for

a further 30 rain at 4 ° on a rotating wheel

Dialysis and Storage

For microinjection purposes we dialyze against 2 liters of 10 mM Tris- HC1, pH 7.6, 150 mM NaC1, 2 mM MgC12, and 0.1 mM DTT at 4 ° overnight with one buffer change For GTPase assays where a low salt concentration

is required (10 mM NaC1), we dialyze against 10 mM Tris-HCl, pH 7.6, 2

mM MgC12, and 0.1 mM DTT Proteins are concentrated to approximately 150/xl in an Amicon Centricon 10 filter device by centrifugation in a fixed angle rotor at 7000 rpm We routinely store the final protein preparations

at approximately 1 mg/ml in 10-/zl aliquots, snap frozen in liquid nitrogen The protein concentration is determined by a [3H]GTP/[3H]GDP binding assay as described below The yield of wild-type proteins as determined by nucleotide binding is in the order of 0.1-0.2 rag/liter of bacterial culture Figure 2 shows a Coomassie-stained gel of GST fusion and thrombin- cleaved RhoA, N25RhoA (see later), Racl, and G25K proteins

Determination of Protein Concentration

Protein concentration is determined by a guanine nucleotide nitrocellulose filter binding assay We use [3H]GTP or [3H]GDP but 32p-labeled nucleotides can also be used Samples of concentrated protein (0.1, 0.2,

Trang 10

8 EXPRESSION AND PURIFICATION [ 1]

and 0.3/zl) are incubated in a total volume of 40/zl of assay buffer (50

mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 5 mM DT]?) containing

10 mM E D T A and 0.5/zl [3H]GTP or [3H]GDP (Amersham, 10 Ci/mmol,

1 mCi/ml) for 10 min at 30 ° Samples are diluted with 1 ml of cold assay buffer (without DTT) and are filtered through prewetted 25-ram nitrocellulose filters (NC45 Schleicher & Schuell 0.45/zm) using a Millipore filtration device The filters are washed three times with 3 ml of cold assay buffer (without DTT) and are allowed to dry in air Radioactivity is determined

by scintillation counting If 1 tool of Rho binds 1 mol of [3H]GTP, then 1 /zg Rho should yield 10 6 dpm (disintegrations per minute) The concentration of the protein sample (mg/ml) is calculated using Eq (1):

In our hands counting efficiency can be as low as 20%

Protein concentration can also be determined by comparing samples with bovine serum albumin (BSA) standards after electrophoresis on a 12% polyacrylamide gel and staining with Coomassie Brilliant Blue R (Sigma) The concentration of Rho proteins determined by this method is 3- to 5-fold higher than that determined by guanine nucleotide binding The estimation of protein concentration by Bradford or Lowry methods gives values approximately 10-fold higher than those determined by guanine nucleotide binding We do not understand the reason for the differences

in the three assays, but a similar discrepancy has been found by others and also with Ras protein preparations We use the guanine nucleotide binding assay as a measure of protein concentration

Protein Stability

We previously reported that wild-type RhoA produced as a nonfusion

protein in a trp promoter expression system was biologically inactive after

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[ 11 Rho/Rac/G25K FROM g coli 9

microinjection into cells 15 The protein was, however, still able to bind guanine nucleotide and to hydrolyze GTP Subsequent experiments revealed that the protein was substantially clipped at its C terminus during the relative long purification procedure required using this system A similar observation is found with Ras expression plasmids; Ki-Ras in particular is highly susceptible to proteolysis at its C terminus in E coli We found, however, that Rho with an amino acid substitution of phenylalanine to asparagine at codon 25 (N25Rho), produced using the same expression system, is biologically active Since N25Rho has a similar nucleotide exchange rate and GTP hydrolysis rate to wild-type Rho and is sensitive

to R h o - G A P , we have used N25 versions of Rho proteins for many of our experiments

We have reexamined the problem of expressing wild-type RhoA using the pGEX-2T expression system described earlier As can be seen from Fig 2, N25Rho migrates slightly slower than wild-type RhoA and produces

a much sharper band We have found that all Ras and Rho-related GTPases are prone to smearing after electrophoresis, particularly if freshly prepared sample loading buffer is not used, and it is likely that the proteins are sensitive to oxidation Even with fresh buffer, however, the smearing observed with wild-type RhoA could not be overcome Despite this, wild- type R h o A purified from the p G E X expression system is only around twofold less active than N25RhoA in the microinjection assay

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10 EXPRESSION AND PURIFICATION [ 1 ]

M u t a n t Rho, Rac, a n d G25K Proteins

We have purified a variety of Rho, Rac, and G25K proteins containing amino acid substitutions using the pGEX-2T vector and the protocol described earlier These include constitutively activated protein with (i) glycine to valine substitutions at codon 14 in RhoA (V14Rho) or codon 12

in Rac (V12Rac) and G25K (V12G25K), equivalent to the oncogenic V12 mutation in Ras; and (ii) glutamine to leucine substitutions at codon 63 in Rho (L63Rho) or codon 61 in Rac (L61Rac), equivalent to the oncogenic L61 mutation in Ras In addition, we have made dominant negative mutations with a threonine to asparagine substitution at codon 17 in Rac (N17Rac) and G25K (N17G25K), equivalent to the dominant negative N17 mutation in Ras The yields of these mutant proteins as determined by nucleotide binding and Coomassie staining of acrylamide gels are shown

in Table I Table I shows that the yields, as judged by nucleotide binding

of N17Racl, V12G25K, and particularly N17G25K, are very low but that the actual concentrations of the proteins, as determined by gel electrophoresis, are clearly much higher In addition, we have found that N17Racl and N17G25K only bind [3H]GDP and not [3H]GTP in the guanine nucleotide filter binding assay This appears to be a common feature of the N17 dominant negative proteins first observed by Cooper and Feig with Rasff Attempts to produce a dominant negative RhoA protein, N19RhoA, in

E coli have so far been unsuccessful The fusion protein is expressed, but after sonication almost all of the protein is found in the pellet (A Ridley, personal communication, 1994) Although N19RhoA can be solubilized from the pellet using detergent, the resulting protein has no detectable biological effect when microinjected into cells

Acknowledgments

We thank Suzanne Brill, Dagmar Diekmann, and Anne Ridley for data on mutant proteins; Catherine Nobes for comparing wild-type and N25RhoA by microinjection; and Mark Shipman for help with figures This work was supported by the Cancer Research Campaign and the Medical Research Council of Great Britain

17 L A Feig and G M Cooper, Mol Cell, Biol 8, 3235 (1988)

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a human fetal brain library 5 These two cDNAs predicted amino acid sequences that were 95% identical However, it was especially interesting that the amino acid sequences for the human GTP-binding protein were 80% identical and 90% similar to the sequence for the Saccharomyces cerevisiae cell division cycle protein, Cdc42 (designated Cdc42Sc), which

had been shown to be essential for proper assembly of the bud site 6 The human cDNAs complement fully temperature-sensitive mutations of the yeast cdc42 Thus, based on the high degree of sequence similarity as well

as the functional complementation, it was concluded that the brain phospho- substrate and the cloned human GTP-binding proteins represent the mammalian (or human) homologs of the yeast cell division cycle protein and

so we have designated the human proteins as Cdc42Hs

1 M J Hart, P G Polakis, T Evans, and R A Cerione, J Biol Chem 265, 5990 (1990)

2 T Evans, M L Brown, E D Fraser, and J K Northup, J Biol Chem 261, 7052 (1986)

3 p G Polakis, R Snyderman, and T Evans, Biochem Biophys Res Commun 160, 25 (1989)

4 K Shinjo, J G Koland, M J Hart, V Narasimhan, D I Johnson, T Evans, and R A Cerione, Proc Natl Acad Sci U.S.A 87, 9853 (1990)

5 S Munemitsu, M A Innis, R Clark, F McCormick, A Ullrich, and P Polakis, Mol Cell Biol 10, 5977 (1990)

6 D I Johnson and J R Pringle, J Cell Biol 111, 143 (1990)

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12 EXPRESSION AND PURIFICATION [2]

T h r e e classes of regulatory proteins for Cdc42Hs have been identified: a GTPase-activating protein ( G A P ) , 7,8 a GDP-dissociation inhibitor ( G D I ) , 9 and a guanine nucleotide exchange factor, the Dbl oncogene product, l°,u The purification of some of these regulatory proteins is considered in other chapters in this volume In o r d e r to fully characterize the mechanisms that underly the regulation of the G T P - b i n d i n g / G T P a s e cycle of Cdc42Hs by these regulatory proteins, it was desirable to develop systems for expressing recombinant forms of wild-type Cdc42Hs as well as different mutated forms

of the GTP-binding protein In addition, because of the possibility that the isoprenylation (geranylgeranylation) of the carboxyl-terminal cysteine of Cdc42Hs may be crucial to its recognition by other regulatory proteins, it seemed important to establish procedures for the expression of Cdc42Hs

in Spodoptera frugiperda (fall armyworm) cells via baculovirus infection,

because it has been well d o c u m e n t e d that insect cell-expressed proteins

(unlike Escherichia coli-expressed proteins) are correctly posttranslationally modified By comparing the purified E coli- and insect cell-ex-

pressed Cdc42Hs proteins, we have shown that the C d c 4 2 H s - G A P as well

as the Dbl oncogene product (i.e., the C d c 4 2 H s - G E F ) are able to interact

functionally with the E coli Cdc42Hs protein and do not appear to require

the presence of an isoprenoid moiety on the GTP-binding protein However, the G D I absolutely requires isoprenylated Cdc42Hs to bind and inhibit

G D P dissociation as well as to elicit the removal of Cdc42Hs from membranes and inhibit its G T P a s e activity

The following sections describe the relatively straightforward methods

that can be used to express Cdc42Hs in S frugiperda cells and to purify

the recombinant GTP-binding protein

P u r i f i c a t i o n of S p o d o p t e r a f r u g i p e r d a - E x p r e s s e d C d c 4 2 H s

Expression o f Cdc42Hs in S frugiperda Cells via Baculovirus Infection

T h e Cdc42Hs protein was first expressed in S frugiperda (Sf21) cells

by subcloning a 660-bp DraI fragment from the full-length CDC42Hs c D N A into the SmaI site of pUC19 (designated p U C - C D C 4 2 ) A 700-bp B a m H I /

7 M J Hart, K Shinjo, A Hall, T Evans, and R A Cerione, J Biol Chem 266, 20840 (1991)

8 E T Barfod, Y Zheng, W.-J Kuang, M J Hart, T Evans, R A Cerione, and A Askenazi,

J Biol Chem 268, 26059 (1993)

9 D Leonard, M J Hart, J V Platko, A Eva, W Henzel, T Evans, and R A Cerione, J

Biol Chem 267, 22860 (1992)

x0 M J Hart, A Eva, T Evans, S A Aaronson, and R A Cerione, Nature 354, 311 (1991)

u M J Hart, A Eva, D Zangrilli, S A Aaronson, T Evans, R A Cerione, and Y Zheng,

J Biol Chem 269, 62 (1994)

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[2] PURIFICATION OF Cdc42Hs 13

EcoRI fragment from p U C - C D C 4 2 was then cloned into the SmaI/BamHI

sites of the baculovirus transfer vector (pACYMP2) 12 Cotransfection of the wild-type baculovirus D N A with the transfer vector p A C Y M P 2 - CDC42 into Sf21 cells was then performed using calcium phosphate 12 to generate recombinant virus that contained the full-length cDNA encoding Cdc42Hs After 2-3 days, the extracellular viral supernatant was harvested from the cells and was then used to infect new Sf21 cells High titer extracellular viral D N A was then harvested and assayed for effective recombination

by Southern blot analysis using 32p-labeled CDC42Hs cDNA as a probe

The virus containing the CDC42Hs cDNA was then isolated using a

dilution/hybridization method, i.e., the Sf21 cells in multiwell plates (-104 cells/well) were infected with different dilutions of the viral supernatant 12 The Sf21 cells that were infected with the pure recombinant virus were used

to purify recombinant Cdc42Hs from the particulate membrane fraction

Purification o f Cdc42Hs from the Membrane Fractions of Sf21 Cells

In order to ensure that the Cdc42Hs purified from S frugiperda cells

is the isoprenylated form, membrane fractions were first prepared The cells from a l-liter spinner flask, grown in Grace's media and 10% fetal calf serum, are pelleted and then lysed by resuspending the pellet in 20

m M Tris-HC1, 100 mM NaC1, 6 mM EDTA, 1 mM dithiothreitol (DTT),

pH 8.0, 0.5 mM phenylmethylsulfonyl fluoride, and 25 /xg/ml (each) of leupeptin and aprotinin The lysate is then homogenized (in a 7-ml glass/ glass homogenizer) and the homogenates are split between two plastic 15-

ml centrifuge tubes and centrifuged at 2500 rpm (4 °) for 10 min in an IEC tabletop centrifuge This step pellets the nucleus and any unbroken insect cells The supernatant is removed and the membrane fraction from the insect cells is then pelleted by centrifugation for 15 min at 12,000 g in a Sorvall SS-34 rotor The membranes are resuspended in 20 mM Tris-HC1,

100 mM NaC1, 1 mM EDTA, 3.75 mM MgCI2, 1 mM DTF, pH 8.0, 3/zM GDP, and 1 /xg/ml (each) of aprotinin and leupeptin, and recentrifuged for 15 min at 12,000 g This step is repeated and then Cdc42Hs is solubilized

by incubating the membranes with - 1 0 ml of 20 mM Tris-HC1, 100 mM NaC1, 1 m M EDTA, 1 mM DTT, 3.75 mM MgCI2, 3 / x M GDP, pH 8.0, 1% sodium cholate, and 1 /xg/ml (each) of aprotinin and leupeptin (this

is designated "solubilization buffer") for 90 min at 4 ° The particulate (membrane) fraction is removed by centrifugation at 100,000 g for 1 hr at

4 ° The supernatant ( - 1 0 ml) is then applied to a 400-ml Ultrogel AcA34 column equilibrated with 20 mM Tris-HC1, 100 mM NaCI, 1 mM EDTA,

12 p M Guy, K L Carraway III, and R A Cerione, J Biol Chem 267, 13851 (1992)

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1 m M DTT, 3.75 m M MgC12, and 1% sodium cholate, pH 8.0 The protein

is eluted with the solubilization buffer at approximately 50 ml per hr The fractions containing Cdc42Hs can be identified by Western blot analysis, using specific antipeptide Cdc42Hs antibodies (raised against peptides rep- resenting amino acid residues 167-183 and 180-191 of the Cdc42Hs protein 4) and by assaying [35S]GTPyS binding) The peak fractions containing Cdc42Hs activity are pooled and dialyzed against 20 m M Tris-HC1, 1 m M EDTA, 0.5% CHAPS, 1 t~g/ml leupeptin, and 5% glycerol, pH 8.0 (two changes of 500 ml each for at least 10 hr) The dialyzed Cdc42Hs is then applied to a Pharmacia Mono Q column, equilibrated with 20 mM Tris- HC1, 1 m M EDTA, 1 m M DTT, pH 8.0, and 0.5% CHAPS The purified Cdc42Hs is eluted from the column using a linear NaC1 gradient (0-300 raM)

At this stage, the purity of the Cdc42Hs preparation can be assessed

by S D S - P A G E and protein (Coomassie blue) staining Typically, the sole band detected is the 22-kDa Cdc42Hs protein If necessary, the peak fractions containing the Cdc42Hs can be concentrated by hydroxyapatite chromatography In such cases, the Cdc42Hs is applied to a 3-ml hydroxyapatite

FIG 1 G D I activity on the S frugiperda- and E coli-recombinant Cdc42Hs (A) The S

frugiperda-expressed Cdc42Hs ( 0 ) or the E coli-recombinant Cdc42Hs (&) was preincubated

with [c~-32p]GTP (7 tzM) for 25 rain at r o o m temperature This incubation period ensures that all of the bound G T P is converted to G D P as a result of the intrinsic GTPase activity

of Cdc42Hs The [c~-32p]GDP-bound Cdc42Hs proteins (~15 ng) were then incubated with the indicated amounts of the M o n o S-purified G D I activity (in the presence of 2.5 m M E D T A )

as described in L e o n a r d et aL 9 A f t e r 6 rain, the samples were then filtered on nitrocellulose

(BA85) filters and the amount of [a-32p]GDP that remained bound to Cdc42Hs (relative to the amount of G D P b o u n d at the start of the assay), as a function of the amount of G D I added to the assay incubation, was determined (B) Cdc42Hs (20 ng) that was purified from

the m e m b r a n e fraction of S frugiperda cells was preincubated with [3H]GDP (7/~M) for 25

min The dissociation of the radiolabeled G D P was measured at the indicated times in the

absence (A) and presence (O) of the GDI R e p r o d u c e d from Leonard et al 9 with permission

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[3] P U R I F I C A T I O N A N D P R O P E R T I E S O F R a c 2 15 column that was equilibrated in 20 m M Tris-HCl, pH 8.0, and 0.5% CHAPS The Cdc42Hs is eluted from the column with 20 m M Tris-HC1, 1 m M EDTA, 1 m M DTT, pH 8.0, plus 100 m M potassium phosphate, 40% glycerol, and 0.5% CHAPS The peak Cdc42Hs fractions can be identified

by Western blot analysis using the anti-Cdc42Hs antibody or by Coomassie blue staining (i.e., as indicated by the presence of a single protein band at

22 kDa)

We had previously demonstrated that the interactions of Cdc42Hs with its GAP (7) or GEF (11) were independent of the presence of an isoprenoid moiety on the GTP-binding protein However, the interactions of the GDI with Cdc42Hs do appear to require that Cdc42Hs is geranylgeranylated Figure 1A shows a comparison of the ability of the R h o - G D I to inhibit the dissociation of radiolabeled GDP from Cdc42Hs expressed and purified from insect cells and E coli Under the conditions of this experiment,

100% of the bound radiolabeled GDP is dissociated from the recombinant Cdc42Hs proteins within 5 min (in the presence of excess EDTA, i.e., no added MgC12 and [EDTA] = 2.5 mM) Likewise, complete dissociation of the bound GDP occurs when the E coli Cdc42Hs is incubated with the

brain GDI, whereas 25% to as much as 50% of the originally bound GDP remains associated when the insect cell-expressed Cdc42Hs is incubated with the brain GDI The fact that only a percentage of the insect cell- expressed Cdc42Hs was sensitive to the GDI stems from the fact that the GTP-binding protein was prepared from whole cell lysates When the same experiments were performed with Cdc42Hs that was purified from the membrane fractions of insect cells, as outlined earlier, the extents of inhibi- tion by the R h o - G D I approached 100% (Fig 1B) These results then indicate that when the Cdc42Hs is purified from insect cell membranes, virtually all of the Cdc42Hs is in the geranylgeranylated form

[3] P u r i f i c a t i o n a n d P r o p e r t i e s o f R a c 2 f r o m H u m a n

L e u k e m i a C e l l s

By T A K A K A Z U M I Z U N O , H I R O Y U K I N A K A N I S H i , and Y O S H I M I T A K A I Introduction

The superoxide-generating N A D P H oxidase system in phagocytes, such

as neutrophils and monocytes, consists of membrane-associated cytochrome b-558, composed of gp91-phox and p22-phox heterodimer, as a terminal

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[3] P U R I F I C A T I O N A N D P R O P E R T I E S O F R a c 2 15 column that was equilibrated in 20 m M Tris-HCl, pH 8.0, and 0.5% CHAPS The Cdc42Hs is eluted from the column with 20 m M Tris-HC1, 1 m M EDTA, 1 m M DTT, pH 8.0, plus 100 m M potassium phosphate, 40% glycerol, and 0.5% CHAPS The peak Cdc42Hs fractions can be identified

by Western blot analysis using the anti-Cdc42Hs antibody or by Coomassie blue staining (i.e., as indicated by the presence of a single protein band at

22 kDa)

We had previously demonstrated that the interactions of Cdc42Hs with its GAP (7) or GEF (11) were independent of the presence of an isoprenoid moiety on the GTP-binding protein However, the interactions of the GDI with Cdc42Hs do appear to require that Cdc42Hs is geranylgeranylated Figure 1A shows a comparison of the ability of the R h o - G D I to inhibit the dissociation of radiolabeled GDP from Cdc42Hs expressed and purified from insect cells and E coli Under the conditions of this experiment,

100% of the bound radiolabeled GDP is dissociated from the recombinant Cdc42Hs proteins within 5 min (in the presence of excess EDTA, i.e., no added MgC12 and [EDTA] = 2.5 mM) Likewise, complete dissociation of the bound GDP occurs when the E coli Cdc42Hs is incubated with the

brain GDI, whereas 25% to as much as 50% of the originally bound GDP remains associated when the insect cell-expressed Cdc42Hs is incubated with the brain GDI The fact that only a percentage of the insect cell- expressed Cdc42Hs was sensitive to the GDI stems from the fact that the GTP-binding protein was prepared from whole cell lysates When the same experiments were performed with Cdc42Hs that was purified from the membrane fractions of insect cells, as outlined earlier, the extents of inhibi- tion by the R h o - G D I approached 100% (Fig 1B) These results then indicate that when the Cdc42Hs is purified from insect cell membranes, virtually all of the Cdc42Hs is in the geranylgeranylated form

[3] P u r i f i c a t i o n a n d P r o p e r t i e s o f R a c 2 f r o m H u m a n

L e u k e m i a C e l l s

By T A K A K A Z U M I Z U N O , H I R O Y U K I N A K A N I S H i , and Y O S H I M I T A K A I Introduction

The superoxide-generating N A D P H oxidase system in phagocytes, such

as neutrophils and monocytes, consists of membrane-associated cytochrome b-558, composed of gp91-phox and p22-phox heterodimer, as a terminal

Trang 19

redox carrier and at least three cytosolic regulatory components 1,2 Two of them, p47-phox and p67-phox, have been identified as the products of the genes causing autosomal recessive type of chronic granulomatous disease 3,4

A series of studies from several laboratories, including our own, have revealed that the third cytosolic component is a member of the Rho-related small GTPases, Rac 5-8 The Rac family consists of highly homologous Racl and Rac2, and our result indicates that both members stimulate the superoxide generation 9

Smg-GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for a group of small GTPases including at least Ki-Ras, Rapl, Rho, and Rac, 1°-13 stimulates the conversion of G D P - R a c to GTP- Rac and thereby stimulates the N A D P H oxidase activity 7'9 In contrast, the

R h o - G D P dissociation inhibitor (GDI), an inhibitory GDP/GTP exchange protein for a group of small GTPases including at least Rho, Rac, and mCdc42 (see [6], this volume), 13-t8 inhibits the conversion of G D P - R a c to

1 A W Segal, J Clin Invest 83, 1785 (1989)

z R A Clark, J Infect Dis 161, 1140 (1990)

3 K J Lomax, T L Leto, H Nunoi, J I Gallin, and H L Malech, Science 245, 409 (1989)

4 T L Leto, K J Lomax, B D Volpp, H Nunoi, J M G Sechler, W M Nauseef, R A Clark, J I Gallin, and H L Malech, Science 248, 727 (1990)

s A Abo, E Pick, A Hall, N Totty, C G Teahan, and A W Segal, Nature 353, 668 (1991)

6 U G Knaus, P G Heyworth, T Evans, J T Curnutte, and G M Bokoch, Science 254,

1512 (1991)

7 T Mizuno, K Kaibuchi, S Ando, T Musha, K Hiraoka, K Takaishi, M Asada, H Nunoi,

I Matsuda, and Y Takai, J Biol Chem 267, 10215 (1992)

s C H Kwong, H L Malech, D Rotrosen, and T L Leto, Biochemistry 32, 5711 (1993)

9 S Ando, K Kaibuchi, T Sasaki, K Hiraoka, T Nishiyama, T Mizuno, M Asada, H Nunoi,

I Matsuda, Y Matsuura, P Polakis, F McCormick, and Y Takai, Z Biol Chem 267,

25709 (1992)

10 T Yamamoto, K Kaibuchi, T Mizuno, H Hiroyoshi, H Shirataki, and Y Takai, J Biol Chem 265, 16626 (1990)

11 K Kaibuchi, T Mizuno, H Fujioka, T Yamamoto, K Kishi, Y Fukumoto, Y Hori, and

Y Takai, 3,1ol Cell Biol 11, 2873 (1991)

12 T Mizuno, K Kaibuchi, T Yamamoto, M Kawamura, T Sakoda, H Fujioka, Y Matsuura, and Y Takai, Proc Natl Acad Sci U.S.A 88, 6442 (1991)

13 K Hiraoka, K Kaibuchi, S Ando, T Musha, K Takaishi, T Mizuno, M Asada, L Menard,

E Tomhave, J Didsbury, R Snyderman, and Y Takai, Biochem Biophys Res Commun

182, 921 (1992)

14 N Ohga, A Kikuchi, T Ueda, J Yamamoto, and Y Takai, Biochem Biophys Res Commun

163, 1523 (1989)

is T Ueda, A Kikuchi, N Ohga, J Yamamoto, and Y Takai, J Biol Chem 265, 9373 (1990)

16 y Fukumoto, K Kaibuchi, Y Hori, H Fujioka, S Araki, T Ueda, A Kikuchi, and Y Takai, Oncogene 5, 1321 (1990)

17 T Sasaki, K Kato, T Nishiyama, and Y Takai, Biochem Biophys Res Commun 194,

1188 (1993)

18 D Leonard, M J Hart, J V Platko, E Alessandra, W Henzel, T Evaans, and R A Cerione, J Biol Chem 267, 22860 (1992)

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[3] PURIFICATION AND PROPERTIES OF Rac2 17

G T P - R a c and thereby inhibits the N A D P H oxidase activity 7'9 T h e r e f o r e , once G D P - R a c is converted to G T P - R a c , S m g - G D S or R h o - G D I does not affect the superoxide generation in our assay system, although other groups have r e p o r t e d that the R a c / R h o - G D I complex stimulates the

N A D P H oxidase activity in the absence of exogenous GTP 5'8

T h e R h o family members, including R a c l and Rac2, have a unique C- terminal amino acid structure of Cys-A-A-Leu (A, aliphatic amino acid), which undergoes postranslational modifications including geranylgeranylation followed by removal of three amino acids and the carboxylmethylation

of the exposed cysteine 19'2° T h e lipid modifications of Rac are essential for its interactions with S m g - G D S and R h o - G D I 9 Moreover, lipid-modified Rac stimulates the N A D P H oxidase activity more efficiently than does

a lipid-unmodified one, 9 although a n o t h e r group has reported that both forms stimulate the N A D P H oxidase activity with similar efficiency 21,22 This chapter describes the assay for the N A D P H oxidase activity, the procedures for the purification of Rac2 from the cytosol fraction of the differentiated HL-60 (human promyelocytic leukemia) cells, and the properties of Rac2

M a t e r i a l s

R P M I 1640 medium and fetal calf serum are purchased from G I B C O -

B R L (Gaithersburg, MD) Sodium cholate, sodium deoxycholate, and L- o~-dimyristoylphosphatidylcholine ( D M P C ) are from Wako Pure Chemicals (Osaka, Japan) N-2-Hydroxyethylpiperadine-N'-2-ethanesulfonic acid ( H E P E S ) , 3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid ( C H A P S ) , and E D T A are from Dojindo Laboratories (Kumamoto, Japan) Dithiothreitol ( D T T ) and E G T A are from Nacalai Tesque (Kyoto, Japan) Phenylmethylsulfonyl fluoride (PMSF), 2-(N-morpholino)ethanesulfonic acid (MES), ferricytochrome c, N A D P H , F A D , catalase, arachidonic acid, and superoxide dismutase (SOD) are from Sigma (St Louis, MO) G D P and guanosine 5'-(3-O-thio)triphosphate ( G T P y S ) are from Boehringer M a n n h e i m (Indianapolis, IN) [35S]GTPyS is from D u Pont- New England Nuclear (Boston, MA) Carboxymethyl (CM)-Sepharose and

M o n o Q H R 5 / 5 are from Pharmacia P-L Biochemicals Inc (Milwaukee,

19 M Katayama, K Kawata, Y Yoshida, H Horiuchi, T Yamamoto, Y Matsuura, and Y Takai, J Biol Chem 266, 12639 (1991)

20 p Adamson, C J Marshall, A Hall, and P A Tilbrook, J Biol Chem 267, 20033 (1992)

21 p G Heyworth, U G Knaus, X Xu, D J Uhlinger, L Conray, G M Bokoch, and J T Curnutte, Mol Biol Cell 4, 261 (1993)

22 M L Kreck, D J Uhlinger, S R Tyagi, K L Inge, and J D Lambeth, J Biol Chem

269, 4161 (1994)

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WI) Hydroxyapatite is from Seikagaku Kogyo Co (Tokyo, Japan) All other chemicals are of reagent grade

HL-60 cells are obtained from the American Tissue Culture Center (Rockville, MD) Recombinant p47-phox and p67-phox are purified from

baculoviruses carrying the cDNAs of p47-phox and p67-phox are from H Nunoi and I Matsuda (Kumamoto University School of Medicine, Kuma- moto, Japan) Recombinant Smg-GDS is purified from Smg-GDS-overex- pressing Escherichia coli u R h o - G D I is purified from the cytosol fraction

of bovine brain 1~ Lipid-modified and lipid-unmodified recombinant Rac2s are purified from the membrane and cytosol fractions of Sf9 cells, respectively, using a baculovirus expression system 9 The baculovirus carrying the cDNA of Rac2 is from P Polakis and F McCormick (Onyx Pharmaceuti- cals, Richmond, CA) GTPTS-Rac2 and G D P - R a c 2 are prepared as described 9

Methods

Purification of Rac2 from Differentiated HL-60 Cells

The various buffers used in the purification of Rac2 are as follows: Buffer A: 10 mM KHzPO4/KzHPO4 at pH 7.5, 1 mM EGTA, 1 mM PMSF, 130 mM NaCI, 5 mM MgCI2, 340 mM sucrose

Buffer B: 10 mM MES at pH 6.8, 1 mM EGTA, 1 mM PMSF, 5 mM 2-mercaptoethanol

Buffer C: 20 mM Tris/HCl at pH 8.0, l mM EDTA, 1 mM DTT, 5

mM MgCI2, 100 mM NaC1, 1% sodium cholate

Buffer D: 20 mM Tris/HC1 at pH 8.0, 0.1 mM EDTA, 1 mM DTT, 3

mM MgC12

Buffer E: 20 mM Tris/HC1 at pH 8.0, 1 mM EDTA, 1 mM DTT, 5

mM MgCI2, 0.5% sodium cholate

Buffer F: 20 mM Tris/HC1 at pH 8.0, 1 mM EDTA, 1 mM DTT, 5

mM MgC12, 0.6% CHAPS

The steps used in the purification of Rac2 are as follows: (1) preparation

of differentiated HL-60 cells; (2) preparation of the cytosol and membrane fractions; (3) CM-Sepharose column chromatography; (4) Ultrogel AcA

44 column chromatography; (5) hydroxyapatite column chromatography; (6) Mono Q HR5/5 column chromatography; and (7) Mono Q HR5/5 column rechromatography

23 T L Leto, M C Garrett, H Fujii, and H Nunoi, J BioL Chem 266, 19812 (1991)

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[3] PURIFICATION AND PROPERTIES OF R a c 2 19

Preparation of Differentiated HL-60 Cells

HL-60 cells are grown in RPMI 1640 medium containing 10% fetal calf serum, 100 rag/liter streptomycin, and 100,000 units/liter penicillin at 37 °

in 5% COj95% air (v/v) The cells (5 x 109 cells) are differentiated into neutrophil-like cells by treatment with 3/zM retinoic acid for 4 days The differentiation is estimated by measuring the expression of C D l l b , a marker antigen of neutrophils

Preparation of Cytosol and Membrane Fractions

All the following procedures are carried out at 0-4 ° The differentiated HL-60 cells (5 x 109 cells) are washed twice with phosphate-buffered saline (PBS) at pH 7.4, suspended in 20 ml of buffer A, and then sonicated for

15 sec three times at 10-sec intervals After removal of unbroken cells and nuclei, the sonicate is layered on 20 ml of buffer A containing 40% sucrose and is centrifuged at 140,000g for I hr The supernatant is further centrifuged

at 200,000g for 1 hr and is used as the cytosol fraction The membrane fraction is collected from the surface of the 40% sucrose layer, rinsed with buffer A, and resuspended with 10 ml of buffer A Both fractions are stored

at - 8 0 ° and are stable for at least several months

CM-Sepharose Column Chromatography

One-third of the cytosol fraction (10 ml, 30 mg of protein) is diluted fivefold with buffer B and applied to a CM-Sepharose column (1.5 x 20 cm) equilibrated with buffer B Elution is performed with 50 ml of buffer

B followed by buffer B containing 300 mM NaCI at a flow rate of 1 ml/ min Fractions of 4 ml each are collected When each fraction is assayed for [35S]GTPyS-binding activity, one broad peak and one sharp peak appear

in fractions 6-25 and 31-40, respectively These peaks correspond to those

of absorbance at 280 nm The active fractions of the first peak are collected

Ultrogel AcA 44 Column Chromatography

The active fractions of the CM-Sepharose column chromatography (80

ml, 16 mg of protein) are pooled and concentrated to approximately 10 ml

by an ultrafiltration cell (Amicon) equipped with a PM-10 filter membrane After the addition of MgC12 and sodium cholate at final concentrations of

5 mM and 1%, respectively, the concentrate is applied to an Ultrogel AcA-

44 column (2 X 80 cm) equilibrated with buffer C Elution is performed with 210 ml of buffer C at a flow rate of 0.275 ml/min Fractions of 2.2 ml each are collected When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear in fractions 50-58 and 62-73 The first peak

Trang 23

20 eXVRZSSION AND PURIFICATION [31 contains heterotrimeric GTPases and the second peak contains small GTPases including Rac2 When each fraction is assayed for the NADPH oxidase activity, a single peak appears, which corresponds to the second peak of [35S]GTPyS-binding activity The active fractions of the NADPH oxidase activity are pooled and purified further

Hydroxyapatite Column Chromatography

The pooled fractions of the Ultrogel AcA-44 column chromatography (26 ml, 10 mg of protein) are diluted to 40 ml with 20 mM Tris/HC1 at pH 8.0 containing 1 mM DTT and are applied to a hydroxyapatite column (1.5

× 6 cm) equilibrated with buffer D containing 10 mM KH2PO4 Elution

is performed first with 160 ml of buffer D containing 10 mM KHzPO4 and 0.6% CHAPS and then with 80 ml of buffer D containing 100 mM KHePO4 and 0.6% CHAPS at a flow rate of 0.67 ml/min Fractions of 4 ml each are collected When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear in fractions 16-40 and 53-56 When each fraction is assayed for the NADPH oxidase activity, two peaks appear in fractions 17-23 as a major peak and in fractions 53-56 as a minor peak The major peak of the NADPH oxidase activity contains Rac2 and the minor peak contains Rac! as estimated by Western blot analysis using their respective antibodies The active fractions of the major peak are pooled and purified further

Mono Q HR5/5 Column Chromatography

The active fractions of the major peak of the hydroxyapatite column chromatography (28 ml, 2.5 mg of protein) are concentrated to approximately 1.5 ml by an ultrafiltration cell equipped with a YM5 filter membrane (Amicon) The concentrate is diluted 10-fold with buffer E and is applied

to a Mono Q HR5/5 column equilibrated with buffer E containing 10 mM NaC1 After the column is washed with 15 ml of the same buffer, elution

is performed with a 30-ml linear gradient of NaC1 (10-500 mM) in buffer

E at a flow rate of 0.5 ml/min Fractions of 0.5 ml each are collected When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear

in fractions 76-88 and 90-100 as shown in Fig 1 When each fraction is assayed for the NADPH oxidase activity, a single peak appears in fractions 88-92 The active fractions of the NADPH oxidase activity are collected and stored at - 8 0 ° The rest of the cytosol fraction is treated in the same way The pooled fractions can be stored for at least 3 months at - 8 0 ° without loss of activity

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01i I

0 "6

FIG 1 Mono Q column chromatography A 5-/A aliquot of each fraction was assayed for

N A D P H oxidase and [35S]GTPyS-binding activities (3, the NADPH oxidase activity; O, the [35S]GTPyS-binding activity

Mono Q HR 5/5 Column Rechromatography

The combined pools of the active fractions of the Mono Q HR5/5 column chromatography (7.5 ml, 120 mg of protein) are dialyzed against buffer E and further applied to a Mono Q HR 5/5 column equilibrated with buffer F After the column is washed with 15 ml of the same buffer, elution is performed with a 30-ml linear gradient of NaC1 (0-500 mM) in buffer F at a flow rate of 0.5 ml/min Fractions of 0.5 ml each are collected When each fraction is assayed for [35S]GTPyS-binding and NADPH oxidase activities, a single peak appears in fractions 50-58 as shown in Fig 2 This

Journal o f Biological Chemistry and the American Society for Biochemistry and Molecular Bi- ology.)

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22 EXPRESSION AND PURIFICATION [3] GTPase is a nearly homogeneous protein with a Mr of about 22,000 and

is identified to be Rac2 by the partial amino acid sequences

Assay for Cell-Free NADPH Oxidase Activity

The cell-free NADPH oxidase activity is assayed by measuring the arachidonic acid-elicited superoxide generation, which is determined by the SOD-inhibitable ferricytochrome c reduction by use of Rac2, p47-phox,

membrane-associated cytochrome b - 5 5 8 24

Preparation of Solubilized Membrane Components

The solubilized membrane components including membrane-associated cytochrome b-558 are prepared as follows The membrane fraction from the differentiated HL-60 cells (10 ml, 36 mg of protein) described earlier

is incubated for 30 min at 4 ° with the same volume of 20 mM glycine/ NaOH at pH 8.0 containing 50% glycerol, 1 mM NAN3, 1.7/xM CaC12, and 2.3% sodium deoxycholate After centrifugation at 100,000g for 1 hr, the extract is diluted 20-fold by water and is used as the solubilized membrane components These membrane components can be stored for at least 3 months at - 8 0 ° without loss of activity Repeated freezing and thawing of this sample should be avoided This sample is free from p47-phox p67-

unidentified endogenous GTPases

Assay

Fourteen nanomolar p47-phox, 24 nM p67-phox, and 2 nM Rac2 or a 5-~1 aliquot of each fraction of the column chromatographies are first incubated in a reaction mixture (300/xl) containing 10 mM MES at pH 6.8, 1 mM EGTA, 1 mM PMSF, 5 mM 2-mercaptoethanol, and 10/xM GTPTS After a 5-rain incubation at 25 °, the mixture is cooled on ice The second reaction mixture (200/zl) containing 30 mM HEPES/NaOH at pH 7.3, 30 mM KH2POa/K2HPO4 at pH 7.0, 240/~M ferricytochrome c, 750 /zM NADPH, 3/zM FAD, 75/zg/ml catalase, 1.5 mM EDTA, 9 mM MgC12, and 6 mM NaN3 is added, and then the solubilized membrane components (60/zl, 5.4/xg of protein) are added to the mixture The second reaction

is initiated by the additional of 10/zl of 700/xM arachidonic acid to give

a final concentration of 12.5 nM and is performed for 15 min at 25 ° The reaction is stopped by the addition of 30/xl of 500/zg/ml SOD The rates

24 E Pick, Y Bromberg, S Shpungin, and R Gadba, J Biol Chem 262, 16476 (1987)

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of the superoxide production are calculated from the absorbance at 550

nm as [/xmol superoxide/min/mg membrane protein], based on Ae550 = 2.1 × 104 M-acm -1 (reduced minus oxidized cytochrome) 24 The reference reaction is performed in the presence of 25/~g/ml SOD During the purification procedures of Rac2, the NADPH oxidase activity is assayed in the presence of 150 nM Smg-GDS When the properties of Rac2 are studied, the NADPH oxidase activity is assayed in the presence of various combinations of 2 nM Rac2, 14 nM p47-phox, 24 nM p67-phox, 10 mM GTPyS,

150 nM Smg-GDS, and 300 nM R h o - G D I

Assay for f~SS]GTPyS-Binding Activity

The [35S]GTPyS-binding activity is assayed by measuring the radioactivity of [35S]GTPTS bound to a small GTPase trapped on nitrocellulose filters (BA-85, Schleicher & Schuell) A 20-/A aliquot of each fraction of the column chromatographies described earlier is incubated for 20 min at 30 °

in a reaction mixture (40/zl) containing 20 mM Tris-HCl at pH 7.5, 10 mM EDTA, 5 mM MgC12, 1 mM DTT, 1 mM DMPC, and 1/zM [35S]GTPTS (3-6 x 103 cpm/pmol) The reaction is stopped by the addition of about

2 ml of an ice-cold stopping solution containing 20 mM Tris/HC1 at pH 7.5, 25 mM MgC12, and 100 mM NaC1, followed by rapid filtration on nitrocellulose filters Filters are washed five times with the same ice-cold stopping solution After filtration, the radioactivity is counted

Properties of Rac2

Activation of NADPH oxidase by the Rac2 purified from differentiated HL-60 cells is summarized in Fig 3A Rac2 stimulates the cell-free NADPH oxidase activity in the presence of p47-phox, p67-phox, and GTPyS Smg-GDS enhances the Rac2-induced NADPH oxidase activity, whereas

R h o - G D I counteracts this stimulatory effect of Smg-GDS A removal

of each component completely abolishes the NADPH oxidase activity The recombinant Rac2, produced in insect ceils using a baculovirus system, shows a similar effect (Fig 3B) Moreover, lipid-modified Rac2 is far more effective than lipid-unmodified Rac2 When the first incubation

is performed with GTPTS-Rac2, Smg-GDS is not required for the NADPH oxidase activation R h o - G D I is unable to inhibit the GTPyS-Rac2-induced NADPH oxidase activation Similar results are also obtained using recombinant Racl 9 Rapl, RhoA, or Ki-Ras do not affect the NADPH oxidase activity 7

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R h o - G D I , and 10/zM GTPyS (B) Effect of recombinant Rac2 The first incubation was peformed in the presence of 14 nM p47-phox, 24 nM p67-phox, 10/xM GTPyS, and various combinations of 12 nM lipid-modified recombinant G D P - or GTPyS-Rac2 (mRac2), 12 nM lipid-unmodified recombinant G D P - or GTPyS-Rac2 (cRac2), 150 nM Smg-GDS, and 300

nM R h o - G D I

C o m m e n t s

Several points need to be considered for the NADPH oxidase assay First, an optimal concentration of arachidonic acid varies depending on preparations of the solubilized membrane components It also varies depending on the concentrations of the proteins, including Rac, p47-phox,

and p67-phox Therefore, it is recommended to determine the optimal concentration of arachidonic acid used for each assay system Second, an excess amount of sodium cholate or CHAPS suppresses the NADPH oxidase activity Therefore, when an aliquot of each fraction of the column chromatographies is assayed for NADPH oxidase activity, the volume of the aliquot should be less than 5/zl

We have purified Rac2 free from R h o - G D I from the differentiated HL-60 cells as an activator of superoxide generation, and have shown that GTPyS-recombinant Rac2 stimulates NADPH oxidase activity irrespective

of the presence or absence of Smg-GDS and R h o - G D I These regulatory proteins just regulate the conversion of GDP-Rac2 to GTP-Rac2 and do not affect the NADPH oxidase activity itself However, other groups have purified Racl and Rac2 as a heterodimer with R h o - G D I from the cytosol fractions of macrophages and neutrophils, respectively, and have shown that the R a c / R h o - G D I complex stimulates the NADPH oxidase activity

in the absence of exogenous GTP, 5's although it has been also shown that

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[41 PURIFICATION OF Rac2 FROM NEUTROPHILS 25 the superoxide-producing activity of the purified R a c / R h o - G D I complex

is increased three-fold by G T P and completely inhibited by G D P 25 T h e discrepancy b e t w e e n the results of ours and other groups concerning the purification of Rac as a m o n o m e r i c form free from R h o - G D I and a hetero- dimeric form complexed with R h o - G D I may be due to the fact that we use detergents during the purification procedures and that other groups do not The reason why R a c l or Rac2 complexed with R h o - G D I is active on the N A D P H oxidase is currently unknown, but we have found that R h o -

G D I preferentially forms a complex with G D P - R a c but weakly forms a complex with G T P - R a c T h e efficiency of R h o - G D I to form a complex with G T P - R a c is about 10% that of R h o - G D I to form a complex with

G D P - R a c 26 T h e R h o - G D l / R a c complex purified from other groups may contain G T P - R a c

We have shown that lipid-modified Rac is far more effective on the activation of N A D P H oxidase than lipid-unmodified Rac However, another group has shown that both forms of Rac are equally effective 21'=

T h e reason for this discrepancy is currently unknown

25 A Abo, M R Webb, A Grogan, and A Segal, Biochem J 298, 585 (1994)

26 T Sasaki, M Kato, and Y Takai, J Biol Chem 268, 23959 (1993)

[4] P u r i f i c a t i o n o f R a c 2 f r o m H u m a n N e u t r o p h i l s

By ULLA G KNAUS and GARY M BOKOCH

T h e Rac2 protein belongs to the R h o family of GTP-binding proteins and is closely related to the R a c l protein (92% identity) Whereas R a c l is ubiquitously expressed, Rac2 is only found in cells of myeloid origin Studies with a h u m a n leukemia cell line (HL-60) showed a seven- to ninefold increase of Rac2 expression on differentiation to a neutrophiMike type, whereas R a c l levels rose only slightly 1 Analysis of Rac protein levels by immunoblotting with specific antibodies revealed also the predominance

of Rac2 in h u m a n neutrophils 2 These data indicate that neutrophils are

an excellent source for the purification of biologically active Rac2, if recombinant sources that allow post-translational processing of GTP-binding pro-

1J Didsbury, R F Weber, G.M Bokoch, T Evans, and R Snyderman, J BioL Chem 264,

Trang 29

[41 PURIFICATION OF Rac2 FROM NEUTROPHILS 25 the superoxide-producing activity of the purified R a c / R h o - G D I complex

is increased three-fold by G T P and completely inhibited by G D P 25 T h e discrepancy b e t w e e n the results of ours and other groups concerning the purification of Rac as a m o n o m e r i c form free from R h o - G D I and a hetero- dimeric form complexed with R h o - G D I may be due to the fact that we use detergents during the purification procedures and that other groups do not The reason why R a c l or Rac2 complexed with R h o - G D I is active on the N A D P H oxidase is currently unknown, but we have found that R h o -

G D I preferentially forms a complex with G D P - R a c but weakly forms a complex with G T P - R a c T h e efficiency of R h o - G D I to form a complex with G T P - R a c is about 10% that of R h o - G D I to form a complex with

G D P - R a c 26 T h e R h o - G D l / R a c complex purified from other groups may contain G T P - R a c

We have shown that lipid-modified Rac is far more effective on the activation of N A D P H oxidase than lipid-unmodified Rac However, another group has shown that both forms of Rac are equally effective 21'=

T h e reason for this discrepancy is currently unknown

25 A Abo, M R Webb, A Grogan, and A Segal, Biochem J 298, 585 (1994)

26 T Sasaki, M Kato, and Y Takai, J Biol Chem 268, 23959 (1993)

[4] P u r i f i c a t i o n o f R a c 2 f r o m H u m a n N e u t r o p h i l s

By ULLA G KNAUS and GARY M BOKOCH

T h e Rac2 protein belongs to the R h o family of GTP-binding proteins and is closely related to the R a c l protein (92% identity) Whereas R a c l is ubiquitously expressed, Rac2 is only found in cells of myeloid origin Studies with a h u m a n leukemia cell line (HL-60) showed a seven- to ninefold increase of Rac2 expression on differentiation to a neutrophiMike type, whereas R a c l levels rose only slightly 1 Analysis of Rac protein levels by immunoblotting with specific antibodies revealed also the predominance

of Rac2 in h u m a n neutrophils 2 These data indicate that neutrophils are

an excellent source for the purification of biologically active Rac2, if recombinant sources that allow post-translational processing of GTP-binding pro-

1J Didsbury, R F Weber, G.M Bokoch, T Evans, and R Snyderman, J BioL Chem 264,

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26 EXPRESSION AND PURIFICATION [4] teins, such as the baculovirus expression system in Sf9 ( S p o d o p t e r a f r u g i -

p e r d a falls armyworm ovary) cells, are not available The Rac2 protein is localized in the cytosolic compartment of unstimulated neutrophils, where

it forms a stable complex with the G D P dissociation inhibitor [rho]GDI 3 Upon stimulation with the chemoattractant fMLP or with phorbol myristate acetate, this complex partially dissociates and Rac2 translocates to the plasma membrane 2 This underscores the importance of avoiding any step during the isolation procedure of neutrophils that could lead to activation and loss of cytosolic Rac2 protein

Biologically active Rac2 protein can be readily detected by its ability

to bind GTP and G D P with high affinity Screening of column fractions for Rac and several related GTP-binding proteins is achieved by [32S]GTPTS binding to the protein under low magnesium conditions, followed by a rapid filtration assay, as described later Western blotting with specific peptide antibodies, directed to the carboxyl terminus of the GTP-binding proteins R a c l , Rac2, and Cdc42Hs (i.e., Santa Cruz Biotech., CA), allows further identification To distinguish Rac2 from the R h o - G T P - b i n d i n g protein, the specific ability of botulinum toxin C3 transferase to ADP-ribosylate

R h o can be utilized 4

A specialized biological role for Rac2 in phagocyte function has been reported Rac2 has been shown to be absolutely required for activation of the N A D P H oxidase 5,6 and for the subsequent generation of reactive oxygen species This function provides another means to detect Rac2 protein during purification by using a cell-free N A D P H oxidase assay system consisting of neutrophil cytosol and membranes 7 or recombinant proteins as described 8 The following purification protocol implements foremost the use of the GTPyS-binding assay to detect active GTP-binding protein, assisted by the more specific methods for Rac2 The procedure is carried out at 4 ° unless otherwise stated

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[4] PURIFICATION OF Rac2 FROM NEUTROPHILS 27

a 30- to 40-fold enrichment in white blood cells A f t e r removal of erythro- cytes by hypotonic lysis, neutrophils are separated from monocytes and platelets by differential centrifugation through F i c o l l - H y p a q u e 9 In a different procedure, whole h u m a n blood can also be used as starting material 1°

T h e neutrophils are treated with 3 m M diisopropyl fluorophosphate for 15 min on ice, washed several times with phosphate-buffered saline (PBS), and subjected to nitrogen cavitation (450 psi, 20 min) in a buffer consisting

of 100 m M KCI, 3 m M NaCI, 1 m M A T P , 3.5 m M MgCI2, 10 m M P I P E S ( p H 7.3), 1 m M phenylmethylsulfonyl fluoride (PMSF), and 100 kallikrein inhibitory units of aprotinin/ml Cavitated cells are collected into sufficient

E G T A to give a final concentration of i mM, centrifuged at low speed (1000 rpm, 10 rain) to r e m o v e u n b r o k e n cells and nuclei, and then fractionated on discontinuous, 15/40/60% sucrose gradients in 25 m M H E P E S (pH 8.0), 1

m M E G T A , 1 m M E D T A buffer T h e cytosol overlay is collected and immediately frozen at - 7 0 ° , whereas the 15 to 40% interface containing plasma m e m b r a n e s is washed, repelleted, and stored in aliquots at - 7 0 ° in

25 m M H E P E S , p H 8.0, and 20 m M sucrose

A n a l y s i s of G u a n i n e N u c l e o t i d e B i n d i n g

Binding of [35S] G T P y S to GTP-binding proteins during all chromatography steps is d e t e r m i n e d with the rapid filtration technique as described, u For the standard assay, 10/xl of column fraction is incubated for 5 rain at

30 ° in 90/xl of reaction mixture containing 50 m M H E P E S (pH 8.0), 1 m M dithiothreitol ( D T T ) , 2 m M E D T A , and 1 /xM [35S]GTPyS (1-2 × 104 cpm/pmol, D u P o n t - N E N ) T h e reaction is terminated by addition of 2 ml

of ice-cold stop mixture [25 m M Tris-HC1 (pH 8.0), 100 m M NaC1, 30

m M MgCle, 2 m M D T T , 1 mg/ml bovine serum albumin] and binding is quantitated by vacuum filtration on BA-85 nitrocellulose filters (Schleicher and Schuell) and liquid scintillation counting (Scint-A XF, Packard) The free magnesium ion concentration u n d e r the conditions described is calculated to be 950 nM 12

9 j T Curnutte, R Kuver, and P J Scott, J Biol Chem 262, 5563 (1987)

10 A J Jesaitis, J R Naemura, R G Painter, L A Sklar, and C G Cochrane, Biochim Biophys Acta 719, 556 (1982)

11 U G Knaus, P G Heyworth, B T Kinsella, J T Curnutte, and G M Bokoch, J BioL Chem 267, 23575 (1992)

12 T Higashijima, K M Ferguson, P C Sternweis, M D Smigel, and A G Gilman, J Biol Chem 262, 762 (1987)

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28 E X P R E S S I O N A N D P U R I F I C A T I O N [4] Purification Procedure

DEAE-Sephacel Anion-Exchange Chromatography

Neutrophil cytosol (200-250 ml) from different donors (4 x 10 l° cell equivalents) is pooled and concentrated 10-fold by Amicon filtration using

a 10,000 MW cut-off filtration membrane The concentrated cytosol is then supplemented to final concentrations of 1 mM 2-mercaptoethanol, 0.1 mM DTT, 0.5 mM PMSF, 1/xM leupeptin, 1 ~ M pepstatin, 100 kallikrein inhibitory units of aprotinin per ml, and 100/~M 1-chloro-3-tosylamido-7-amino- 2-heptanone (TLCK) After a subsequent 10-fold dilution in TEDMPM buffer containing 1.5 mM ATP, the cytosol is applied at a flow rate of 25 ml/hr to a column (2.5 x 25 cm) of DEAE-Sephacel (Pharmacia LKB Biotechnology Inc.) equilibrated with the same buffer The column is washed with TEDMPM/1.5 ATP buffer until the monitored absorbance at

280 nm reaches the baseline and eluted with a linear gradient of NaC1 (280

ml, 0 ~ 180 mM NaC1) in TEDMPM/0.1 mM ATP After completion of the gradient, the column is further eluted with 150 ml of 1 M NaC1 in

T E D M P M buffer Fractions of 3.5 ml are collected and assayed for GTPyS- binding activity The flow through and two overlapping peaks in the salt gradient (100-170 mM NaC1) show GTPyS binding (Fig 1) When assayed for N A D P H oxidase activity in a cell-free system, as mentioned earlier, the first part of the GTPyS-binding peak (100-130 mM NaC1) correlates with oxidase stimulatory activity The second part of the biphasic GTPyS- binding peak represents the Rac-related proteins Rho (detected by ADP- ribosylation with botulinum toxin C3 ADP-ribosyltransferase) and Cdc42Hs (detected by Western blotting) To assure clean separation of Rac2 from these proteins, only the early fractions of the first peak should

be pooled for further purification

Sephacryl S-200 HR Gel Filtration

The pooled DEAE-Sephacel fractions are concentrated to 600/~1 and applied to a Sephacryl S-200 H R column (1.5 x 120 cm, Pharmacia LKB

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[4] PURIFICATION OF R a c 2 FROM NEUTROPHILS 2 9

Biotechnology Inc.) equilibrated with 100 m M NaCI in T E D M P M buffer/ 0.15 m M ATP T h e column is eluted at a flow rate of 13 ml/hr, and fractions

of 2 ml are collected T h e column should be preevaluated with molecular weight standards u n d e r the same gel filtration conditions On this column

G T P T S binding and oxidase stimulatory activity coelute in a single peak with a relative molecular weight of 160,000

Mono Q Anion-Exchange Chromatography

T h e peak fractions of the gel filtration column are pooled, concentrated

to 2 ml, adjusted to 10 m M NaC1 with T E D M M , and injected onto a

M o n o Q H R 5/5 column connected to a F P L C system (Pharmacia L K B Biotechnology, Inc.) T h e column is then washed with T E D M M and eluted with a shallow linear NaCI gradient (25 ml, 0 ~ 120 m M NaCI in T E D M M ) , followed by a steeper linear NaC1 gradient (10 ml, 120 ~ 250 m M NaC1

in T E D M M ) and a 1 M NaC1 wash (5 ml, in T E D M M ) at a flow rate of 0.5 ml/min Fractions (1 ml) are collected and assayed for GTPTS binding

A single peak can be detected at 90-110 m M NaC1 in the salt gradient

Heptylamine-Sepharose Chromatography

T h e peak M o n o Q fractions are pooled, adjusted to 0.2% cholate, and injected onto a heptylamine-Sepharose column (1.5 x 20 cm, F P L C sys-

Trang 34

in the gradient are combined, dialyzed, and subjected to phenyl-Superose chromatography

tern), 13 equilibrated with T E D M M containing 100 m M NaCl, and 0.2% sodium cholate (v/v) T h e column is then washed with 50 ml of equilibration buffer at a flow rate of 0.2 ml/min T h e elution is p e r f o r m e d with two successive linear cholate gradients in T E D M M buffer (10 ml, 0.2 ~ 1.0% sodium cholate (v/v); 25 ml, 1.0 ~ 1.6% sodium cholate), followed by two cholate step gradients (15 ml 1.6% sodium cholate and 30 ml 2.0% sodium cholate in T E D M M buffer) A t the same time a negative NaC1 gradient

in T E D M M buffer (250 ~ 0 m M NaCI) is performed Fractions of 2 ml are collected and assayed for [35S]GTPTS-binding Active fractions, detected at 1.2-1.6% sodium cholate (Fig 2), are combined and concentrated A f t e r extensive dialysis in T E D M M buffer, the pool exhibits stimulatory activity

in the cell-free N A D P H oxidase assay

Phenyl-Superose Chromatography

The dialyzed heptylamine-Sepharose pool is diluted with an equal

a m o u n t of T E D M M buffer containing 1.5 M a m m o n i u m sulfate and 5% ethylene glycol A f t e r injection onto a phenyl-Superose H R 5/5 F P L C column (Pharmacia L K B Biotechnology Inc.) equilibrated with the same

13 s Shaltiel, in "Methods in Enzymology" (W B Jakoby and M Wilchek, eds.), Vol 34, p

126 Academic Press, New York, 1974

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[41 PURIFICATION OF R a c 2 FROM NEUTROPHILS 31

buffer, the column is washed with 8 ml of equilibration buffer until the absorbance baseline is obtained The elution is performed with a simultane- ous reverse linear gradient of ammonium sulfate in T E D M M (12 ml, 1.5 ~ 0 M ammonium sulfate) and a linear ethylene glycol gradient (12 ml, 5 ~ 50% ethylene glycol) This is immediately followed by a 3-ml wash with T E D M M buffer/50% ethylene glycol and 5 ml TEDMM/60% ethylene glycol The elution is performed at a flow rate of 0.1 ml/min, and fractions of 0.5 ml are collected A major peak of GTPyS binding is detectable in fractions eluted with 50-60% ethylene glycol These fractions are combined, concentrated, and dialyzed extensively in T E D M and are stored

in aliquots at - 7 0 ° The amount of active, GTP-binding Rac2 can be determined by [3SS]GTPyS binding using the rapid filtration assay

Results and C o m m e n t s

The purity of the Rac2 preparation achieved with the just outlined purification procedure is shown in Fig 3 Each chromatography pool and the final concentrated Rac2 preparation are electrophoresed and silver

1 2 3 4 5 6

FIG 3 S D S - P A G E of Rac2 purification from human neutrophil cytosol at each step of chromatography Samples containing pooled peak fractions were electrophoresed on a 13% acrylamide gel and silver stained Lane I, molecular mass markers (kDa, Bio-Rad); land 2, first part of GTPyS binding in salt gradient (100-130 mM NaC1), DEAE-Sephacel; lane 3, Sephacryl S-200 HR; lane 4, Mono Q H R 5/5; lane 5, heptylamine-Sepharose; and lane 6, concentrated peak, phenyl-Superose HR 5/5

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32 EXPRESSION AND PURIFICATION [4] stained Rac2 is obtained with greater than 90% purity and migrates with

a relative molecular weight of 22,000 In some preparations, a second band, migrating at 21,000, is visible Tryptic peptide sequences of this band show identity with Rac2 A possible explanation is the occurrence of proteolytic breakdown or different post-translational processing of this lower band The actual yield of pure Rac2 from the starting material is difficult to calculate exactly because of the presence of multiple GTP-binding proteins

in the cytosol and an overlap in binding activity During later chromatography steps, an increase in the apparent recovery of Rac2 can be observed This is because of the separation of Rac2 from its complex with the cytosolic protein [rho]GDI, which has the property of inhibiting GDP dissociation and thus guanine nucleotide exchange Western blot analysis with an affinity-purified peptide antibody directed against [rho]GDI (generated to aa 17-28) shows complete dissociation of this complex after the fourth chromatographic step u Most detergents, as well as several lipids, are able to disrupt the interaction of Rac2 with [rho]GDI 3 Therefore it might be useful

to include l% sodium cholate in the reaction mixture for [35S]GTPyS- binding assays After release from the [rho]GDI protein, the hydrophobicity

of Rac2 results in high unspecific binding of the protein Therefore the protein should be kept in detergents as long as possible Judged by Western blot analysis with Rac2 antibodies, cytosol of 1 × 10 l° cell equivalents contains 0.5-0.6 mg Rac2 Each chromatographic step will result in protein loss in the range of 30-45%, with the last phenyl-Superose step being significantly higher The Rac2 protein is very labile at higher temperatures, low magnesium concentrations, and repeated freeze-thaw cycles

This purification protocol should be easily applicable to isolate Rac from other cellular sources It should also be noted that two slightly different procedures for Rac2 purification from differentiated HL-60 cells as well as neutrophils have been reported 14,15

17 T Mizuno, K Kaibuchi, S Ando, T Musha, K Hiraoka, K Takaishi, M Asada, H Nunoi,

I Matsuda, and Y Takai, J Biol Chem 267, 10215 (1992)

15 C H Kwong, H L Malech, D Rotrosen, and T L Leto, Biochemistry 32, 5711 (1993)

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[5] PURIFICATION OF R a c - G D I 33

[5] Purification of Rac-GDP Dissociation Inhibitor

Complex from Phagocyte Cytosol

By ARIE ABO

I n t r o d u c t i o n

Several small m o l e c u l a r mass G T P - b i n d i n g p r o t e i n s h a v e b e e n isolated

b y s e q u e n t i a l c o l u m n c h r o m a t o g r a p h y p r o c e d u r e s a n d have s h o w n to re- solve into m o l e c u l a r masses o f 2 0 - 3 0 k D a 1 T h e G T P a s e R a c has b e e n isolated f r o m platelets, d i f f e r e n t i a t e d H L - 6 0 cells, a n d n e u t r o p h i l s as a

2 1 - k D a m o n o m e r 1-3 H o w e v e r , in contrast, a t t e m p t s to r e c o n s t i t u t e the

N A D P H oxidase activity d e m o n s t r a t e d a r e q u i r e m e n t of a novel cytosolic

c o m p o n e n t w h i c h u p o n purification resolves into a h e t e r o d i m e r i c c o m p l e x

o f p r o t e i n s identified as p21 R a c l a n d R h o - G D I ( G D P dissociation inhibi-

t o r ) Y M o r e o v e r , studies h a v e s h o w n t h a t the entire p21 R a c p o o l is in a

c o m p l e x with R h o - G D I o f a m o l e c u l a r mass o f 4 5 - 5 0 k D a which m o s t likely r e p r e s e n t s the physiological f o r m o f R a c a n d R h o proteins 6 O n

n e u t r o p h i l stimulation, p21 R a c dissociates f r o m R h o - G D I a n d subse-

q u e n t l y t r a n s l o c a t e s to the p l a s m a m e m b r a n e 6'7 Interestingly, o t h e r m e m - bers o f t h e R a c / R h o family including R h o , Rac2, a n d C D C 4 2 H copurified with R h o - G D I f r o m n e u t r o p h i l cytosol as a h e t e r o d i m e r 8'9 Pick a n d co-

w o r k e r s 1° h a v e s h o w n t h a t R a c - G D I h e t e r o d i m e r s can be readily d i s r u p t e d

b y d e t e r g e n t to f o r m a m o n o m e r i c R a c a n d can reassociate with R h o - G D I

w h e n the d e t e r g e n t is r e m o v e d A d e t e r g e n t t h a t was i n c l u d e d in the early purification p r o c e d u r e s is m o s t likely responsible f o r t h e disruption of the

1 K Hiraoka, K Kaibuchi, S Ando, T Musha, K Takaishi, T Mizuno, M Asada, L Menard,

E Tomhav, J Didsbury, R Snyderman, and Y Takai, Biochem Biophys Res Commun

4 A Abo and E Pick, J BioL Chem 266, 23577 (1991)

5 A Abo, E Pick, A Hall, N Totty, C G Teahan, and A W Segal, Nature 353, 668 (1991)

6 A Abo, M R Webb, A Grogan, and A W Segal, Biochem J 298, 585 (1994)

7 M T Quinn, T Evans, L R Loetterle, A J Jesaitis, and G M Bokoch, J Biol Chem

268, 20983 (1993)

8 C H Kwong, H L Malech, D Rotrosen, and T L Leto, Biochemistry 32, 5711 (1993)

9 N Bourmeyster, M J Stasia, J Garin, J Gagnon, P Boquet, and P V Vignais, Biochemistry

31, 12863 (1992)

10 E Pick, Y Gorzalczany, and S Engel, Eur J Biochem 217, 441 (1993)

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R a c - G D I complex which led to the isolation of the monomeric form of p21 Rac

R h o - G D I inhibits the GDP dissociation from post-translationally modified Rho and Rac proteins and forms a complex only with the GDP-bound form of these proteins 11 In contrast, the isolated Rac/GDI complex purified from macrophage cytosol could activate the oxidase in the absence of GTP, suggesting that the Rac is in the GTP-bound form when complexed in GDI This is in agreement with reports demonstrating that R h o - G D I can inhibit the intrinsic and GAP-stimulated GTPase activity of Rac and Rho pro- teinsJ 2-14 It is not clear what is the physiological relevance of these multi- tude roles which R h o - G D I plays in regulating Rac and Rho proteins This chapter describes a rapid method for purification of R a c - G D I complex from phagocyte cytosol

Source of Phagocyte Cytosol

We used peritoneal macrophages from guinea pigs as the initial source

of cytosol; however, cytosol can also be prepared from HL-60 cells or neutrophils as described in this series

Isolation of Peritoneal Macrophages from Guinea Pigs

The isolation of macrophages from guinea pigs is a reliable procedure and can serve as a continuous source of relatively large amounts of cells (200 × 106 macrophages/animal); however, it requires animal facilities and can be often unpleasant

Hartley strain guinea pigs (250-300 g) are injected with 10 ml of steri- lized mineral oil into the peritoneum cavity The animal is held vertically

by one person and is injected by another person with great caution to avoid internal organ damage The animals are sacrificed after 6 days and the peritoneum exudate is harvested by washing the peritoneum cavity with phosphate-buffered saline (PBS) The macrophages are suspended at a concentration of 10 s cells/ml in ice-cold sonication buffer consisting of 8

mM Na phosphate buffer, pH 7.0, 130 mM NaCI, 340 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2/zM leupeptin The cells are

11 y Hori, A Kikuchi, M Isomura, M Katayama, Y Miura, H Fujioka, K Kaibuchi, and

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[5] PURIFICATION OF Rac-GDI 35 sonicated and cell debris and nuclei are removed by centrifugation at 200g

at 4 ° for 10 rain The particulate fraction is removed from the cytosol by centrifugation at 165,000g for 1 hr at 4 ° in a Type 65 fixed-angle rotor (Beckman) The resulting supernatant is what I will refer to throughout the chapter as cytosol and the sediment represents the membrane Mem- branes and cytosol are stored at - 7 0 °, and the cytosol serves as a source for the purification of R a c - G D I proteins

HL-60 Cell line

HL-60 cells are a leukemic cell line derived from patients with promyelocytic leukemia and can be obtained from the American Type Culture Collec- tion (Rockville, MD) These cells can be grown in large batches in suspension and are readily harvested by centrifugation In addition, the cells can

be differentiated into neutrophils or macrophages by simply treating them with an agent such as dimethyl sulfoxide (DMSO)

HL-60 cells are grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine (Irvine Scientific, Santa Ana, CA) at 0.5-2 x 106 cells/ml in 5% CO2/air The cells can be differentiated by various agents to differentiate the cells to neutrophil like The addition of 1.25% DMSO causes 80-90% of the cells to differentiate into neutrophil- like cells within 4-5 days Cells are harvested by centrifugation, washed twice with PBS, and resuspended in sonication buffer at 1-2 × l0 s cells/

ml Cytosol and membrane can be prepared in exactly the same manner

as described earlier for macrophages or for neutrophils as described in this series

Neutrophils

Preparation of neutrophils and subcellular fractionation of these cells are described in detail in this series

Detection Assays for Rac-GDI

The most important preliminary step is to develop an appropriate assay which will facilitate the detection of the R a c - G D I complex during the purification procedure A simple way to follow these two proteins is by Western blot analysis with antibodies against Rac proteins which are available commercially (Santa Cruz Biotechnology, Inc.) and from groups active

in the field The dilution, incubation time, and cross-reactivity should be determined and optimized prior to the purification In addition, it is important to note that neutrophils have a high level of expression of a R h o -

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3 6 EXPRESSION AND PURIFICATION [5]

GDI homolog 15 (70% homology, known also as D4) which cross-reacts with the polyclonal antibody derived from the recombinant R h o - G D I Thus, it

is advisable to probe the fractions only with p21rac antibodies during the first few purification steps Partially purified proteins should be also ana- lyzed on S D S - P A G E and visualized by silver staining

An alternative assay is the reconstitution of cell-free NADPH oxidase which is described in detail in this series To follow this activity, each fraction should be tested for the ability to activate the NADPH oxidase when supplemented with the other oxidase components: p47-phox, p67-

Purification of Rac-GDI Complex

All chromatographic procedures can be performed in a system containing two pumps, controller, injector, detector used to follow the absorbance at 280 nm, and fraction collector We have used the HPLC system (Waters Milford, MA) and the FPLC system (Pharmacia LKB Biotechnol- ogy Inc.) In addition, all purification steps are conducted at 4°; the columns are immersed in ice during the separations The purification scheme is outlined in Fig 1

a Ammonium Sulfate Precipitation

Ammonium sulfate precipitation is a convenient first step; it allows the processing of a large volume of extract and can be directly applied to phenyl- Sepharose with no buffer exchange The ammonium sulfate conditions are first optimized for the required protein by the precipitation of small amounts

of cytosol under different concentrations of ammonium sulfate The optimi- zation of R a c - G D I precipitation is described in detail elsewhere 16 We found that a 37% saturation of ammonium sulfate is sufficient to separate about 30% of the protein and to recover R a c - G D I in the supernatant Ammonium sulfate (37% saturation) is added to 10-20 ml of phagocyte cytosol (1-2 × 108 cell equivalents/ml) in small amounts over a period of

30 rain The mixture is stirred on ice for 1 hr and is centrifuged for 15 rain

at 200,000g in a TLX Beckman ultracentrifuge The pellet should contain

described in this series The supernatant containing the R a c - G D I proteins

is concentrated by reprecipitating with 85% saturation of ammonium sulfate The mixture is stirred for an additional hour on ice and is sedimented by

is j M Lelias, C N Adra, G M Wulf, J C Guillemot, M Khagad, D Caput, and B Lira,

Proc Natl Acad Sci U.S.A 911, 1479 (1993)

16 E Pick, T Kroizman, and A Abo, J lmmunoL 143, 4180 (1989)

Tiêu đề	Small GTPases and Their Regulators, Part B
Tác giả	Alan Hall, W. E. Balch, Channing J. Der
Trường học	Emory University Medical School
Chuyên ngành	Biochemistry
Thể loại	scientific article
Thành phố	Atlanta

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Số trang	418
Dung lượng	7,28 MB