small gtpases and their regulators, part d

Preface In 1955 we edited three volumes of Methods in Enzymology (255, 256, 257) dedicated to small GTPases. Since then this field has exploded, and these monomeric, regulatory proteins are now firmly established as a common focus of interest in a wide variety of research areas including cell and developmental biology, immunology, neurobiology, and, more re- cently, microbiology. After talking with colleagues, it became apparent that all three volumes needed to be significantly updated. We have, there- fore, attempted to identify the major new areas and themes that have emerged. This volume covers the Rho GTPase family. These proteins are key regulators of the actin cytoskeleton, and since the last volume on the subject there has been significant progress in identifying and characterizing the biochemical pathways associated with the three best characterized members of this family, Rho, Rac, and Cdc42. In the past five years, interest has also widened to a much broader community, as it has become clear that Rho GTPases also participate in the regulation of many other signaling path- ways, notably activation of the JNK and p38 MAP kinase pathways and of transcription factors such as SRF and NF-KB. This ability to coordinately regulate changes in the actin cytoskeleton with changes in gene transcription and other associated activities appears to be conserved from yeast to mammals. When the last volumes were published, the large diversity of both down- stream targets and upstream guanine nucleotide exchange factors that inter- act with Rho GTPases was not fully appreciated. Not surprisingly, therefore, these figure more prominantly this time around. Also, although it was thought likely that Rho GTPases might participate in many processes de- pendent on the organization of filamentous actin, it has now been directly shown that these proteins control cell movement, phagocytosis, growth cone guidance, and cytokinesis. An additional exciting new development has been the identification and characterization of numerous proteins en- coded by pathogenic bacteria that directly affect the activity of mammalian Rho GTPases. We very much hope that this and the accompanying volumes covering the Ras family (Volumes 332 and 333) and the small GTPases involved in membrane trafficking (Volume 329) will provide a useful source of practical information for anyone entering the field. None of this would have been XV xvi PREFACE possible without the talents and commitment of all our colleagues who have contributed to these volumes. We are indebted to them. ALAN HALL WILLIAM E. BALCH CHANNING J. DER Contributors to Volume 325 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. KARON ABE (38), Lineberger Comprehensive Cancer Center, University of North Caro- lina, Chapel Hill, North Carolina 27599 KLAUS AKTORIES (12), Institut far Pharma- kologie und Toxikologie, Albert-Ludwigs- Universitiit Freiburg, D-79104 Freiburg, Germany MUTSUKI AMANO (14), Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan ANSER C. AZIM (22), Division of Hematology, Brigham and Women's, Hospital, Harvard Medical School, Boston, Massachusetts 02115 DIANE L. BARBER (30), Departments of Sto- matology and Surgery, University of Cali- fornia, San Francisco, California 94143 KURT L. BARKALOW (22, 31), Division of He- matology, Brigham and Women's Hospital Harvard Medical School, Boston, Massa- chusetts 02115 DAFNA BAR-SAGI (29), Department of Molec- ular Genetics and Microbiology, State Uni- versi O, of New York, Stony Brook, New York 11794-5222 GARY M. BOKOCH (28), Departments" of Im- munology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037 GIDEON BOLLAG (5, 6), Onyx Pharmaceuti- cals, Richmond, California 94806 DANIEL BROEK (4), Department of Biochem- istry and Molecular Biology, Keck School ()f Medicine, University of Southern Califor- nia, Los Angeles, California 90033 SIIARON L. CAMPBELL (3), Department of Bio- chemistry and Biophysics. University of North Carolina, Chapel Hill, North Caro- lina 27599-7260 ix EMMANUELLE CARON (41), MRC Laboratory for Molecular Cell Biology, University Col- lege London, London WC1E 6BT, En- gland, United Kingdom CHRISTOPHER L. CARPENTER (18), Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts" 02215 FLAVIA CASTELLANO (25), Institut Curie- Recherche, CNRS UMR 144, 75248 Paris Cedex 05, France CHESTER E. CHAMBERLAIN (35), Department of Cell Biology, The Scripps Research Insti- tute, La Jolla, California 92037 PHILIPPE CHAVRIER (25), lnstitut Curie-Re- cherche, CNRS UMR 144, 75248 Paris Cedex 05, France EDWIN CHOY (10), Department of Medicine, Massachusetts" General Hospital, Boston, Massachusetts 02114 JOHN G. COLLARD (26, 36), Division of Cell Biology, The Netherlands" Cancer Institute, 1066 CX Amsterdam, The Netherlands ANNE M. CROMPTON (5), Onyx Pharmaceuti- cals, Richmond, California 94806 GIOVANNA M. D'ABACO (37), Cancer Re- search Campaign for Cell and Molecular Biology, Chester Beatty Laboratories, Insti- tute of Cancer Research, London S W3 6JB, England, United Kingdom BALAKA DAS (4), Department of Biochemis- try and Molecular Biology, Keck School of Medicine, University of Southern Califor- nia, Los Angeles, California 90033 SHERYL P. DENKER (30), Department of Sto- matology, University of California, San Francisco, California 94143 X CONTRIBUTORS TO VOLUME 325 CHANNING J. DER (38), Lineberger Compre- hensive Cancer Center, The University of North Carolina, Chapel Hill, North Caro- lina 27599 JOHN F. ECCLESTON (7), Division of Physical Biochemistry, National Institute for Medical Research, London NW7 1AA, England, United Kingdom EVA E. EVERS (36), Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands TOREN FINKEL (27), National Heart, Lung, and Blood Institute, Laboratory of Molec- ular Biology, National Institutes of Health, Bethesda, Maryland 20892-1650 ALYSON E. FOURNIER (42), Department of Neurology, Yale University School of Medi- cine, New Haven, Connecticut 06520 ANDREA FRIEBEL (8), Max von Pettenkofer- Institut, Ludwig Maximilians Universitiit, 80336 Munich, Germany MICHAEL A. FROHMAN (17), Department of Pharmacology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-8651 YtXIN Fu (44), Section of Microbial Pathogen- esis, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812 KEIGI FUJIWARA (33), Department of Struc- tural Analysis, National Cardiovascular Center Research Institute, Osaka 565- 8565, Japan YUKO FUKATA (14), Department of Cell Phar- macology, Nagoya University School of Medicine, Nagoya AICHI 466-8550, Japan JORGE E. (}ALAN (44), Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medi- cine, New Haven, Connecticut 06536-0812 PETER GIERSCHIK (16), Department of Phar- macology and Toxicology, University of Ulm, D-89081 Ulm, Germany GASTON G. M. HABETS (5), Onyx Pharmaceu- ticals, Richmond, California 94806 KLAUS M. HAHN (35), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 ALAN HALL (41), MRC Laboratory for Mo- lecular Cell Biology, University College London, London WCIE 6BT, England, United Kingdom ANDREW D. HAMILTON (34), Department of Chemistry, Yale University, New Haven, Connecticut 06511 JAEWON HAN (4), Department of Vascular Bi- ology, The Scripps Research Institute, La JoUa, California 92037 WOLF-DIETRICH HARDY (8), Max von Pet- tenkofer-lnstitut, Ludwig Maximilians Uni- versiti#, 80336 Munich, Germany MATTHEW J. HART (6), Onyx Pharmaceuti- cals, Richmond, California 94806 JOHN H. HARTWIG (22, 31), Division of Hema- tology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massa- chusetts 02115 PATRICK HEARING (29), Department of Mo- lecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222 MARK R. HOLT (32), Physiology Department, University College London, London WC1E 6J J, England, United Kingdom JON P. HUTCHINSON (7), Division of Physical Biochemistry, National Institute for Medical Research, London NW7 1AA, England, United Kingdom DARIA ILLENBEROER (16), Department of Pharmacology and Toxicology, University of Ulm, D-89081 Ulm, Germany TOSHIMASA ISHIZAKI (24), Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606-8315, Japan LENNERT JANSSEN (26), Division of Cell Biol- ogy, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands DANIEL G. JAY (43), Department of Physiol- ogy, Tufts University School of Medicine, Boston, Massachusetts 02111 CONTRIBUTORS TO VOLUME 325 xi GARETH E. JONES (40), Randall Centre for Molecular Mechanisms of Cell Fanction, King's College London, London SE1 1UL, England, United Kingdom Kozo KAIBUCHI (14), Department of Cell Pharmacology, Nagoya University School of Medicine, Nagoya AICHI 466-8550, Ja- pan and Division of Signal Transduction, Nara Institute of Science and Technology, lkoma, Nara 630-0101, Japan ROBERT G. KALB (42), Department of Neurol- ogy, Yale University School qf Medicine, .New Haven, Connecticut 06520 YASUNORI KANAItO (17), Department of Pharmacology, Tokyo Metropolitan Msti- tute of Medical Science, Tokyo 113-8613, Japan YUMJKO KANO (33), Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan KAzuo KATOH (33), Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan CHARLES C. KING (15, 28), Department of Im- munology, The Scripps Research Institute, La .lol&, Cal(fornia 92037 UEEA G. KNAUS (15), Department of Immu- nology, The Scripps Research Institute, La Jolla, California 92037 ANNA KOFFER (32), Physiology Department, University College London, London WC1E 6J J, England, United Kingdom VADIM S. KRAYNOV (35), Department of Cell, Biology, The Scripps Research Institute, La Jolla, Califi)rnia 92037 IAN O. MACARA (1), The Markey Center fi)r Cell Signaling, University of Virginia, Char- lottesville, Virginia 22908 LAURA M. MACHESKY (20), Division of Mo- lecular Cell Biology. School of Biosciences, University of Birmingham, Birmingham B15 2TT, England, United Kingdom AKIKO MAMMOTO (9), Department of Molec- ular Biology and Biochemistry, Osaka Uni- versity Graduate School of Medicine, Fac- uhv of Medicine, Osaka 565-0871, Japan DANNY MANOR (13), Division o¢" Nutritional Sciences, Cornell University, Ithaca, New York 14853 FRITS MICHIELS (26), Galapagos Genomics, 2333 AL Leiden, The Netherlands MICttAEL MOOS (11), lnstitutfiir Medizinische Mikrobiologie, Universitiit Mainz, D-55101 Mainz, Germany ANDREW J. MORRIS (17), Department of Phar- macology and institute for Cell and Devel- opmental Biology, State University of New York, Stony Brook, New York 11794-8651 RAYMOND MOSTELLER (4), Department of Biochemistry and Molecular Biology, Keck School of Medicine, University qf Southern Cal(fi~rnia, Los Angeles, California 90033 R. DYCHE MULLINS (20), Department of' Cel- hdar and Molecular Pharmacology, Univer- sity of California School of Medicine, San Francisco, California 94143 ROBERT K. NAKAMOIO (2), Department of Molecular Physiology and Biological Phys- ics, University of Virginia, Charh)ttesvilh', Virginia 22908-07.36 SHUH NAI~.UMIYA (24). Department of Phar- macology, Kyoto Univers'ity Faculty of Medicine, Kyoto 606-8.315, Japan CIIERYL L. NEUDAUER (1)~ The Markey Cen- ter for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908 MARGARE l'A NIKOLIC (19), Molectdar Neuro- biology Group, King's College, London, Enghmd, United Kingdom CATHERINE D. NOBES (39), MRC Laboratory for Molecular Cell Biology and Department of Anatomy and Developmental Biology, University College London, London WCI E 6BT, England, United Kingdom GARRY NOLAN (26), Stanford University School of Medicine, Stanford, California 94305 MICHAEL F. OLSON (37), Cancer Research Campaign for Cell and Molecular Bioh)gy, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, En- gland, United Kingdom xii CONTRIBUTORS TO VOLUME 325 JAYESI-I C. PATEL (41), MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, England, United Kingdom DANIELLE PEVERLY-MITCHELL (5), Onyx Pharmaceuticals, Richmond, California 94806 MARK PHILIPS (10), Departments of Medicine and Cell Biology, New York University School of Medicine, New York, New York 10016 PAUL W. READ (2), Department of Molecu- lar Physiology and Biological Physics, Uni- versity of Virginia, Charlottesville, Virginia 22908-0736 ABINA M. REILLY (15), Department oflmmu- nology, The Scripps Research Institute, La Jolla, California 92037 XIANG-DONG REN (23), State University of New York, Stony Brook, New York 11794-8165 ANNE J. RIDLEY (40), Ludwig Institute for Cancer Research, London W1P 8BT, En- gland, United Kingdom KATRIN RITI~INGER (7), Division of Protein Structure, National Institute for Medical Research, London NW7 1AA, England, United Kingdom WILLIAM ROSCOE (6), Onyx Pharmaceuticals, Richmond, California 94806 KENT L. ROSSMAN (3), Department of Bio- chemistry and Biophysics, University of North Carolina, Chapel Hill, North Caro- lina 27599-7260 LURAYNNE C. SANDERS (28), Department of Immunology, The Scripps Research Insti- tute, La Jolla, California 92037 TAKUYA SASAKI (9), Department of Biochem- istry, The University of Tokushima, School of Medicine, Kuramoto, Japan GUDULA SCHMIDT (12), Institut far Pharma- kologie und Toxikologie, Albert-Ludwigs- Universitiit Freiburg, D-79104 Freiburg, Germany FRIEDER SCHWALD (16), Department of Phar- macology and Toxicology, University of Ulm, D-89081 Ulm, Germany MARTIN ALEXANDER SCHWARTZ (23), The Scripps Research Institute, La Jolla, Califor- nia 92037 SA'I'D M. SEBTI (34), Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 HIROAKI SHIMOKAWA (14), Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medi- cine, Fukuoka 812-8582, Japan PATRICIA A. SOLSKI (38), Lineberger Compre- hensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 ILONA STEPHAN (16), Department of Pharma- cology and Toxicology, University of Ulm, D-89081 Ulm, Germany STEPHEN M. STRITrMATrER (42), Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06520 DANIEL M. SULLIVAN (27), National Heart, Lung, and Blood Institute, Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892-1650 MARC SYMONS (5), Onyx Pharmaceuticals, Richmond, California 94806 KAZUO TAKAHASHI (9), Second Department of Internal Medicine, Chiba University Medical School, Chiba 260-0856, Japan YOSHIMI TAKAI (9), Department of Molecular Biology and Biochemistry, Osaka Univer- sity Graduate School of Medicine~Faculty of Medicine, Osaka 565-0871, Japan LAURA J. TAYLOR (29), Department of Molec- ular Genetics and Microbiology, State Uni- versity of New York, Stony Brook, New York 11794-5222 JEAN P. TEN KLOOSTER (36), Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands KIMBERLEY TOLIAS (18), Division of Signal Transduction, Beth Israel Deaconess Medi- cal Center, Boston, Massachusetts 02215 CONTRIBUTORS TO VOLUME 325 Xlll LI-HUEI TSAI (19), Howard Hughes Medical Institute, Department of Pathology, Har- vard Medical School, Boston, Massachu- setts 02115 MASAYOSHI UEHATA (24), Drug Discovery Laboratories, WelFide (Yoshitomi) Corpo- ration, Osaka 573-1153, Japan RoB A. VAN DER KAMMEN (26, 36), Division of Cell Biology, The Netherlands' Cancer Institute, 1066 CX Amsterdam, The Nether- lands' CHRISTOPH VON EICHEL-STREIBER (11), Ver- fligungsgebziude fiir Forschung und Ent- wicklung, lnstitut far Medizinisch Mikrobi- ologie und Hygiene, Johannes Gutenberg- Universitiit, 55101 Mainz, Germany AMY B. WALSH (29), Department of Molecu- lar Genetics and Microbiology, State Uni- versity of New York, Stony Brook, New York 11794-5222 ERIC V. WONG (43), Department of Physiol- ogy, Tufts University School of Medicine, Boston, Massachusetts 02111 WEIHONG YAN (30), Department of Stoma- tology, University of California, San Fran- cisco, California 94143 YUE ZHANG (17), Department of Pharmacol- ogy and Institute for Cell and Develop- mental Biology, State University of New York, Stony Brook, New York 11794-8651 DANIEL ZICHA (40), Imperial Cancer Re- search Fund, London WC2A 3PX, En- gland, United Kingdom SALLY H. ZIGMOND (21), Biology Depart- ment, University of Pennsylvania, Philadel- phia, Pennsylvania 19104-6018 [ 1 ] CHARACTERIZATION OF TC10 3 [1] Purification and Biochemical Characterization of TC 1 0 By CHERYL L. NEUDAUER and IAN G. MACARA Introduction TC10 is a member of the Rho family of small GTPases. We have previously characterized the biochemistry, cellular effects, and effector in- teractions of TC10.1 This study established TC10 as a distinct member of the Rho family most closely related to Cdc42. In NIH 3T3 cells, the ectopic expression of hemagglutinin (HA)-tagged, gain-of-function TC10 induces long filopodia and loss of stress fibers. TC10 interacts with a subset of those effector proteins that bind to Cdc42. TC10 also interacts with several distinct proteins that are specific for TC10. 2 This article describes the methods used for the mutagenesis of TC10 and the set of vectors used for the purification of recombinant proteins and mammalian expression of TC10. It also de- scribes the biochemical characterization of TC10 and various methods used to study the interaction of TC10 with putative effector proteins. Mutagenesis and Subcloning of TC10 We have tested several methods for the introduction of point mutations and have found megaprimer polymerase chain reaction (PCR) to be a relatively consistent, cost effective, and reliable technique. 3 Briefly, an inter- nal primer is designed that contains the nucleotide substitution(s). An initial round of PCR is performed with this primer and a primer to either the 5' or 3' end of the sequence. In general, we include a BamHI site in the 5' primer and an EcoRI site in the 3' primer to facilitate subcloning. Restric- tion enzymes usually cut the ends of PCR products inefficiently and are therefore digested with high concentrations of BamHI and EcoRI at 37 ° for greater than 4 hr. To facilitate the expression of TCI0 and other proteins in bacteria, yeast, and mammalian cells, we have designed a set of vectors with similar I C. L. Neudauer, G. Joberty, N. Tatsis, and I. G. Macara, Curr. Biol. 8, 1151 (1998). 2 G. Joberty, unpublished results (1999). 3 S. Batik, in "'PCR Protocols: Current Methods and Applications" (B. A. White, ed.), p. 277. Humana Press, Totowa, NJ, 1993. Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. MET1 tODS IN ENZYMOLOGY, VOL. 325 0076-6879/00 $30.00 4 PURIFICATION, MODIFICATION, AND REGULATION [ 1] cloning sites (Table I). The majority of these vectors produce N-terminally tagged fusion proteins; C-terminal tagging of the small GTPases is usually avoided as most of these proteins undergo posttranslational modification (e.g., prenylation, carboxymethylation) at their C termini. Each vector contains a BamHI site in the same reading frame as pGEX-2T (Amersham Pharmacia, Piscataway, N J; Fig. 1). The pK series of vectors derives expression from a cytomegalovirus (CMV) promoter and contains splice donor and acceptor sites upstream of the initiation codon to increase the efficiency of mRNA export from the nucleus. The vectors contain a simian virus 40 (SV40) origin, so they will replicate in COS-7 cells (which contain the SV40 large T antigen). They are designed for high-level expression in transient transfections and do not contain a eukaryotic selectable marker. This set of vectors allows for the rapid characterization of TC10 or other proteins by prokaryotic expression and purification and by mammalian expression and immunoprecipitation, immunoblotting, or immunofluorescence. The purification methods are listed in Table I. The antibodies used and their concentrations for immu- noblots or immunofluorescence are listed in Table II. TABLE I VECTOR SUMMARY Parent vector Vector Tag Purification Expression pQE70 pQNzz ZZ lgG Sepharose Prokaryotic Oiagen pRK7 Mammalian pRK7 pKH3 Triple HA 12CA5 with Sigma protein Mammalian A-Sepharose pRK7 pKMyc Myc 9El0 with Amersham Phar- Mammalian macia GammaBind Plus Sepharose pRK7 pKFLAG FLAG Sigma anti-FLAG Mammalian M2-agarose pRK7 pRK7-GFP GFP Santa Cruz anti-GFP with Mammalian Sigma protein A Sepharose pRK7 pKNzz ZZ IgG Sepharose Mammalian pGBT9 pGBT10 GAL4 DNA-bind- Yeast Clontech ing domain pVP16 pVP16-CP GAL4 activation Yeast domain [ 1] CHARACTERIZATION OF TC10 pGEX-2T Smal pQNzz NcoI r "'D B amHI NotI EcoRI BgllI CC ATG~ GCJG 'CGG CCG C~A ATT C~G ATC T I pRK7 HindllI PstI SalI XbaI BamHI EcoRI ClaI ~'CTG CAG"GTC GAC'~FCT AGA'~GA TcciccG GG~'~'~'IA~ PKH3 BamHI EcoRI ClaI iGGA TCCUGAA TFCtAtAT CGA T i PKMyc Sinai iNheI Notl EcoRI ClaI CGG ~3CT AGC t GG~3 CGG CCG C~lqGAA TTCnATC GAT i pKFLAG BamHI EeoRI ClaI Sill tGGA TCC~tGAA TI"C~AtAT CGA qtGG CCG CCA TGG CC~ PRKT-GFP BamHI EcoRI ClaI ~~GA GAA ~TC pKNzz BamHI Notl EeoRI ClaI ~GCIG CGG CCG CbA ATT C~A~ pGBT10 BamHI AatI1 EcoRI iGGA TCCIIGAC GTCI~ VP16-CP BamHI AatlI EcoRI IGGA TCCJIGAC GTCtIGGT ACC I FIG. 1. Multiple cloning sites of expression vector set. These vectors were designed to placc the BamHI cloning site in the same reading frame as pGEX-2T (Amersham Pharmacia, shown as reference). [...]... and GST-Cdc42(wt) are expressed 30 PURIFICATION, MODIFICATION, AND REGULATION [3] A Vav2 ,iL A " 191- 402 HP.I Dbs A A A ~623-832 623- 967 B kDa MW Vav2 DH Dbs DH Dbs DH/PH 50 35 ii!i!i! !ii!!iii~iiii: 28 ~i~ii~i!i~i!ii~i:~ 20 FI~ 1 Bacterial expressed and purified Vav2 DH and Dbs DH and DH/PH domains (A) The domain structures of full-length Vav2 and Dbs are shown Also indicated are the DH and D. .. pET28a (Novagen) The Vav2 and Dbs D H domains were designed to encode the entire D H domain and extend into the linker region between the D H and the PH domains (Fig 1A) This was done to avoid truncation of any structured elements that may be present at the carboxy terminus of the D H domain The Dbs D H / P H domain was extended a few residues beyond what is predicted to be the PH domain carboxy-terminal... exchange, the DH/PH tandem domains may constitute the complete catalytic unit Unquestionably, the ability to express and purify active forms of Dblrelated proteins and their DH and DH/PH domains will greatly facilitate structural and biochemical studies of these proteins This article presents methods for the expression and purification of active DH and DH/PH domains from the Dbl family proteins, Dbs and Vav2,... D H / P H DOMAINS 29 E x p r e s s i o n a n d Purification of Vav2 DH D o m a i n a n d Dbs DH a n d D H / P H D o m a i n s in Escherichia coli A c D N A fragment encoding the human Vav2 D H domain (residues 191-402), murine Dbs D H domain (residues 623-832), and D H / P H domains (residues 623-967) was generated by polymerase chain reaction and inserted into the N c o I / X h o I sites of the bacterial... repeat (SPEC) (B) S D S - P A G E and Coomassie staining of purified recombinant bacterial-expressed Vav2 DH and Dbs DH and D H / P H domains Lane 1, molecular weight markers; lane 2, Vav2 DH (3 /~g); lane 3, Dbs DH (3 p~g); and lane 4, Dbs D H / P H (3 ~g) and initially purified on glutathione agarose essentially as described 24 The G S T - G T P a s e s are then further purified on an S-200 column... hydrolysis by the complex GTP-loaded complexes were incubated at 22°, aliquots were taken at specified time points, the reaction was quenched, the protein was precipitated by the addition of perchloric acid, and the nucleotide content was determined Diamonds, FLAGRhoA/His6-RhoGDI: triangles, His6-RhoA/FLAG-RhoGDI; and squares, RhoA/RhoGDI with affinity tags removed 24 PURIFICATION, MODIFICATION, AND. .. When compared to the GEF activity of the isolated DH domain, the DH/PH bidomains of Dbl and Trio exhibited greatly enhanced catalytic activity toward Cdc42 and Racl, respectively.5'1~ Furthermore, the structure of the SOS1 DH/PH bidomain shows a possible structural interdependence between the DH and the PH domains for catalysis 13 Taken together, these observations suggest that although the DH domain alone... D H / P H domain regions from Vav2 and Dbs that were cloned and expressed in Escherichia coli to produce the recombinant proteins used in this study: calponin homology domain (CH), acidic domain (AD), Dbl homology domain (DH), linker region (L), pleckstrin homology domain (PH), cysteine-rich domain (CRD), serine-rich region (SR), Src homology 2 domain (SH2), Src homology 3 domain (SH3), and spectrin... molecule This surface is composed of residues in and surrounding helices 1, 9, and 10 in the Trio D H domain structure and helices A, I, J, and K in the structure of/3-Pix, which corresponds to the conserved regions 1 and 3 (CR1 and CR3) and regions carboxy-terminal to CR3 in the primary sequence) Whereas the structures of the D H domains have revealed the putative Rho GTPase-binding surface, it is still... R h o G D I complex is therefore useful for signal transduction studies in which the investigator wishes to deliver highly concentrated, posttranslationally modified RhoA into cellular systems, without the worry of detergents and rapid nucleotide loss or hydrolysis to study downstream signaling effects [3] Bacterial Expressed DH and DH/PH Domains By KENT L ROSSMAN and SHARON L CAMPBELL Introduction . [a-3ep]GDP (equivalent to 1 tzl GTP), and 25 mM MOPS, pH 7.1, and 1 mM EDTA to 50/zl and incubate on ice for 20 min. Add 1/xl 1 M MgC12 and incubate on ice for an additional 10 rain. Store loaded. GST-TC10 and a cleaved effector protein can be added to the beads at the same time.) Wash the beads three to five times with binding buffer. Add 10 /zl 2× SDS-PAGE sample buffer to the beads, and. and 0.1% (v/v) BSA), 20 /xl of the buffer in which the effector is diluted, and 2.5/xl of diluted, loaded TC10 and incubate on ice for 20 min. Add 2.5/xl of the buffer in which the GAP is diluted