Small GTPases and Their Regulators Part D PDF
Preview 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 ECAFERP 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 GNINNAHC J. DER Contributors to Volume 325 elcitrA numbers era ni parentheses gniwollof the names of .srotubirtnoc snoitailiffA listed era .tnerruc KARON ABE (38), Lineberger Comprehensive ELLEUNAMME CARON (41), MRC Laboratory Cancer Center, University of North Caro- for Molecular Cell Biology, University Col- lina, Chapel Hill, North Carolina 27599 lege London, London WC1E 6BT, En- gland, United Kingdom KLAUS AKTORIES (12), Institut far Pharma- kologie und Toxikologie, Albert-Ludwigs- REHPOTSIRHC L. CARPENTER (18), Division of Universitiit Freiburg, D-79104 Freiburg, Signal Transduction, Beth Israel Deaconess Germany Medical Center, Boston, Massachusetts" MUTSUKI AMANO (14), Division of Signal 02215 Transduction, Nara Institute of Science and FLAVIA CASTELLANO (25), Institut Curie- Technology, Ikoma, Nara 630-0101, Japan Recherche, CNRS UMR 144, 75248 Paris ANSER C. AZIM (22), Division of Hematology, Cedex ,50 France Brigham and Women's, Hospital, Harvard CHESTER E. CHAMBERLAIN (35), Department Medical School, Boston, Massachusetts of Cell Biology, The Scripps Research Insti- 02115 tute, La Jolla, California 92037 DIANE L. BARBER (30), Departments of Sto- matology and Surgery, University of Cali- PHILIPPE CHAVRIER (25), lnstitut Curie-Re- fornia, San Francisco, California 94143 cherche, CNRS UMR 144, 75248 Paris Cedex ,50 France KURT L. BARKALOW (22, 31), Division of He- matology, Brigham and Women's Hospital EDWIN CHOY (10), Department of Medicine, Harvard Medical School, Boston, Massa- "sttesuhcassaM General Hospital, Boston, chusetts 02115 Massachusetts 02114 DAFNA BAR-SAGI (29), Department of Molec- JOHN G. COLLARD (26, 36), Division of Cell ular Genetics and Microbiology, State Uni- Biology, The Netherlands" Cancer Institute, versi ,O of New York, Stony Brook, New 1066 CX Amsterdam, The Netherlands York 11794-5222 ANNE M. CROMPTON (5), Onyx Pharmaceuti- GARY M. BOKOCH (28), Departments" of Im- cals, Richmond, California 94806 munology and Cell Biology, The Scripps Research Institute, La Jolla, California GIOVANNA M. D'ABACO (37), Cancer Re- 92037 search Campaign for Cell and Molecular Biology, Chester Beatty Laboratories, Insti- GIDEON BOLLAG (5, 6), Onyx Pharmaceuti- tute of Cancer Research, London S W3 6JB, cals, Richmond, California 94806 England, United Kingdom DANIEL BROEK (4), Department of Biochem- istry and Molecular Biology, Keck School BALAKA DAS (4), Department of Biochemis- ()f Medicine, University of Southern Califor- try and Molecular Biology, Keck School of nia, Los Angeles, California 90033 Medicine, University of Southern Califor- nia, Los Angeles, California 90033 SIIARON L. CAMPBELL (3), Department of Bio- chemistry and Biophysics. University of SHERYL P. DENKER (30), Department of Sto- North Carolina, Chapel Hill, North Caro- matology, University of California, San lina 27599-7260 Francisco, California 94143 ix X CONTRIBUTORS TO VOLUME 325 CHANNING J. DER (38), Lineberger Compre- KLAUS M. HAHN (35), Department of Cell hensive Cancer Center, ehT University of Biology, ehT Scripps hcraeseR ,etutitsnI La North ,aniloraC Chapel Hill, North -oraC Jolla, California 73029 lina 99572 ALAN HALL (41), MRC Laboratory for -oM JOHN F. ECCLESTON (7), Division of Physical lecular Cell Biology, University College ,yrtsimehcoiB National etutitsnI for lacideM London, London WCIE 6BT, England, ,hcraeseR London NW7 1AA, England, detinU Kingdom detinU Kingdom ANDREW D. HAMILTON (34), Department of EVA E. EVERS (36), Division of Cell ,ygoloiB ,yrtsimehC Yale University, New Haven, ehT Netherlands Cancer ,etutitsnI 1066 CX tucitcennoC 11560 Amsterdam, ehT sdnalrehteN JAEWON HAN (4), Department of ralucsaV Bi- TOREN FINKEL (27), National Heart, Lung, ology, ehT Scripps Research Institute, La and Blood Institute, Laboratory of Molec- ,aUoJ California 73029 ular Biology, National Institutes of ,htlaeH WOLF-DIETRICH HARDY (8), Max von Pet- ,adsehteB Maryland 0561-29802 ,tutitsnl-refoknet Ludwig Maximilians -inU ALYSON E. FOURNIER (42), Department of ,#itisrev 80336 Munich, Germany Neurology, Yale ytisrevinU School of -ideM MATTHEW J. HART (6), Onyx -ituecamrahP ,enic New Haven, Connecticut 02560 ,slac Richmond, California 60849 ANDREA FRIEBEL (8), Max nov Pettenkofer- JOHN H. HARTWIG (22, 31), Division of Hema- Institut, Ludwig Maximilians ,tiitisrevinU tology, Brigham and Women's ,latipsoH 63308 Munich, Germany Harvard Medical School, Boston, Massa- MICHAEL A. FROHMAN (17), Department of sttesuhc 51120 Pharmacology and Institute for Cell and Developmental Biology, State ytisrevinU of PATRICK HEARING (29), Department of Mo- lecular Genetics and Microbiology, State New York, Stony Brook, New York ytisrevinU of New York, Stony Brook, New 1568-49711 York 2225-49711 YtXIN Fu (44), Section of Microbial -negohtaP ,sise Boyer Center for Molecular ,enicideM MARK R. HOLT (32), Physiology ,tnemtrapeD Yale University School of Medicine, New ytisrevinU egelloC London, London WC1E Haven, Connecticut 2180-63560 J6 ,J England, United Kingdom KEIGI FUJIWARA (33), Department of -curtS JON P. HUTCHINSON (7), Division of Physical tural Analysis, National ralucsavoidraC ,yrtsimehcoiB National etutitsnI for lacideM Center Research Institute, Osaka 565- ,hcraeseR London NW7 1AA, England, ,5658 Japan detinU Kingdom YUKO FUKATA (14), Department of lleC Phar- DARIA ILLENBEROER (16), Department of macology, Nagoya University School of Pharmacology and Toxicology, ytisrevinU ,enicideM Nagoya AICHI ,0558-664 Japan of Ulm, 18098-D Ulm, Germany JORGE E. (}ALAN (44), Section of Microbial TOSHIMASA ISHIZAKI (24), Department of ,sisenegohtaP Boyer Center for Molecular ,ygolocamrahP Kyoto ytisrevinU Faculty of ,enicideM Yale University School of -ideM ,enicideM Kyoto ,5138-606 Japan ,enic New Haven, Connecticut 2180-63560 LENNERT JANSSEN (26), Division of Cell Biol- PETER GIERSCHIK (16), Department of -rahP ogy, ehT sdnalrehteN Cancer ,etutitsnI 6601 macology and Toxicology, University of CX Amsterdam, ehT sdnalrehteN ,mlU 18098-D Ulm, Germany DANIEL G. JAY (43), Department of Physiol- GASTON G. M. HABETS (5), Onyx -uecamrahP ogy, Tufts University School of ,enicideM ,slacit Richmond, California 60849 Boston, sttesuhcassaM 11120 CONTRIBUTORS TO VOLUME 325 xi GARETH E. JONES (40), Randall Centre for YNNAD MANOR (13), Division "¢o Nutritional Molecular Mechanisms of Cell Fanction, Sciences, Cornell University, Ithaca, New King's College London, London SE1 1UL, York 14853 England, United Kingdom FRITS MICHIELS (26), Galapagos Genomics, Kozo KAIBUCHI (14), Department of Cell 2333 AL Leiden, The Netherlands Pharmacology, Nagoya University School MICttAEL MOOS (11), lnstitutfiir Medizinische of Medicine, Nagoya AICHI 466-8550, Ja- Mikrobiologie, Universitiit Mainz, D-55101 pan and Division of Signal Transduction, Mainz, Germany Nara Institute of Science and Technology, WERDNA J. MORRIS (17), Department of Phar- lkoma, Nara 630-0101, Japan macology and institute for Cell and Devel- ROBERT G. KALB (42), Department of Neurol- opmental Biology, State University of New ogy, Yale University School qf Medicine, York, Stony Brook, New York 1568-49711 .New Haven, Connecticut 06520 DNOMYAR MOSTELLER (4), Department of YASUNORI KANAItO (17), Department of Biochemistry and Molecular Biology, Keck Pharmacology, Tokyo Metropolitan Msti- School of Medicine, University qf Southern tute of Medical Science, Tokyo ,3168-311 ,ainr~if(laC Los Angeles, California 90033 Japan R. DYCHE SNILLUM (20), Department of' Cel- OKJMUY KANO (33), Department of Structural hdar and Molecular Pharmacology, Univer- Analysis, National Cardiovascular Center sity of California School of Medicine, San Research Institute, Osaka 565-8565, Japan Francisco, California 94143 KAzuo KATOH (33), Department of Structural ROBERT K. NAKAMOIO (2), Department of Analysis, National Cardiovascular Center Molecular Physiology and Biological Phys- Research Institute, Osaka 565-8565, Japan ics, University of Virginia, Charh)ttesvilh', SELRAHC C. KING (15, 28), Department of Im- Virginia 63.70-80922 munology, The Scripps Research Institute, SHUH AYIMU.~IAN (24). Department of Phar- La .lol&, Cal(fornia 92037 macology, Kyoto Univers'ity Faculty of UEEA G. KNAUS (15), Department of Immu- Medicine, Kyoto ,513.8-606 Japan nology, The Scripps Research Institute, La CIIERYL L. NEUDAUER (1)~ The Markey Cen- Jolla, California 92037 ter for Cell Signaling, University of Virginia, ANNA KOFFER (32), Physiology Department, Charlottesville, Virginia 22908 University College London, London WC1E MARGARE A'l CILOKIN (19), Molectdar Neuro- 6J ,J England, United Kingdom biology Group, King's College, London, VADIM S. VONYARK (35), Department of Cell, Enghmd, United Kingdom Biology, The Scripps Research Institute, La ENIREHTAC D. NOBES (39), MRC Laboratory Jolla, Califi)rnia 92037 for Molecular Cell Biology and Department NAI O. MACARA (1), The Markey Center fi)r of Anatomy and Developmental Biology, Cell Signaling, University of Virginia, Char- University College London, London WCI E lottesville, Virginia 22908 6BT, England, United Kingdom LAURA M. YKSEHCAM (20), Division of Mo- GARRY NOLAN (26), Stanford University lecular Cell Biology. School of Biosciences, School of Medicine, Stanford, California University of Birmingham, Birmingham 50349 B15 2TT, England, United Kingdom MICHAEL F. OLSON (37), Cancer Research AKIKO OTOMMAM (9), Department of Molec- Campaign for Cell and Molecular Bioh)gy, ular Biology and Biochemistry, Osaka Uni- Chester Beatty Laboratories, Institute of versity Graduate School of Medicine, Fac- Cancer Research, London SW3 6JB, En- uhv of Medicine, Osaka 565-0871, Japan gland, United Kingdom xii CONTRIBUTORS TO VOLUME 325 I-ISEYAJ C. PATEL (41), MRC Laboratory for MARTIN ALEXANDER SCHWARTZ (23), ehT Biology, University Cell Molecular egelloC sppircS hcraeseR ,etutitsnI La ,alloJ -rofilaC London, London WC1E 6BT, England, ain 73029 detinU Kingdom D'I'AS M. SEBTI (34), Discovery Drug ,margorP DANIELLE PEVERLY-MITCHELL (5), Onyx .H and Center Lee Cancer Moffitt hcraeseR ,slacituecamrahP Richmond, California Institute, University of South Florida, 60849 Tampa, Florida 21633 MARK PHILIPS (10), Departments of enicideM HIROAKI AWAKOMIHS (14), hcraeseR etutitsnI and Cell Biology, New York ytisrevinU of Angiocardiology and ralucsavoidraC School of Medicine, New York, New ,cinilC Kyushu School University of -ideM York 61001 ,enic Fukuoka ,2858-218 Japan PAUL W. READ (2), Department of Molecu- ral Physics, Biological and Physiology -inU PATRICIA A. IKSLOS (38), Lineberger -erpmoC ytisrev of ,ainigriV ,ellivsettolrahC ainigriV hensive Cancer ,retneC ytisrevinU of North 6370-80922 ,aniloraC Chapel Hill, North Carolina 99572 ABINA M. REILLY (15), Department oflmmu- nology, ehT Institute, Research Scripps La STEPHAN ILONA (16), Department of -amrahP ,alloJ California 73029 cology and Toxicology, University of ,mlU XIANG-DONG REN (23), State University of 18098-D Ulm, Germany New York, Stony Brook, New York NEHPETS M. STRITrMATrER (42), Department 5618-49711 of Neurology, Yale University School of ANNE J. RIDLEY (40), Ludwig Institute for ,enicideM New Haven, Connecticut 02560 recnaC ,hcraeseR London W1P 8BT, En- DANIEL M. SULLIVAN (27), National ,traeH ,dnalg Kingdom United Lung, and Blood Institute, Laboratory of KATRIN RITI~INGER (7), Division of Protein Molecular Biology, National Institutes of ,erutcurtS National Institute for Medical ,htlaeH Maryland Bethesda, 0561-29802 ,hcraeseR London NW7 1AA, England, detinU Kingdom MARC SYMONS (5), Onyx ,slacituecamrahP Richmond, California 60849 WILLIAM ROSCOE (6), Onyx ,slacituecamrahP Richmond, California 60849 KAZUO TAKAHASHI (9), Second Department KENT L. ROSSMAN (3), Department of Bio- of Internal Medicine, Chiba University chemistry and Biophysics, University of Medical School, Chiba ,6580-062 Japan North Hill, Chapel Carolina, North -oraC TAKAI YOSHIMI (9), Department of raluceloM lina 0627-99572 Biology and Biochemistry, Osaka -revinU LURAYNNE C. SANDERS (28), Department of sity Graduate School of ytlucaF~enicideM Immunology, ehT Scripps Research Insti- of ,enicideM Osaka ,1780-565 Japan ,etut La Jolla, California 73029 LAURA J. TAYLOR (29), Department of -celoM TAKUYA SASAKI (9), Department of Biochem- ular sciteneG State and Microbiology, -inU ,yrtsi ehT ytisrevinU of Tokushima, School ytisrev of New York, Stony Brook, New of ,enicideM Kuramoto, Japan York 2225-49711 GUDULA SCHMIDT (12), Institut far Pharma- JEAN P. NET KLOOSTER (36), Division of lleC kologie und Toxikologie, Albert-Ludwigs- Biology, ehT Cancer Netherlands ,etutitsnI tiitisrevinU Freiburg, D-79104 ,grubierF 6601 CX Amsterdam, ehT sdnalrehteN ynamreG FRIEDER DLAWHCS (16), Department of -rahP KIMBERLEY TOLIAS (18), Division of Signal macology and Toxicology, University of ,noitcudsnarT Deaconess Israel Beth -ideM ,mlU Ulm, D-89081 Germany lac ,retneC Boston, sttesuhcassaM 51220 CONTRIBUTORS TO VOLUME 325 Xlll LI-HUEI TSAI (19), Howard Hughes Medical versity of New York, Stony Brook, New Institute, Department of Pathology, Har- York 11794-5222 vard Medical School, Boston, Massachu- ERIC V. WONG (43), Department of Physiol- setts 02115 ogy, Tufts University School of Medicine, IHSOYASAM UEHATA (24), Drug Discovery Boston, Massachusetts 02111 Laboratories, WelFide (Yoshitomi) Corpo- WEIHONG YAN (30), Department of Stoma- ration, Osaka 573-1153, Japan tology, University of California, San Fran- RoB A. NAV RED NEMMAK (26, 36), Division cisco, California 94143 of Cell Biology, The Netherlands' Cancer YUE ZHANG (17), Department of Pharmacol- Institute, 1066 CX Amsterdam, The Nether- ogy and Institute for Cell and Develop- 'sdnal mental Biology, State University of New CHRISTOPH NOV EICHEL-STREIBER (11), Ver- York, Stony Brook, New York 1568-49711 fligungsgebziude fiir Forschung und Ent- DANIEL ZICHA (40), Imperial Cancer Re- wicklung, lnstitut far Medizinisch Mikrobi- search Fund, London WC2A 3PX, En- ologie und Hygiene, Johannes Gutenberg- gland, United Kingdom Universitiit, 55101 Mainz, Germany YLLAS H. ZIGMOND (21), Biology Depart- AMY B. WALSH (29), Department of Molecu- ment, University of Pennsylvania, Philadel- lar Genetics and Microbiology, State Uni- phia, Pennsylvania 19104-6018 ] 1 [ NOITAZIRETCARAHC FO TC10 3 [1] Purification and Biochemical Characterization of TC 0 1 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 ofT C10 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,a n 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). S. 3 Batik, in "'PCR Protocols: Current Methods and Applications" (B. A. White, ed.), p. 277. Humana Press, Totowa, NJ, 1993. thgirypoC © 0002 yb cimedacA sserP llA rights fo noitcudorper ni yna mrof .devreser 1TEM SDOt NI ,YGOLOMYZNE .LOV 523 00/9786-6700 00.03$ 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 YRAMMUS Parent vector Vector Tag noitacifiruP noisserpxE pQE70 pQNzz ZZ lgG Sepharose Prokaryotic negaiO pRK7 -- -- nailammaM pRK7 pKH3 Triple HA 5AC21 with protein Sigma Mammalian A-Sepharose pRK7 cyMKp cyM 9El0 with Amersham Phar- Mammalian macia GammaBind Plus Sepharose pRK7 pKFLAG FLAG amgiS anti-FLAG Mammalian esoraga-2M pRK7 pRK7-GFP GFP Santa Cruz anti-GFP with Mammalian amgiS protein A Sepharose pRK7 pKNzz ZZ IgG Sepharose nailammaM pGBT9 pGBT10 GAL4 DNA-bind- Yeast hcetnolC gni 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 A T G ~ GJCG 'CGG CCG C~A ATT C~G ATC I T pRK7 HindllI PstI SalI XbaI BamHI EcoRI ClaI ~ ' C T G CAG"GTC GAC'~FCT AGA'~GA TcciccG GG~'~'~'IA~ PKH3 BamHI EcoRI ClaI TFCtAtAT TCCUGAA iGGA CGA i T PKMyc Sinai iNheI Notl EcoRI ClaI CGG ~3CT AGC t 3~GG CGG CCG C~lqGAA TTCnATC GAT i pKFLAG BamHI EeoRI ClaI Sill tGGA AAGt~CCT TAtA~C"IT CGA qtGG CCG CCA TGG CC~ PRKT-GFP BamHI EcoRI ClaI ~ ~ G A GAA ~TC pKNzz BamHI Notl EeoRI ClaI ~ G C I G CGG CCG CbA ATT C ~ A ~ pGBT10 BamHI AatI1 EcoRI iGGA TCCIIGAC G T C I ~ VP16-CP BamHI AatlI EcoRI GTCtIGGT TCCJIGAC IGGA ACC I .GIF .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).
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