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Small GTPases and Their Regulators Part F PDF

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Preface As with the Rho and Rab branches of the Ras superfamily of small GTPases, research interest in the Ras branch has continued to expand dramatically into new areas and to embrace new themes since the last Methods ni Enzymology Volume 255 on Ras GTPases was published in 1995. First, the Ras branch has expanded beyond the original Ras, Rap, and Ral members. New members include M-Ras, Rheb, Rin, and Rit. Second, the signaling activities of Ras are much more diverse and complex than appreciated previously. In particular, while the Raf/MEK/ERK kinase cascade remains a key signaling pathway activated by Ras, it is now appreci- ated that an increasing number onfo n-Raf effectors also mediate Ras family protein function. Third, it is increasingly clear that the cellular functions regulated by Ras go beyond regulation of cell proliferation, and involve regulation of senescence and cell survival and induction of tumor cell invasion, metastasis, and angiogenesis. Fourth, another theme that has emerged is regulatory cross talk among Ras family proteins, including both GTPase signaling cascades that link signaling from one family member to another, as well as the use of shared regulators and effectors by different family members. Concurrent with the expanded complexity of Ras family biology, bio- chemistry, and signaling have been the development and application of a wider array of methodology to study Ras family function. While some are simply improved methods to study old questions, many others involve novel approaches to study aspects of Ras family protein function not studied previously. In particular, the emerging application of techniques to study Ras regulation of gene and protein expression represents an important direction for current and future studies. Consequently, Methods ni Enzy- mology, Volumes 332 and 333 cover many of the new techniques that have emerged during the past five years. We are grateful for the efforts of all our colleagues who contributed to these volumes. We are indebted to them for sharing their expertise and experiences, as well as their time, in compiling this comprehensive series of chapters. In particular, we hope these volumes will provide valuable references and sources of information that will facilitate the efforts of newly incoming researchers to the study of the Ras family of small GTPases. CHANNING J. DER ALAN HALL WILLIAM E. BALCH iiix Contributors to Volume 332 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. NATALIE G. AHN (31), Department of Chem- versity School of Medicine, .tS Louis, Mis- istry and Biochemistry, Howard Hughes souri 63110 Medical Institute, University of Colorado, CHERNOFF JONATHAN (22), Division of Basic Boulder, Colorado 80309 Science, Fox Chase Cancer Center, Phila- GORDON ALTON (23), Celgene Corporation delphia, Pennsylvania 11191 Signal Research Division, Department of YONO-JIG CHO (18), Vanderbilt-Ingram Can- Imformatics and Functional Genomics, San cer Nashville, Tennessee Center, 8386-23273 Diego, California 12129 YUN-JUNG CHOI (7, 19), Onyx Pharmaceuti- DOUGLAS A. ANDRES (14, 15), Department of cals, Richmond, California 60849 Biochemistry, University of Kentucky, EDWIN CHOY (3), Departments of Medicine Lexington, Kentucky 4800-63504 and Cell Biology, New York University M. ARBOLEDA JANE (27), Onyx Pharmaceuti- School of Medicine, New York, New cals, Richmond, California 60849 York 61001 AMI ARONHEIM (20), Department of Molecu- MELANIE H. COBB (29), Department of Phar- lar Genetics, The B. Rappaport Faculty of macology, University of Texas Southwest- Medicine, Israel Institute of Technology, ern Medical Center, Dallas, Texas -53257 Haifa Israel 31096, 1409 BRYDON L. BENNEqT (32), Pharmaceu- Signal JOHN COLICELLI (10), Department of Biologi- Inc., ticals, San Diego, California 12129 cal Chemistry and Molecular Biology Insti- W. BISHOP ROBERT (8), Department of Tumor tute, UCLA School of Medicine, Los Biology, Schering Plough Research Insti- Angeles, California 59009 tute, Kenilworth, New Jersey 33070 ADRIENNE D. Cox (1, 23), Department of BOETTNER BENJAMIN (11), Cold Spring Har- Radiation Oncology and Pharmacology, bor Laboratory, Cold Spring Harbor, New University of North Carolina School of Medicine, Chapel Hill, North Carolina York 42711 99572 GIDEON BOLLAG (7, 19), Onyx Pharmaceuti- ROGER J. DAVIS (24), Howard Hughes Medi- cals, Richmond, California 60849 cal Institute, Department of Biochemistry MICHELLE A. BOODEN (4), Lineberger Com- and Molecular Biology, University of Mas- prehensive Cancer Center, CB-7295, Uni- Medical School, sachusetts Program in Mo- versity of North Carolina, Chapel Hill, lecular Medicine, Worcester, Massachu- North Carolina 27599 setts 50610 JANICE E. Buss (4), Department of Biochemis- CHANNING J. DER (1, 17), Lineberger Com- try, Biophysics, and Molecular Biology, prehensive Cancer Center, Department of Iowa State University, Ames, Iowa 11005 Pharmacology, University of North Caro- ANDREW D. CATLING (28), Department of Mi- lina, Chapel Hill, North Carolina 27599 crobiology and Cancer Center, University STEVEN F. DOWDY (2), Departments of Pa- of Virginia Health Sciences Center, Char- thology and Medicine, Howard Hughes lottesville, Virginia 4370-80922 Medical Institute, Washington University MEENA A. CHELLAIAH (2), Renal Division, School of Medicine, .tS Louis, Missouri Barnes-Jewish Hospital, Washington Uni- 01136 ix X CONTRIBUTORS TO VOLUME 332 DEREK EBERWEIN (27), Bayer Corporation, KIRAN J. KAUR (21), Department of Cell Biol- West Haven, Connecticut 5714-61560 ogy, University of Southwestern Med- Texas ical Center, Dallas, Texas 09357 TTOCS T. EaLEN( 28), Department of Microbi- ology and Cancer Center, University of BRIAN K. KAY (6), Department of Pharmacol- Charlottes- Center, Sciences Health Virginia ogy, University of Wisconsin, Madison, ville, Virginia 4370-80922 Wisconsin 2351-60735 AKIRA KIKUCHI (9), Department of Biochem- JAMES J. FIORDALISI (1), Departments of Ra- Hiroshima istry, University School of Medi- diation, Oncology, and Pharmacology, Uni- cine, Hiroshima ,1558-437 Japan versity of North Carolina, Chapel Hill, North Carolina 27599 PAUL T. KIRSCHMEIER (8), Department of Tu- mor Biology, Plough Schering Research In- DANIEL G. GIOELI (26), Department of Micro- stitute, Kenilworth, New Jersey 33070 biology and Cancer Center, University of MARC KNEPPER (19), Advanced Medicine, Charlottes- Center, Sciences Health Virginia Inc., San Francisco, California 94080 ville, Virginia 22908 SHINYA KOYAMA (9), Department of Bio- ERICA A. GOLEMIS (5, 22), Division of Basic chemistry, Hiroshima University School of Science, Fox Chase Cancer Center, Phila- Medicine, Hiroshima ,1558-437 Japan delphia, Pennsylvania 11191 PENG LIANG (18), Vanderbilt-lngram Cancer SAID A. GOUELI (25), Signal Transduction Center, Nashville, Tennessee 8386-23273 Group, and Research Development Depart- DAN LIU (13), Verna and MartsM cLean De- Promega ment, Madison, Corporation, Wis- partment of Biochemistry and Molecular consin ,11735 Department and of Pathology Biology, Baylor College of Medicine, Hous- and Laboratory Medicine, University of ton, Texas 03077 Wisconsin School of Medicine, Madison, MARK LYNCH (7), Bayer Research Center, Wisconsin 11735 West Haven, Connecticut 61560 GASTON G. HABETS (19), Onyx Pharmaceuti- JOHN F. LYONS (27), Onyx Pharmaceuticals, cals, Richmond, California 60849 Richmond, California 60849 HERRMANN CHRISTIAN (11), Max Planck In- NYLODNEWG M. MAHON (16), Department of stitute for Molecular Physiology, 44227 Microbiology and Molecular Genetics, Dortmund, Germany UMDNJ-New Jersey Medical School, BARBARA HIBNER (27), Bayer Corporation, Newark, New Jersey 4172-30170 West Haven, Connecticut 5714-61560 MARTIN MCMAHON (30), Cancer Research Institute and Department of Cellular and KEITH A. HRUSKA (2), Renal Division, Molecular Pharmacology, University of Barnes-Jewish Hospital, Washington Uni- California San Francisco/Mt. Zion Com- versity School of Medicine, .tS Louis, Mis- prehensive Cancer Center, San Francisco, souri 01136 California 51149 BRUCE W. JARVIS (25), Signal Transduction OLGA V. MITINA (22), Department of Molecu- Group, Research and Development Depart- lar Biology and Medical Biotechnology, Promega ment, Corporation, Madison, Wis- Russian State Medical University, Mos- consin 11735 cow, Russia HAKRYUL Jo (18), Vanderbilt-lngram Cancer BRION W. MURRAY (32), Agouron Pharma- Center, Nashville, Tennessee 8386-23273 San Diego, California ceuticals, 8041-12129 RONALD L. JOHNSON II (1), Departments of NAHREINI THERESA STINES (31), Department Radiation, Oncology, and Pharmacology, of Chemistry and Biochemistry, Howard University of North Carolina at Hill, Chapel Institute, Hughes Medical University of Col- Chapel Hill, North Carolina 27599 orado, Boulder, Colorado 80309 CONTRIBUTORS TO VOLUME 332 xi LEAHCIM ALABDEIN (7), Bayer Research Cen- TEBARAG G. YBOT (5), Division of Basic Sci- ter, West Haven, Connecticut 61560 ence, Fox Chase Cancer Center, Philadel- ENNA K. HTRON (7), Onyx Pharmaceuticals, phia, Pennsylvania ,11191 and Cell and Mo- Richmond, California 60849 lecular Biology Group, University of Pennsylvania School of Medicine, Philadel- NOEK-NIJ PAl (8), Department of Tumor Biol- phia, Pennsylvania 4606-40191 ogy, Schering Plough Research Institute, Kenilworth, New Jersey 33070 SALOHCIN S. IKSNlWLOT (31), The Graduate College, Princeton University, Princeton, MARK SPILIHP (3), Departments of Medicine New Jersey 08544 and Cell Biology, New York University School of Medicine, New York, New L. DRAREG TNIASSUOT III (23), Distinguished York 61001 Medical Scholar Program, University of North Carolina School of Medicine, Chapel TTOCS SREWOP (17), Tularik Genomics, Hill, North Carolina 27599 Greenlawn, New York 11740 NILYA S. LILK0 (1), Department of Pharma- NYREHTAK A. RESING (31), Department of cology, University of North Carolina at Chemistry and Biochemistry, University of Chapel Hill, Chapel Hill, North Carolina Colorado, Boulder, Colorado 80309 99572 SINNED Z. IKASAS (32), Signal Pharmaceuti- ADNIL NAV TSLEA (11), Cold Spring Harbor Inc., cals, San Diego, California 12129 Laboratory, Cold Spring Harbor, New OKIHEKAT IKUZASAS (19), Medical Institute York 42711 of Bioregulation, Kyushu University, Fuku- ANIMADA INABKA-ORECOV (2), Departments oka Japan 812, of Pathology and Medicine, Howard SNAH J. REFFEAHCS (28), MDC, Gruppe .W Hughes Medical Institute, Washington Uni- Birchmeier, Berlin, 13125 Germany versity School of Medicine, .tS Louis, Mis- AYLI G. IIKSIIRBERES (22), Division of Basic souri 01136 Science, Fox Chase Cancer Center, Phila- GNAW GNIY (10), Department of Biological delphia, Pennsylvania 11191 Chemistry and Molecular Biology Institute, LEINAJ M. SDLEIHS (17), Department of Phar- UCLA School of Medicine, Los Angeles, macology, Lineberger Comprehensive Can- California 59009 cer Center, University of North Carolina, LEAHCIM J. REBEW (26, 28), Department of Chapel Hill, North Carolina 5927-99572 Microbiology and Cancer Center, Univer- IJNES AWASARIHS (19), Medical Institute of sity of Virginia Health Sciences Center, Bioregulation, Kyushu University, Fukuoka Charlottesville, Virginia 4370-80922 ,218 Japan NHOJ K. KCIWTSEW (23), Corporation Celgene UOHZ GNAYGNOS (12, 13), Verna and Marts Signal Research Division, Department of McLean Department of Biochemistry and San Signaling, Diego, Cell California 12129 Molecular Biology, Baylor College of Medi- LEAHCIM A. ETIHW (21), Department of Cell cine, Houston, Texas 03077 Biology, University of Texas Southwestern NHOJ T. YENKCITS (4), Department of Cell Medical Center, Dallas, Texas 09357 Biology, Neurobiology, and Anatomy, Uni- NAI P. DAEHETIHW (16), Department of Mi- versity of Cincinnati Medical Center, Cin- crobiology and Molecular Genetics, cinnati, Ohio 1250-76254 UMDNJ-New Jersey Medical School, ANIAJ NITROMUS (19), Onyx Pharmaceuticals, Newark, New Jersey 4172-30170 Richmond, California 60849 ALAN J. HSRAMTIHW (24), Howard Hughes CRAM SNOMYS (7), The Picower Institute for Medical Institute, Department of Biochem- Medical Research, Manhasset, New York istry and Molecular Biology, University of 03011 Massachusetts Medical School, Program in xii CONTRIBUTORS TO VOLUME 332 raluceloM ,enicideM ,retsecroW -uhcassaM PORATNOM IAHBAMAY (6), School of Bio- sttes 50610 ,ygolonhcet Suranaree University of -hceT (8), DAVID WHYTE Sugen Inc., South naS nology, Institute of Agricultural Technol- ,ocsicnarF California 08049 ogy, Nakhon Ratchasima ,00003 Thailand Jueiz L. REHCABSmW (29), Department of MAJA CIVECEZ (26), Department of -iborciM ,ygolocamrahP University of South- Texas and Cancer ology ,retneC ytisrevinU of -riV western Medical Center, Dallas, Texas ginia Health Sciences Center, -settolrahC 1409-53257 ,elliv Virginia 80922 DLAWSO NOSLIW (8), Department of Tumor Biology, Schering Plough Research Insti- GNOH ZHANG (18), Can- Vanderbilt-Ingram ,etut New Kenilworth, Jersey 33070 rec ,retneC ,ellivhsaN 8386-23273eessenneT [ ]1 NAILAMMAM NOISSERPXE SROTCEV ROF Ras 3 [i] Mammalian Expression Vectors for Ras Family Proteins: Generation and Use of Expression Constructs to Analyze Ras Family Function yB II, JAMES J. FIORDALISI, RONALD L. JOHNSON AYLIN S. I, JLKO, GNINNAHC J. DER, and ENNEIRDA D. Cox Introduction Cell-based assays are useful for the characterization of Ras family struc- ture-function relationships, identification of upstream regulators and down- stream effectors, characterization of signaling inputs and outputs, analysis of the role of Ras family proteins in normal and aberrant cellular metabo- lism, and evaluation of potential anticancer agents. Common to all such studies is the need to express the protein(s) of interest within a cell. This is accomplished through the use of plasmid vectors into which are placed the coding sequences of the proteins to be studied, and which can then be introduced into cells by a variety of methods. Protein expression plasmid vectors contain signal sequences required for transcription and translation of the target protein (i.e., promoter elements, polyadenylation sites, etc.) as well as origins of replication for maintenance of the plasmid. Expression vectors have been developed with a variety of features, including selectable markers and sequences encoding epitope tags that are recognized by specific antibodies, which facilitate the subsequent analysis of protein expression and function. Not all vectors function equally well in different assay systems, even if the sequences being expressed are identical. Similarly, not all proteins are expressed equally well in the same vector. Moreover, the reasons for these differences are not well understood and can be determined only by trial and error. Therefore, choosing the optimum vector for a given protein and assay system can be an empirical and time-consuming endeavor. Undoubt- edly, such factors as the identity of the cell line, the gene of interest, the biological readout, as well as others all contribute to variability in the usefulness of the vector. In this chapter, we attempt to provide readers with a starting point from which to choose the most appropriate vector for their particular proteins of interest and intended uses. We present some observations concerning the strengths and weaknesses of several mammalian protein expression vectors, both commercially available and "homemade." Because there are many vectors currently in use, as well as new vectors and assay systems thgirypoC © 1002 yb cimedacA sserP llA sthgir fo noitcudorper ni yna mrof .devreser SDOHTEM NI ,YGOLOMYZNE .LOV 233 00/9786-6700 00.53$ 4 PROTEIN EXPRESSION AND PROTEIN-PROTEIN INTERACTIONS [ 1] continually being developed, it is not possible to present a comprehensive physical or functional evaluation of all vectors under all circumstances. In this work we identify and discuss most of the major factors that should be considered. In addition to discussing the advantages and disadvantages of particular features of mammalian protein expression vectors, we also compare and contrast them functionally with respect to biological readouts commonly used in the study of Ras protein function, including protein expression, signaling activity in enzyme-linked transcriptional trans-activa- tion reporter assays, and transforming ability in fibroblast focus-forming assays. In all cases we use activated, oncogenic Ras proteins as the model system. Because the choice of vector will be influenced by, among other things, the ease with which protein-coding sequences can be introduced into them, we also discuss several techniques for generating and manipulating protein expression constructs. Finally, we discuss several methods for intro- ducing plasmid DNA into mammalian cells, including transfection with a variety of reagents and infection using retroviral packaging vectors. Properties to Consider in Choosing a Vector Promoter In choosing a mammalian protein expression vector (Table Ii-8), the most important factor to consider is whether the plasmid will express the protein of interest to the desired level in the cell type to be used. Sometimes the highest possible protein expression levels are desired, usually in order to maximize the biological effect being studied. In other cases, lower levels are desired, usually either to achieve more physiologically relevant levels or to minimize toxicity. Protein expression is controlled primarily by the transcriptional promoter region of the vector, which contains elements necessary for transcription (such as binding sites for transcription factors that recruit RNA polymerase) and translation (especially the Kozak se- t M. A. White, C. Nicolette, A. Minden, A. Polverino, L. Van Aelst, M. Karin, and M. H. Wigler, Cell 80, 533 (1995). 2 R. R. Mattingly, A. Sorisky, M. R. Brann, and I. G. Macara, MoL Biol. Cell. 14, 7943 (1994). 3 j. p. Morgenstern and H. Land, Nucleic Acids Res. 18, 1068 (1990). 4 W. .S Pear, G. P. Nolan, M. L. Scott, and D. Baltimore, Proc. Natl. Acad. LcS U.S.A. 90, 8392 (1993). 5 I. Whitehead, H. Kirk, C. Tognon, G. Trigo-Gonzalez, and R. Kay, J. Biol. Chem. 270, 18388 (1995). 6 C. L. Cepko, B. Roberts, and R. C. Mulligan, Cell 37, 1053 (1984). 7 j. A. Southern, D. F. Young, F. Heaney, W. K. Baumgartner, and R. E. Randall, J. Gen. Virol. 72, 1551 (1991). 8 A. Yen, M. Williams, J. D. Platko, C. Der, and M. Hisaka, Eur. J. Cell Biol. 65, 103 (1994). [ ]1 NAILAMMAM NOISSERPXE SROTCEV ROF Ras 5 quence )9 of the coding sequence. Most promoters found in expression vectors are derived from viral promoters that induce the high rates of protein expression necessary for viral replication. The cytomegalovirus (CMV) promoter, the mouse mammary tumor virus long terminal repeat promoter (MMTV LTR), and the Moloney murine leukemia virus promoter LTR (Mo-MuLV LTR) are commonly used viral promoters. The CMV promoter generally works well in cell lines derived from primate tissues such as human embryonic kidney cells (HEK293), human breast epithelial cells (T-47D, MCF-7, and MCF-10A), and monkey kidney cells (COS-7), but works less well in cells of rodent origin, such as mouse fibroblasts (NIH 3T3, Ratl, and Rat2) and rat intestinal epithelial cells (RIE-1). The reverse is true of the MMTV LTR and the Mo-MuLV LTR promoters. Naturally, there are always exceptions to such a rule; for exam- ple, we have found that pZIP-NeoSV(X)l-based constructs work well in T-47D cells but not in 293 or COS cells. Protein expression levels should always be confirmed directly for each expression construct in the cells of interest, using Western blot analysis or a similar method. Constitutive versus Inducible Protein Expression Although most vectors express proteins in a constitutive fashion, protein expression in some vectors is controlled by promoters that contain inducible elements that bind either repressor proteins or inducers that can be inacti- vated or induced, respectively, by exposure to exogenously added inducing agents. Until then, protein expression does not occur. We have more experi- ence with dexamethasone-inducible vectors 3 (Table I); other common in- ducible elements are responsive to tetracycline, 11'°1 isopropyl-/3-o-thiogalac- topyranoside (IPTG), 21 and ecdysone (see Ref. 13 and [19] in this volumel4). Inducible protein expression is desirable if the protein of interest is toxic or otherwise growth inhibitory to the cell, in which case, stable transfection of cells with a vector expressing this protein constitutively would be impossi- ble. Moreover, any transient or temporally distinct cellular phenotype caused by the expression of the protein can be evaluated better if protein expression can be turned on and off relatively rapidly. 9 M. Kozak, Nucleic Acids Res. 9, 5233 (1981). 01 L. Chin, A. Tam, J. Pomerantz, M. Wong, J. Holash, N. Bardeesy, Q. Shen, R. O'Hagan, J. Pantginis, H. Zhou, J. W. Horner II, C. Cordon-Cardo, G. D. Yancopoulos, and R. A. DePinho, Nature (London) 400, 468 (1999). 11 H. S. Liu, C. H. Lee, C. F. Lee, I. J. Su, and T. Y. Chang, BioTechniques 24, 624 (1998). 21 M. A. Wani, X. Xu, and P. J. Stambrook, Cancer Res. 54, 2504 (1994). 31 M. J. Calonge and J. Massague, J. BioL Chem. 274, 33637 (1999). 41 G. G. Habets, M. Knepper, J. Sumortin, Y.-J. Choi, T. Sasazuki, S. Shirasawa, and G. Bollag, Methods Enzymol. 332 [19] 2001 (this volume). 6 [1] PROTEIN EXPRESSION AND PROTEIN-PROTEIN INTERACTIONS I 2~ m D~ ~ ~ooo .~'~ ~~ O o/ Z 0 ZZO O m O a--, m ~.~ d O t.) a O < <<<<<<~<<<< rn M O m o E~ << e~ o, >. [1] MAMMALIAN EXPRESSION VECTORS FOR Ras 7 ~ . ~ o .~ o ~,-, ~" O.Oc~ c.,) "~~. ~°° ~ .,~>.~=~ ~ .~..~ o " " ~- ~" , ..~..' ~- • ¢.) 0 .,~ ~l ~c ¢) 0" ~ '~ ~.~ o ~o0 =l ,.~ ~ -"~"~ 0 ~ .0~ ~=~ ~_ ~jo 0 ~o ~ ~.~ = ~=°o-~ ~.~ ~ ~ ~ = o ~ ~c ~'~" ~ ~.~,-o ,~ ~ ~ ~ = ~ ~ ~ ~ , ~ 0 .~ ~.a.I ~ ~-, 0 o ~=.--~ ~ ~ ~=~.~ ~-."~ .~ v ~ ~ ~ £.) ~ 0 "~ "d 0 ~ ~I ~-~ ~-~ ~ -~ o> ~ ~ .~: ~ ~ I ~-0.= "~ ~° .~ c-~ ~ .~ ~c~ ~ ~ .~ ~ o= o =.~ ~.~ x >~ >'~, ~ ~ ~ o ~o ~ ~'0,~ ~ ~.~ ~'~ ~ o

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