Small GTPases and Their Regulators Part F

512 Pages·1995·9.576 MB·English

by  Balch W.E., Channing J.D., Hall A.

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
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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
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