Table Of ContentCurrent Topics in
Microbiology
123 and Immunology
Editors
A. Clarke, ParkvilleNictoria . R W. Compans,
Birmingham/Alabama . M. Cooper, Birmingham/Alabama
H. Eisen, Paris . W. Goebel, Wiirzburg . H. Koprowski,
Philadelphia . F. Me1chers, Basel . M. Oldstone,
La Jolla/California.' R Rott, GieBen . P.K Vogt, Los Angeles
H. Wagner, Ulm . 1. Wilson, La Jolla/California
Retroviruses 4
Edited by P.K. Vogt and H. Koprowski
With 23 Figures
Springer-Verlag
Berlin Heidelberg NewY ork Tokyo
Professor Dr. PETER K. VOOT
University of Southern California
School of Medicine
Department of Microbiology
2025 Zonal Avenue HMR 401
Los Angeles, CA 90033, USA
Professor Dr. HILARY KOPROWSKI
The Wistar Institute
36th Street at Spruce
Philadelphia, PA 19104, USA
ISBN-13: 978-3-642-70812-1 e-ISBN-13: 978-3-642-70810-7
DOl: 10.1007/978-3-642-70810-7
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2123/3130-543210
Table of Contents
J.S. BRUGGE: Interaction of the Rous Sarcoma Virus
Protein pp60src with the Cellular Proteins pp50 and
pp90. With 4 Figures . . . . . . . . . . . .. 1
D.W. STACEY: Microinjection Studies of Retroviral
Polynucleotides. With 9 Figures ........ 23
B.M. SEFTON: The Viral Tyrosine Protein Kinases.
With 4 Figures 39
C. VAN BEVEREN and I.M. VERMA: Homology Among
Oncogenes. With 6 Figures .......... 73
Indexed in Current Contents
List of Contributors
BRUGGE, J.S., Department of Microbiology, State Universi
ty of New York at Stony Brook, Stony Brook,
NY 11794, USA
SEFTON, B.M., Molecular Biology and Virology Laborato
ry, The Salk Institute, P.O. Box 85800, San Diego,
CA 92138, USA
STACEY, D.W., Department of Cell Biology, Roche Institute
of Molecular Biology, Roche Research Center, Nutley,
NJ 07110, USA
V AN BEVEREN, C., Molecular Biology and Virology Labora
tory, Salk Institute for Biological Studies, P.O.
Box 85800, San Diego, CA 92138-9216, USA
VERMA, I.M., Molecular Biology and Virology Laboratory,
Salk Institute for Biological Studies, P.O. Box 85800, San
Diego, CA 92138-9216, USA
Interaction of the Rous Sarcoma Virus Protein
pp60src with the Cellular Proteins pp50 and pp90
J.S. BRUGGE
Introduction 1
2 Identification of the Complex 2
3 Interaction of pp50 and pp90 with Other Oncogene Products 5
4 Specificity of the Interaction Between pp60"', pp50, and pp90 5
4.1 pp50 and pp90 Bind to Newly Synthesized Molecules of pp60"c 5
4.2 pp90 and pp50 are Complexed with pp60src Molecules Which Are Not Associated with the
Plasma Membrane 6
4.3 pp60"c Associated with pp90 and pp50 Does Not Contain Phosphotyrosine 6
5 Protein Kinase Activity of pp60src Bound to pp50 and pp90 7
6 Characterization of the pp90 and pp50 Proteins 7
6.1 pp90 7
6.2 pp50 9
7 Sites of pp60src Which Interact with pp90 and pp50 11
7.1 Analyses of Viruses Carrying Mutations Within the src Gene 11
7.2 Analyses of the Complex Using Antibodies to Specific Regions of pp60src 11
8 Model for the Interaction Between pp50 and pp90 12
9 Possible Functional Roles of the Interaction Between pp50, pp60"c, and pp90 13
9.1 Transport of pp60src to the Plasma Membrane 14
9.2 Attachment of Myristate to pp60"c 15
9.3 Phosphorylation of pp50 16
9.4 Regulation of the Phosphotransferase Activity of pp60src 17
10 Interaction ofpp90 and pp50 with Nonviral Proteins 18
11 Future Directions 19
References 20
1 Introduction
Oncogenic retroviruses cause multiple and profound alterations in the morphol
ogy, metabolism, and growth control of cells. In most retrovirus infected cells,
all of these complex changes in the cellular phenotype are mediated by a single
virus-encoded gene product, referred to as the transforming protein. Rous sar
coma virus (RSV) has proven to be an ideal system for the analysis of events
which occur following oncogenic transformation by retroviruses. RSV induces
rapid sarcoma production after injection of virus in vivo and efficient and rapid
transformation of cells in culture (HANAFUSA 1977). The transforming gene
Department of Microbiology, State University of New York at Stony Brook, Stony Brook, NY
11794, USA
Current Topics in Microbiology and Immunology, Vol. 123
© Springer-Verlag Berlin· Heidelberg 1986
2 I.S. Brugge
of RSV encodes a protein of Mr 60000 which has been designated pp60src
(BRUGGE and ERIKSON 1977; PURCHIO et al. 1978). Genetic studies of viruses
encoding mutant src gene products which induce a partially transformed pheno
type suggest that interactions between pp60src and multiple cellular targets are
required to elicit a fully transformed phenotype (review, SEFTON and HUNTER
1984). This review will discuss one such interaction between pp60Src and host
cell proteins. This interaction occurs between newly synthesized molecules of
pp60src and two cellular proteins of Mr 90000 (Pp90) and 50000 (Pp50). The
kinetics and localization of this interaction suggest that the cellular pp90 and
pp50 proteins are involved in some aspect of the processing of pp60src before
it reaches its residence in the plasma membrane (COURTNEIDGE and BISHOP
1982; BRUGGE et al. 1983). pp90 and pp50 have also been shown to associate
with many retrovirus-encoded transforming proteins other than pp60src (LIpSICH
et al. 1982; ADKINS et al. 1982). This suggests that pp50 and pp90 may play
a common role in the events which take place after transformation by at least
one class of retrovirus transforming proteins.
The RSV -transforming protein has been shown to possess a tyrosine-specific
phospho transferase activity (COLLETT and ERIKSON 1978; LEVINSON et al. 1978;
HUNTER and SEFTON 1980). All of the other retrovirus transforming proteins
which interact with pp50 and pp90 also possess this enzymatic activity and
share structural homology with pp60src. This review will summarize the informa
tion which has accumulated on the nature of this complex and its protein compo
nents and discuss the possible functions of the interaction between pp50 and
pp90 and viral transforming proteins which possess tyrosine-specific protein
kinase activity.
2 Identification of the Complex
All of the initial experiments performed to characterize the pp60src protein were
carried out using an antisera obtained from rabbits bearing tumors induced
by RSV. This antisera was not monospecific for pp60src and consequently precip
itated multiple protein species other than pp60src. The immunoprecipitation of
most of these proteins could be blocked by preabsorption of the antiserum
with the RSV structural proteins, indicating that these proteins were related
to viral structural proteins expressed in the tumor cells. However, two proteins
of Mr 90000 and Mr 50000 were consistently precipitated from RSV-trans
formed chicken cells by the preabsorbed serum from tumor-bearing rabbits
(TBR serum) (see Fig. 1 and SEFTON et al. 1978; BRUGGE et al. 1981; OPPERMANN
et al. 1981 b). Three possible explanations for the precipitation of these proteins
by TBR serum were the following: (a) The pp50 and pp90 proteins were structur
ally related to pp60 as precursors or cleavage products; (b) TBR serum con
tained unique antibody molecules which recognized pp90 and pp50; (c) pp50
and pp90 were associated in a protein complex with pp60 and thus coprecipitated
with antibody to pp60.
Possibility (a) was ruled out by analysis of peptides derived by partial or
complete digestion of pp50, and pp60, and pp90 using a variety of proteolytic
Interaction of the Rous Sarcoma Virus Protein pp60"c with the Cellular Proteins pp50 and pp90 3
1 2 3 4
-76
Fig. 1. Immunoprecipitation of pp60, pp50,
and pp90 from 32P-labeled RSV transformed
chicken cells. RSV (Schmidt-Ruppin subgroup
A) transformed cells were labeled with 32p 4 h.
Cell lysates were prepared, and the proteins
were immunoprecipitated by the antisera below
and analyzed on 7.5% SDS-polyacrylamide
gels as described (BRUGGE and ERIKSON 1977).
Lane 1, monoclonal antibody to pp90; lane 2,
monoclonal antibody to pp60"'; lane 3, tumor
bearing pp60""c, rabbit (TBR) serum; lane 4,
control rabbit serum. Pr76 is the viral gag-gene
translation product
enzymes (SEFTON et al. 1978; BRUGGE et al. 1981; OPPERMANN et al. 1981 b).
These studies did not reveal any structural similarities between the three pro
teins. Possibility (b) was ruled out because pp50 and pp90 were not precipitated
from uninfected cells or cells infected with viruses containing large deletions
in the src genes (BRUGGE et al. 1981).
Support for the last alternative, that pp50 and pp90 were associated in
a complex with pp60, was obtained from several lines of evidence: (a) Sedimenta
tion analysis of RSV-transformed cell lysates indicated that pp60 existed in
two forms, the majority of the protein sedimented as a monomer 60000-dalton
protein while a small percentage sedimented more rapidly in glycerol gradients
(Fig. 2; BRUGGE etal. 1981). The pp50 and pp90 proteins were found to cosedi
ment and coprecipitate with this faster sedimenting form of pp60. (b) Polyclonal
antiserum prepared against pp90 immunoprecipitated pp60 and pp50 from the
same gradient fractions which allowed precipitation of pp60, pp50, and pp90
using TBR serum (BRUGGE et al. 1981). (c) Monoclonal antibodies directed
against pp90 coprecipitated pp60 and pp50 from RSV -transformed cells and
4 J .S.B rugge
·90
-60
-50
Fig. 2. Sedimentation analysis of 3SS-methionine labeled RSV-transformed chicken-celllysates. SR
RSV (subgroup A) transformed chicken cells were labeled with 35S-methionine for 24 h. A cell
lysate was prepared and sedimented on a 10%-30% glycerol gradient as described (BRUGGE et al.
1981). Alternating gradient fractions were immunoprecipitated with antibody against pp60"'. Sedi
mentation was from left to right
monoclonal antibodies to pp60 (LIPSICH et al. 1983) also precipitated pp90 and
pp50 (Fig. 1).
Taken together, these data provide strong evidence that pp60 is associated
with pp90 and pp50 in lysates from RSV -transformed cells. Further character
ization of this complex revealed that the interaction between these three proteins
is very stable in vitro. The pp50: pp60src: pp90 complex is not dissociated by
incubation with high salt (2M NaCl, reducing agents, or metal-chelating agents);
however, long-term incubation in the absence of salt dissociates the protein
complex (BRUGGE et al. 1981).
The pp50 and pp90 proteins were detectable in pp60src immunoprecipitates
of cells infected with all nondefective strains of RSV; however, there were slight
variations between virus strains in the levels of pp90 and pp50 precipitation
(BRUGGE et al. 1983). Cells infected with mutant viruses displaying a tempera
ture-dependent transformed phenotype showed elevated levels of pp50 and pp90
when incubated under either permissive or nonpermissive conditions with the
highest levels being detected at the nonpermissive temperature (BRUGGE et al.
1981, 1983). Sedimentation analysis of lysates from cells infected with these
mutant viruses indicated that the majority of the src protein was complexed
with pp50 and pp90 at the nonpermissive temperature. These results indicate
that mutations in the src gene which affect the transforming activity of pp60
also affect the binding of pp50 and pp90. This property was found in cells
infected with a variety of ts mutants isolated using different methods of muta
genesis.
Interaction of the Rous Sarcoma Virus Protein pp6()",c with the Cellular Proteins pp50 and pp90 5
3 Interaction of pp50 and pp90 with Other Oncogene Products
Many of the retrovirus-encoded oncogene products have been shown to carry
an associated tyrosine-specific protein kinase activity. pp50 and pp90 have been
shown to co-immunoprecipitate with many of these transforming proteins in
cluding the fps gene products from Fuginami (LIPSICH et al. 1982) and PRell
sarcoma virus (ADKINS et al. 1982), the yes gene product from Yamaguchi 73
sarcoma virus (LIPSICH et al. 1982), and the fes gene product from Snyder
Theilen feline sarcoma virus (ZIEMIECKI, unpublished results). It has also been
observed that mutant viruses carrying ts defects in the fps (S. MARTIN and
T. PAWSON, unpublished results) andfes gene products (R. SNYDER, unpublished
results) show elevated levels of pp50 and pp90 binding. MATHEy-PREVOT et al.
(1984) have isolated a revertant line of Fuginami virus-transformed cells in
which the majority of the p130Jps protein is associated in a complex with pp50
and pp90. This evidence indicates that several unique tyrosine-specific protein
kinase transforming proteins bind to the same cellular proteins, and suggests
that these proteins may playa common role in their interaction with all tyrosine
kinase transforming proteins. It is clear from immunoprecipitation experiments
using monoclonal antibodies to pp90 that the association with these proteins
is specific and not merely due to nonspecific interactions 'with protein present
in celllysates.
4 Specificity of the Interaction Between pp6O"rc, pp50, and pp90
The sedimentation analysis of pp60src on glycerol gradients revealed that only
a small percentage of pp60src was bound to pp50 and pp90. These complex
associated pp60src molecules could represent either a specific population which
enter a nondissociable, dead-end complex with pp50 and pp90 or pp60src mole
cules which were associated in a short-lived complex during some phase of
their cellular lifetime. The evidence described below favors the latter possibility,
that the interaction between pp90, pp50, and pp60src is transient and involves
all molecules of newly synthesized pp60src.
4.1 pp50 and pp90 Bind to Newly Synthesized Molecules of pp60src
The first hint that pp50 and pp90 bind to newly synthesized pp60src molecules
was the evidence that the percentage of radiolabeled pp60src bound to pp50
and pp90 varied with the length of incorporation of 35S-methionine into RSV
transformed cells. Analysis of cells labeled for short periods (2-5 min) indicated
that more than 90% of radiolabeled src was associated with pp50 and pp90.
The percentage of the radiolabeled form of complex-associated pp60src decreased
with increasing labeling periods until a steady-state percentage of 1 %-5% was
reached in 10-h labeling periods (BRUGGE et al. 1983). This suggested that newly
synthesized molecules of pp60src preferentially bind to pp90 and pp50. This
possibility was further examined using pulse-chase experiments. The majority
