Download Simian Virus 40 Large T Antigen and p53 Are

Vol.
2. 1 15-127,
February
Cell Growth
1991
Simian Virus 40 Large
Microtubule-associated
Transformed
Cells1
T Antigen
Proteins
Division
of Molecular
Virology
Department
of Microbiology
[S. A. M., S. K. A., E. T. S., J. S. B.] and
and Immunology
[R. G. C.], Baylor
College
of Medicine,
Houston,
Texas 77030, and Department
of Biochemistry
and Molecular
Biology, The University
of Texas M. D. Anderson
Cancer
Center, Houston,
Texas 77030 [G. L. D.]
possesses
is a prototype
potent
its oncogenic
types
(2-4).
DNA
transforming
activity
virus
oncogene
potential,
in a wide
(1). It
as evidenced
by
of cultured
cell
variety
and in different
target
organs
in transgenic
T-ag is a remarkably
multifunctional
protein,
activities
ranging
cellular
Abstract
The cellular proteins that interact with simian virus 40
large T antigen (T-ag) must be identified
in order to
understand
T-ag effects on cellular growth control
mechanisms.
A protein extraction
procedure
utilizing
single-phase
concentrations
of 1-butanol recovered
a
complex composed
of T-ag, p53, and other Mr 35,000
60,000 proteins from suspension cultures of the simian
virus 40-transformed
mouse cell line mKSA. Partial
protease mapping showed each of the associated
proteins to be unique. Automated
microsequence
analysis of the NH2-terminal
30 amino acids of the M
56,000 protein purified after coprecipitating
with T-ag
and p53 identified
it as the
subunit of mouse tubulin.
The existence of a complex containing
tubulin, T-ag,
and p53 was confirmed
by reciprocal
immunoblotting
experiments.
Both T-ag and p53 were coprecipitated
by
three different
monoclonal
antibodies
directed against
tubulin, and conversely,
monoclonal
antibodies
specific
for T-ag or p53 coprecipitated
tubulin. Mixing
experiments
and extractions
in the presence of purified
tubulin indicated that the complex existed in situ prior
to cell lysis. Both p53 and T-ag copurified
with
microtubules
through two cycles of temperaturedependent
disassembly
and assembly. Both T-ag and
p53 were localized
to microtubules
in the cytoplasm
of
mKSA cells by immunoeledron
microscopy.
Treatment
of mKSA cells with 10 iM colchicine
followed
by lysis
in 0.1% Nonidet
P-40 resulted
in increased
amounts
of
solubilized
T-ag and p53. Both T-ag and p53 were also
associated with microtubules
in three other simian
virus 40-transformed
mouse cell lines growing as
monolayers,
confirming
the generality
of the
association.
An interaction
of T-ag and p53 with
microtubules
may be important
in the intracellular
transport
of these proteins
and may affect cellular
signal transduction
or growth control.
115
and p53 Are
in
Introduction
SV404 T-ag
Steve A. Maxwell,2
Sharla K. Ames, Earl T. Sawai,
Glenn L Decker, Richard G. Cook, and Janet S. Butel3
& Differentiation
from
replication
transformation
(1,
of the viral
5-8).
The
proteins
with
the molecular
oncogenesis.
The intracellular
to
path-
role(s) in cell
effects
their
Identification
which
T-ag interacts
should
basis of T-ag function
in
distribution
in its multifunctionality.
genome
regulatory
way(s) subverted
by T-ag must play central
growth
control
in view of the dramatic
redirection
may have on cell phenotype.
of the cellular
help elucidate
mice
with
of T-ag
Although
T-ag
may be involved
is found
predom-
mnantly (‘95%)
in the nucleus
of infected
and transformed
cells, a portion
gets transported
to the plasma
membrane
(5, 6). Evidence
for functionality
of the nonnuclear
population
has been provided
by SV4O variants
that encode
nuclear
transport-defective
forms of T-ag.
Nonkaryophilic
T-ag
mutants
were
able
to
transform
established
cell lines but not primary
cells (9-11)
and
cooperated
with the ras oncogene
and polyoma
middle
T-ag in transformation
of primary
cells (12, 13). The
elevated
expression
of pmT-ag
in actively
dividing
cells
as compared
to quiescent
cells
vation that transformation
inhibited
in the presence
lymphocytes
(16),
(14,
15),
plus
the
obser-
of primary
cells by SV4O is
of SV4O-specific
cytotoxic
T-
substantiate
a functional
role
for
cy-
toplasmic
T-ag or pmT-ag or both.
One cellular target for T-ag is cellular
protein
p53, an
apparent
tumor
suppressor
gene product
(17-21)
intimately
involved
in T-ag-mediated
22, 23). A membrane
form
transformation
of p53 has been
the surface
of SV4O-transformed
cells
by
surface
radioiodination
experiments
(24-26)
sensitive
3H-labeled
protein
A binding
assay
plasma
mouse
membrane
cells
and in the
by reactivity
during
of
mitosis
both
plasma membrane
with antiidiotypic
detergent
and
on
situ cell
and by a
(27), at the
in
transformed
by immunocytochemistry
At least two subpopulations
guished
based
upon
differences
of pmT-ag
can be extracted
nonionic
normal
(1, 6,
detected
(28),
of Raji B lymphoma
anti-CR2
antibodies
cells
(29).
of pmT-ag
can be distinin solubility.
One class
from
membranes
by the
NP4O, whereas
a second
form,
which
is modified
by palmitylation,
can only be extracted
from
NP4O-insoluble
material
with the zwitterionic
detergent
Received
1
This
work
HD21483
CA08564
10/18/90.
was
and
from
supported
by
National
in
part
by
Research
Research
Service
Grants
CA22555
and
Awards
CA09197
and
Empigen
BB (25, 30, 31). This latter form is believed
to
be associated
with the plasma membrane
lamina, a struc-
the NIH.
Present address:
Department
of Thoracic
Surgery,
M. D. Anderson
Hospital
Cancer Center, Houston,
TX 77030.
3 To
whom
requests
for reprints
should
be addressed
at Division
of
Molecular
Virology,
Baylor College of Medicine,
One Baylor Plaza, Houston, TX 77030.
2
The abbreviations
used are: SV4O, simian virus 40; T-ag, large T antigen;
pmT-ag,
plasma membrane-associated
fraction
of T-ag; NP4O, Nonidet
P-40; BSA, bovine
serum albumin;
IEM, immune
electron
microscopy;
4
SDS,
sodium
dodecyl
sulfate.
1 16
SV4O
Tag
and
p53
Bind
Microtubuk’s
A
3
2
1
B
4
124
w
:
Tag
,
95k
ii
-
-
6ok
56k
5Ok
“45k
i:..
p53)
“35k
Fig. 1
Coprecipitation
of M, .35,000-60,000
proteins
from butanol
extracts
of sSlabeled
mKSA
cells using monoclonal
antibodies
against
T.ag or p53.
A, mKSA ((115 metabolically
labeled
with [‘5S]methionine
were lysed in 1% NP4O (Lanes
I and 2) or were
extracted
with 2.5% butanol
at 37’C
(Lanes 3
and 4). Proteins were immunoprecipitated
with p53 monoclonal
antibody
200.47 (Lanes 2 and 4) or null antibody
m73 (Lanes
1 and 3). B, ‘5S-labeled
mKSA
((‘115 were
extracted
with
2.5%
butanol
at 37’C,
and solubilized
proteins
were
precipitated
with
monoclonal
antibody
m73 (Lane
1), T-ag
mono(lonal
antibody
SDS.polya
rylamide
45,000,
and 35,000
ture that directly
PAb43O
gels. and
proteins.
underlies
(Lane
2), or p53
labeled
bands
the lipid
monoclonal
were
antibodies
detected
bilayer
proteins
200.47
autoradiography.
of the plasma
membrane
and connects
it to the cytoskeleton
These
subpopulations
of pmT-ag
may interact
tinct membrane
or cytoskeletal
ing biological
effects.
by
(30,
with
and exert
32).
dis-
differ-
(Lane
3) and
PAb421
Arrowheads,
(Lane
4).
positions
Precipitated
of T.ag,
p53,
proteins
and
M,
were
60,000,
separated
in 8%
56,000,
50,000,
of proteins,
ranging in size from M, 35,000 to M, 60,000,
differs
from that routinely
obtained
from NP4O detergent
extracts
of [35S]methionine-labeled
mKSA cells using the
same p53 monoclonal
antibody
(Fig. 1A, Lane 2). Identical proteins
were
precipitated
from
the butanol
extracts
identify
other
proteins
that interact
and
membrane-associated
T-ag and
a protein
extraction
technique
utilizing
using another
p53 monoclonal
antibody,
PAb421
(Fig.
1B, Lane 4), and a T-ag monoclonal
antibody,
PAb43O
(Fig. 18, Lane 2). Specificity
of recognition
of the Mr
1-butanol.
Single-phase
concentrations
of butanol
facilitated solubilization
of a complex
of 1-ag with cellular
proteins
ranging
in size from M, 35,000 to Mr 60,000 (31,
33). We identify
here the M, 56,000
component
of the
complex
as the fi subunit
of tubulin and describe
a series
tibodies
was demonstrated
by lack of precipitation
of the
complex
using a null antibody
(Fig. 1A, Lane 3; Fig. 1B,
Lane 1 ). Similar
patterns
of the M, 35,000-60,000
com-
of experiments
bodies
In a program
with
cytoplasmic
PS3, we adapted
both
to
that
document
1-ag and p53 with
a specific
association
of
microtubules
in viva and in vitro.
Results
Coprecipitation
of Mr 35,00060,000
Cellular
Proteins
with Monoclonal
Antibodies
against 1-ag or p53 from
Butanol Extracts of mKSA Cells. We previously
reported
the coprecipitation
of at least five cellular
proteins
with
1-ag and p53 in monoclonal
antibody
immunoprecipitates from butanol
extracts
of mKSA cells (31, 33). These
proteins
were coprecipitated
with monoclonal
antibodies
against
either
the NH2 or COOH
terminus
of 1-ag as well
as with several
monocbonal
antibodies
against
p53.
Typical
profiles
of proteins
precipitated
with the p53
monocbonal
antibody
200.47
from
butanob
extracts
of
mKSA
cells metabolically
labeled
with
[‘5Sjmethionine
are shown
(Fig. in, Lane 4; Fig. 1B, Lane 3). The pattern
35,000-60,000
ponents
proteins
were
observed
by the reactive
using
different
monoclonal
an-
monoclonal
anti-
against
1-ag or p53 (Fig. 1A, Lane 4; Fig. 1 B, Lanes
2-4) (31).
The M, 95,000
protein
present
in the complexes
was
identified
as 1-ag
anti-I-ag
serum
monoclonal
by immunoblotting
(33).
antibody
p53
was
assays
usually
immunoprecipitates
not
using
rabbit
apparent
from
in
butanol
extracts
of 55S-labeled
mKSA
cells, although
it could
be
observed
in immunoprecipitates
from butanol
extracts
of
32P-labeled
mKSA
cells.
Immunoblotting
analyses
using
rabbit
anti-p53
serum
of total
protein
extracts
of unla-
beled mKSA cells showed
a significant
solubilized
by butanol
treatment
(data
amount
of p53
not shown).
The
other
M,
45,000-60,000
proteins
did not appear
to be
related
to I-ag
or to p53, based
upon
immunoblotting
assays
(31, 33). Partial
protease
mapping
experiments
using V8 protease
also indicated
no similarity
with
p53
(Fig. 2; compare
Lanes 1 and 2 with Lanes 3-10).
Each of
the M, 45,00060,000
proteins
appeared
to be unique,
Cell
1
2
3
4
5
6
7
Growth
8
& Differentiation
117
910
6Ok
45k)’
Es
,
2
a
.i
Ip
I
‘*‘
Fig. 2.
Partial
protease
mapping
of 35S-labeled
M, 45,000-60,000
proteins
coprecipitating
5, 8, and 9) or 1.0 g (Lanes 2, 3, 6, 7, and 10). Authentic
p53 (Lanes
1 and 2), M, 56,000
and 8), and M, 45,000
(Lanes 9 and 10) proteins
were
digested
as described
in “Materials
SDS-polyacrylamide
gel. Molecular
weight
markers
are indicated
on the left.
as partial
structural
V8 protease
mapping
similarity
among
them
revealed
(Fig. 2).
no
apparent
Identification
of the Mr 56,000 Coprecipitating
Protein
as the fi Subunit
of Tubulin.
We initiated
attempts
to
identify
the Mr 35,00060,000
proteins
coprecipitating
with
1-ag
and
p53
by microsequencing.
Monocbonal
antibody
200.47
immunoprecipitates
from
butanol
extracts of mKSA cells were disrupted
in NP4O-urea
buffer
(34), and eluted
proteins
were
purified
by two-dimensional
electrophoresis.
Only
the Mr 56,000
and 45,000
proteins
were efficiently
solubilized
and resolved
under
these conditions
(Fig. 3A). Purified
proteins
were electro-
phoretically
blotted
from
Immobibon
PVDF
paper
automated
microsequence
the
and
two-dimensional
were
subjected
analysis.
A blocked
gel onto
directly
to
NH2 ter-
minus
prevented
microsequencing
of p45. However,
30
amino-terminal
residues
of p56
were
sequenced.
A
search
of GenBank
sequences
identified
strong
homology of the p56 sequence
with the fi subunit
of tubulin
isoforms
3 and 4 (Fig. 3B).
A Complex
Present
tification
bulin
monoclonal
the nature
Reciprocal
of 1-ag,
from
antibodies
p53,
anti-p53,
butanol
extracts
and Tubulin
Is
The iden-
from mKSA Cells.
and the availability
allowed
and antitubulin
of mKSA
of antitu-
us to investigate
of tubulin
interaction
with
immunobbotting
experiments
out. Anti-I-ag,
itates
Composed
in Butanol
Extracts
of p56 as tubulin
cells
1-ag and p53.
were
carried
immunoprecipwere
separated
with T-ag and p53. V8 protease
was used at 0.1 pg (Lanes
1, 4,
(Lanes 3 and 4), M, 60,000
(Lanes 5 and 6), M, 50,000
(Lanes 7
and Methods,”
and partial
fragments
were
resolved
on a 15%
by gel electrophoresis,
and the proteins
were
analyzed
by immunobbotting
using rabbit anti-I-ag
serum (Fig. 4A)
or a monocbonal
antibody
(N.356)
specific
for the fi
subunit
of tubulin
(Fig. 48). The I-ag
monoclonal
antibodies
PAb43O
and PAb419
precipitated
I-ag (Fig. 4A,
Lanes 4 and 5), and the p53 monoclonal
antibodies
200.47
and PAb421
coprecipitated
I-ag
due to complex
formation
with p53 (Fig. 4A, Lanes 6 and 7). Three different monoclonal
antibodies
against
tubulin
coprecipitated
I-ag (Fig. 4A, Lanes 8-JO).
Conversely,
monocbonal
antibodies
against
either
I-ag or p53 coprecipitated
tubulin
(Fig. 4B, Lanes 2 and 3). Control
tubulin
was precipitated
by a monoclonal
antibody
against the a subunit
of tubulin
(Fig. 4B, Lane 4). Null antibodies
did not precipitate
1-ag
(Fig. 4A,
Lanes
1-3)
or tubulin
(Fig. 4B, Lane
1). No
quantitative
comparisons
could
be made from these precipitation
experiments,
because
the tubulin
antibody
was
not
used
in excess.
However,
it was
evident
that
complex
composed
of I-ag, p53, and tubulin
was present
in butanol
extracts
from mKSA cells.
Mixing
experiments
were
performed
to ascertain
whether
the complex
existed
in situ
or was formed
postlysis
in the butanol.
If complex
formation
occurred
postlysis,
excess unlabeled
proteins
should
compete
with
the labeled
proteins
and diminish
the amount
tubulin
and other
proteins
coprecipitated
antibodies.
mKSA cells were
metabolically
[35Sjmethionine.
Labeled
cells were then
of labeled
by p53 or I-ag
labeled
with
mixed
in vary-
a
1 18
SV4O
T-ag
and
p53
A
,-
Bind
5.9
Microtubules
6.87.2
V
V
9.3
V
presence
of 1 or 3 tg of tubulin
or 1 tg of BSA (Fig. 5).
Although
a decrease
in recoverable
complex
was apparent from
samples
prepared
in the presence
of 10 ag of
tubulin,
a similar
decrease
occurred
when
10 tg of BSA
were present
in the extraction
buffer
(Fig. 5), so this was
presumably
a nonspecific
effect.
These results strongly
suggest that the protein
complex
exists
prior
to cell lysis and does
not form
during
the
extraction
process
by nonspecific
adherence
to tubulin.
V
P56
60b.
-
-
5Ok’
4-4p45
.
45k
Localization
IEM. As tubulin
pS(.
luhulun
3suhunii:
M
_X_E_1_V_H_tLI_Q_A_G_Q_C_G_N_Q_I_G_.A_K_F
51
-R-E-I-V-tl-
21
i)c(,
I uhui,n
isuhunit
[.1
W
L -Q-A-G-Q-C-G-N-Q-I-(-.\--K-
:iii
-E-V-I-S-D-E-X-G-l
-E-V-I-S-D-E-H-G-I
fig. 3.
Purification
and amino
acid sequencing
of M, 45,000
and 56,000
proteins
coprecipitating
with
T-ag and p53. A, butanol-solubilized
pro.
teins from
mKSA
cells were
immunoprecipitated
with
monoclonal
antibody
200.47,
and
Precipitated
proteins
were
eluted
with
urea-NP4O
buffer.
Soluble
proteins
were
purified
by two-dimensional
gel electrophoresis
and were blotted
onto Immobilon
PVDF membranes.
Membrane
strips containing
blotted
M, 45,000
and 56,000
proteins
were subjected
to microsequence
analysis.
B, NH2-terminal
sequence
comparison
of the
l subunit
of tubulin
and M, 56,000
proteins.
X. unidentified
brackets,
tentative
identification.
C, one-dimensional
gel pattern
munoprecipitate
from which
p56 was purified.
residue;
of rn-
ing ratios with unlabeled
mKSA cells, the mixtures
were
extracted
with
butanol,
and the extracts
were
immunoprecipitated
using excess
antibody
(PAb43O
or 200.47).
The amount
of labeled
complex
remained
the same
when
cell lysates were prepared
in the presence
of equal
numbers
or a 5-fold
excess
of unlabeled
cells (data not
shown).
The absence
of any competition
by unlabeled
cell
proteins
of the recovery
of labeled
complex
by
immunoprecipitation
strongly
suggested
that the complex existed
prior to cell lysis.
In a second
experiment,
we used
partially
purified
tubulin
as a specific
competitor
for the M, 56,000
component
of the complex.
The tubulin
was prepared
by two
cycles
of repolymerization
from suspension
human
lung
carcinoma
cells of line H69. Methionine-labeled
mKSA
cells were extracted
in 2.5%
butanol
containing
0, 1, 3,
or 10 tg of tubulin.
Duplicate
samples
were extracted
in
the presence
of 1 or 10
of BSA to control
for any
nonspecific
effects
that might
be encountered
due to the
presence
of extraneous
protein
in the extraction
mixture.
This experiment
was designed
to determine
whether
the
components
in the complex
were nonspecifically
sticking
to tubulin
or were
associating
with
tubulin
during
the
extraction
procedure.
If the complex
were
generated
during
extraction,
a decrease
in the recovery
of labeled
tubulin
in the complex
would
be expected
as more
competitor
unlabeled
tubulin
was added.
As seen in Fig.
5, no decrease
in the recovery
of labeled
components
in
the complex
was observed
in either
anti-I-ag
or anti-p53
immunoprecipitates
when
extracts
were prepared
in the
of SV4O 1-ag
and p53 to Microtubules
by
appeared
to be complexed
with I-ag or
p53
or both in extracts
of transformed
cells, we examined
whether
those
proteins
were associated
with
intact
microtubules.
EM was performed
to visualize
the cytoskeletal components
to which
1-ag and pS3 might
be bound.
A number
of permeabilization
and fixation
conditions
were
evaluated
in an attempt
to balance
acceptable
retention
of ultrastructural
integrity
with optimal
immunolabeling
of I-ag.
Under
conditions
that favored
ultrastructural
preservation
and labeling
of tubulin
by the
YLY2
antibody
(35), I-ag did not retain
adequate
antigenicity.
As I-ag
is predominantly
a nuclear
antigen
with
only a small
plasma
membrane-associated
fraction
(6),
the subpopulation
available
for microtubule
binding
is
expected
to be of low abundance.
Similarly,
p53 is found
mainly
in nuclei
and also would
not be expected
to
exhibit
an abundant
microtubule-associated
subpopulation.
Therefore,
conditions
were
used
that
produced
optimal
I-ag
labeling
with
an observable
cytoskeleton.
The antitubulin
antibody
did not react well under
these
conditions
but was included
in double-labeling
reactions
to verify
that the observed
structures
were microtubules.
mKSA
cells were
treated
with
0.1%
NP4O
in a harsh
permeabilization
procedure
to remove
all soluble
and
membrane
components
of the cytoplasm.
Cytoskeletal
preparations
were
then
processed
for IEM and
embedded
in Lowicryl
as described
in “Materials
and
An irrelevant
primary
antibody,
m73, was used as a
control
for specificity
and failed
to bind to any of the
microtubule
subsets,
such as centriolar,
mitotic
spindle,
or cytoplasmic
microtubules,
in immunolabeling
experiments
performed
on thin sections
(Fig. 6A).
Double
Iabeling
of thin sections
of mKSA
cells with
mouse
antip53
and rat antitubulin
antibodies
was detected
with 15nm gold-goat
anti-mouse
and 10-nm
gold-goat
anti-rat
secondary
antibody
probes.
The probes
for p53 and
tubulin
cobocalized
to microtubules
of the centriolar
region (Fig. 6B) as well as to mitotic
spindles
and unassigned
microtubules,
such as those
in Fig. 6A. Similar
experiments were done
using anti-I-ag
and antitubulin
primary
antibodies.
Cobocalization
of both probes
to microtubular
structures
of many types indicated
that I-ag was associated with cytoplasmic
microtubules
(Fig. 6, C and D), as
well as with the mitotic
spindle.
The low levels
of label
over
the microtubules
are believed
to be significant,
considering
that I-ag
and p53 are present
in the cytoplasm
in very small amounts
and that the negative
controls displayed
very rare labeling
reactions.
Little extranuclear
binding
ofT-ag
or tubulin
antibodies
occurred
on thin sections
of mKSA
cells that,
prior
to
permeabilization,
were
cultured
in the presence
of 10
LM colchicine
for 3 h followed
by incubation
on ice for
1 5 mm (data not shown).
Cytoskeletons
were not readily
visible
in sections
of nonpermeabilized
cells. Such sam-
Cell
A
I
2
3
4
5
Growth
& Differentiation
B
6
7
8
9
I
10
234
p
Tag
I
-
.
,
Fig. 4.
Coprecipitation
of T-ag,
p53,
and
tubulin
from
butanol
extracts
of suspension
cultures
of mKSA
.
cells.
.::=:
ttuIin
A, immunoblot
analysis
using
rabbit
anti-T-
ag serum
of proteins
in immunoprecipitates
from
butanol
extracts
of unlabeled
mKSA
cells. Antisera
used for immunoprecipitation
were
the following:
monoclonal
antibodies
against
rotavirus
VP7 (Lanes
1 and 2) or m73 (Lane 3), T-ag monoclonal
antibodies
PAb43O
(Lane 4) and PAb419
(Lane 5), p53
monoclonal
antibodies
200.47
(Lane 6) and PAb421
(Lane 7), and monoclonal
antibodies
against
the o subunit
oftubulin
(Lane 8), the $ subunit
of tubulin
(Lane 9), or one cross-reactive
with both subunits
(YLY2; Lane 10). Note the coprecipitation
of T-ag by pS3 and tubulin
antibodies.
Bands corresponding
to mouse
lgG chains
from the immunoprecipitates
are evident
in some
lanes. B, proteins
precipitated
with m73 (Lane 1), PAb43O
(Lane 2), 200.47
(Lane
3), and anti-a
tubulin
subunit
(Lane 4) monoclonal
antibodies
were
immunoblotted
using monoclonal
antibody
N.356
against
the
subunit
of tubulin.
Note the coprecipitation
of tubulin
by T-ag and p53 antibodies.
The positions
of 1-ag (M, 95,000)
and tubulin
(M, 56,000)
are indicated.
pIes,
however,
did
show
I-ag
label
in the
nucleus
(data
not shown). Thus, IEM provided
ultrastructural
evidence
that both 1-ag and p53 are associated
with a variety of
microtubules
in mKSA cells.
Temperature-dependent
Cycle Purification
of Tubulin
and Associated
Proteins. Temperature-dependent
cycle
purification
bule-associated
to quantitate
of tubulin
is a means
of identifying
microtuproteins.
This approach
was performed
the interaction
of 1-ag and p53 with tubulin
and to further
rule out artifactual
be induced
by butanol.
Two
associations
that might
cycles
of temperature-
dependent
polymerization
and depolymerization
were
carried
out as described
in “Materials
and Methods.”
Approximately
45% of tubulin
polymerized
into microtubules
in the first cycle
of temperature-dependent
purification
(Fig. 7A; compare
Lanes 1 and 2; see quantitation data in Table
1, step 1). From the microtubules
that
polymerized
in the first cycle,
only a portion
depolymerized
during
incubation
on ice, as evidenced
by the
large amount
of tubulin
(‘-60%)
that remained
in the
cold-stable
fraction
(pellet)
after the depolymerization
step (Fig. 7A, Lane 3; Table
1, step 2). Approximately
30% of the depolymerized
tubulin
repolymerized
into
microtubules
1, step 3).
in the
second
cycle
(Fig.
7A,
Lane
5; Table
ulin
Both I-ag and pS3
that polymerized
and second cycles
1). The stoichiometry
label
in the polymerized
remained
associated
with the tubinto microtubules
during
the first
(Fig. 7, B and C, Lanes 2 and 5; Table
of I-ag
tubules
label
(pellet
relative
to tubulin
fractions)
through
the two
cycles of purification
varied by less than 2-fold
(2.3 versus
4.4; Table 1), whereas the ratio of tubulin:p53
labels did not change
(14.3 versus
13.8). This relatively
constant
stoichiometry
of association
through
two
rounds
of polymerization
suggests a specific
interaction
of I-ag
and p53 with microtubules.
It is striking that about 80%
of I-ag and p53 was associated
with polymerized
microtubules
after the first cycle,
although
only 45% of the
tubulin
polymerized.
Similar
to tubulin,
only a fraction
of
I-ag and p53 was released
from the microtubule
pellet
upon
incubation
in the cold (Fig. 7, B and C, Lanes 3;
Table
1).
Samples
were taken prior
cycle in vitro repolymerization
to and following
reaction
and
med by EM in an effort to visualize
dried onto Parlodion-coated
nickel
1-ag.
grids,
were
immunolabeled
for I-ag
and tubulin
negatively
stained
for electron
microscopy.
taken 3 mm after initiation
of repolymerization
re-formed
microtubules
that
immunolabeled
the secondwere exam-
Samples were
and the grids
and were
Samples
contained
for I-ag
119
120
SV4O
T-ag
and
p53
Bind
Microtubules
Antibody
M73
tgTubulin(BSA)
Lane
PAb43O
0
1
3
1O Q 1
1
2
3
4
5
3
7
6
200.47
10 (1)(1Q)H
1 3 1O
8 9 10 11121314
*,
97
-
80
-
55
43
.
-
-
-
$Sa
-
-
36-
.
Fig. S.
La k of competition
by unlabeled
purified
tubulin
during
hutanol
extractions.
Spinner
niKSA cells were
metabolically
labeled
with
1 mCi
of [355]
methionine
or 1 .5 h at 37’C.
Aliquots
of 3 X 106 labeled
cells were pelleted
and resuspended
in 2.5% butanol
or 2.5% butanol
plus various
concentrations
of unlabeled
competitor
protein.
Cells
were
extracted
as described
(31). Extracts
were
immunoprecipitated
with
monoclonal
antibodies,
and the
imrnunopre(ipitates
were
washed
with
buffer
[150 mxi NaCI,
1%
NP4O,
0.5%
sodium
deoxycholate,
0.1%
SDS, 50 rnsi Tris (pH 8.0)], were disrupted,
and were analyzed
by SDS-polyacrylarnide
gel electrophoresis.
Extractions
made
with 2.5% butanol
in the absence
of any competitor
protein
are shown
in Lanes 1, 5, and 1 1. Extractions
were
made
with
2.5%
butanol
in the presence
of 1 ig of unlabeled
tubulin
(Lanes 2, 6, and 12), 3 ig of unlabeled
tubulin
(Lanes
3, 7, and 1 3), or 10 g of unlabeled
tubulin
(Lanec 4, 8, and 14). Butanol
extractions
were made in the presence
of 1 and 10 g, respectively,
of BSA, a nonspecific
competitor
protein
(Lanes
9 and 10). Extracts
were
immunoprecipitated
with control
monoclonal
antibody
m73 (Lanes
1-4), antiSV4O T-ag rnonodonal
antibody
PAb43O
(Lanes
5-10),
or anti-p53
monoclonal
antibody
200.47
(Lanes
11-14).
Molecular
weight
markers
are shown
on
the left.
(Fig. 8A), as well
yet repolymerized.
as some tubulin
(see inset) that had not
Control
antibody
m73 failed
to label
the tubular
structures
(Fig. 8B). Samples
taken
prior
to
repolymerization
showed
cobocalization
of I-ag
and
tubulin
probes
to small globular
structures,
presumably
the tubulin
heterodimer
complexes
(Fig. 8B, inset).
Therefore,
ultrastructural
studies
confirmed
that I-ag copurified
with
tubulin
through
two cycles
of purification
and revealed
that I-ag
was associated
with
both
the
depolymerized
form of tubulin
and intact
microtubules.
Effect of Colchicine
on Extraction
of 1-ag and p53.
Chemicals
such as colchicine,
which
affect the formation
of microtubules
in vivo, would
he predicted
to alter the
solubility
of cytoplasmic
I-ag and p53. Thus, an increase
in the amount
of soluble
I-ag and p53 might be observed
in colchicine-treated
cells, as compared
to control
cells,
due to the disruption
of microtubules.
Cultures
of mKSA
cells were incubated
in 10 iM colchicine
for 3 h at 37#{176}C.
Colchicine-treated
and untreated
mKSA cells then were
incubated
at 37#{176}Cin 0.1%
NP4O in microtubule
stabilization
buffer
for 15 mm, conditions
that would
leave
nuclei
and cytoskeletal
structures
intact and would
solu-
bilize
predominantly
membrane
and
cytosolic
compo-
nents. Total soluble
proteins
in the extracts
were precipitated
with
trichloroacetic
acid and analyzed
by immunobbotting.
More
tubulin,
I-ag,
and p53 were
extracted
with 0.1% NP4O from colchicine-treated
mKSA cells than
from untreated
cells (Table
2). The biggest
change
was
observed
with
p53 (2.4-fold
more
p53 was recovered
after colchicine
treatment).
Colchicine-treated
cells observed
by
IEM
using
double
immunogold
labeling
showed
a loss of microtubular
structures
and of 1-ag and
tubulin
labels (data not shown).
T-ag
Tubulin
and p53 Are Associated
with Microtubules
and
in Other SV4O-transformed
Cell Lines. It was
important
to establish
p53 with microtubules
mouse
fibroblast
cell
that the association
of I-ag
and
was not limited
to mKSA cells. A
line (WIB1a)
and a mouse
mam-
Cell
fig. 6.
Localization
of SV4O 1ag and p53 to mKSA
cytoskeletons
by
EM using
double
immunogold
labeling.
Cytoskeletons prepared
from
mKSA
cells
by extraction
in 0.1% NP4O were
processed
for electron
microscopy
and
were
embedded
in
Lowicryl,
as detailed
in “Mate-
,:
.-,...,I,’
Growth
& Differentiation
,,
fr
.
:
rials and Methods.”
Thin sections
were
immunolabeled.
Fifteennrn gold beads linked
to an antimouse
secondary
antibody
mark
the presence
of anti-T-ag
or antipS3
beads
tubulin
antibodies;
denote
the
antibodies.
10-nm
gold
presence
of
A, control
Ia-
beling
experiments
using
the
m73
antibody.
Microtubules
were not labeled.
Bar, 0.2 gm. B,
double
labeling
with
anti-p53
(PAb421,
arrowhead)
and antitubulin
(YL’/2, arrow)
antibodies
demonstrated
p53
associated
with
microtubules.
Bar, 0.1 zm.
C and
0, anti-T-ag
)PAb4O5,
arrowhead)
and antitubulin
(YLY2,
arrow)
antibodies
localized
1-ag
to microtubules.
Bars, 0.1 zm.
Cytoplasmic
(A),
centriolar
(B
and C), and rnitotic
spindle
(0)
microtubules
are shown.
‘1
B
A
123
12345
C
12345
4_5
I’b’Pp.
tubulifl’1
r
T1
.5.
Fig. 7.
Analysis
of temperature-dependent
cycle-purified
microtubules
for the presence
of 1-ag and p53. Total protein
in the supernatant
(Lanes
1 ( and
pellet
(Lanes
2) from
the high-speed
centrifugation
of the first-cycle
polyrnerized
microtubules,
the cold-stable
pellet
(Lanes
3), and the supernatant
(Lanes 4) and pellet
(Lanes 5) from the second
polymerization
cycle were
irnmunohlotted
using monoclonal
antibody
YLY2 against
tubulin
(A), rabbit
anti1-ag serum
(B), or rabbit
anti-p53
serum
(C). The positions
oftuhulin
)M, 56.000).
intact
1-ag )M, 95.000),
and p53 are indicated.
Some degradation
of Tag is evident
in B.
121
122
SV4O
T-sg
Table
1
and
p5
Bind
Association
Mi
r(itul)ules
1)1 tuhulin,
T-ag,
and
3
through
two
cycles
of temperature-dependent
(I(’polymerizatiOn
and
polymerization
of mk
rotubules
About
6 X 10#{176}
niKSA
( (‘(IS Wi.’Ii.’
Soni(,lt(’(l
in nii rotubule
stabilization
buffer
and were
incubated
on ice. Clarified
supernatant
plus GTP (unassembled
tubulin(
was in(uhated
at .37#{176}C or 30 mm. Polyrnerized
microtul)lIk’s
were l)elleted
(step 1). The pellet
from step 1 was sonicated
and incubated
on ice,
followed
1)5 C (‘ntntugatR)n
at .50,000
X g for 3() nlin.
The pelleted
( old-stable
microtubules
(step 2) represent
the population
that failed
to dissociate
when
the l)ell(’t
obtained
miii
the first cy Ic of polymerization
was in uhated
on ice. Clarified
supernatant
from step 2 was incubated
at 37’C
for 30
mm, and repolynlerize(l
mi( rotubules
were
f)(’llet(’(l
(step 3). For analysis
of total proteins
by immunoblotting,
pellets
were solubilized
in SDS disruption
I)utter;
supernatant
tra( finns were’ first pr(’( il)itate(l
with trichloroa
(‘ti(
a( id and then dissolved
in SDS disruption
buffer.
Purifi(ati(in
Step
1 : First-
ycle
polynierii,i-
tii)ii
Step
Fr 1( tiiin
analyze(l
of nli(rotuhuk’s
2: Cold-stable
riii
r(itu-
(iiantitation
Tubulin
Ratios
)cpni)”
T-ag”
p53
Sux’rnatant
P(’ll(’t
Pellet
8937
7402
45 16
646
3218
2688
Supernatant
Pellet
2 1 28
992
157
226
of cpm
Tubulin/T-ag
Tubulin/p53
1 30
518
39 1
1 3.8
2.3
1 .7
68.7
14.3
1 1.5
72
1 3.6
4.4
I)Ul(’S
St(’1) 3: Se ond-yck’
ization
l)o)YIller-
,‘ Protein
l)ands
from the ininsunoblots
were (letermined
by liquid
S( intillation
I) The
majority
of T-ag in the transformed
1 in the ( ( le purifi(afii)n
)r’dUr#{128}’).
shown
in Fig. 7 were
spectros
opy. Total
cells (>90%)
was
excised
,mnd solubilized
in 0.1 N KOH
cpm per immunoblot
band are shown.
pelleted
with other
particulate
materials
for
1 h with
when
the
microtubules.
mKSA cells,
Following
microtubules
fied through
one cycle
13.8
shaking
initial
at room
cell
lysate
the
from
temperature,
and
251 cpm
was
(prior
to step
described
line were
for
pun-
clarified
protocol
each cell
of depolymenization
and polym-
enization.
Total
proteins
in the supennatant
and pellet
fractions
of the polymerization
reaction
mixture
were
analyzed
by immunobbotting
for tubulin,
I-ag,
and p53
(Fig. 9). Results
similar
to those observed
for suspension
mKSA cells were obtained
with the two transformed
cell
lines growing
as monolayers.
Both I-ag and p53 copunified with
tubulin.
These
data show
that the association
of 1-ag and p53 with
microtubules
is not restricted
to
growth
of cells in suspension
culture.
It was noted
that
the amount
of I-ag cosedimenting
with
in vitro polymenized
microtubules
was significantly
greater
from
the
19C and WIB1a
SV4O-transformed
cell lines than from
the mKSA cells.
Another
SV4O-transformed
mouse
cell line,
analyzed
by IEM using
double
labeling
as
above
for mKSA cells. Gold
beads were rarely
on sections
I ig. 8.
Association
i)f
SV4O 1-ag with
iiie
rotubules
two cycles
of l)urif
ation
by in vitro assembly-disassembly.
we’re l)rePIrt’d
for in sitre polymerization
experiments
‘Materials
and
Methods.’
Samples
were
withdrawn
during
stained
the second
( ycle,
with
arnrnonium
f)olynlerized
after
mKSA
cells
as descrilx’d
in
at various
finies
were
imniunolaheled,
and were
niolybdate.
.-\, a sample
removed
negatively
from
the
repolymerization
reaction
after
3 mm of assembly
at 37’C
contained
microtubuk’s
as svell as unassembled
tuhulin.
lnimuncabeling
was done
using PAb4O5
anti-I-ag
nionoclonal
antibody
(arrowhead).
Note labeling
of
1-ag on reassembled
tul)ules.
Bar, 0. 1 m.
B, immunogold
labeling
of
a duplicate
sample
(sanie
as in A) was
carried
out using
an irrelevant
antibody,
ni7 3, as a ( ontrol.
Bar, 0.2 pm. Inset,
depolymerized
tuhulin
withdrawn
immediately
prior to initiation
of the second
cycle
of polyrnerization
was labeled
with anfi-T-ag
(PA64OS,
,irrowhe,id)
and antitul)ulin
)YL’/2, arrow)
aiitibodit’s.
Bar, 0.1 Mm.
mary
were
epithelial
analyzed
cell line (19C),
for copurification
both
transformed
of I-ag
and
by SV4O,
p53 with
treated
with
irrelevant
control
F9I,
was
described
observed
antibodies;
the
m73 control
failed
to react with all subsets
of microtubules, showing
the specificity
ofthe
procedure
(Fig. bA).
Antibodies
for 1-ag (Fig. lOB) and for p53 (Fig. 1OC)
cobocalized
with tubulin
antibodies
on microtubules
(Fig.
10, B and
C). IEM observations,
coupled
with
cycle
purification
of microtubules,
suggest
that the association
ofT-ag
and p53 with microtubules
is a common
phenom-
enon
in SV4O-transformed
origin
or culture
cells,
regardless
of tissue
of
conditions.
Discussion
Single-phase
concentrations
of butanol
have facilitated
the recovery
of a complex
composed
of I-ag, p53, tububin, and several
unidentified
proteins
from
SV4O-transformed
mouse
cells. The M, 56,000
component
of the
complex
was identified
as tububin
in this study
by amino
acid sequence
analysis
and by immunological
reactions
using antitubulin
monoclonal
antibodies.
The I-ag/p53/
tububin
complex
is not recovered
using
conventional
methods
of protein
extraction
that utilize
detergents
(31).
The less deleterious
effect of single-phase
concentrations
Cell
Table 2
p53 from
Effect
mKSA
of colchicine
cells
treatment
mKSA
cells were
incubated
colchicine
for 3 h at 37#{176}C.Cells
microtubule
stabilization
buffer
NP4O extracts
were
precipitated
by immunoblofting
as described
..
on solubility
of tubulin,
1-ag,
and
inthe
presence
or absence
of 10 MM
were
then extracted
with 0.1%
NP4O in
for 15 mm. Total soluble
proteins
in the
with
trichloroacetic
acid and analyzed
in “Materials
and Methods.”
Quantitation
(cpm)’
Ratios:
treated
Solubilized
Untreated
cells
protein
Tubulin
1-ag
p53
a
Protein
cpm
3274
944
182
bands
were
Colchicine-treated
cells
from
determined
immunoblots
cells/untreated
cells
4804
1152
443
were
as described
1.47
1.22
2.40
excised
in Table
and
1, footnote
solubilized,
and
251
a.
of butanol
on native protein
conformation
(36) may allow
for recovery
of protein
complexes
that are usually
disrupted
by
detergent
during
initial
cell
extraction
procedures.
The precise
origin
of the protein
complex
recovered
by butanob
from
the transformed
cells
is not known.
Previous
experiments
(31) found
that at 37#{176}Cbutanol
sobubilized
pml-ag
as well as a number
of proteins
resistant to extraction
by detergent.
Optimal
conditions
for
release
of the complex
resulted
in the leakage
of some
cytoplasmic
proteins
(31). It is possible
that the complex
may reside
within
or on the periphery
of the plasma
membrane,
in association
with cytoplasmic
structures,
or
may reflect
transient
interactions
among
proteins
being
shuttled
within
the cell.
Identification
of other
cellular
A
.
[)ifferentiation
123
components
present
within
the complex
may help clarify
its subcellular
origin.
Deppert
and his colleagues
(30, 32,
37) have reported
that a fraction
of pml-ag
is tightly
bound
to an NP4O-insobubbe
framework
in the plasma
membrane,
designated
the plasma
membrane
lamina.
We have confirmed
the existence
of an NP4O-insobuble,
Empigen
BB-sobubbe
population
of pml-ag
(31). Butanob
extraction
recovered
a species
of T-ag that was positioned
so as to be less accessible
to surface
iodination
than NP4O-sobubbe
pml-ag
(31). It may be that the butanol-solubbe
fraction
of I-ag
is related
to the plasma
membrane
bamina-associated
fraction
of I-ag that is predominantly
interior
in the cell (37).
The multimenic
protein
complex
appears
to exist in
situ prior to cell bysis. The possibility
that the observed
complex
was an artifact
induced
by butanob
was ruled
out previously
by various
control
experiments
(31). The
coprecipitating
M, 35,000-60,000
proteins
remained
relatively
constant
to one another
under
various
conditions
(31,
33).
Competition
experiments
performed
here
showed
that extraction
of labeled
cells in the presence
of either
unlabeled
cells or unlabeled
tububin
did not
diminish
the recovery
of the labeled
complex.
Additional
approaches
that did not involve
butanol
(also reported
here) substantiated
a specific
interaction
of I-ag and p53
with
microtububes.
Both 1-ag and p53 copunified
with
microtubules
through
two
cycles
of temperature-dependent
depolymenization
and
polymerization,
and
treatment
of mKSA cells with colchicine
prior to extraction under
conditions
that preserve
nuclei
and cytoskebetal filaments
other
than
microtubules
resulted
in increased
release
of I-ag
and p53. Thus,
both
I-ag
and
C
B
12
Growth
34
12
34
tubulin’
.. .
9.
Copurification
of 1-ag and p53 with single-cycle
purified
microtubules
from 5V40-transformed
pelleted
(Lanes 2 and 4) microtubule
fractions
from the first cycle of temperature-dependent
purification
fibroblast
cell line WIB1a
(Lanes
1 and 2) and SV4O-transformed
mammary
epithelial
cell line 19C
immunoblotted
with
antitubulin
monoclonal
antibody
YLY2 (A), rabbit
anti-I-ag
serum
(B), or rabbit
56,000),
1-ag )M, 95,000),
and p53 are indicated.
Fig.
mouse
cell lines. Supernatant
(Lanes
I and 3) and
of tubulin
were obtained
from SV4O-transformed
(Lanes
.3 and 4). Total
protein
in the samples
was
anti-p53
serum
(C). The positions
of tubulin
(M,
124
SV4O
I-ag
md
Bind
Mi
rotuhules
fit the criteria
of microtubule-associated
proteins.
IEM corroborated
these biochemical
analyses
and established an association
of I-ag and p53 with intact
microtubules
in situ. We do not yet know the precise
relationship between
the T-ag/p53/tubulin
complex
recovered
by butanol
and the T-ag/p53/microtubule
interactions
visualized
by IEM.
A specific
interaction
in vitro between
SV4O small
antigen
and tubulin
has been observed
(38). We have
not rigorously
ruled
out the presence
of small t antigen
in the butanob
extracts,
but so far we have not detected
it. Small
t antigen
was not evident
in immunobbots
of
solubilized
microtubule-punified
proteins
using
polycbonab rabbit
anti-I-ag
serum,
nor was it observed
in
immunobbots
of protein
complexes
immunoprecipitated
from
butanol
extracts
of transformed
cells (33). Small
antigen
is not responsible
for the protein
interactions
described
here. The T-ag/tububin
complexes
can be immunoprecipitated
using monocbonal
antibodies
directed
against
COOH-terminal
sequences
of I-ag not shared
by
small t antigen
(33). Furthermore,
the
EM studies
reported
here that revealed
I-ag associated
with microtubules
utilized
a COOH-terminal-specific
anti-I-ag
antibody.
The observed
association
of p53 with cellular
microtubules
is intriguing.
Wild-type
p53 exhibits
transcniptional
activating
ability
(39, 40). As a negative
growthregulatory
protein,
p53 must exert some influence
on the
cell cycle.
It has been
suggested
(41) that cell cycle
transitions
in eukaryotic
cells are controlled
by p34, the
product
of the cdc2 gene. The activity
of p34, the catalytic subunit
of a protein
kinase,
is regulated
by phosphorybation.
Microtubube
dynamics
during
the
interphase-metaphase
transition
in Xenopus
eggs have been
shown
to be regulated
by cdc2 kinase
(42). Human
p53
is phosphorylated
by p34f(2
(43 44) and it has recently
p53
been
reported
that
p53
can associate
with
p34k2
(44
45). It is possible
that the protein
complex
described
here, which
contains
a M, 35,000
member
is important
in cell growth
control.
The association
of I-ag and p53 may be functionally
important
in the transport
of these
proteins
within
the
cell and to the plasma
membrane.
Transport
motor
proteins such as kinesin
and dynein
facilitate
movement
of
vesicles
and proteins
on microtubules
(46). This mechanism provides
an attractive
explanation
for the transport
of I-ag to the plasma
membrane.
Previous
studies
have
shown
that I-ag
does
not enter
the cellular
secretory
pathway
(47-49).
Nothing
is known
about
the intraceblular transport
of p53. It is possible
that 1-ag is following
a pathway
fig. 10.
Denionstration
of microtubule
association
SV4O 1-ag and p53
in SV4O-translormed
F9T cells by IEM.
F9T cells expressing
1-ag were
extracted
s ith 0. l%
NP4O and were’ prepared
for ele tron microscopy.
Thin sections
of cytoskelefons
were
inimunolabeled
with antitubulin
and
anti-T-ag,
with anti-tubulin
and anfi-p53,
or with a control
antibody.
Bars,
0.1 m.
A, (ontrol
experiments,
in which
samples
were
labeled
with
ii73
as the primary
anfil)ody,
showed
no labeling
of microtubules.
B, anfi-Tag (PAb4OS.
,irr(iesheaci)
and antitubulin
(YL’/2, arr(iw)
antibodies
colocalized to nix rotubules
after simultaneous
double
immunogold
labeling.
C,
anti-p53
(PAb42
1 , arrowhead)
and antitubulin
(YLV2,
,irrow(
antibodies
colocalized
to rnicrofuhules
by double
labeling.
Mitotii.
spindle
(A ( and
niicrotuhuk’s
near the spindle
(B and C) are shown.
utilized
by cellular
growth-regulatory
proteins
such
as p53.
Other
enzymes
and proteins
bound
to
microtububes
and functioning
in some
aspect
of cell
metabolism
(50) may be targets
affected
by I-ag or p53.
It is interesting
that a correlation
has been
observed
between
the association
of Rous sarcoma
virus
p60vsr(
with cytoskebetal
structures
and transforming
activity
(51).
It will be important
to define
the domain(s)
on I-ag
and p53 that interact
either
with
tububin
or with
some
microtubule-associated
protein.
This will help determine
the functional
importance
of the microtubube
association.
Mapping
studies
using deletion
mutants
of I-ag
are in
progress,
as are attempts
to identify
other
members
of
Cell Growth
the T-ag/p53/tubulmn
complex
SV4O-transformed
recovered
by butanol
Partial
compared
from
cells.
partial
Materials
and Methods
Cell Lines. SV4O-transformed
cell lines mKSA (14, 25,
SV4O-transformed
BALB/c
52) and
BALB/c
mouse
WTB1a
mouse
mammary
Rabbit
polyclonal
antisera
53),
the
epithelial
cell line 1 9C (54, 55), and SV4O-transformed
carcinoma
cells were used for metabolic
protein extraction
experiments.
Antisera.
fibroblast
(14,
F9T teratolabeling
and
against
gel-pun-
fied SV4O I-ag (56) and p53 (31) have been described.
Monoclonal
antibodies
recognizing
sites on the NH2 tenminus of 1-ag are designated
PAb419
and PAb43O (57,
58). Those specific
for carboxy-terminal
epitopes
on Iag are PAb4O5 and PAb423
(57, 58). Monoclonal
antibodies used to detect p53 were PAb421 (57) and 200.47
(59). Monoclonal
antibody
against
tubulin
(YL#{189})was
obtained
commercially
from Seralab (Accurate
Chemical
and
Scientific
Corp.,
Westbury,
NY),
and
monoclonal
antibodies
specific for either the a subunit
(N.357) or the
3 subunit
(N.356)
of tubulin
were purchased
from Amensham (Arlington
Heights,
IL). Control
monoclonal
antibodies
were directed
against
adenovinus
E1A protein
(m73) (60) or against rotavinus SAl 1 VP7 (provided
by M.
K. Estes, Baylor College
of Medicine).
Metabolic
Labeling,
Protein
Extraction,
and Protein
Analyses. Approximately
5-7 x 10 cells were incubated
for
3 h in 1-2
ml
of methionine-free
minimal
125
Proteolytic
Mapping.
Complexed
proteins were
for structural
relatedness
by the V8 protease
peptide
mapping
technique
of
Cleveland
et a!.
(64). [35S]Methionine-Iabeled
partial
peptides
were nesolved in a 15% SDS-polyacrylamide
gel (65).
Temperature-dependent
Cycle Purification
of Tubulin.
Tubulin
was purified
by a modification
of the temperatune-dependent
depolymenization
and polymerization
method
of Vallee (66). Approximately
6 x 108 cells were
sonicated
in 2-3 ml of microtubule
stabilization
buffer
[0.1 M 2-(N-morpholino)ethanesulfonic
acid (pH 6.9), 1
mM ethylenebis(oxyethylenenitnilo)tetraacetic
acid, 0.5
mM MgCl2, 1 mM GTP, 200 tM leupeptin,
1% Inasylol,
1
mM phenylmethylsulfonyl
fluoride]
with
two
15-s
bursts
on ice. After incubation
on ice for 15 mm, the lysate was
clarified
of particulate
debris by centnifugation
at 50,000
x g for 1 h at 2#{176}C.
The supernatant
plus GTP (containing
unassembled
tubulin)
was then incubated
at 37#{176}C
for 30
mm to allow polymerization
of tubulin
into microtubules
(first cycle). The polymenized
microtubules
were pelleted
at 50,000 x g at 25#{176}C
for 1 h. The pellet was solubilized
in SDS disruption
buffer [0.125 M Iris hydrochloride
(pH
6.8), 2% SDS, and 5% fl-mercaptoethanol]
for electrophoresis and immunoblotting.
The supernatant
(containing tubulin
that
failed
to polymerize
during
the
incuba-
tion at 37#{176}C)
was adjusted
to 20% tnichloroacetic
acid
and was incubated
on ice for 30 mm; aggregated,
insoluble protein was then pelleted
at 23,000 x g for 20 mm.
The
essential
& Differentiation
protein
pellet
was
washed
95%
For
proteins,
ization, a pellet from the first-cycle
polymerization
reaction was sonicated
in microtubule
stabilization
buffer and
phosphorylated
cells
were
in-
cubated
for 3 h in phosphate-free
minimal
essential
medium
containing
2% dialyzed
bovine
serum and 500
zCi of 32P (ICN Biomedicals)
per ml. Radiolabebed
suspension
cells were pelleted
and completely
drained
of
radioactive
medium.
Cells were lysed immediately
in
NP4O lysis buffer
[1% NP4O, 0.05 M Iris hydrochloride
(pH 8), 1%apnotinin,and
200 IzM Ieupeptin]
(61)on were
treated with 2.5% butanol
in 0.01 M phosphate-buffered
saline
at 37#{176}C
for bO mm
(at 4 x b0
cells/mi)
as described
previously
(31, 33). Proteins
were then immunoprecipitated and analyzed
by SDS gel electrophoresis
(31, 56,
62).
Immunoblot
Assays. Immunobbotting
was performed
using a modification
of the procedure
of SlagIe et a!. (63).
Immunoprecipitated
proteins on total solubilized
proteins
were resolved
in 10% polyacrylamide
gels and were
electrophonetically
blotted
onto
nitnocellulose.
Blots
were preincubated
for 1 h at 37#{176}C
in blocking
buffer
(0.1% NP4O, 1% dried skim milk, and 0.25% gelatin
in
Iris-buffered
saline).
Treated
blots
37#{176}C
with shaking in a 1 :500 dilution
or monoclonal
antibody
in blocking
serum
diluted
in blocking
buffer
nitrocellulose
strips (treated
least 1 h at 37#{176}C
to reduce
were
rinsed
incubated
gel
lane
Vigorously
sodium
saline
well
with
at 37#{176}C
with
in blocking
three
deoxycholate,
at 37#{176}C.
incubated
was
preincubated
buffer
After
in 1%
and
with
with blocking
buffer)
nonspecific
binding.]
blocking
1
at
of rabbit antiserum
buffer. [Rabbit anti-
and
1 h, blots
for at
Blots
then
0.5 jzCi of 125l-Iabeled
buffer.
times
were
were
were
protein
A/
washed
NP4O, 0.1% SDS, 0.5%
NaCI in Iris-buffered
M
For a second
was
cycle
incubated
disruption
ethanol,
dried,
and disrupted
immunoblotting.
of
SDS
with
fetal bovine serum and
(1000-1150
Ci/mmol;
Inc., Irvine, CA) per ml.
analysis
in
twice
medium
containing
2% dialyzed
200
zCi
of [355]methionine
Tran35S-Iabel;
ICN Biomedicals,
of depolymenization
on ice for 30 mm
buffer
and
for
polymer-
to depolymenize
micro-
tubules.
Aggregates
of cold-insoluble
material
were removed by centnifugation
at 50,000 x g for 30 mm at 2#{176}C.
GTP was added
to 1 mii to the clarified
supennatant,
which
was
then
incubated
at 37#{176}Cfor 30 mm.
Microtu-
bule pellets and the supennatants
were analyzed
for total
protein as described
for the first-cycle
purification.
Purification
of Proteins for Automated
Microsequence
Analysis. 35S-Iabeled
protein
complex
components
were
immunoprecipitated
with monoclonal
antibody
200.47
and were resolved
by two-dimensional
electrophoresis
(34). Precautions
were taken during
preparation
of immunoprecipitates
and gel electrophonesis
to minimize
possible
NH2-terminal
blocking
on denivatization
of
amino acid side chains.
Immunoprecipitates
were disrupted
in NP4O-unea
buffer (34) and were incubated
at
room temperature
for 2 h. First-dimension
separation
was
performed
in the
presence
of 0.1
mrs’i sodium
thio-
glycolate
to sequester
any oxidants
on free radicals generated during gel polymerization
(67). For second-dimension
analysis,
isoelectnic
focusing
gels
were
equilibrated
in sample
buffer at room temperature
for 30 mm. Focused proteins
were then resolved
on 10% gels that had
been allowed
to polymerize
overnight
in the presence
of
0.1 mM sodium
thioglycolate.
Electrophonesis
running
buffers also contained
sodium thioglycolate.
Electrophoresis was performed
at 45 V constant
voltage overnight.
Proteins
were then blotted
overnight
at 0.5 A onto an
Immobilon
toradiognaphy.
PVDF membrane
and
The area containing
were
localized
pnctein
was
by auexcised,
126
SV4O
1-ag
and
p53
Bind
Microtubules
and the blotted
protein
was sequenced
directly
on a
PVDF membrane
in an Applied
Biosystems
model 477A
protein
sequencer.
Approximately
50 pmol of purified
protein were recovered
from butanol
extracts of 8 x i0
mKSA
cells.
Lowicryl
with
K4M
Embedding.
Cells
stabilization
buffer
microtubule
were
washed
once
followed
by a 15-
References
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1989.
into complex
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mm treatment
with 0.1% NP4O in the same buffer. Cells
were washed
twice with either 0.1 M sodium
phosphate
buffer
or 0.1 M piperazine-N,N’-bis(2-ethanesulfonic
acid) monosodium
salt buffer
and were then fixed in
buffer containing
2% glutaraldehyde
and 2% formaldehyde for 30 mm at room temperature.
Further processing
was done as described
by Decker et a!. (68). Briefly, cells
were pelleted
in a microcentnifuge
immediately
after
addition
to fixative.
Fixed cell pellets
were washed
in
buffer and dehydrated
in dimethylformamide
in steps of
50, 70, and 95% for 5 mm each and 100% for 15 mm.
The pellets were then transferred
to a 1:1 mixture
of
4. Sepulveda,
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for
15-60
mm
and
were
infiltrated
with Lowicryl
for 60 mm, all at room temperature. Samples
were rinsed
twice
with Lowicryl,
embedded
in Beem capsules,
and polymenized
at room
temperature
via UV light (69).
IEM. Fifteen-nm
gold-goat
anti-mouse
and 10-nm goldgoat anti-rat
conjugates
were purchased
from Janssen
Life Science
Products
(Piscataway,
NJ). For immunolabeling experiments,
the antitubulin
antibody
was diluted
1:2, and all other antibodies
were diluted
1 :20 in phosphate-buffered
saline containing
0.1% Triton X-100 and
0.5% Tween
20. Thin sections
of Lowicryl-embedded
cell pellets were collected
on Panlodion-coated
nickel
grids. Immunolabeling
was performed
according
to the
procedure
of Decker et a!. (68). Grids were floated on a
drop of phosphate-buffered
saline with 0.1% Triton
X100 and
0.5%
Tween
20 for
5-15
mm
to block
nonspe-
cific reactions.
All antibody
dilutions
and the first wash
also were performed
using this buffer.
After blocking,
sections
were incubated
for 60 mm with a primary
antibody or, for double-labeling
experiments,
with a mixture
of mouse
and rat primary
antibodies.
Grids were jet
washed
with 10-15 ml of buffer/grid
and were drained
on filter paper.
Incubations
with secondary
antibodygold
conjugates
also
were
carried
out
for
60
mm.
For
double-labeling
experiments,
a 10-nm gold-goat
anti-nat
probe was mixed
with a 15-nm gold-goat
anti-mouse
probe for simultaneous
reactions.
After incubation
with
probe, grids were jet washed with water. Immunolabeled
grids were stained
either
with osmium
tetroxide
and
tannic
acid
(70)
on with
4%
ethanolic
unanyl
acetate
and
tissue-specific
elements
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SV4O
Biochim.
moved
from
polymerization,
were taken
the reaction
immediately
and repolymenized
3 mm after assembly
prior
microtubule
was initiated.
were
ne-
to the final
samples
Samples
were adsorbed
to Panlodion-coated
nickel
grids for 3
mm, and the grids were drained
on filter paper, immunolabeled
as described
for thin-sectioned
samples,
and
negatively
stained with 1% ammonium
molybdate.
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