(Received for publication, May 28, 1997, and in revised form, July 3, 1997)
From the Departments of Mutations in the tumor suppressor gene
APC invariably lead to the development of colorectal
cancer. The vast majority of these mutations are nonsense or
frameshifts resulting in nonfunctional, truncated APC protein products.
Eleven cyclin-dependent kinase (CDK) consensus
phosphorylation sites have been identified in the frequently deleted
carboxyl-terminal region of APC; loss of these phosphorylation sites by
mutation could therefore compromise the ability of APC to inhibit cell
growth. This report demonstrates that immunoprecipitates of
full-length, but not truncated, APC protein include a mitosis-specific
kinase activity in vivo. Biochemical and Western analysis
of these immunoprecipitates confirms the presence of the CDK
p34cdc2. We also show that APC is a substrate for recombinant
human p34cdc2-cyclin B1. Modification of APC by p34cdc2
implicates phosphorylation as a mechanism for regulating APC function
via a link to the cell cycle.
The tumor suppressor protein
APC1 is inactivated by
mutation in over 80% of all colorectal cancers, both sporadic and
familial. Loss of APC function is considered the initial and
rate-limiting mutation in the multistep model of colon carcinogenesis
(1). APC is a cytoplasmic protein of 2844 amino acids with a predicted molecular mass of 312 kDa (2, 3) and without extensive homology to
other known proteins. Functional roles for APC have been suggested by
its association with other proteins whose characteristics are better
delineated. APC directly associates with the adherens junction constituent and signaling protein Germline alteration of APC results in an inherited
predisposition to colorectal cancer known as familial adenomatous
polyposis coli (2, 3). In one patient, a frameshift mutation has been identified at nucleotide 7935 of the APC cDNA that
results in the loss of 200 amino acids from the carboxyl terminus of
APC (15). Expression of a similarly truncated mutant protein in cells
lacking normal APC protein is sufficient to down-regulate the
HCT116 cells were obtained from the American
Type Culture Collection (ATCC) and cultured in McCoy's 5A medium
supplemented with 10% fetal bovine serum. SW480 cells (ATCC) were
cultured in Leibovitz's L15 medium supplemented with 10% fetal bovine
serum. For synchronization of cells at the G1/S boundary,
and in M-phase, nonconfluent cells were incubated for 20 h in 0.2 mM mimosine or 1.0 µg/ml nocodazole, respectively.
Synchronization was confirmed separately by FACS analysis (data not
shown) and was typically greater than 85% G1/S or 95% M. HCT116 cells were in vivo labeled for 4 h in
phosphate-free medium supplemented with 2.0 mCi of [32P]orthophosphate per 100-mm plate.
Cells were rinsed with
phosphate-buffered saline and lysed in Nonidet P-40 wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5%
Nonidet P-40) supplemented with protease inhibitors (2.0 µg/ml each
of pepstatin A, leupeptin, and aprotinin, 1 mM EDTA, and
0.2 mM phenylmethylsulfonyl fluoride), and protein was
quantitated using a detergent-compatible assay (Bio-Rad). Cell lysates
were precleared with normal mouse IgG (Sigma) and immunoprecipitated as
described (18) using normal mouse IgG, the monoclonal antibody APC-Ab5
(Oncogene Science), or the monoclonal antibody cdc2-Ab2 (Oncogene
Science). Immunocomplexes were isolated with Protein A-agarose, washed
liberally with wash buffer, and used in the H1 kinase assays or Western
analyses.
In vitro H1 kinase assays were
performed using immunoprecipitates in 30.0 µl of kinase buffer (50 mM HEPES (pH 7.0), 10 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 100 µM ATP, and 5.0 µCi of [ APC protein was resolved as described (18)
on 3.0% lo-melt agarose gels. Other proteins were resolved by
SDS-PAGE. After transfer to nylon membranes, proteins were visualized
by Western blotting using the monoclonal antibodies APC-Ab1 (Oncogene
Science), APC-Ab2 (Oncogene Science), or cdc2 (17) (Santa Cruz
Biotechnology) and enhanced chemiluminescence (Amersham).
The FLAG-APC protein was generated
by subcloning nucleotides 6422-8532 of the full-length APC
cDNA into the pFLAG-1 (International Biotechnologies, Inc.)
expression vector. Expression of the FLAG-APC protein is induced by
addition of isopropyl-1-thio- The final 200 carboxyl-terminal residues of APC contain two
consensus phosphorylation sites usually targeted by the CDKs (Fig. 1A). Analysis of the peptide
sequence of the full-length APC molecule revealed an additional nine
consensus sites, for a total of 11 (Fig. 1B); each of these
is conserved in the predicted mouse and rat APC sequences (data not
shown). The majority of these sites are located in the region that is
frequently deleted in mutant APC proteins. Populations of asynchronous,
G1/S boundary-synchronized, and M-phase-synchronized HCT116
colon carcinoma cells (which express a normal, full-length APC protein)
were labeled with [32P]orthophosphate in vivo
to look for cell cycle specificity in the incorporation of the isotope
in immunoprecipitated APC protein. APC was found to be phosphorylated
in each of these cell populations (Fig. 1C), with no
apparent cell cycle-specific incorporation of phosphate into the
protein.
We subsequently employed the histone H1 kinase assay to evaluate
potential CDK modification of APC. Histone H1 is an in vivo and in vitro substrate of CDKs, with the amount of phosphate
incorporated in the protein a quantitative indicator of CDK activity.
Immunoprecipitates from lysates of asynchronous populations of HCT116
cells contain an H1 kinase activity that coprecipitates with
APC-specific antibodies but not with the control antibody (Fig.
2). To ascertain whether this kinase
activity is cell cycle-regulated, HCT116 cells were synchronized into
G1/S boundary and M-phase populations, and the experiments
were repeated. The APC-associated kinase activity was most pronounced
during M-phase, with only a low level of activity found in
G1/S. Such mitosis-restricted H1 kinase activity is most characteristic of the CDK p34cdc2-cyclin B complex. In
vitro kinase assays using p34cdc2-specific
immunoprecipitates isolated from similar cell lysates revealed that the
kinase activity associated with APC and that of p34cdc2 are
most active in the same cell cycle periods. This cell cycle-specific kinase activity is not the result of differential expression of the APC
protein, since the protein is present in equivalent quantities throughout the cell cycle as shown by anti-APC immunoblots (Fig. 2B), nor is it a consequence of a generalized reduction in
cellular H1 kinase activity in the examined populations of cells (data not shown). A similar series of H1 kinase assays were performed using
immunoprecipitates from the colon carcinoma cell line SW480. This cell
line produces an APC protein truncated at residue 1338 (20) and is
predicted to contain only one CDK consensus site. The SW480 IgG- and
p34-specific immunoprecipitates display associated H1 kinase activity
profiles similar to those of HCT116. However, the immunoprecipitates of
truncated APC molecules show greatly reduced levels of kinase activity
when isolated from asynchronous and mitotic cell lysates (Fig.
2A), establishing a positive correlation between the
presence of APC-associated H1 kinase activity and the carboxyl-terminal
region of APC, which contains 10 additional CDK consensus
phosphorylation sites.
The p34cdc2-cyclin B complex is active during M-phase; its
association with APC may be one source of the H1 kinase activity
associated with APC in this segment of the cell cycle. To test this,
immunoprecipitates from nocodazole-synchronized HCT116 cell lysates
were incubated with increasing concentrations of olomoucine, a
nucleotide analog that specifically and competitively inhibits several
CDKs, including p34cdc2 (14). As expected, olomoucine inhibited
the phosphorylation of H1 histone catalyzed by the p34cdc2
immunoprecipitates and a purified recombinant p34cdc2-cyclin B1
enzyme complex (Fig. 3). The
APC-associated kinase activity also was inhibited by olomoucine at a
concentration equivalent to that required for inhibition of
p34cdc2 (Fig. 3), suggesting the physical association of an
active and inhibitable CDK with APC.
Finally, to determine whether APC and p34cdc2 are associated
within the same complex, APC protein was immunoprecipitated from lysates of asynchronous, G1/S boundary, and M-phase HCT116
cells and analyzed by Western blotting for associated p34cdc2
(Fig. 4). Immunoprecipitations with a
control antibody (IgG) from mitotic lysates or APC-specific antibodies
from asynchronous and G1/S lysates failed to detect
associated p34cdc2. Immunoprecipitation of APC from M-phase
lysates, however, revealed that p34cdc2 coimmunoprecipitated
with APC. The presence of p34cdc2 consensus sites in APC, its
association with an olomoucine-inhibitable H1 kinase activity, and the
physical interaction of APC and p34cdc2 strongly indicate that
the H1 kinase activity present in APC immunoprecipitates is
attributable to p34cdc2 kinase.
These data implicate APC as a catalytic target of p34cdc2,
although it is possible that other proteins interacting with APC could also be phosphorylated by p34cdc2 in addition to, or instead
of, APC. Microtubules, for example, associate both with APC (8, 9) and
p34cdc2 (21, 22). Alternatively, the presence and activity of
p34cdc2 may be required for the activation of another
coimmunoprecipitated kinase which may modify APC. Therefore, the
capacity of APC to act as a direct substrate of the human
p34cdc2-cyclin B1 active kinase complex was evaluated. A
fragment of APC encoding the residues 2141-2844 was cloned
in-frame with a FLAG epitope (FLAG-APC) and introduced into bacteria.
This region of APC contains 8 of the 11 putative CDK consensus sites
(Fig. 5A). Bacterially
expressed FLAG-APC protein was recovered by immunoprecipitation and
incubated with recombinant human p34cdc2-cyclin B1. The
FLAG-APC protein was phosphorylated by the kinase (Fig. 5, B
and C).
Although p34cdc2 is best recognized for its role in the
G2/M transition, we have now shown that its activity is
associated with APC during M-phase of the cell cycle. This kinase not
only phosphorylates APC in vitro but likely targets this
tumor suppressor as a substrate in vivo. Our data (Fig.
1C), as well as that of another recent study (17), have
demonstrated that APC is phosphorylated throughout the cell cycle and
that APC becomes hyperphosphorylated during M-phase, suggesting that
additional kinases may be responsible for phosphorylating APC. Indeed,
APC is known to be phosphorylated by two other kinases in
vitro: protein kinase A and glycogen synthase kinase-3 Substrates of the CDK family include diverse proteins, both nuclear and
cytoplasmic, that are critically involved in the regulation or
mechanics of the cell cycle (24). Furthermore, their phosphorylation by
CDKs often dictates functional status. By extension, the
phosphorylation of APC by p34cdc2 may result in similar cell
cycle-regulated modulations of APC-mediated tumor suppression by
altering a domain of APC that is frequently deleted by mutation. It
remains to be determined whether the loss of CDK consensus
phosphorylation sites is sufficient to disrupt APC function.
We thank Geoff Joslyn for the production of
the FLAG-APC expression construct, Daniel Hassett and Mike Howell for
their assistance in preparing the recombinant APC proteins, and Peter
Stambrook for critically reviewing the manuscript.
Molecular Genetics,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-catenin (4, 5), a protein of
unknown function designated EB1 (6), and the Drosophila tumor suppressor discs large (DLG) protein (7). APC localizes to the
microtubular network in vivo, while purified APC proteins promote the polymerization of tubulin into microtubules in
vitro (8, 9). The regions that interact with these proteins have been localized to the carboxyl half of APC and include the regions deleted by somatic and germline mutations of APC (10). Such mutations disrupt the ability of APC to regulate cytoplasmic levels of
-catenin (11, 12) and consequently lead to the constitutive activation of
-catenin/Tcf signaling (13, 14). Other effects of APC
mutation are less understood.
-catenin/Tcf signal (14). Interestingly, the patient with this
germline mutation has a phenotype similar to that of other patients
whose mutations have been shown to disrupt this down-regulation function. Therefore, this minimal region of APC must also harbor additional elements that facilitate the function or stability of this
tumor suppressor. In addition to binding the DLG protein, these 200 amino acids in the predicted human APC protein sequence include two
phosphorylation sites that are identical to the consensus target
sequence for the cyclin-dependent kinase (CDK) family of serine and threonine kinases. Such kinases catalyze the addition of
phosphate to their substrates in a cell cycle-specific manner. APC, a
phosphoprotein, has been shown to be modified exclusively on serine and
threonine residues (16, 17); therefore this modification could be the
result of CDK activity. In this report, we investigated the
relationship between APC phosphorylation and the CDKs.
Cell Culture
-32P]ATP using
10.0 µg of histone H1 (Boehringer Mannheim) as a substrate. For
olomoucine inhibition reactions, H1 kinase assays were performed with
the concentration of ATP decreased to 15 µM (19).
Olomoucine (Sigma) was added from a 10 mM stock to the
indicated concentrations prior to incubation. As a control, the
activity of 1.0 unit of recombinant human p34cdc2-cyclin B1
enzyme (New England Biolabs) was assayed in the presence of increasing
concentrations of olomoucine. Reactions were incubated at 30 °C for
10 min and terminated by the addition of an equal volume of 2 × SDS sample buffer and heating to 100 °C. Phosphorylation of H1 was
visualized by autoradiography following electrophoretic resolution on
10% polyacrylamide gels in the presence of SDS (SDS-PAGE). In
vitro phosphorylation of immunologically isolated bacterial proteins was performed with 5.0 units recombinant human
p34cdc2-cyclin B1 enzyme (New England Biolabs) in kinase buffer
for 10 min at 30 °C and terminated as described previously.
-D-galactopyranoside and
consists of the FLAG epitope grafted to APC residues 2141-2844. This
construct was introduced into Escherichia coli and
expressed, and protein was purified by immunoprecipitation using normal
mouse IgG or the FLAG-PROBE monoclonal antibody (Santa Cruz
Biotechnology).
Fig. 1.
Location of CDK consensus phosphorylation
sites in APC. A, sequence of the final 244 amino acids of
APC. The sequences that match the consensus site for CDK
phosphorylation ((S/T)PX(R/K)) are
underlined and bold. The star
designates the site of the most carboxyl-terminal truncating mutation
of APC, a four-nucleotide deletion that results in a novel reading
frame (lower sequence) and the creation of a stop codon 14 residues downstream (15). B, map of APC protein. The
numbers above the protein indicate the residue number in
increments of 500 amino acids; the black arrows indicate the
locations of the CDK consensus phosphorylation sites. The majority of
these sites are located in the region of APC frequently deleted by
mutation (10). N, amino terminus; C, carboxyl
terminus. C, APC is a phosphoprotein.
[32P]orthophosphate-labeled asynchronous (AS),
G1/S boundary (G1/S), or mitotic (M)
HCT116 cell lysates harvested from 100-mm plates were used for
immunoprecipitations with normal mouse IgG (IgG) or an
APC-specific antibody (APC) followed by resolution on an SDS-denaturing agarose gel. The left panel displays
autoradiography, the right a Western blot of the same gel
using the monoclonal antibody APC-Ab1 as a probe. There is no
quantitative difference in the phosphorylation of APC in the three
different cell populations. Numbers indicate approximate
molecular masses in kDa.
[View Larger Version of this Image (24K GIF file)]
Fig. 2.
Association of APC with a cell
cycle-regulated kinase activity. A, in vitro
histone H1 kinase assays. Phosphorylation of histone H1 was used to
measure the level of kinase activity associated in vivo with
the complexes immunoprecipitated with normal mouse IgG
(IgG), a p34cdc2-specific antibody (p34),
or an APC-specific antibody (APC) from HCT116 or SW480 cell
lysates. AS, asynchronous; G1/S, mimosine-treated cells; M, nocodazole-treated cells. B, Western
blot of APC immunoprecipitated from the same three cell populations
from cell lines HCT116 and SW480 using APC-Ab1 as a probe. All
immunoprecipitations were performed on normalized quantities of
protein, 0.50 mg per assay. APC, full-length APC protein;
APCmut, mutant truncated APC protein. Numbers
indicate approximate molecular masses in kDa.
[View Larger Version of this Image (67K GIF file)]
Fig. 3.
Olomoucine inhibits APC-associated kinase
activity. Recombinant p34cdc2-cyclin B1 enzyme (p34
enzyme) or p34-specific immunoprecipitates (p34 IP) and
APC-specific immunoprecipitates (APC IP) from HCT116 mitotic
cell lysates were used in H1 kinase assays in the presence of
increasing concentrations of olomoucine. Each data point represents the
mean ± S.D. of five independent kinase experiments and is derived
from the quantitation of H1 autoradiography relative to an uninhibited
control (represented as 0 µM olomoucine). Values for each
point are: p34 enzyme, 100, 94 ± 14, 76.6 ± 5.1, 65.4 ± 14.1, 49.7 ± 28.2, 26.8 ± 14.3, 24.5 ± 8.8, 11.6 ± 2.4; p34 IP, 100, 86.7 ± 18.7, 73.4 ± 26.2, 55.9 ± 17.1, 46.5 ± 19.1, 29.6 ± 14.6, 34.8 ± 3.5, 23.6 ± 9.5; APC IP, 100, 82.6 ± 22.2, 66.2 ± 19.7, 74.2 ± 25.4, 45.1 ± 18.4, 30.7 ± 3.6, 30.9 ± 6.7, 24.0 ± 12.9. Quantitation was performed with a Molecular
Dynamics PhosphorImager.
[View Larger Version of this Image (16K GIF file)]
Fig. 4.
APC associates with p34cdc2 in
vivo. Immunoprecipitates isolated with normal mouse IgG
(IgG), a p34cdc2-specific antibody (p34),
or an APC-specific antibody (APC) from asynchronous
(AS), mimosine-treated (G1/S), or
nocodazole-treated (M) HCT116 cell lysates were resolved on
a 10% acrylamide gel by SDS-PAGE and analyzed by Western blotting
using the monoclonal antibody cdc2 (17). The number on the
right indicates approximate molecular mass in kDa.
[View Larger Version of this Image (45K GIF file)]
Fig. 5.
In vitro phosphorylation of APC by
p34cdc2. Control antibody (IgG) and FLAG-PROBE
(FLAG) immunoprecipitates from uninduced () and
isopropyl-1-thio-
-D-galactopyranoside-induced (+)
lysates of bacteria harboring the FLAG-APC expression construct were
incubated with recombinant human p34cdc2-cyclin B1 complex in
the presence of [
-32P]ATP and resolved by SDS-PAGE on
an 8% gel. A, representation of bacterially expressed
FLAG-APC protein in comparison with full-length APC.
Asterisks denote CDK consensus phosphorylation sites, and numbers indicate residue position. B, Western
analysis using the monoclonal antibody APC-Ab2. C,
autoradiograph of the same blot. Numbers on the
right indicate approximate molecular masses in kDa.
[View Larger Version of this Image (69K GIF file)]
(23).
Phosphorylation of APC by these kinases regulates the binding,
turnover, and subsequent degradation of
-catenin in association with
APC and therefore controls
-catenin-transduced signaling. The
interaction of
-catenin with APC is unchanged in the
G1/S boundary and M-phase populations of HCT116 cells, suggesting that modification of APC by p34cdc2 does not
directly affect this association
quantitatively2 although the
rate of APC-mediated
-catenin degradation may be influenced. It also
remains possible that phosphorylation of APC at CDK consensus sites
alters its ability to interact with other proteins, and that a cell
cycle-specific change in such interactions mediates some of the
tumor-suppressing effects of APC.
*
This work was supported by the Council for Tobacco Research,
USA, Inc. (to J. G.), American Gastroenterological Association Foundation (to J. G.), Elsa U. Pardee Foundation (to J. G.),
National Institutes of Health Grant CA-63507 (to J. G.), Albert J. Ryan Foundation (to C. T.), and American Cancer Society (to
A. M. L. and J. J. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
513-558-0088; Fax: 513-558-8474; E-mail:
grodenjl{at}ucbeh.san.uc.edu.
1
The abbreviations used are: APC, adenomatous
polyposis coli; CDK, cyclin-dependent kinase; DLG, discs
large protein; PAGE, polyacrylamide gel electrophoresis.
2
C. Trzepacz and J. Groden, unpublished
data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.