(Received for publication, November 3, 1995; and in revised form, January 25, 1996)
From the
The MCC gene was isolated from the human chromosome
5q21 by positional cloning and was found to be mutated in several
colorectal tumors. In this study, we prepared specific antibodies and
detected the MCC gene product as a cytoplasmic 100-kDa
phosphoprotein in mouse NIH3T3 cells. Immunoelectron microscopic
analysis showed that the MCC protein is associated with the plasma
membrane and membrane organelles in mouse intestinal epithelial cells
and neuronal cells. The amount of the MCC protein remained constant
during the cell cycle progression of NIH3T3 cells, while its
phosphorylation state changed markedly in a cell cycle-dependent
manner, being weakly phosphorylated in the G/G
and highly phosphorylated during the G
to S
transition. Overexpression of the MCC protein blocked the serum-induced
cell cycle transition from the G
to S phase, whereas a
mutant MCC, initially identified in a colorectal tumor, did not exhibit
this activity. These results suggest that the MCC protein may play a
role in the signaling pathway negatively regulating cell cycle
progression.
Cell proliferation is controlled by both positive and negative regulators. A number of genes encoding positive regulators have been identified as oncogenes(1) . Most of the oncogene products are deregulated forms of cellular proteins that participate in the signal transduction cascade from the cell membrane receptors to the nucleus. On the other hand, several genes encoding negative regulators have been identified as tumor suppressor genes. Like the oncogene products, the tumor suppressor gene products include proteins localized in the plasma membrane, cytoplasm, and nucleus(2) . The best studied tumor suppressor gene products, pRB, p53, and WT1, are nuclear transcription factors, while DCC (deleted in colorectal cancer) has the attributes of a cell surface receptor. NF (neurofibromatosis) 1 and 2 share structural similarities with GTPase-activating protein and the ERM family proteins (3, 4, 5) , respectively, presumably functioning at the cytoplasmic face of the plasma membrane. In contrast to these proteins, the predicted amino acid sequences of MCC (mutated in colorectal cancer) and APC (adenomatous polyposis coli) are unique and have little sequence similarity to other proteins, providing few clues to its mechanism of action(6, 7, 8) .
The MCC gene was isolated from the human chromosome 5q21 by positional cloning and was found to be mutated in several colorectal tumors(6) . In addition, this gene was first suggested to be implicated in the development of familial adenomatous polyposis. However, further studies revealed that inactivation of another gene isolated from 5q21, APC, is responsible for the genesis of familial adenomatous polyposis(7, 8, 9, 10) . Moreover, APC has been found to be somatically mutated in the majority of sporadic colorectal tumors(11, 12) .
The MCC and APC genes are predicted to encode proteins of 829 and
2843 amino acids, respectively, with little homology to other known
proteins(6, 7, 8) . Interestingly, both
proteins contain several regions that have a high probability of
forming coiled-coil structures. Indeed, the APC protein has been shown
to form a stable homodimer via the amino-terminal part of the
molecule(13, 14) . Cell fractionation experiments and
immunohistochemical analysis suggested that APC is present as insoluble
aggregates in the cytoplasm (15) . Furthermore, it has recently
been reported that APC is associated with an adherence junction
protein, -catenin, suggesting that APC is involved in cell
adhesion(16, 17) . On the other hand, the function of
the MCC protein is still unknown. In the present study, we identified
and characterized the product of the MCC gene. We found that
the MCC gene product is a 100-kDa phosphoprotein localized in
the cytoplasm. Furthermore, we show that this protein has the potential
to negatively regulate the cell cycle transition from the G
to S phase.
Figure 1:
Identification of MCC. A, lanes 1-3, in vitro translation of MCC. A plasmid vector containing the MCC cDNA
downstream of the T7 promoter was transcribed and translated in
vitro in the presence of [S]methionine as
described under ``Experimental Procedures.'' In vitro translation products of MCC are shown in lane 1.
MCC was then immunoprecipitated with anti-MCC antibodies (lane
2) or anti-MCC antibodies that had been preadsorbed with an excess
of peptide antigen (lane 3). Lanes 4-12,
identification of MCC in vivo. COS-7 cells transfected with
the expression vector alone (pME18S vector) (lane 4) or MCC expression plasmid (pME18S-MCC) (lanes 5 and 6) were labeled with [
S]methionine for
4 h. NIH3T3 cells (lanes 7-12) were also labeled with
[
S]methionine (lanes 7-10) or
[
P]phosphate (lanes 11 and 12)
for 4 h. MCC was immunoprecipitated from cell lysates with anti-MCC
antibodies. Lanes 6, 8, 10, and 12,
anti-MCC antibodies were preadsorbed by an excess of the antigenic
peptide. The immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis followed by autoradiography. B, partial
peptide mapping analysis of MCC. MCC that had been immunoprecipitated
from [
S]methionine-labeled NIH3T3 cells (lanes 1-3) and that generated by in vitro translation (lanes 4-6) were excised from the gel,
partially digested with V8 protease (lanes 1 and 4, 2
µg/ml; lanes 2 and 5, 20 µg/ml; lanes 3 and 6, 200 µg/ml), and analyzed by SDS-polyacrylamide
gel (15%) electrophoresis.
Figure 4:
Level of expression and phosphorylation of
MCC during cell cycle progression. A, NIH3T3 cells were
growth-arrested by culturing in DMEM containing 0.5% calf serum for 48
h, and then stimulated with DMEM containing 10% calf serum to induce
synchronous cell cycle progression. At the times indicated after serum
stimulation, the cell cycle profile was determined by
fluorescence-activated cell sorting analysis of cellular DNA content.
The relative percentages of G, S, and G
or M
cells are listed at the bottom. B, NIH3T3 cells
synchronized as in A were harvested at the indicated times and
subjected to Western blotting analysis with anti-MCC antibodies. C, NIH3T3 cells synchronized as in A were labeled
with [
P]phosphate for 2 h before harvesting. MCC
was immunoprecipitated with anti-MCC antibodies and analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Randomly growing NIH3T3 cells were also
analyzed.
Figure 2:
Subcellular localization of MCC. NIH 3T3
cells labeled with [S]methionine (lanes 1 and 2) were separated into crude membrane (lanes 3 and 4), cytoplasmic (lanes 5 and 6),
and nuclear (lanes 7 and 8) fractions as described
under ``Experimental Procedures,'' and lysates of these
fractions were subjected to immunoprecipitation with anti-MCC
antibodies. The crude membrane fraction derived from subcellular
fractionation was resuspended in 0.1% SDS (lanes 9 and 10), 1% Nonidet P-40 (lanes 11 and 12), or
0.5 M NaCl (lanes 13 and 14). After 30 min
on ice, the insoluble material was pelleted by centrifugation, and the
supernatants (lanes 9, 11, and 13) and
pellets (lanes 10, 12, and 14) were
subjected to immunoprecipitation with anti-MCC
antibodies.
In the mouse intestinal epithelium, MCC was localized mainly along lateral cell borders of the epithelial cells (Fig. 3A). The cytoplasm of the epithelial cell showed weak immunoreactivity for MCC, whereas the apical brush border and nuclei showed no significant labeling. Immunoelectron microscopic analysis using colloidal gold-conjugated second antibodies clearly demonstrated a close association of MCC with lateral plasma membranes of the intestinal epithelial cells (Fig. 3B). Some gold particles were scattered in the cytoplasmic matrix. By contrast, control sections stained with anti-MCC antibodies that had been preabsorbed with antigenic peptide showed no immunoreactivity either in the immunofluorescence (Fig. 3C) or immunoelectron microscopic (Fig. 3D) analyses.
Figure 3: Localization of MCC in the mouse intestinal epithelium (A-D) and cerebellar cortex (E-H). A, C, E, and G, immunofluorescence microscopic analyses. B, D, F, and H, immunoelectron microscopic analyses using colloidal gold-conjugated secondary antibodies. A, lateral cell borders of the intestinal epithelial cells showed intense immunoreactivity for MCC. The cytoplasm of the epithelial cell is weakly positive. B, a number of gold particles were associated with the interdigitated lateral plasma membranes of the epithelial cells. Some gold particles were scattered in the cytoplasmic matrix. E, MCC is highly expressed in the molecular layer (m) of the cerebellar cortex. Purkinje cells (arrows) and cells in the granular layer (g) were also immunopositive. F, immunogolds were associated with the plasma membrane (arrows) and membrane organelles (arrowheads) of Purkinje cell (p) and nerve fibers (n). C, D, G, and H, as a negative control, anti-MCC antibodies that had been preabsorbed with antigenic peptide were used for staining. Scale bars: 10 µm in A, C, E and G; 100 nm in B, D, F, and H.
In the mouse cerebellar cortex, the molecular layer showed high MCC expression (Fig. 3E). Purkinje cells and granular layer cells were also immunoreactive for the anti-MCC antibodies. Immunoelectron microscopic analysis showed that MCC is mainly associated with the plasma membrane and membrane organelles in the neuronal cell bodies and nerve fibers in the molecular layer and granular layer cells (Fig. 3F). Some gold particles were also distributed within the cytoplasmic matrix of the neuronal cells, whereas no MCC was detected in the nuclei of the cerebellar cortex. Sections stained with anti-MCC antibodies that had been preabsorbed with antigenic peptide showed no immunoreactivity either in the immunofluorescence (Fig. 3G) or immunoelectron microscopic (Fig. 3H) analyses.
Figure 5:
Effect of overexpression of MCC on cell
cycle progression. Serum-starved NIH3T3 cells were microinjected with
the MCC or -galactosidase expression plasmid. Cells were
re-stimulated with serum and then visualized for MCC,
-galactosidase, or BrdUrd incorporation. The histogram shows the
percentage of BrdUrd-positive NIH3T3 cells expressing the injected
normal or mutant MCC cDNA. At least 90 cells were scored for each
experiment. MCC-R506Q, the mutant MCC cDNA encoding Gln in
place of 506-Arg; MCC-K233T-E234A, the mutant MCC cDNA
encoding Thr and Ala in place of Lys-233 and Glu-234, respectively. The
MCC cDNA was also microinjected into cells that had been arrested at
G
/S by aphidicolin treatment.
In the present study, we detected the MCC gene product as a 100-kDa phosphoprotein localized in the cytoplasm. This finding is consistent with the expected characteristics of the predicted amino acid sequence of the MCC protein, i.e. MCC consists of 829 amino acids and does not have any obvious membrane-spanning region or nuclear localization signals(6) . In several experiments, slowly migrating proteins were detected in addition to the main band of 100 kDa. It remains to be determined whether these are generated by modification or splicing, or whether these are related but different proteins.
Cell fractionation
experiments showed that most of the MCC protein is present in the
cytosol, i.e. the 100,000 g supernatant
fraction, but a proportion of MCC was also detected in the crude
membrane fraction. Immunoelectron microscopic analysis demonstrated
that MCC is associated with the plasma membrane and membrane organelles
in the mouse intestinal epithelial cells and neuronal cells. However,
treatment of the insoluble crude membrane fraction with 1% Nonidet P-40
did not solubilize MCC, suggesting that MCC is not a membrane protein,
but rather is complexed in an insoluble aggregate. This is consistent
with the fact that the MCC protein contains heptad repeats throughout
almost the entire length of the molecule. These features are similar to
those of the APC gene product; APC also possesses heptad repeats in the
amino-terminal region and is present in an insoluble
aggregate(15) . Importantly, APC has been suggested to form a
parallel, helical homodimer, as expected for a coiled
coil(13) . In addition, the wild type APC associates with
truncated mutant APC proteins in colorectal cancer
cells(14, 15) . However, our preliminary experiments
failed to detect homodimerization of MCC. Additionally, MCC did not
heterodimerize with APC.
While the amount of MCC was constant during
cell cycle progression, its phosphorylation state changed markedly in a
cell cycle-dependent manner, being weakly phosphorylated in
G/G
and highly phosphorylated during the
G
to S transition. These findings suggest that the function
of MCC is regulated by phosphorylation, similar to the case of pRB
whose function is inhibited by phosphorylation during the G
to S phase
transition(22, 23, 24, 25, 26, 27, 28, 29) .
Although MCC possesses several consensus sequences for phosphorylation
by protein kinase C and casein kinase II, identification of the
responsible kinases remains to be achieved.
Our microinjection
experiments showed that overexpression of MCC blocks serum-induced cell
cycle progression from the G to S phase. Thus, MCC may play
a role in the signaling pathway negatively regulating cell cycle
progression. In this regard, it is interesting that the MCC gene was previously found to be mutated in several colorectal
tumors(6) , although mutation of the APC gene is far
more frequent. In addition, it has been reported that the MCC loci frequently undergoes a loss of heterozygosity in esophageal
and lung cancers and that this occurs without significant correlation
to alterations in APC(30, 31) . Intriguingly,
a mutant version of MCC (MCC-R506Q), corresponding to that identified
in a colorectal tumor(6) , did not exhibit any cell
cycle-blocking activity, suggesting the biological importance of this
MCC mutation. Additionally, MCC-K233T/E234A, which was engineered to
have mutations in a region possessing an amino acid similarity to the G
protein-coupled m3 muscarinic acetylcholine receptor(6) , also
failed to induce cell cycle arrest. This finding suggests that this
region of MCC may play an important role in regulating cell cycle
progression.
Among the known tumor suppressor gene products,
p53(32, 33) , pRB(34, 35) , and WT1 (36, 37) have been shown to have a potential to
negatively regulate cell proliferation. These are all nuclear proteins
and are believed to regulate the expression of the genes whose products
are important for negative growth control. Recently, one of the
important targets of p53 has been identified as the gene encoding the
CDK inhibitor
p21(38, 39, 40, 41, 42) .
In addition to these tumor suppressor gene products, several different
classes of negative regulators of cell growth have also been reported,
including secreted proteins such as the transforming growth factor
proteins (reviewed in (43) ), a growth arrest-specific
membrane protein Gas1(44) , prohibitin(45) , and a
Ras-related transformation suppressor gene product K-Rev-1/Rap
1(46) . Compared to these negative regulators, MCC is unique in
its structure and subcellular localization. Additionally, MCC is highly
expressed in differentiated murine tissues, (
)such as
neuronal cells (Fig. 3), suggesting that MCC may play a role
besides cell cycle regulation. Thus, detailed analysis of the function
of MCC may give new insight into the mechanism of the regulation of
cell growth and differentiation.