Increased nuclear translocation of catalytically
active PKC-
during mouse colonocyte hyperproliferation
Shahid
Umar1,
Joseph H.
Sellin1,2, and
Andrew P.
Morris1,2
1 Department of Integrative Biology, Pharmacology, and
Physiology, and 2 Department of Internal Medicine, Division of
Gastroenterology, Hepatology, and Nutrition, University of Texas Health
Science Center at Houston, Medical School, Houston, Texas 77030
 |
ABSTRACT |
Protein kinase (PK) C-
is implicated in the control of colonic epithelial cell proliferation
in vitro. However, less is known about its physiological role in vivo.
Using the transmissible murine colonic hyperplasia (TMCH) model, we
determined its expression, subcellular localization, and kinase
activity during native crypt hyperproliferation. Enhanced mitosis was
associated with increased cellular 72-kDa holoenzyme (PKC-
,
3.2-fold), 48-kDa catalytic subunit (PKM-
, 3- to 9-fold), and 24-kDa
membrane-bound fragment (Mf-
, >10-fold)
expression. Both PKC-
and PKM-
exhibited intrinsic kinase
activity, and substrate phosphorylation increased 4.5-fold. No change
in cellular PKC-
/PKM-
expression occurred. The subcellular distribution of immunoreactive PKC-
changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas
proliferating cell nuclear antigen-positive cells exhibited elevated
cytoplasmic, lateral membrane, and nuclear staining. Subcellular
fractionation revealed increased PKC-
and PKM-
expression and
activity within nuclei, which preferentially accumulated PKM-
. These
results suggest separate cellular and nuclear roles, respectively, for
PKC-
in quiescent and mitotically active colonocytes. PKM-
may
specifically act as a modulator of proliferation during TMCH.
protein kinase C; cellular mitosis; mouse colon
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INTRODUCTION |
THE INTESTINAL
MUCOSA is a highly dynamic epithelium in which
proliferatory cells within lower regions of the crypts migrate along a
longitudinal axis to replace their senescent counterparts at the
mucosal surface. Progression along this axis is associated with
increased differentiation and phenotypic maturation. This process of
mucosal homeostasis is normally tightly regulated to ensure that
proliferative activity 1) balances the requirements for
functionally mature cell types and 2) maintains mucosal
integrity as senescent cells apoptose/exfoliate into the lumen.
However, during mucosal hyperplasia and neoplasia a dramatic imbalance occurs, with proliferation predominating. Indeed, hyperproliferation and a concomitant shift in the number (hyperplasia) of immature colonocytes into compartments close to the mucosal surface are frequent
cytokinetic abnormalities observed in the macroscopically normal mucosa
of patients with colorectal neoplasia (33,
35, 42). Malignant transformation is a
multistage process characterized both by activating mutations in
protooncogenes (32) and by genetic alterations that
inactivate tumor suppressor genes (45). In addition,
accumulating evidence from both human (21,
23, 25) and animal (5,
15, 46) studies suggests that epigenetic changes in protein kinase (PK) C-dependent cellular signal transduction are integral to this process (reviewed in Ref. 48).
PKC is a multigene family consisting of at least 11 distinct
lipid-regulated protein-serine/threonine kinases that play pivotal roles in signal transduction and growth control (8). Each
isoform possesses unique structural properties as well as a distinct
tissue and cell distribution, supporting the concept that individual isoforms perform specific roles in cellular signaling. The isoforms can
be divided into three groups: 1) classical PKCs that are
regulated by Ca2+ and diacylglycerol (DAG) (
,
1,
2, and
), 2) novel PKCs that are Ca2+
independent but activated by DAG (
,
,
,
, and µ), and
3) atypical PKCs (aPKCs) that are both Ca2+ and
DAG independent (
,
, and
). A number of studies have
shown a reduction in PKC activity in human colonic adenocarcinomas
compared with normal adjacent mucosa (23, 25,
26). Furthermore, several laboratories using the
procarcinogen 1,2-dimethylhydrazine (DMH) or its more proximate
metabolite azoxymethane (AOM) to induce experimental colon cancer
have demonstrated changes in PKC within both premalignant and malignant
colonocytes. During the premalignant phase, translocation of
PKC to the particulate fraction is observed, followed by subsequent
decreases in the total biochemical activity (5,
15). It remains uncertain, however, whether initial
activation or later downregulation is the most important PKC-signaling
event promoting neoplastic change or whether these events
reflect an unsuccessful attempt by these cells to negatively
modulate proliferatory signals generated by activated oncogenic
pathways. Because transmissible murine colonic hyperplasia (TMCH) has
been identified as a condition that promotes DMH-induced carcinogenesis
in the mouse (2, 4) and PKC-
signaling has
been linked to cellular growth control, the escape of cells from
apoptosis, and anchorage-independent growth (7,
8, 50, 52), we used this model
to record the expression levels, location, and biochemical activation
status of this kinase. Our results suggest a physiological role for
PKC-
and, more importantly, its proteolytic fragment PKM-
in
nuclear signaling events regulating hyperproliferation within the
native colonic mucosa.
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MATERIALS AND METHODS |
Antibodies.
Polyclonal rabbit anti-PKC-
anti-peptide antibodies were procured
from Santa Cruz Biotechnology (SC-216/7282; Santa Cruz, CA) and Upstate
Biotechnology (06-475; Lake Placid, NY) and were also received as a
gift (C-14) from Dr. Todd Sacktor, State University of New York
Downstate Medical Center (Brooklyn, NY) (see Table 1).
Monoclonal anti-PKC-
antibody was purchased from Transduction Laboratories (San Diego, CA), and proliferating cell nuclear antigen (PCNA) antibody was purchased from Signet Laboratories (Dedham, MA).
FITC- and Texas red-conjugated antibodies for immunofluorescence studies were procured from Molecular Probes (Eugene, OR).
In vivo model for hyperplasia.
TMCH was developed in Swiss-Webster mice (15-20 g; Harlan Sprague
Dawley, Houston, TX) by oral inoculation with a 16-h culture of
Citrobacter rodentium, formerly
Citrobacter freundii biotype 4280 (3). Age- and sex-matched control mice received sterile culture medium only. Infected and normal mice were housed separately in
microisolator cages in different rooms of the animal house facility. To
determine the changes in the gross morphology of the
Citrobacter-infected colonic mucosa, animals were killed by cervical dislocation after 2 wk of infection and their distal colons
were removed, flushed with saline, embedded in OCT compound (Miles,
IN), and cryopreserved in liquid nitrogen before sectioning and
staining with hematoxylin and eosin.
Immunofluorescence.
Distal colonic samples from normal and Citrobacter-infected
animals were attached to a paddle and immersed in Ca2+-free
standard Krebs buffered saline (in mM: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4, 1.8 Na2HPO4,
10 glucose, and 10 EDTA) at 37°C for 10-20 min, gassed with 5%
CO2-95%O2. The crypts were then separated from
the surrounding connective tissue/muscle layers by mechanical vibration
for 30 s into ice-cold KCl HEPES saline (in mM: 100 K-gluconate,
20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 5 Na-pyruvate) and 0.1% BSA, resembling the intracellular medium. Suspended crypts were then deposited (1,200 rpm for 1 min) onto
poly-L-lysine-coated microscope slides using a Shandon Cytospin cell preparation system. Immunolocalization studies were carried out by permeabilizing the crypts for 5 min in PBS containing 0.5% Triton X-100 (PBS-Triton). Nonspecific sites were blocked for
1 h with 3% BSA in PBS-Triton (blocking solution). Crypts were
stained for the PKC-
isoform using polyclonal antibody diluted in
blocking solution at 1:100 and 1:500, respectively. After incubation with appropriate antibodies either at room temperature for 1 h or
at 4°C overnight, the slides were washed and incubated with goat
anti-rabbit secondary antibody conjugated with either Texas red (PCNA)
or FITC (PKC) and diluted in blocking solution at 1:500 for 1 h at
room temperature or overnight at 4°C. Between washes, slides were
incubated for 30 min in PBS containing 1% BSA. Control slides were
incubated without the primary antibody or with isoform-specific primary
antibody in the presence of appropriate antigenic peptide. Fluorescence
was viewed using a Noran confocal laser scanning microscope (Noran
Instruments, Middleton, WI) equipped with an argon laser and
appropriate optics and filter modules for Texas red/FITC detection.
Digital images were obtained at ×400, ×800 and ×1,200 using a
high-numerical-aperture lens (Nikon ×40, 1.4 N/A). A z-axis
motor attached to the inverted microscope stage was calibrated to move
the plane of focus at 0.4-µm steps through the sample. Collected 8- or 16-bit color green-encoded images at 512 × 480 resolution were
then stored on a mass storage device (removal rewriteable optical hard
disk) and later volumetrically reconstructed using the
Image-1/Metamorph 3-D module (Universal Imaging, West Chester, PA).
Preparation of nuclear extracts.
Nuclear extracts were prepared from the distal colons of normal and
Citrobacter-infected mice essentially as described by Zhang
and colleagues (51) with some modifications. Briefly, tissues were cut and rinsed in saline A [in mM: 20 Tris · HCl, (pH 7.0), 137 NaCl, and 5 KCl] and homogenized in
buffer A [in mM: 15 Tris · HCl (pH 7.0), 60 KCl, 15 NaCl, 2 EDTA, 0.5 EGTA, 1 dithiothreitol (DTT), 0.15 spermine, 0.5 spermidine, 0.4 PMSF, and 2 benzamidine], 0.25 M sucrose, and 1 µg/ml each of chymostatin, leupeptin, and pepstatin A. The homogenate
was mixed with 2 vols of buffer B (buffer A with
2.3 M sucrose), layered on top of buffer C (buffer
A with 1.8 M sucrose), and centrifuged at 25,000 rpm for 60 min at
4°C in an SW27 rotor. The nuclear pellets were resuspended in
buffer D [in mM: 100 KCl, 10 Tris · HCl (pH 8.0), 2 MgCl2, 0.1 EDTA, 1 DTT, 0.4 PMSF, and 2 benzamidine and 1 µg/ml each of chymostatin, leupeptin, and pepstatin A]. The
suspension was extracted with 0.1 vol of 4 M
(NH4)2SO4 on a rotator for 30 min and then centrifuged at 30,000 rpm for 45 min in an SW40 rotor. The
protein in the supernatant was precipitated with 0.3 g/ml (NH4)2SO4, pelleted, resuspended in
buffer E [in mM: 20 HEPES (pH 7.8), 100 KCl, 0.2 EDTA, 0.5 DTT, 0.5 PMSF, and 2 benzamidine and 1 µg/ml each of chymostatin,
leupeptin, and pepstatin A] and dialyzed against buffer E
for 4-6 h. The dialysates were centrifuged for 5 min to remove the
precipitates. Protein concentrations were determined, and extracts were
used immediately for immunoprecipitation as described in PKC-
activity assay or frozen in liquid nitrogen and stored at
70°C.
Tissue preparation and Western blot analysis.
Normal and Citrobacter-infected animals were killed by
cervical dislocation 0, 1, 3, 6, 9, 12, and 15 days after infection, and their colons were removed and flushed with ice-cold PBS.
Homogenates were prepared from whole distal colon as well as isolated
crypts from a set of three animals for each infection in a buffer
consisting of 50 mM Tris · HCl, pH 7.5, 0.25 M sucrose, 2 mM
EDTA, 1 mM EGTA, 0.5% Triton X-100, 25 µg/ml each of leupeptin,
aprotinin, and pepstatin, 1 µg/ml soybean trypsin inhibitor, 50 µM
sodium fluoride, 50 µg/ml PMSF, and 10 mM
-mercaptoethanol. The
homogenate was centrifuged at 15,000 g for 15 min, and clear
supernatant was saved as total tissue extracts. The soluble and
particulate fractions from normal and Citrobacter-infected
animals were prepared by homogenizing the whole distal colon or
isolated crypts in the homogenizing buffer without Triton X-100.
Cytosolic and membrane fractions were separated by sedimentation at
100,000 g in a centrifuge (TL-100, Beckman Instruments,
Fullerton, CA) for 30 min. The resulting pellet was extracted in
homogenizing buffer containing 1% Triton X-100, and the membrane
subcellular fraction was recovered as the supernatant after
centrifugation. Total tissue extracts or subcellular fractions
(50-100 µg protein/lane) were subjected to 10% SDS-PAGE and
electrotransferred onto the nitrocellulose membrane. Transfer
efficiency was evaluated by backstaining gels with Coomassie blue
and/or by reversible staining of the electrotransferred protein
directly on the nitrocellulose membrane with Ponceau S solution; no
variability in transfer was noted. Membranes were blocked with 5%
nonfat dried milk in 20 mM Tris · HCl and 137 mM NaCl, pH 7.6 (TBS) for 1 h at room temperature and then at 4°C overnight. PKC
isoforms were detected by incubating the nitrocellulose membranes for
2 h with anti-PKC isoform antibodies at 0.5-1.0 µg/ml in
TBS containing 0.1% Tween 20 (TBS-Tween; Sigma Chemical). After being
washed in TBS-Tween, membranes were incubated with horseradish
peroxidase-conjugated goat-anti-rabbit IgG diluted 1:5,000 in 1%
milk-TBS-Tween. After washes, immunoreactivity was detected
using the enhanced chemiluminescence detection system (Amersham,
Arlington Heights, IL) according to the manufacturer's instructions.
PKC-
activity assay.
PKC-
activity assay was carried out after immunoprecipitating either
nucleus-free cellular or purified nuclear isolated crypt extracts from
the distal colon of normal and infected mice. In brief, extracts were
normalized for protein concentration and precleared for 1 h at
4°C with 30 µl of protein A-coated Sepharose beads.
Immunoprecipitation was performed at 4°C by incubating the fractions
for 2 h with polyclonal anti-PKC-
antibody and then for 1 h with 50 µl of protein A/G-Sepharose beads. Control experiments were
performed by carrying out the immunoprecipitations in the presence of
the immunizing peptides for PKC-
. After extensive washing, the
immunoprecipitate was resuspended in 50 µl of a buffer containing 50 mM Tris · HCl (pH 7.5), 5 mM MgCl2, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM each of sodium
orthovanadate, sodium pyrophosphate, and NaF. The kinase reaction for
autophosphorylation was started by addition of 50 µM
[
-32P]ATP at 37°C, and the phosphorylation was
stopped by addition of 50 µl of reducing Laemmli sample buffer.
Substrate phosphorylation was started by addition of 50 µM
[
-32P]ATP at 37°C using PKC-
pseudosubstrate
derivative (ERMRPRKRQGSVRRRV) as substrate for PKC-
in the presence
or absence of inhibitor peptide. Stock solutions of lipids in ethanol
and dilutions in kinase buffer were freshly prepared for each
experiment from lyophilized aliquots. Phosphorylation was stopped by
addition of reducing Laemmli buffer, and proteins were separated on
7.5% or 10% SDS-PAGE, transferred onto the nitrocellulose membrane,
and analyzed by autoradiography as well as by Western blotting.
 |
RESULTS |
Establishment of model.
TMCH is an infectious disease of mice caused by Citrobacter
rodentium and characterized by significant epithelial cell
proliferation within the mucosa of the descending colon with or without
an inflammatory axis depending on the genetic background
(3). In adult Swiss-Webster mice, crypt hyperplasia is not
associated with significant changes in inflammatory status, and, at the
time of the maximal hyperproliferatory response (2 wk after infection),
this strain of bacteria no longer colonized the gut (1).
Isolated distal colonic crypts from postinfection day 12 Citrobacter-inoculated mice showed increased abundance of
PCNA (6-fold after normalization with
-actin, n = 6 mice) compared with controls (Fig.
1A), and immunofluorescent PCNA staining within the normal crypt base was redistributed throughout the TMCH crypt axis (Fig. 1B). We have reported (43a) that
apoptosis, a measure of programmed cell death, was similar in both
normal and Citrobacter-infected crypts.

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Fig. 1.
Measurement of proliferating cell nuclear antigen (PCNA)
abundance in cellular extracts of distal colonic crypts isolated from
normal (N) and day 12 transmissible murine colonic
hyperplasia (TMCH) (H) mice. A: Western blot analysis using
anti-PCNA monoclonal antibody normalized to -actin (see
MATERIALS AND METHODS). TMCH was associated with a 6-fold
increase in cellular PCNA expression. B: in the same sample,
the longitudinal axis of TMCH crypts revealed enhanced PCNA
immunofluorescence staining (n = 7 individual
experimental observations).
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PKC-
abundance and activity changes occur during mucosal
hyperproliferation.
During TMCH, enhanced proliferative activity within colonic crypts is
linked to selective activations in two [conventional (c) (PKC-
1)
and novel (n) (PKC-
)] of seven phorbol ester-sensitive PKC isoforms
detected in the colonic mucosa (43b). In this study, we sought
to determine whether the expression levels and activation status of
aPKC-
changed.
aPKC-
isoform expression in purified crypt extracts during TMCH.
Colonic crypts express PKC-
(38, 44). The
availability of isozyme-specific antisera for PKC-
detection
prompted us to measure the expression levels of this aPKC.
Triton-solubilized extracts were prepared from isolated, purified
crypts from animals 0, 1, 3, 6, 9, 12, and 15 days after Citrobacter infection and analyzed by Western blotting with
a panel of isozyme-specific polyclonal anti-PKC-
antibodies.
Specificity was determined by competitive blotting with corresponding
immunizing peptides, and the protein concentrations were normalized by
densitometry to
-actin (to account for differences in gel loading).
Basal levels of aPKC-
expression in isolated crypt extracts were
similar to those of brain [ratio = 1.0 ± 0.1 (SD);
n = 6 animals in duplicate] and were characterized by
the presence of more than one immunoreactive species (Fig.
2).

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Fig. 2.
Mucosal hyperproliferation was associated with increased
cellular atypical (a) protein kinase C (PKC)- expression.
Left, representative Western blot of isolated crypt cellular
extracts collected from normal (N) and day 12 TMCH (H) mouse
distal colon. Immunoreactivity was recorded at 72/76 (bands
A, A*), 46/48 (bands B, B*), and
26/28 (bands C, C*) kDa.
Right, blots made over the first 15 days after
Citrobacter infection were stripped and probed for
housekeeping protein -actin to create a bar graph of relative
optical density (Rel. O.D.; each time point represents the mean ± SD of 2 or 3 experiments performed in duplicate). All
immunoreactive bands increased their cellular expression during TMCH;
some bands were barely detectable (A*, B*) or
undetectable (C, C*) in normally proliferating
crypts. Specificity was confirmed with a panel of anti-PKC- specific
peptide antibodies and their corresponding immunogenic peptides (Table
1).
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The C-20 rabbit anti-rat PKC-
polyclonal antibody (Table
1) used for most of the Western blotting
and immunofluorescence aspects of this study detected an immunospecific
band at 72 kDa (band A, Fig. 2, left) which was
accompanied by a very much fainter band at 76 kDa in
hyperproliferating, but not normal, crypt extracts (band A*,
Fig. 2, left). Additionally, immunoreactive doublets were
recorded at 46/48 kDa and 26/28 kDa (bands B/B* and
C/C*, Fig. 2, left). The relative expression of
each band (~72, <48, and <29 kDa), increased significantly during
TMCH (Fig. 2, right). Values quantified
relative to
-actin at postinfection day 12 at the time of
the peak hyperproliferatory response were 72 (band A = 3.2-fold), 46 (band B = 3.3-fold), 48 (band B* = 9-fold), 26 (band C = >10-fold), and 28 (band
C* > 10-fold) kDa (P < 0.01, n = 6 animals in duplicate). The time course of increased expression was
the same for all bands. However, bands B*, C, and
C* were only detected after postinfection day 6,
and they continued to rise in parallel with bands A and
B (Fig. 2, right). Band A* was too
faint to detect on most gels and was not subjected to further analysis.
Compared with crude brain homogenates run on the same gel (data not
shown), overall levels of PKC-
holoenzyme were similar and
hyperproliferating colonocyte PKM-
levels were 5.5-fold higher.
The authenticity of the immunodetected bands at 72, 48, and <29 kDa
was confirmed by using a panel of different PKC-
antibodies together
with the relevant competing peptide (Table 1). All three molecular mass
species (72, 46/48, and 26/28 kDa) were recognized both by a goat
anti-rat COOH-terminal polyclonal antibody that cross-reacts with
murine PKC-
and the closely related
-isoform (C-20, Table 1) and
by a more selective rabbit anti-rat COOH-terminal antibody (C-15, Table
1), with no reported cross-reactivity with cPKC-
or the murine
aPKC-
/
homologues (9, Table 1). However, band A* was
not detected by a similar and closely related anti-rat PKC-
peptide
antibody (C-14, Table 1) mapped to a slightly shorter region of the
COOH-terminal domain (37). When extracts were probed with
an NH2-terminal goat anti-human PKC-
selective antibody, non-cross-reactive with PKC-
(29), only the 72-kDa band
was detected (N-17, Table 1). Immunocompetition with recombinant peptides against which C-20 and N-17 antibodies were made confirmed the
immunospecificity of the 72-, 46/48-, and 26/28-kDa bands, respectively
(Table 1). Further evidence supporting the notion that the 46/48-kDa
immunoreactive species were closely related to the 72-kDa holoenzyme
was found when using the rabbit C-14 anti-rat PKC-
COOH-terminal
antibody, which has been shown to react with both PKC-
holoenzyme
and its catalytically active 51-kDa proteolytic breakdown product,
PKM-
(37). In this instance, only band A (72 kDa) and bands B and B* (46/48 kDa) were detected.
Because PKC-
closely resembles PKC-
, we next used anti-PKC-
antibody to check whether PKC-
was contributing to PKM expression during TMCH. When nitrocellulose membranes probed with PKC-
C-20 antibody (Fig. 3A) were
stripped and probed for PKC-
with a monoclonal antibody specific for
its catalytic domain, very modest (<1.1-fold, n = 3)
increases in 72-kDa PKC-
abundance were observed (Fig. 3B). However, no PKM-
was detected in normal or
hyperproliferating crypts; PKC-
was not further investigated.

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Fig. 3.
Mucosal hyperproliferation did not affect PKC-
expression. C-20 anti-PKC- (A)- and anti-PKC-
(B)-specific antibodies were used to probe normal (N) and
day 12 (H) crypt cellular extracts by Western
blotting (see MATERIALS AND METHODS). PKC-
immunoreactivity increased during TMCH, whereas PKC- expression
exhibited only a very modest change. No detectable PKM-
(bands B, B*) was recorded during
TMCH.
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The crypt epithelium therefore contained both PKC-
holoenzyme and
PKM-
, together with a smaller immunoreactive species related to the
COOH-terminal catalytic domain of PKC-
. Our studies with immunospecific cPKC and nPKC antibodies failed to resolve
similar-molecular-weight bands (data not shown). The crypt-specific
rise in aPKC-
cellular expression indicated that, along with
cPKC-
and nPKC-
isoforms (43b), PKC-
may participate in
Citrobacter-induced colonocyte hyperproliferation.
Effect of mucosal hyperproliferation on PKC-
translocation.
Because alterations in the subcellular compartmentalization of PKCs can
be a surrogate and/or marker of activation, the distribution of PKC-
was assessed in the soluble (cytosolic) and particulate subcellular
fractions of colonocytes at 12 days after Citrobacter infection. Analysis of the complex pattern of subcellular
PKC-
partitioning by Western blotting of isolated crypt extracts
from normal and day 12 Citrobacter-infected
animals revealed (Fig. 4,
left) that the membrane-to-cytoplasmic partitioning ratio
(Rm:c) for PKC-
bands A and B/B*
decreased during TMCH when PKC-
expression increased. Values
relative to normally proliferating crypts were 2.4 ± 0.1-, 2.1 ± 0.1-, and 3.0 ± 0.2-fold lower, respectively, and
were significantly different (Fig. 4, right;
P < 0.05, n = 6 animals in duplicate).
In contrast, band C and C* Rm:c
increased >10- and 30-fold, respectively (Fig. 4, left).
This complex pattern of decreased PKC-
holoenzyme and PKM-
membrane translocation together with almost complete band C
membrane association was unexpected considering that movement of PKC
from cytosol to intracellular membranes is assumed to predict
activation. We therefore reasoned that increased cellular expression of
PKC-
during TMCH was associated with the redistribution of this
isoform to other locales within the cell.

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Fig. 4.
Mucosal hyperproliferation was associated with selective
alterations in PKC- immunoband membrane association.
Left, representative Western blot of PKC-
immunoreactivity in membrane-to-cytosolic (m:c) fractions of normal (N)
and day 12 TMCH (H) distal colonic crypts. Right,
corresponding histogram of m:c partitioning ratio of PKC-
bands A (72 kDa), B/B* (46/48 kDa),
and C/C* (26/28 kDa). Although the membrane
association of bands A and B/B*
decreased during TMCH, bands C/C* were almost entirely
membrane associated (values were averaged from 3 independent
experiments ± SD).
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Nuclear PKC-
/-
partitioning during crypt hyperproliferation.
Substantial evidence indicates a role for PKC in linking cell plasma
membrane receptor signaling, particularly PKC-
, to events occurring
at the genome level (10, 49). Because
cellular mitosis is a nuclear event, activated PKCs involved in the
regulation of colonic proliferation may redistribute into this
organelle during TMCH. Purified nuclei and nucleus-free cellular
fractions from normal and day 12 Citrobacter-inoculated mouse distal colons were probed for
PKC-
and PKC-
expression using the anti-PKC-
and C-20
anti-PKC-
antibodies (Fig. 5).

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Fig. 5.
PKC- is nuclear translocated in hyperproliferating
crypts. Anti-PKC- (A)- and C-20 anti-PKC-
(B)-specific antibodies were used to probe whole cell
(lane 1), nuclear free supernatant (lane 2), and
purified nuclear (lane 3) extracts by Western blotting (see
MATERIALS AND METHODS). Significant PKC- nuclear
immunoreactivity was not detected. However, 72- and 46/48 kDa
immunoreactive PKC- bands were present in the nuclear extracts and
levels were higher than in nucleus-free cellular extracts (similar
amounts of protein were run on the gels). All bands were also competed
out with 1:1 molar immunizing peptide (+P). C: when PKC-
immunoreactivity in purified nuclear extracts from normal (N) and
day 12 TMCH (H) crypts were compared, the expression of all
detected PKC- immunobands increased. Hyperproliferating crypt nuclei
accumulated more of the 46-kDa form of PKC- (band B) than
either 48-kDa (band B*) or 72-kDa (band A) forms.
Band B was itself resolved into 2 separate bands on 7% gels
(shown).
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PKC-
immunoreactivity was barely detectable in Western blotted
purified nuclear extracts in hyperproliferating mucosa (Fig. 5A). However, strong immunoreactivity was observed for
PKC-
in hyperproliferating crypt nuclei probed with the C-20
antibody (Fig. 5B). Both PKC-
band
A (72 kDa) and bands B*/B (46/48 kDa) were
detected; band C (<29 kDa) was seen only faintly on
overexposed gels. When similar amounts of protein were run on the gel,
the levels of both PKC-
and PKM-
were higher in purified
hyperproliferating crypt nuclei than in total cell extracts. This
difference was most pronounced for PKM-
, in which nuclear levels
were 2.2 ± 0.1-fold higher than total cellular levels (Fig.
5B). Specificity was confirmed by competitive blotting in
the presence of immunizing peptide (Fig. 5, A and
B). When nuclear levels of both PKC-
holoenzyme and
PKM-
species were compared between normal and TMCH crypts, hyperproliferation was clearly associated with elevated nuclear accumulation of both bands A and B (Fig.
5C). On the same Western blots, an additional immunoreactive
species running at 42 kDa was also recorded. These findings
demonstrated that a substantial amount of both PKC-
and PKM-
was
localized to the nucleus and that partitioning of these species into
the nucleus accompanied the rise in cellular PKC-
and PKM-
expression recorded during TMCH (Fig. 2). However, these findings did
not determine whether nuclear accumulated PKC-
or PKM-
was
catalytically active. The same C-20 antibody was used to
immunoprecipitate PKC-
in cellular extracts for the subsequent
measurement of enzymatic activity.
Immunoprecipitable PKC-
enzyme activity.
Activation of PKC-
is accompanied by intramolecular
autophosphorylation (24, 43). To determine
whether nuclear translocated PKC-
/PKM-
was catalytically active,
autophosphorylation of PKC-
was measured in protein
G-agarose-recovered immune complexes. Figure
6 is a representative blot showing both
the immunoprecipitation protocol used and our control for C-20
anti-PKC-
antibody specificity.

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Fig. 6.
PKC- immunoprecipitated from nucleus-free
(A) and purified nuclear (B) fractions from
normal (N) and day 12 TMCH (H) crypts exhibited subtle
alterations in molecular mass. After immunoprecipitation with the C-20
anti-PKC- antibody in the absence ( ) or presence (+) of immunizing
peptide, PKC- immunoreactivity was detected by Western blotting with
the same antibody. Although the apparent molecular mass of the 72-kDa
band did not differ between fractions, the 48-kDa form of PKC-
preferentially accumulated in the nuclear-free cytoplasm and the 46-kDa
form of PKC- preferentially accumulated in the nucleus, which
resolved as a doublet on 7% gels (shown). All PKC- bands were
competed away with immunizing peptide; the remaining signal ( )
represents IgG heavy-chain fragments. This protocol was adapted for the
measurement of intrinsic and extrinsic kinase activity.
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After immunoprecipitation and transfer onto nitrocellulose membranes,
PKC-
-immunoreactive bands A and B* were
detected by Western blotting with the same antibody (Fig. 6). In
nucleus-free cellular immunoprecipitates, bands recorded at 72 and 48 kDa were competed away with immunizing peptide (Fig. 6A,
n = 6 animals in duplicate). In purified nuclear
immunoprecipitates (Fig. 6B), bands were detected at 72 and
46/48 kDa that were likewise lost on competition with 1:1 molar
immunizing peptide (n = 6 animals in duplicate). This
immunoprecipitation protocol confirmed our earlier findings that both
PKC-
holoenzyme and, more importantly, a PKM-
-like species were
accumulated in hyperproliferating nuclei. This protocol was modified to
measure the autophosphorylation status of the PKC-
holoenzyme
(band A) and PKM-
(band B*/B) (Fig.
7).

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Fig. 7.
Modulation of PKC- activity during TMCH.
Left, PKC- immunoprecipitated in nucleus-free cellular
(A) and purified nuclear (B) fractions from
day 12 TMCH crypts were used to estimate autophosphorylation
levels (see MATERIALS AND METHODS). After nitrocellulose
membrane autoradiography (i), PKC- was Western blotted
(ii). Intrinsic kinase activity corresponding to nonnuclear
(Ai, 72 and 48 kDa) and nuclear (Bi, 72 and 46 kDa) forms of PKC- were clearly detectable and were significantly
reduced when the immunoprecipitation protocol was performed in presence
(+) of immunizing peptide. Right, immunoprecipitated PKC-
from identical fractions was used to assay extrinsic kinase activity.
Reconstituted kinase was incubated with modified PKC-
pseudosubstrate peptide (alanine to serine modification) in presence or
absence of PKC- inhibitor peptide and [ -32P]ATP
(see MATERIALS AND METHODS). Substrate phosphorylating
activity, calculated as picomoles per minute per milligram of protein,
was found to increase 4.5- and 3.4-fold in the cellular and purified
nuclear extracts, respectively (values are means ± SD from 3 individual experiments).
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To detect intrinsic immunokinase activity, the immunoprecipitates were
incubated with [
-32P]ATP and phosphatidylserine before
SDS-PAGE and transfer to nitrocellulose membrane (see MATERIALS
AND METHODS). Shown in Fig. 7 are autoradiographs of nucleus-free
cellular and purified nuclear immunoprecipitates from TMCH crypts
together with their corresponding Western blots (Fig. 7,
left). Both sources exhibited active kinase at 72 kDa, corresponding to PKC-
holoenzyme. However, the intrinsic activity of
bands B/B* was different in that signal was recorded at 48 kDa in nucleus-free cellular extracts and at 46/48 kDa in purified nuclear extracts. This difference was mirrored by PKM-
mobility differences on corresponding Western blots. Thus crypt
hyperproliferation was accompanied by activation of PKC-
holoenzyme
with an identical mobility in both subcellular compartments and
compartment-specific PKM-
activation shown by subtle changes in gel
mobility. The autophosphorylation levels of both bands were
considerably reduced when immunoprecipitation was performed in the
presence of the immunizing peptide (Fig. 7, left;
n = 6 animals in duplicate). When the amount of
autophosphorylation per
-isoform molecule was compared, 48-kDa
cytoplasmic PKM-
was more autonomously active than lower-mass
nuclear PKM-
. No difference was seen for the holoenzyme.
In addition to autophosphorylation, substrate phosphorylation of
PKC-
pseudosubstrate peptide derivative by immunoprecipitated PKC-
was recorded (Fig. 7, right). TMCH was associated
with a 4.5-fold increase in PKC-
activity in nucleus-free cellular
extracts from isolated crypts (n = 3) and a
corresponding 3.4-fold increase in PKC-
activity in nuclear extracts
from the same source (n = 3, extracts made from
day 12 TMCH animals). These studies confirmed the presence
of catalytically active 72-kDa PKC-
and 46/48-kDa PKM-
in
purified nuclear fractions isolated from hyperproliferating colonocytes.
Immunofluorescence staining of PKC-
.
We next examined the immunolocalization of PKC-
in isolated crypts
to understand whether increased cellular expression, decreased cytosolic membrane partitioning, and increased nuclear kinase activity
during TMCH were paralleled by changes in PKC-
subcellular distribution (Fig. 8).

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Fig. 8.
Confocal volumetric reconstruction (100 × 0.4-µm
z-axis planes) of cellular PKC- staining in isolated
crypts from normal (A) and day 12 TMCH (B).
Crypts from both sources were simultaneously fixed,
permeabilized, incubated with anti-PKC- C-20 antibody (Table 1), and
processed for immunofluorescence (see MATERIALS AND
METHODS). Normal crypts (A) exhibited cellular
staining predominantly within the neck region (arrow),
whereas TMCH crypts (B) exhibited enhanced cellular staining
throughout the crypt axis. Neither normal (C) nor
hyperproliferating (D) crypts exhibited any signal when the
primary antibody was omitted from the staining protocol. Digital
contrast levels were not changed during image capture; crypts were
isolated from 6 normal and 6 day 12 TMCH animals and were
used for Figs. 9-11. Magnification, ×400.
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Confocal microscopy with the C-20 PKC-
antibody (Table 1) and
FITC-conjugated secondary antibody (see MATERIALS AND
METHODS) revealed that PKC-
staining in volumetrically
reconstructed normal mucosal crypts was highest in the apical and
basolateral subcellular poles of colonocytes located within the crypt
neck (Fig. 8A). In contrast, in hyperproliferating TMCH
mucosa, subcellular PKC-
staining was evenly distributed throughout
the length of the crypt (Fig. 8B). Concurrently processed
crypts in which the primary antibody had been omitted failed to exhibit
staining (Fig. 7, C and D). Because all crypts
were processed and imaged identically, with no alteration in
post-image-capture signal gain, changes in the overall levels of
PKC-
subcellular staining in hyperproliferating crypts correlated
very well with increased levels of cellular PKC-
expression reported
by our Western blotting approaches (see Figs. 2, 3, and 6).
At higher magnification, crypts isolated from the normal mucosa also
exhibited low levels of cytoplasmic PKC-
staining (Fig. 9). In this instance, PKC-
epithelium-specific cell labeling was confined to within 50 µm of the
base of the normal crypt (Fig. 9A), corresponding to the
region of PCNA immunostaining (Fig. 1), beyond which it diminished to
background levels (Fig. 9A). In contrast, the
elevated cytoplasmic PKC-
staining observed in hyperproliferating
crypts did not vary greatly between these regions (Fig. 9B).
Quantification revealed that the total (0.5 µm) z-axis
plane immunofluorescent signal recorded 50 µm from the base of
simultaneously processed normal and hyperproliferating crypts was
105 ± 15 and 218 ± 26 pixel intensities, respectively. At
100 µm from the base of the crypt, this signal decreased to 45 ± 5 pixel intensities in normal crypts but remained elevated at
197 ± 17 pixel intensities in hyperproliferating crypts
(n = 20 individual crypts assayed in tandem from 6 mice). In addition, crypt surface-apposed bipolar cells of neuronal
origin also exhibited PKC-
immunoreactivity (Fig. 9A).
The latter cell population was recorded in all crypts at low frequency
(2-4 cells/crypt) and was not addressed further. When
quantified, control crypts for both conditions immunostained with
FITC-conjugated secondary antibody and nonspecific IgG primary antibody
exhibited low levels of background immunostaining (25 ± 18 pixel
intensities, n = 60 crypts from 12 animals). Similar
values were recorded when the primary antibody was omitted or when
immunizing peptide was used to compete the primary antibody signal
(data not shown).

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Fig. 9.
Higher-magnification (×800) confocal volumetric
(100 × 0.4-µm z-axis plane) reconstructions of
PKC- immunoreactive staining in the basal cellular regions
of normal (A) and day 12 TMCH (B)
crypts. Simultaneous staining was performed; normal crypts exhibited
weak cellular cytoplasmic immunoreactivity at their base, shown more
clearly in the corresponding orthogonal z-y plane
(Ai), but staining was considerably reduced 100 µm
proximal to this region (Aii). In contrast,
hyperproliferating crypts exhibited higher overall levels of cellular
PKC- staining at the crypt base (Bi) and in all regions
proximal to this area (Bii). Crypt-apposed bipolar cells of
neuronal origin were also present at low but equal density in both
preparations (A). The staining pattern was eliminated by
omitting the primary antibody. Asterisks indicate points along the
longitudinal crypt axis where z-y images were
taken.
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Quantification of PKC-
immunofluorescent signal in the neck regions
of normal and hyperproliferating crypts revealed another difference in
PKC-
subcellular location (Fig. 10).
Subcellular PKC-
immunofluorescence in colonocytes located within
the neck region of normal crypts was evenly distributed between the
subapical and subbasolateral cellular poles (Fig. 10A),
whereas in hyperproliferating crypt neck regions PKC-
immunostaining
was preferentially lost from the subbasolateral cellular region (Fig.
10B). Higher-spatial-resolution imaging (×800
magnification) within the neck region revealed that the basolateral
subcellular pole staining in normal crypts was associated with as yet
undefined filamentous subcellular structures (Fig.
11).

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Fig. 10.
Left, average PKC- immunoreactive signal
recorded from 3 confocal mid-z-axis (0.4 µm)
x-y planes within the neck region of
simultaneously processed normal (A) and day 12 TMCH (B) crypts. High levels of PKC- staining were
detected within the subcellular apical and basolateral poles of normal
crypt colonocytes (cell position determined from crypt neck toward
base, in A, 1 13). In contrast, hyperproliferating crypt
neck regions exhibited decreased subcellular basal pole labeling (cell
position in B, 1-9). Right, corresponding
bar graphs of subcellular staining intensity collected with a
5.2-µm2 (26 × 26 bit) window placed over the apical
(Ap), basolateral (Bas), and nuclear (Nuc) regions of individual cells
within the neck region. Digitized fluorescence intensity values in this
representative experiment are expressed as means ± SE for paired
observations.
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Fig. 11.
Three representative single confocal planes of cellular
PKC- immunoreactivity in the neck region of the normal crypt at
×1,200 magnification. Planes were collected at 33 (A), 0 (B), and +35 (C) µm relative to the
mid-z crypt axis. High levels of PKC- immunofluorescent
staining recorded at lower magnification in Figs. 7 and 9A
were resolved as a filamentous subcellular staining pattern. In
addition, infrequent bipolar cells of neuronal origin are stained.
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Figure 11 shows three vertical 1-µm image planes taken at +5, +38,
and +73 µm from the coverslip surface. Filamentous subcellular PKC-
immunostaining was most apparent in the vertical
(z-axis) image plains close to the coverslip (+5 µm, Fig.
11A) and bath-apposed crypt surfaces (+73 µm, Fig.
11C), which bisect larger areas of subbasolateral plasma
membrane cytoplasm. The mid-z-axis plane (+38 µm,
Fig. 11B) revealed that filamentous immunostaining extended throughout the interior volume of the crypt. Very little nuclear staining was apparent.
When single x-y axis planes from the upper one-third or neck
region of the TMCH crypts (12 days after Citrobacter
infection) were analyzed at ×800 magnification, a different staining
pattern was recorded (Fig. 12).
Aggregation of PKC-
with subcellular basolateral microfilamentous
structures was not as apparent. However, clear cytoplasmic,
perinuclear, and nuclear PKC-
immunostaining was recorded (Fig. 12,
A-C), as well as accumulation of PKC-
within the
lateral but not basolateral plasma membrane (Fig. 12D). The nuclear accumulated PKC-
signal was clearly higher than that recorded in normal crypts, in which the nuclear volume was devoid of
signal (Fig. 11B). Discrete hot spots within the
hyperproliferating nuclei were detected (Fig. 12), suggesting
aggregation of PKC-
into specific subnuclear structures. This
pattern of PKC-
immunostaining was extended throughout the crypt
axis and was similar to that recorded in the crypt base
(n = 6 animals in triplicate).

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Fig. 12.
Representative single mid-z-axis confocal
plane of PKC- immunoreactivity recorded in the mid/neck region of a
day 12 TMCH crypt. In images collected at ×800
magnification, diffuse cytoplasmic PKC- immunoreactivity is
evident, particularly within the subcellular apical pole (A,
arrow). Areas within the same crypt at higher ×1,200
magnification (B-D) clearly showed prominent
perinuclear and punctate nuclear immunoreactivity (B and
D, arrow) together with accumulation of signal at or below
the lateral plasma membrane (A and B,
arrow-§). Note that hyperproliferating crypts failed to
exhibit the same filamentous subcellular PKC- staining as seen in
the corresponding neck regions of normal crypts (Fig. 11).
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Thus complex alterations in colonocyte subcellular PKC-
distribution
that occurred as a consequence of crypt hyperproliferation were
characterized as 1) loss from filamentous subbasolateral membrane cytosolic structures in crypt neck cells and 2)
enhanced accumulation within the cytoplasm, at lateral plasma membrane and perinuclear sheaf, and within the nucleus. Thus the expression and
subcellular distribution profile of PKC-
recorded in TMCH crypts
corroborated the biochemical analysis of PKC-
expression and
function outlined above (Figs. 2-6).
 |
DISCUSSION |
Previous studies have documented the distribution and biochemical
activation status of PKC isoforms within the normally proliferating colonic epithelium. A gradient of PKC activity between crypt base and
apex has been correlated with isoform-specific expression and
subcellular location in an attempt to determine cellular function at
different points along the crypt base-to-surface axis. Early studies
reporting mass PKC activity found highest levels within the crypt base
(14), whereas subcellular fractionation and immunological techniques revealed that most crypt-expressed PKCs are localized in the
cellular cytoplasm in the lower proliferatory regions and only become
membrane associated in mid to higher crypt regions (28).
This leads to a translocation vs. activation paradox: PKC subcellular
distribution in proliferatory crypt regions is opposite to that
expected if membrane association predicts activation and an involvement
in proliferatory growth control. However, this limited viewpoint may be
an oversimplification of the true signaling status of the kinase
because 1) it is based almost entirely on the PKC
membrane-associating effects of phorbol esters (52), a
model not directly relevant to atypical PKC activation; 2)
it is becoming increasingly recognized that membrane association alone
does not always predict substrate phosphorylation (52); and 3) PKCs exert their signaling effects at many locales
within the cell through interactions with other proteins
(29). Thus, in addition to participation in cell signaling
cascades between the plasma membrane, internal membranes, and nucleus,
direct PKC involvement in the nuclear responses of both mitogenic and
differentiating factors was suggested recently (40). Our
findings in the TMCH model directly address this question.
Cellular roles of aPKC-
in colonic epithelial proliferation.
This isoform has received considerable recent interest. PKC-
has
been implicated in the control of cellular mitogenic signaling and
survival (16, 20, 34) and has
been reported to play a pivotal role in tumor necrosis factor-
activation of nuclear factor-
B (17), an inducible
transcriptional activator that participates in the control of cell
proliferation and maintenance, as well as in inflammatory
response and viral gene expression. The active component of fetal
bovine serum-induced signaling in vitro, identified as
lysophosphatidic acid, has also been shown to stimulate both
Ras-dependent and -independent mitogen-activated protein (MAP) kinase
cell signaling via phosphatidylinositol 3-kinase and aPKC-
activation (38). The role of this isoform as a
downstream effector of p21ras, [RAS/RAF-MAP
kinase kinase (MEK)-MAP kinase cascade] has been confirmed by the
observations that PKC-
directly phosphorylates and activates MEK,
resulting in the subsequent activation of MAP kinase (6,
12, 17, 39). Studies by Berra
and colleagues (7) showed that the activation of PKC-
holoenzyme is critical for mitogenic signaling during maturation of
Xenopus laevis oocytes (7).
Complex pattern of PKC-
expression and subcellular membrane
partitioning accompanies TMCH.
More than one immunoreactive PKC-
band was detected in normally
proliferating and hyperproliferating isolated crypt extracts. Possible
explanations for the appearance of multiple bands include the presence
of closely related species or posttranslational modification of the
parental holoenzyme. To address this concern, a panel of
-immunospecific antibodies were used. All three bands [holoenzyme, band A = 72 kDa; PKM-
, band B/B* = 46/48
kDa, and membrane-bound fragment (Mf)-
, band
C/C* = 26/28 kDa] were recognized by two of three COOH-terminal
antibodies, including one reported to be specific for PKC-
over the
closely related PKC-
(9). The appearance of a
catalytically active PKM-
fragment was noted previously by others
after heterologous PKC-
overexpression in vitro (31).
We hypothesize that band B* represents a closely related
homologue or posttranslational modification to band B and
that both B bands are proteolytic breakdown products of band A. In common with PKM-
, the immunoreactive <29-kDa PKC-
fragment routinely detected with COOH-terminal-specific antibodies
(Fig. 2) was not observed when the NH2-terminal antibody
was used for Western blotting (N-15, Table 1). Because our studies
found that this lightest fragment was almost entirely membrane
associated (Fig. 4) and was a fragment of the enzyme's catalytic
domain, we designated this fragment as Mf-
.
Bands A and B exhibited markedly decreased
membrane association during crypt hyperproliferation (Fig. 4,
A/B) at a time when overall cellular levels of both species
rose dramatically (Fig. 2), whereas band C was undetectable
in normally proliferating crypts but, during TMCH, increased its
expression in parallel with the other bands and was almost entirely
membrane associated (Fig. 4C). Given that cellular levels of
PKC-
increased in proliferating cells at a time when
membrane-associated forms of this kinase were dramatically lowered, we
investigated other possible cellular locales for the kinase. We found
that in contrast to the majority of all other PKCs detected in crypts
(43b), both PKC-
holoenzyme and PKM-
were nuclear translocated
during TMCH (Figs. 5-7, 12). These observations indicated to us
that either form of PKC-
, if catalytically active in the nucleus,
could be hypothesized to play a direct role in regulating crypt
colonocyte hyperproliferation.
Differences in PKM-
gel mobility correlated with nuclear
accumulation of active PKM-
during TMCH.
Phosphorylation has been found to be an important mechanism for
regulating PKC activity. Two separate phosphorylation events appear to
be required. The first event involves a critical threonine residue in
the activation loop. This residue in PKC-
was recently shown to be
phosphorylated in vivo by phosphoinositide-dependent protein kinase-1
(12, 27). After phosphorylation,
autophosphorylation of the holoenzyme at critical serine and threonine
residues within the catalytic domain occurs to fully activate the
kinase (24, 43). To elucidate the activation
status of PKC-
both within nucleus-free cytoplasm and within the
nucleus of normal and TMCH colonocytes, immunoprecipitated enzyme
from both subcellular pools was assayed for intrinsic kinase activity.
TMCH increased the autophosphorylation status of both cytoplasmic and
nuclear PKC-
/PKM-
, and these changes were reflected by increased
-peptide substrate phosphorylation in nuclear extracts. These in
vivo findings were corroborated by previous reports that described
small (8- to 10-fold; Ref. 36) to very large (200-fold; Ref. 11)
increases in phospholipid-stimulated nuclear PKC activity after
treatment of isolated nuclei with exogenous activators and by the fact
that truncated forms of PKC encompassing the kinase domain are
exclusively localized to the nucleus (18).
When related
-isoform Western blot and autophosphorylation data were
compared, we found that protein expression and intrinsic kinase
activity correlated for both nuclear and cytoplasmic holoenzyme and for
cytoplasmic PKM-
. However, nuclear accumulated PKM-
paradoxically
exhibited lower autonomous activity during TMCH, suggesting that some
degree of inactivation had occurred. Because PKC phosphorylation leads
to both activation and an increase in apparent molecular mass, the
recorded decrease in PKM-
mobility on Western-blotted nuclear
extracts may reflect this phenomenon. The function of
-isoform
COOH-terminal domain autophosphorylation remains unknown. Our data
suggests that posttranslational modification of nuclear PKM-
may be
integral to modulation of the proliferatory signal during TMCH.
Changes in PKC-
subcellular distribution reflect biochemical
changes in PKC-
activity recorded in TMCH crypts.
Our immunofluorescence studies (Figs. 8-12) clearly demonstrated
that crypt hyperproliferation was matched by enhanced immunoreactive PKC-
cell staining throughout the longitudinal axis of the crypt (Fig. 8) and by the accumulation of PKC-
signal within the cellular cytoplasm, at the nuclear envelope, and within the nucleus itself (Figs. 8, 9, and 12). Although there was incomplete overlap, PKC-
staining was observed in all crypt areas in which PCNA immunoreactivity (Fig. 1) occurred. Because the COOH-terminal antibody used for these
studies detected all three PKC-
species (C-20, Table 1), we were
unable to determine PKC-
/PKM-
/Mf-
specific
subcellular location information. However, given that during TMCH only
Mf-
increased its membrane association, whereas the
other species exhibited decreased membrane translocation (Fig. 4), a
significant fraction of the lateral membrane PKC-
immunoreactivity
in TMCH crypts (Figs. 9 and 12) may be represented by this
catalytically inert fragment. A corollary of this hypothesis was that
PKC-
staining at this subcellular location represented a byproduct of enzyme activation/proteolysis and not active plasma
membrane-associated kinase. Increases in nuclear envelope/nuclear
staining recorded in TMCH crypts (Fig. 12) suggest that either PKC-
holoenzyme or PKM-
, but not Mf-
, was targeted to this
location. In fact, both may achieve this through sequences within the
COOH-terminal kinase domain (18).
Finally, the immunostaining pattern detected in normal and TMCH crypts
differed in another respect. The neck regions of normal crypts
exhibited clear cellular apical and basolateral pole immunostaining (Figs. 8 and 10) localized mainly to subcellular filamentous structures (Fig. 11). However, colonocyte hyperproliferation and the repopulation of this crypt region with immature cells resulted in the specific loss
of the cellular basolateral pole staining pattern (Fig.
10B). It is interesting to speculate whether increased
Mf-
expression (Fig. 2) reflects either the proteolysis
of cytoskeletally aggregated or membrane-activated PKC-
and whether
either of these cytoplasmic pools contributes to nuclear translocated
PKC-
/PKM-
. It is possible that these different morphological
findings reflect separate physiological roles for PKC-
in quiescent
crypt neck and mitotically active crypt base colonocytes.
Model for subcellular PKC-
activation during TMCH.
Figure 13 shows our working hypothesis
of how during TMCH decreased levels of catalytically active,
membrane-associated PKC-
/PKM-
(Figs. 4 and 6) can occur together
with elevated cellular PKC-
activity (Fig. 7), PKC-
expression
(Fig. 2), and enhanced cell division (Fig. 1). Differences in PKC-
holoenzyme, PKM-
, and Mf-
fragment expression and
membrane association represent steady-state conditions present within
hyperproliferating colonocytes. In the model described in Fig. 13,
Mf-
describes a catalytically inert membrane proteolytic
breakdown product of cytoplasmic holoenzyme PKC-
. Because activation
of PKC and translocation to cell membranes is thought to be a
prerequisite for proteolytic cleavage in vivo, PKM-
formed by the
proteolysis of membrane-activated PKC-
(37) describes a
constitutively active nuclear targeted kinase during TMCH. Subtle
differences in gel mobility and autonomous activity between cytoplasmic
and nuclear forms of autophosphorylated PKM-
identify postactivation
cleavage of the PKC-
holoenzyme as an important regulatory component
of nuclear PKM-
accumulation. However, at present we cannot also
exclude the possibility that nuclear accumulated PKM-
may not have
originated from the peripheral cytoplasm but rather was made within the
endoplasmic reticulum and trafficked into the nucleus after initiation
of translation from an internal ATG. Further studies are
necessary to determine whether the activation of nuclear
PKC-
/PKM-
preceded or followed nuclear translocation.
Supporting our model, chronic activation of PKC-dependent mitogenic
signaling in vivo has been shown to lead to increased cytoplasmic PKC
expression (47), and, similarly, the in vivo effects of a
number of mitogenic growth factors have been correlated with
increased cytoplasmic PKC levels during periods of increased substrate phosphorylation (13, 22).
Furthermore, chronic PKC-
activation during neuronal long-term
potentiation has been correlated with loss of membrane-associated
PKC-
and the cellular production of PKM-
(37). Thus
we hypothesize that the translocation/activation paradox applied to
PKC-
in hyperproliferating crypts can be explained by a switch from
PKC-
holoenzyme signaling at subcellular membranes toward direct
effects of PKM-
within the nucleus.
Physiological implications of PKC-
/PKM-
induction in
hyperproliferating crypts.
As outlined in the introductory paragraphs of this article, PKC-
has
been implicated in colonic cancer progression. In both human and
experimental colon cancers PKC-
expression has been shown to be
decreased (52). Moreover, downregulation has been correlated with adenoma-to-carcinoma transition in AOM-treated mice.
Subsequently, a variety of chemoprotective agents have been shown to
preserve PKC-
expression in this and related carcinogenic models
(48). In addition to the role of PKC-
in
p21ras oncogenic signaling, PKC-
has also been reported
to positively regulate the transcriptional activity of the tumor
suppressor gene p53 in vitro and in vivo (44), mutations
which prevent cells from initiating growth arrest or apoptosis. The
loss of these latter regulatory mechanisms is also an established event in neoplastic transformation and provides cellular clues for another role of PKC-
in native colonocytes. In the TMCH model
increased crypt cell number can be correlated with the fact that
apoptosis fails to increase (43a) and thus match the sixfold
enhancement in PCNA expression (Fig. 1). Therefore, in addition to
regulating TMCH, alterations in PKC-
signaling may also correlate
with the ability of mitotically active colonocytes to escape apoptotic cell death. Recent reports have implicated a prosurvival role of
cellular PKC-
signaling, which must be inhibited for apoptosis to
proceed (19). These phenomena, when coupled with other
epigenetic signaling events, may be responsible for altering the
phenotype of the hyperproliferative mucosa sufficiently to promote
neoplastic transformation. This may explain why TMCH greatly reduces
the time requirement for cancer formation in mice exposed to DMH/AOM and other DNA-hypermethylating colonic carcinogens (1,
4). Together, these studies imply that PKC-
may
directly modulate gene expression during TMCH through signaling
pathways that involve the activation of nuclear PKC-
/PKM-
.
Efforts are underway in our laboratory to identify specific nuclear
events and/or specific transcription factors associated with
catalytically active PKC-
to understand the physiological mechanisms
for Citrobacter-induced mucosal hyperproliferation at the
genome level.
 |
ACKNOWLEDGEMENTS |
This work was supported by funds from the Cystic Fibrosis
Foundation and the American Institute for Cancer Research.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: Andrew
P. Morris, Dept. of Internal Medicine, Division of
Gastroenterology, Hepatology, and Nutrition, Univ. of Texas Health
Science Center at Houston, Medical School, Houston, TX 77030 (E-mail:
amorris{at}girch1.med.uth.tmc.edu).
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. §1734 solely to indicate this fact.
Received 9 November 1999; accepted in final form 1 February 2000.
 |
REFERENCES |
1.
Barthold, SW.
The microbiology of transmissible murine colonic hyperplasia.
Lab Anim Sci
30:
167-173,
1980[Medline].
2.
Barthold, SW,
and
Beck D.
Modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia.
Cancer Res
40:
4651-4655,
1980.
3.
Barthold, SW,
Coleman GL,
Jacoby RU,
Livingstone EM,
and
Jonas AM.
Transmissible murine colonic hyperplasia.
Vet Pathol
15:
223-236,
1978[Abstract].
4.
Barthold, SW,
and
Jonas AM.
Morphogenesis of early 1,2-dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacter freundii.
Cancer Res
37:
4352-4360,
1977[Abstract].
5.
Baum, CL,
Wali RK,
Sitrin MD,
Bolt MJ,
and
Brasitus TA.
1,2-Dimethylhydrazine-induced alterations in protein kinase C activity in the rat preneoplastic colon.
Cancer Res
50:
3915-3920,
1990[Abstract].
6.
Berra, E,
Diaz-Meco MT,
Lozano J,
Frutos S,
Municio MM,
Sanchez P,
Sanz L,
and
Moscat J.
Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.
EMBO J
14:
6157-6163,
1995[Abstract].
7.
Berra, E,
Diaz-Meco MT,
Dominguez I,
Municio M,
Sanz L,
Lozano J,
Chapkin RS,
and
Moscat J.
Protein kinase C zeta isoform is critical for mitogenic signal transduction.
Cell
74:
555-563,
1993[ISI][Medline].
8.
Blobe, GC,
Stribling S,
Obeid LM,
and
Hannun YA.
Protein kinase C isoenzymes: regulation and function.
Cancer Surv
27:
213-268,
1996[ISI][Medline].
9.
Borner, C,
Guadagno SN,
Fabbro D,
and
Weinstein IB.
Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.
J Biol Chem
267:
12892-12899,
1992[Abstract/Free Full Text].
10.
Buchner, K.
Protein kinase C in the transduction of signals toward and within the cell nucleus.
Eur J Biochem
228:
211-221,
1995[ISI][Medline].
11.
Buckley, AR,
Crowe PD,
and
Russell DH.
Rapid activation of protein kinase in isolated rat liver nuclei by prolactin, a known hepatic mitogen.
Proc Natl Acad Sci USA
85:
8649-8653,
1988[Abstract].
12.
Chou, MM,
Hou W,
Johnson J,
Graham LK,
Lee MH,
Chen CS,
Newton AC,
Schaffhausen BS,
and
Toker A.
Regulation of protein kinase C zeta by PI 3-kinase and PDK-1.
Curr Biol
8:
1069-1077,
1998[ISI][Medline].
13.
Costa-Casnellie, MR,
Segel GB,
and
Lichtman MA.
Concanavalin A and phorbol ester cause opposite subcellular redistribution of protein kinase C.
Biochem Biophys Res Commun
133:
1139-1146,
1985[ISI][Medline].
14.
Craven, PA,
and
DeRubertis FR.
Subcellular distribution of protein kinase C in rat colonic epithelial cells with different proliferative activities.
Cancer Res
47:
3434-3438,
1987[Abstract].
15.
Craven, PA,
and
DeRubertis FR.
Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis.
Cancer Res
52:
2216-2221,
1992[Abstract].
16.
Diaz-Meco, MT,
Municio MM,
Frutos S,
Sanchez P,
Lozano J,
Sanz L,
and
Moscat J.
The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C.
Cell
86:
777-786,
1996[ISI][Medline].
17.
Diaz-Meco, MT,
Dominguez I,
Sanz L,
Dent P,
Lozano J,
Municio MM,
Berra E,
Hay RT,
Sturgill TW,
and
Moscat J.
PKC zeta induces phosphorylation and inactivation of I kappa B-alpha in vitro.
EMBO J
13:
2842-2848,
1994[Abstract].
18.
Elder, H,
Ben-Chaim J,
and
Livneh E.
Deletions in the regulatory or kinase domains of protein kinase C-
cause association with the cell nucleus.
Exp Cell Res
202:
259-266,
1992[ISI][Medline].
19.
Frutos, S,
Moscat J,
and
Diaz-Meco MT.
Cleavage of
PKC but not
/
PKC by caspase-3 during UV-induced apoptosis.
J Biol Chem
274:
10765-10770,
1999[Abstract/Free Full Text].
20.
Gomez, J,
Pitton C,
Garcia A,
Martinez de Aragon A,
Silva A,
and
Rebollo A.
The zeta isoform of protein kinase C controls interleukin-2-mediated proliferation in a murine T cell line: evidence for an additional role of protein kinase C epsilon and beta.
Exp Cell Res
218:
105-113,
1995[ISI][Medline].
21.
Guillem, JG,
O'Brian CA,
Fitzer CJ,
Forde KA,
LoGerfo P,
Treat M,
and
Weinstein IB.
Altered levels of protein kinase C and Ca2+-dependent protein kinases in human colon carcinomas.
Cancer Res
47:
2036-2039,
1987[Abstract].
22.
Halsey, DL,
Girard PR,
Kuo JF,
and
Blackshear PJ.
Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens.
J Biol Chem
262:
2234-2263,
1987[Abstract/Free Full Text].
23.
Kahl-Rainer, P,
Karner-Hanusch K,
Weiss J,
and
Marian B.
Five of six protein kinase C isoenzymes present in normal mucosa show reduced protein levels during tumor development in the human colon.
Carcinogenesis
15:
779-784,
1994[Abstract].
24.
Keranen, LM,
Dutil EM,
and
Newton AC.
Protein kinase C is regulated in vivo by three functionally distinct phosphorylations.
Curr Biol
5:
1394-1403,
1995[ISI][Medline].
25.
Kopp, R,
Noelke B,
Sauter G,
Schildberg FW,
Paumgartner G,
and
Pfeiffer A.
Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas.
Cancer Res
51:
205-210,
1991[Abstract].
26.
Kusunoki, M,
Hatada T,
Sakanoue Y,
Yanagi H,
and
Utsunomiya J.
Correlation between protein kinase C activity and histopathological criteria in human colorectal adenoma.
Br J Cancer
65:
673-676,
1992[ISI][Medline].
27.
Le Good, JA,
Ziegler WH,
Parekh DB,
Alessi DR,
Cohen P,
and
Parker PJ.
Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1.
Science
281:
2042-2045,
1998[Abstract/Free Full Text].
28.
Mellor, H,
and
Parker PJ.
The extended protein kinase C superfamily.
Biochem J
332:
281-292,
1998[ISI][Medline].
29.
Mochly-Rosen, D,
and
Gordon AS.
Anchoring proteins for protein kinase C: a means for isozyme selectivity.
FASEB J
12:
35-42,
1998[Abstract/Free Full Text].
30.
Olson, EN,
Burgess R,
and
Staudinger J.
Protein kinase C as a transducer of nuclear signals.
Cell Growth Differ
4:
699-705,
1993[ISI][Medline].
31.
Ono, Y,
Fuji T,
Ogita K,
Kikkawa K,
Igarashi K,
and
Nishizuka Y.
Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.
Proc Natl Acad Sci USA
86:
3099-3103,
1989[Abstract].
32.
Pretlow, TP,
Brasitus TA,
Fulton NC,
Cheyer C,
and
Kaplan EL.
K-ras mutations in putative pre-neoplastic lesions in human colon.
J Natl Cancer Inst
85:
2004-2007,
1993[Abstract].
33.
Ponz de Leon, M,
Roncucci L,
Di Donato P,
Tassi L,
Smerieri O,
Amorico MG,
Malgoli G,
De Maria D,
Antonioli A,
and
Chahin NJ.
Pattern of epithelial cell proliferation in colorectal mucosa of normal subjects and of patients with adenomatous polyps or cancer of the large bowel.
Cancer Res
48:
4121-4126,
1988[Abstract].
34.
Powell, CT,
Gschwend JE,
Fair WR,
Brittis NJ,
Stec D,
and
Huryk R.
Over expression of protein kinase C-zeta (PKC-zeta) inhibits invasive and metastatic abilities of Dunning R-3327 MAT-LyLu rat prostate cancer cells.
Cancer Res
56:
4137-4141,
1996[Abstract].
35.
Risio, M,
Lipkin M,
Candelaresi GL,
Bertone A,
Coverlizza S,
and
Rossini FP.
Correlations between rectal mucosa cell proliferation and the clinical and pathological features of non-familial neoplasia of the large intestine.
Cancer Res
51:
1917-1921,
1991[Abstract].
36.
Rogue, P,
Labourdette G,
Masmoudi A,
Yoshida Y,
Huang FL,
Huang KP,
Zwiller J,
Vincendon G,
and
Malviya AN.
Rat liver nuclei protein kinase C is the isozyme type II.
J. Biol Chem
265:
4161-4165,
1990[Abstract/Free Full Text].
37.
Sacktor, TC,
Osten P,
Valsamis H,
Jiang X,
Naik MU,
and
Sublette E.
Persistent activation of the zeta isoform of protein kinase C in the maintenance of long-term potentiation.
Proc Natl Acad Sci USA
90:
8342-8348,
1993[Abstract/Free Full Text].
38.
Saxon, ML,
Zhao X,
and
Black JD.
Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.
J Cell Biol
126:
747-763,
1994[Abstract].
39.
Schonwasser, DC,
Marais RM,
Marshall CJ,
and
Parker PJ.
Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.
Mol Cell Biol
18:
790-798,
1998[Abstract/Free Full Text].
40.
Selbie, LA,
Schmitz-Peiffer C,
Sheng Y,
and
Bident TJ.
Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells.
J Biol Chem
268:
26296-26302,
1993[Abstract/Free Full Text].
41.
Takeda, H,
Matozaki T,
Takada T,
Noguchi T,
Yamao T,
Tsuda M,
Ochi F,
Fukunaga K,
Inagaki K,
and
Kasuga M.
PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor.
EMBO J
18:
386-395,
1999[Abstract/Free Full Text].
42.
Terpstra, OT,
Blankenstein VM,
Dees J,
and
Eilers GAM
Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or cancer.
Gastroenterology
92:
704-708,
1987[ISI][Medline].
43.
Tsutakawa, SE,
Medzihradszky KF,
Flint AJ,
Burlingame AL,
and
Koshland DE, Jr.
Determination of in vivo phosphorylation sites in protein kinase C.
J Biol Chem
270:
26807-26812,
1995[Abstract/Free Full Text].
43a.
Umar, S,
Scott J,
Sellin JH,
Dubinsky WP,
and
Morris AP.
Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl
secretion.
Am J Physiol Gastrointest Liver Physiol
278:
G753-G764,
2000[Abstract/Free Full Text].
43b.
Umar, S,
Sellin JH,
and
Morris AP.
Murine colonic mucosa hyperproliferation. II. PKC-
activation and cPKC-mediated cellular CFTR overexpression.
Am J Physiol Gastrointest Liver Physiol
278:
G765-G774,
2000[Abstract/Free Full Text].
44.
Verstovsek, G,
Byrd A,
Frey MR,
Petrelli NJ,
and
Black JD.
Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes.
Gastroenterology
115:
75-85,
1998[ISI][Medline].
45.
Vogelstein, B,
and
Kinzler KW.
The multi-step nature of cancer.
Trends Genet
9:
138-141,
1993[ISI][Medline].
46.
Wali, RK,
Frawley BP, Jr,
Hartmann S,
Roy HK,
Khare S,
Scaglione-Sewell BA,
Earnest DL,
Sitrin MD,
Brasitus TA,
and
Bissonnette M.
Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta.
Cancer Res
55:
5257-5264,
1995[Abstract].
47.
Warner, JA,
and
Mac Glashan DW, Jr.
Protein kinase C (PKC) changes in human basophils IgE-mediated activation is accompanied by an increase in total PKC activity.
J Immunol
142:
1669-1677,
1989[Abstract/Free Full Text].
48.
Weinstein, IB.
Growth factors, oncogenes, and multistage carcinogenesis.
J Cell Biochem
33:
213-226,
1987[ISI][Medline].
49.
Wooten, MW,
Zhou G,
Wooten MC,
and
Seibenhener ML.
Transport of protein kinase C isoforms to the nucleus of PC12 cells by nerve growth factor: association of atypical zeta-PKC with the nuclear matrix.
J Neurosci Res
49:
393-403,
1997[ISI][Medline].
50.
Youmell, M,
Park SJ,
Basu S,
and
Price BD.
Regulation of the p53 protein by protein kinase C alpha and protein kinase C zeta.
Biochem Biophys Res Commun
265:
514-518,
1998.
51.
Zhang, DE,
Hoyt PR,
and
Papacostantinou J.
Localization of DNA protein-binding sites in the proximal and distal promoter regions of the mouse alpha-fetoprotein gene.
J Biol Chem
265:
3382-3391,
1990[Abstract/Free Full Text].
52.
Zhou, G,
Wooten MW,
and
Coleman ES.
Regulation of atypical zeta-protein kinase C in cellular signaling.
Exp Cell Res
214:
1-11,
1994[ISI][Medline].
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