Download Increased nuclear translocation of catalytically active PKC

Am J Physiol Gastrointest Liver Physiol
279: G223–G237, 2000.
Increased nuclear translocation of catalytically active
PKC-␨ during mouse colonocyte hyperproliferation
SHAHID
UMAR,1 JOSEPH H. SELLIN,1,2 AND ANDREW P. MORRIS1,2
1
Department of Integrative Biology, Pharmacology, and Physiology, and 2Department of Internal
Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Texas Health
Science Center at Houston, Medical School, Houston, Texas 77030
Received 9 November 1999; accepted in final form 1 February 2000
protein kinase C; cellular mitosis; mouse colon
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,
THE INTESTINAL MUCOSA
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@
girch1.med.uth.tmc.edu).
http://www.ajpgi.org
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 (␦, ε, ␩, ␪, and
␮), 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
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0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society
G223
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Umar, Shahid, Joseph H. Sellin, and Andrew P. Morris. Increased nuclear translocation of catalytically active
PKC-␨ during mouse colonocyte hyperproliferation. Am J
Physiol Gastrointest Liver Physiol 279: G223–G237, 2000.—
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 -␨, ⬎10fold) expression. Both PKC-␨ and PKM-␨ exhibited intrinsic
kinase activity, and substrate phosphorylation increased 4.5fold. 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.
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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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.
Antibodies. Polyclonal rabbit anti-PKC-␨ anti-peptide antibodies were procured from Santa Cruz Biotechnology (SC216/7282; Santa Cruz, CA) and Upstate Biotechnology (06475; 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
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MATERIALS AND METHODS
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-␮m
steps through the sample. Collected 8- or 16-bit color greenencoded 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 䡠 HCl, (pH 7.0), 137 NaCl,
and 5 KCl] and homogenized in buffer A [in mM: 15 Tris 䡠 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 ␮g/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 䡠 HCl (pH 8.0), 2 MgCl2, 0.1 EDTA, 1 DTT, 0.4
PMSF, and 2 benzamidine and 1 ␮g/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 ␮g/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 䡠 HCl, pH 7.5, 0.25 M
sucrose, 2 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 25
␮g/ml each of leupeptin, aprotinin, and pepstatin, 1 ␮g/ml
soybean trypsin inhibitor, 50 ␮M sodium fluoride, 50 ␮g/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
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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.
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-ε)] 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.
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 antiPCNA 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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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 ␮g 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 䡠 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 ␮g/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 ␮l 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 ␮l 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 ␮l of a buffer containing 50 mM Tris 䡠 HCl (pH 7.5), 5
mM MgCl2, 1 mM PMSF, 20 ␮g/ml leupeptin, 20 ␮g/ml
aprotinin, and 1 mM each of sodium orthovanadate, sodium
pyrophosphate, and NaF. The kinase reaction for autophosphorylation was started by addition of 50 ␮M [␥-32P]ATP at
37°C, and the phosphorylation was stopped by addition of 50
␮l of reducing Laemmli sample buffer. Substrate phosphorylation was started by addition of 50 ␮M [␥-32P]ATP at 37°C
using PKC-ε pseudosubstrate derivative (ERMRPRKRQGSVRRRV) as substrate for PKC-␨ in the presence or absence of
inhibitor peptide. Stock solutions of lipids in ethanol and
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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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).
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
Table 1. Summary of PKC-␨ band detection by the
PKC-␨ peptide antibodies used for Western blotting
Antibody
Band A*/A
(⬃72 kDa)
Band B*/B
(⬃48 kDa)
Band C*/C
(⬍29 kDa)
⫹Antigenic
Peptide
C-20
N-17
C-15
C-14
⫹/⫹
⫹
⫹/⫹
⫹
⫹/⫹
⫺
⫹/⫹
⫹
⫹/⫹
⫺
⫹/⫹
⫺
⫺
⫺
N/P
N/P
Affinity-purified polyclonal peptide antibodies were used; n ⫽ 6
animals/antibody in which response was confirmed. ⫹ Antigenic
peptide, detection response of Western blot with 2:1 molar excess
antigenic peptide; C-20, Sc-216 antibody mapped to COOH-terminal
domain of rat protein kinase (PK) C-␨; N-17, Sc-7282 antibody
mapped to NH2-terminal region of human PKC-␨; C-15, 06-473
antibody mapped to amino acids 577–592 of human PKC-␨ with no
cross-reactivity with PKC-␫ or mouse PKC-␭ homologues (9); C-14,
COOH-terminal antibody mapped to amino acids 578–592 of rat
PKC-␨ shown to immunologically detect holoenzyme and PKM-␨;
N/P, experiments not performed.
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 ⫽ ⬎10fold), 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 COOHterminal domain (37). When extracts were probed with
an NH2-terminal goat anti-human PKC-␨ selective an-
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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 antiPKC-␨ specific peptide antibodies and
their corresponding immunogenic peptides (Table 1).
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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.
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 nucleusfree cellular fractions from normal and day 12
Citrobacter-inoculated mouse distal colons were probed
for PKC-ε and PKC-␨ expression using the anti-PKC-ε
and C-20 anti-PKC-␨ antibodies (Fig. 5).
PKC-ε immunoreactivity was barely detectable in
Western blotted purified nuclear extracts in hyperpro-
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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tibody, 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.
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-ε 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
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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
liferating 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
Fig. 5. PKC-␨ is nuclear translocated in
hyperproliferating crypts. Anti-PKC-ε
(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-ε 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 72kDa (band A) forms. Band B was itself
resolved into 2 separate bands on 7%
gels (shown).
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.
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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 membraneto-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).
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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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.
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).
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 difFig. 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-ε 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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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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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
Fig. 8. Confocal volumetric reconstruction (100 ⫻ 0.4-␮m 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.
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 ␮m
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 ␮m) z-axis plane immunofluorescent signal recorded 50 ␮m from the base of simultaneously processed normal and hyperproliferating
crypts was 105 ⫾ 15 and 218 ⫾ 26 pixel intensities,
respectively. At 100 ␮m 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
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ferent 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 lowermass nuclear PKM-␨. No difference was seen for the
holoenzyme.
In addition to autophosphorylation, substrate phosphorylation of PKC-ε 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).
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
G231
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).
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). Higherspatial-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).
Figure 11 shows three vertical 1-␮m image planes
taken at ⫹5, ⫹38, and ⫹73 ␮m from the coverslip
surface. Filamentous subcellular PKC-␨ immunostaining was most apparent in the vertical (z-axis) image
plains close to the coverslip (⫹5 ␮m, Fig. 11A) and
bath-apposed crypt surfaces (⫹73 ␮m, Fig. 11C), which
bisect larger areas of subbasolateral plasma membrane cytoplasm. The mid-z-axis plane (⫹38 ␮m, 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).
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
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Fig. 9. Higher-magnification (⫻800) confocal volumetric (100 ⫻ 0.4-␮m 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
␮m 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). Cryptapposed 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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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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Fig. 10. Left, average PKC-␨ immunoreactive signal recorded from 3 confocal
mid-z-axis (0.4 ␮m) 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, 1313). 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-␮m2 (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.
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 con-
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
G233
trol. 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.
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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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) ␮m 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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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
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 ε-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
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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-␨
PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
Fig. 13. Proposed model for subcellular PKC-␨ activation during
TMCH. Mf, membrane-bound fragment.
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 PKCdependent 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 longterm 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 activ-
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(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 membraneactivated PKC-␨ (37) describes a constitutively active
nuclear targeted kinase during TMCH. Subtle differences in gel mobility and autonomous activity between
G235
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PKC-␨ ACTIVATION DURING EPITHELIAL HYPERPROLIFERATION
This work was supported by funds from the Cystic Fibrosis Foundation and the American Institute for Cancer Research.
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