(Received for publication, November 29, 1995; and in revised form, February 13, 1996)
From the
In a prior study, we have shown that stable transfection of
expression plasmids for protein kinase C1 (PKC
1) or PKC
2
into differentiated colon cancer cells led to elevated levels of
PKC
1 or PKC
2 protein and PKC
kinase activities in the
transfectants, without altering PKC
levels. PKC
is not found
in these cells, so the major modulation was in PKC
. PKC
transfectant cells exhibited blocked differentiation, increased growth
rate in athymic mice, and restoration of the basic fibroblast growth
factor response pathway. In this study, we have extended the analysis
of these PKC
transfectants to the mitogen-activated protein kinase
ERK3. Analysis of cell lysates on the mitogen-activated protein kinase
substrate myelin basic protein by in gel kinase assay showed increased
activity at 63 kDa, the size of ERK3, in each of two PKC
1 and each
of two PKC
2 transfectants compared with the vector control
transfectant. ERK3 was expressed at equal abundance in PKC
1,
PKC
2, and control transfectant cells as demonstrated by Western
blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody.
However, a >10-fold increase in ERK3 activity in each PKC
transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal
antibody followed by either immune complex kinase assay or by in gel
kinase assay. Thus, while overexpression of transfected PKC
does
not lead to overexpression of ERK3, it does lead to constitutive
activation of ERK3. A causal link between PKC
overexpression and
ERK3 activation was established because
12-O-tetradecanoylphorbol-13-acetate treatment down-regulated
both PKC and ERK3 activities in both PKC
1 transfectants. ERK3
activity was found in nuclear and membrane fractions in each PKC
transfectant, in contrast to controls, perhaps accounting for
constitutive activation of ERK3 in cells with elevated levels of
PKC
1 or PKC
2.
In previous studies, we found that differentiation of colon
carcinoma cells down-regulated the abundance and activity of PKC (
)while not altering levels of other PKC isozymes present in
these cells. Each of two independently cloned colon carcinoma lines
that display differentiation characteristics of mature
fluid-transporting colon enterocytes down-regulated PKC
activity
and abundance 5-10-fold compared with each of two
undifferentiated lines(1) . Neither differentiated line was
capable of transmitting mitogenic signals from basic fibroblast growth
factor to p57
(1) . Both differentiated and
undifferentiated lines maintained equal levels of the PKC isozymes
,
, and
, and none of the four lines exhibited either
PKC
or PKC
(1) , limiting at least the major, if not
the entire, PKC modulation to the PKC
isozyme. We transfected both
splice variants of PKC
(2) , PKC
1 and PKC
2, into
one of these differentiated colon carcinoma lines, HD3. The HD3
transfectant cells with increased PKC
1 and PKC
2 expression
but unaltered PKC
levels were blocked in differentiation, had
constitutively activated both the ERK1-related p57
and
ERK1, restored the basic fibroblast growth factor response pathway, and
had acquired the capacity for rapid growth in athymic mice(3) .
In this study, we have extended our studies of PKC
transfectants to the MAP kinase ERK3. ERK3 is structurally related to
the more well studied ERK1 and ERK2, with a 43% overall
homology(4) , but little is known of the function or activation
of ERK3. Two human homologues of the rat erk3 gene have been
cloned, one of 63 kDa with a 73% amino acid identity (5) and,
more recently, a 97-kDa homologue that has a 98% homology to rat Erk3
through the first 500 amino acids followed by a unique 178-amino acid
extension(6) . 97-kDa ERK3 has been shown to have kinase
activity on histone H1 when fibroblasts are stimulated with serum or
phorbol ester, but not insulin, insulin-like growth factor 1, or
epidermal growth factor(6) .
ERK1 and ERK2 are activated by
phosphorylation on tyrosine and threonine in a TEY site in subdomain
VII (7, 8, 9, 10) by a well known
pathway through MEK and MEK kinase (summarized in (11) ). In
contrast, ERK3 has an SEG site at the homologous site in subdomain VII (4) and may be activated in vivo through different
MEKs and MEK kinases or other kinases. We have asked in this study
whether elevated levels of PKC1 and PKC
2, which block
differentiation in colon cancer cells(3) , alter the activation
or abundance of ERK3.
Figure 1:
In gel kinase assay showing major band
of activity at 63 kDa in each PKC transfectant compared with
vector control cells. Molecular mass markers of 44, 71, 110, and 200
kDa are indicated by dashes.
Figure 2:
Immunoprecipitation of equal amounts of
ERK3 protein from each of two PKC1 and each of two PKC
2
transfectants and one vector control transfectant cell line. Antibody
was not used in excess. Immunoprecipitates were analyzed by Western
blotting with anti-ERK3 antibody and
I-protein A. The arrow marks the position of ERK3. The lighter bands are
immunoglobulin heavy chain and light chain. Molecular mass markers of
30, 44, 70, 110, and 200 kDa indicated at the right. V, vector
control transfectant cell line.
Figure 3:
Western blotting of cell lysates
identifies equal levels of ERK3 protein in each of two PKC1 and
each of two PKC
2 transfectants and one vector control transfectant
cell line using anti-ERK3 mAb. Detection was with
I-protein A following secondary antibody. The area around
the 63-kDa band only is shown. The arrow indicates ERK3
protein. V, vector control transfectant cell
line.
ERK3
was immunoprecipitated from each PKC and control transfectant, and
ERK3 activation was then analyzed in two ways. An immune complex kinase
experiment was performed using MBP as the substrate. Both PKC
1 and
both PKC
2 transfectant lines exhibited 13-fold more ERK3 activity
than the vector control in triplicate assays (Fig. 4). ERK3
immunoprecipitates were also analyzed by in gel kinase assay on
immobilized MBP. This assay allows one to visualize the kinase reaction
product at the molecular mass of the kinase, confirming the identify of
the MAP kinase immunoprecipitated by anti-ERK3 mAb as the p63 form of
ERK3 (Fig. 5). Kinase product was only visualized in
immunoprecipitates from the PKC
transfectant lines and was at
least 10-fold control levels. Similar activation of ERK3 in PKC
transfectants was also observed using the pan-ERK antiserum in two
additional immunoprecipitation experiments (data not shown). Therefore,
these experiments show that increased levels of PKC
1 and PKC
2
correlate in each of four transfectant lines with activation of ERK3,
but not with an increase in ERK3 abundance.
Figure 4:
Immune
complex kinase reaction demonstrates 13-fold increased activity of ERK3
in each of two PKC1 and each of two PKC
2 transfectants
compared with one vector control transfectant cell line. Means ±
S.E. of three separate determinations are shown. The actual total cpm
incorporated per PKC
transfectant ranged from 12,000 to 13,000. Vec, vector control transfectant cell
line.
Figure 5:
Immunoprecipitation of ERK3 followed by
analysis of ERK3 activity on in gel kinase assay on MBP showing
detectable activity of ERK3 only in PKC1-1 and PKC
2-2
transfectants, but not in vector control transfectants. Molecular mass
markers of 44, 71, 110, and 200 kDa are indicated by dashes. V, vector control transfectant.
Figure 6:
Down-regulation of PKC activity in each of
two PKC2 transfectants inhibits ERK3 MBP kinase activity in in gel
kinase assay. Cells were treated with either 100 ng/ml TPA or 0.1%
dimethyl sulfoxide, the diluent. The arrow indicates ERK3
activity. Molecular mass markers of 44, 71, 110, and 200 kDa are
indicated by dashes. Vec, vector control transfectant
cell line.
It has been reported that PKC2
found in colonocytes is not down-regulated efficiently by TPA, in
contrast to other PKC isozymes (14) . We were not able to
decrease p63
activity, as measured by in gel kinase
assay, or ERK3 activity, as assayed by immune complex kinase assay
after immunoprecipitation, by TPA treatment of PKC
2 transfectant
cells. Since neither activity was affected, however, this experiment
again correlated ERK3 activity with p63
activity (data
not shown).
Figure 7:
PKC transfectants contain activated
ERK3 kinase in nuclear fractions. In gel kinase assay was carried out
on MBP of cytosolic (C), nuclear (N), and membrane (M) fractions from PKC
transfectants and vector controls (CONT). Molecular mass markers of 44, 71, 110, and 200 kDa are
indicated by dashes. The arrow indicates activated
ERK3 kinase.
Figure 8:
PKC transfectants contain activated
ERK3 activities in their nuclear and membrane fractions. Unidentified
MBP kinases of 105 and 130 kDa are also seen in the membrane fractions
of only PKC
transfectants. Molecular mass markers of 44, 72, 110,
and 200 kDa are indicated at the left. The arrow indicates
activated ERK3 activities. Vec, vector control transfectant
cell line.
Constitutive activation of ERK3 might be related to the
enhanced levels of ERK3 found in nuclear and membrane fractions of
PKC transfectant cells. The nuclear and membrane location of some
of the ERK3 activity makes it unlikely that the MBP kinase activity
immunoprecipitated with both ERK3-specific mAb and pan-ERK antiserum ( Fig. 4and Fig. 5) was a 63-kDa proteolytic fragment of
PKC
contaminating both immunoprecipitations.
One of properties of the p44/p42 isoforms is
their capacity for activation by the phorbol ester class of PKC
activators (11, 16) . We have shown here and in an
earlier study (3) that overexpression of one PKC isoform,
PKC
, in colon cancer cells blocks differentiation and activates
not only the well studied MAP kinase ERK1, but also ERK3 and the
ERK1-related p57
. Thus, permutation in only one PKC
isozyme, PKC
, activates several MAP kinases, each of which may
have different sites of action, either within the nucleus to activate
different sets of transcription factors or at the cytoskeleton to
phosphorylate microtubule-associated proteins, destabilizing
microtubules and thus cytoskeletal structure and
organization(11, 16) . Several MAP kinases have been
isolated from various species from Drosophila to Xenopus to human, and these can be divided into different families
depending on DNA sequence homologies. MAP kinase families are
characterized by high homology between family members; for example,
ERK1 exhibits 90% sequence homology to ERK2(4) . However, these
``classical'' MAP kinases exhibit much lower sequence
homology to other MAP kinases. ERK1 has 43% homology to the ERK3
family(4) ; 40% homology to Jun kinases and p38
,
which are activated by cellular stress such as protein synthesis
inhibitors, osmotic shock, or UV
light(11, 17, 18) ; and 40% homology to
ERK5(19) , a newly cloned, larger MAP kinase. These different
MAP kinase families are expressed preferentially in various cell types,
suggesting that they may have different roles in proliferation,
differentiation, or embryonic development. Little is known about the
activation or role in cell proliferation or differentiation of any of
the ERK3 family members. 97-kDa ERK3 has been shown to have kinase
activity on histone H1 when fibroblasts are stimulated with serum or
phorbol ester, but not insulin, insulin-like growth factor 1, or
epidermal growth factor(6) . ERK1 and ERK2 and the Jun kinases
are activated by phosphorylation on tyrosine and threonine in a
TXY site in subdomain VII(7, 8, 9, 10, 11, 17, 18) by a
well known pathway through specific MEKs and MEK kinases(11) .
In contrast, ERK3 has an SEG site at the homologous site in subdomain
VII (4) and may be activated by other kinases in vivo.
There are at least 12 PKC members classified into three groups:
Ca-, phosphatidylserine-, and
diacylglycerol-dependent conventional PKC isoforms (
,
I,
II, and
); Ca
-independent novel PKC
isoforms (
,
, µ,
, and
); and
Ca
- and diacylglycerol-independent,
phosphatidylserine-dependent atypical PKC isoforms (
,
, and
). Roles for PKC isoforms may depend on cell type, and the ratios
of the different PKC isozymes within a cell also may alter a
cell's response to PKC activators. Craven and DeRubertis (20) have found that treatment of rats with a colon carcinogen
led to a relative increase in PKC
expression, together with a
decrease in PKC
expression in colon epithelial cells, leading to
an increased PKC
/PKC
ratio. Marian and co-workers (21) have measured PKC isozyme protein levels in normal human
colonocytes, human benign colon tumors, and human malignant colon
tumors and found a decrease in PKC isozyme levels in all benign and
malignant tumor cells, with much less decrease in PKC
, leading to
an enrichment of PKC
levels in colon tumors relative to the other
isozymes detectable in colon tumor tissue:
,
,
,
,
and
. The benign tumor cells with increased PKC
/PKC
ratio were responsive to TPA, a mitogen for colon adenoma
cells(21, 22) . The loss in most PKC isozymes on the
protein level in colon tumor tissue is consistent with earlier reports
that colon tumors express less total PKC mRNA than normal tissue (23) and, more recently, less PKC
and less PKC
mRNAs(24) .
We found in earlier studies that differentiation
of colon carcinoma cells to fluid-transporting enterocytic-like cells
led to a 5-10-fold decrease in PKC abundance and activity,
with no change in abundance of the other PKC isozymes detectable,
,
, and
(1) . These studies, like the studies
cited above, point to a role for enhanced levels of PKC
relative
to the other isozymes in colon tumor progression. Transfection of
either PKC
isoform, PKC
1 or PKC
2, into such
differentiated colon carcinoma cells restored the undifferentiated
phenotype and proliferative response to basic fibroblast growth factor
and allowed more rapid growth in athymic mice (3) . The
mechanism, at least in part, for this blocked differentiation was a
constitutive activation of both ERK1 and the ERK1-related
p57
(3) . In the current study, a third MAP
kinase, ERK3, was found also to be constitutively activated by
overexpression of PKC
1 or PKC
2. ERK3 was found associated
with the nucleus and membrane in PKC
transfectant cells. Nuclear
location may allow the activated ERK3 to act as a transcription factor
kinase, while membrane location may allow association with the
microtubule cytoskeleton. ERK1 and ERK2 are associated with the
microtubule cytoskeleton in NIH3T3 fibroblasts(25) . MAP
kinases are known to phosphorylate microtubule-associated proteins,
causing microtubule instability(11) , perhaps leading to the
cellular and nuclear shape changes that occur during S phase and
mitosis.
Our studies have correlated increased expression of
PKC1 and PKC
2 with increased proliferation in colon cancer
due to activation of multiple MAP kinases. Others have found
constitutive activation of MAP kinases in human renal
cancers(26) . In a series of 25 cases of paired normal kidney
tissue and renal carcinomas, constitutive activation of MAP kinases, as
determined by the appearance of phosphorylated forms of ERK2, was found
in 48% of the cases(26) . Others have found PKC
to be
elevated in activity in invasive gastric cancers(27) .
PKC
2 is required for the proliferation of K562 human
erythroleukemia cells (28) . Overexpression of PKC
2 in
HL-60 promyelocytic leukemic cells enhances their proliferation and
makes them resistant to TPA-induced differentiation(29) .
PKC
2 translocates to the nuclear envelope and phosphorylates lamin
B in cells treated with mitogenic stimuli, but not with differentiation
inducers like TPA(29) . Thus, proliferation of HL-60 cells is
correlated with nuclear translocation of PKC
2 and its
phosphorylation of nuclear envelope lamin B, part of the nuclear
membrane breakdown that occurs when cells enter mitosis. It would be of
interest to determine whether nuclear forms of ERK3 could be
responsible for this phosphorylation. Thus, overexpression of PKC
2
has, in several cell types, been associated with increased
proliferation and blocked differentiation.
The role of PKC1 is
more problematic. Other investigators have shown that expression of
elevated levels of PKC
1 in uncloned HT29 colon carcinoma cells (30) and SW480 colon carcinoma cells (31) inhibited
tumorigenicity and cell growth. The uncloned HT29 line and the SW480
line are both undifferentiated and highly tumorigenic lines. In our
studies, PKC
1 was overexpressed following stable transfection of a
poorly tumorigenic, differentiated cell line with low PKC
levels (1) , and the transfectants simply had restored PKC
1
levels to those characteristic of undifferentiated parental
cells(3) , not significantly above this level as in the other
cited studies. In this and earlier studies, we have shown that at least
part of the mechanism of action of PKC
in colon cancer cells is
constitutive activation of multiple MAP kinases. It will now be
necessary to determine the spectrum of substrates for the ERK1-like
p57
, ERK3, and ERK1 in colon carcinoma cells in both the
nucleus and cytoskeleton. In this way, it may be possible to identify
the proximal effectors of PKC
.