(Received for publication, August 30, 1995; and in revised form, October 24, 1995)
From the
The expression of an oncogenic ras gene in
epidermal keratinocytes stimulates the tyrosine phosphorylation of
protein kinase C
and inhibits its enzymatic activity (Denning, M.
F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H.(1993) J. Biol.
Chem. 268, 26079-26081). Keratinocytes expressing an
activated ras
gene secrete transforming growth
factor
(TGF
) and have an altered response to differentiation
signals involving protein kinase C (PKC). Because the neoplastic
phenotype of v-ras
expressing keratinocytes can
be partially mimicked in vitro by chronic treatment with
TGF
and the G protein activator aluminum fluoride
(AlF
), we determined if TGF
or
AlF
could induce tyrosine phosphorylation
of PKC
. Treatment of primary keratinocyte cultures for 4 days with
TGF
induced tyrosine phosphorylation of PKC
, whereas
AlF
only slightly stimulated PKC
tyrosine phosphorylation. The PKC
that was tyrosine-phosphorylated
in response to TGF
had reduced activity compared with the
nontyrosine-phosphorylated PKC
. Treatment of keratinocytes
expressing a normal epidermal growth factor receptor (EGFR) with
TGF
or epidermal growth factor for 5 min induced PKC
tyrosine
phosphorylation. This acute epidermal growth factor treatment did not
induce tyrosine phosphorylation of PKC
in keratinocytes isolated
from waved-2 mice that have a defective epidermal growth
factor receptor. In addition, the level of PKC
tyrosine
phosphorylation in v-ras
-transduced keratinocytes
from EGFR null mice was substantially lower than in
v-ras
transduced wild type cells, suggesting that
activation of the EGFR is important for PKC
tyrosine
phosphorylation in ras transformation. However, purified EGFR
did not phosphorylate recombinant PKC
in vitro, whereas
members of the Src family (c-Src, c-Fyn) and membrane preparations from
keratinocytes did. Furthermore, clearing c-Src or c-Fyn from
keratinocyte membrane lysates decreased PKC
tyrosine
phosphorylation, and c-Src and c-Fyn isolated from keratinocytes
treated with TGF
had increased kinase activity. Acute or chronic
treatment with TGF
did not induce significant PKC
translocation in contrast to the phorbol ester
12-O-tetradecanoylphorbol-13-acetate, which induced both
translocation and tyrosine phosphorylation of PKC
. This suggests
that TGF
-induced tyrosine phosphorylation of PKC
results from
the activation of a tyrosine kinase rather than physical association of
PKC
with a membrane-anchored tyrosine kinase. Taken together,
these results indicate that PKC
activity is inhibited by tyrosine
phosphorylation in response to EGFR-mediated signaling and activation
of a member of the Src kinase family may be the proximal tyrosine
kinase acting on PKC
in keratinocytes.
The protein kinase C (PKC) ()family of
serine/threonine protein kinases is a central component of
phospholipase-coupled growth factor receptor signaling
pathways(1) . The enzymatic activities of several PKC isoforms
are regulated by the allosteric activators diacylglycerol and
Ca
, which are elevated in response to growth factor
receptor activation(2) . The G protein Ras is also a component
of the mitogen-activated protein kinase signaling pathways for growth
factors such as epidermal growth factor (EGF) and platelet derived
growth factor (PDGF). In addition, PKC
can directly affect the
mitogen-activated protein kinase pathway by phosphorylating and
stimulating the autokinase activity of Raf-1(3) . Ras can act
either as a regulator or effector of PKC function, but the molecular
mechanisms involved are
unclear(4, 5, 6, 7, 8) .
In epidermal keratinocytes, Ras can have both positive and negative
effects on PKC signaling. Neoplastic mouse keratinocytes expressing an
activated ras allele have increases in
phosphatidylinositol turnover, diacylglycerol levels, and
calcium-dependent PKC activity(9, 10) . Activation of
PKC
, the only calcium-dependent PKC isoform expressed in
keratinocytes, is correlated with the expression of granular layer
differentiation markers in normal keratinocytes and in
v-ras
expressing keratinocytes, where granular
layer differentiation markers are
up-regulated(10, 11) . Keratinocytes expressing a
v-ras
oncogene also have decreased
Ca
-independent PKC activity and have a block in their
ability to commit to Ca
and TPA-induced terminal
differentiation(10, 12, 13, 14) .
The block in differentiation response may be due to the tyrosine
phosphorylation and inactivation of PKC
because the kinase
inhibitor staurosporine blocks PKC
tyrosine phosphorylation and
induces terminal differentiation of neoplastic
keratinocytes(5) . In addition, v-ras
transformation of keratinocytes up-regulates epidermal growth
factor receptor (EGFR) ligand expression and induces the secretion of
transforming growth factor
(TGF
)(15, 16, 17) . Several studies have
indicated that increased secretion of TGF
can substitute for ras
mutations in the altered response to
differentiation signals (17) and can initiate skin
carcinogenesis(18, 19) .
In this study, we further
characterized the signal transduction pathways regulating PKC
tyrosine phosphorylation in murine keratinocytes. We demonstrate that
TGF
is able to induce tyrosine phosphorylation of PKC
resulting in inhibition of its activity. Although tyrosine
phosphorylation of PKC
in intact cells requires a functional EGFR,
it is not directly mediated by EGFR and may involve a member of the Src
kinase family. Tyrosine phosphorylation of PKC
by the EGFR pathway
demonstrates a perturbation of PKC signaling by TGF
that may
contribute to the process of neoplastic transformation in epithelial
cells.
Immunoprecipitations were performed as
described previously(5) . Briefly, the cells were washed with
phosphate-buffered saline and scraped into immunoprecipitation lysis
buffer, and equal amounts of protein were immunoprecipitated with 20
µl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology) and
either 2.5 µg anti-phosphotyrosine antibody or 0.5-1 µl
of anti-PKC antibody. The c-Src and c-Fyn immunoprecipitations
were performed with 2 µg of antibody from Santa Cruz Biotechnology,
Inc. (sc-19 and sc-16 respectively) and 50 µl protein A-Sepharose
from Sigma. For some phosphotyrosine immunoprecipitations, an
agarose-conjugated anti-phosphotyrosine monoclonal antibody from
Upstate Biotechnology Inc. was used. For immunoblotting, the
immunoprecipitates were washed three times with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS,
and 1% sodium deoxycholate) and boiled in 20 µl of SDS sample
buffer(25) .
PKC assays of tyrosine-phosphorylated and nontyrosine-phosphorylated
PKC were performed as described previously with minor
modifications (5) . Primary keratinocytes were cultured in 10
ng/ml TGF
for 4 days to induce the tyrosine phosphorylation of a
fraction (
50%) of PKC
. Lysates were prepared (n = 3) and immunoprecipitated with an anti-phosphotyrosine
antibody for 2.5 h. The phosphotyrosine-cleared lysates were taken for
immunoprecipitation of nontyrosine-phosphorylated PKC
. Both
phosphorylated and nonphosphorylated samples were immunoprecipitated
for PKC
overnight in the presence of 30 mMp-nitrophenylphosphate to release phosphotyrosine
containing proteins from the anti-phosphotyrosine antibody. The
immunoprecipitates were washed once with immunoprecipitation lysis
buffer and twice in 50 mM Tris-HCl, pH 7.4, and resuspended in
25 µl of Tris-HCl and 5 mM 2-mercaptoethanol. PKC assays
were performed on the immunoprecipitated PKC
in the presence or
the absence of 1 µM TPA as described by Nakadate et
al.(26) except that the PKC substrate peptide
[ser
] (Life Technologies, Inc.) was the
phosphate acceptor, and 20%/80% phosphatidylserine/phosphatidylcholine
vesicles were the phospholipid. After the enzyme reaction, the agarose
beads were spun, and 30 µl of supernatant was spotted for
scintillation counting. The PKC
remaining on the beads was boiled
in SDS sample buffer and analyzed by anti-PKC
immunoblot to
correct for differences in the amount of PKC
in the assay.
Figure 1:
Tyrosine phosphorylation of PKC in
response to TGF
. In A, primary mouse keratinocytes were
cultured in the presence of 10 ng/ml TGF
or 1 µM AlCl
, 1 mM NaF
(AlF
) for 4 days, and lysates (700 µg
protein) were immunoprecipitated with an anti-phosphotyrosine antibody.
The immunoprecipitates were immunoblotted with an anti-PKC
antibody. In B, 10 ng/ml of TGF
was added to cultures of
primary keratinocytes for the indicated times, and lysates (550 µg
of protein) were prepared for PKC
immunoprecipitation. The
immunoprecipitates were immunoblotted against an anti-phosphotyrosine
antibody and restained with an anti-PKC
antibody. Similar results
were obtained in one additional experiment.
Because
TGF was a more effective inducer of PKC
tyrosine
phosphorylation than AlF
, we further
analyzed TGF
-induced PKC
tyrosine phosphorylation. The
kinetics of TGF
-induced PKC
tyrosine phosphorylation were
biphasic (Fig. 1B). Tyrosine phosphorylation of
PKC
was increased approximately 8-fold after a 5-min TGF
treatment (10 ng/ml), returned toward basal levels by 1-2 h, and
increased again from 4 and 6 h (9-11-fold), remaining elevated
(4-fold) even after 24 h of TGF
treatment. The total levels of
PKC
did not change appreciably during this time. Acute and chronic
treatment with EGF also induced PKC
tyrosine phosphorylation (see Fig. 3A).
Figure 3:
Mutant alleles of the EGFR inhibit
PKC tyrosine phosphorylation. A, primary keratinocytes
were isolated from mice having a wild type EGFR (BALB/c, +/+
and +/wa-2) and from waved-2 mice homozygous for
the mutant EGFR (wa-2/wa-2). The keratinocytes were
cultured and infected with v-ras
for 5 days (v-ras), treated with 10 ng/ml EGF for 4 days (EGF 4D), or 5
min (EGF 5 M), and PKC
was immunoprecipitated from
cellular lysates (750 µg of protein). The immunoprecipitates were
immunoblotted with an anti-phosphotyrosine antibody (Anti
p-Tyr) and anti-PKC
antibody (Anti PKC
).
Similar results were obtained in two additional experiments. B, primary keratinocytes were isolated from mice having a wild
type EGFR (+/+) and from EGFR null mice (-/-).
The keratinocytes were initially cultured in 1 ng/ml keratinocyte
growth factor for 3 days, switched to unsupplemented medium, and either
treated with 100 nM TPA for 10 min (TPA) or infected
with v-ras
for 4 days (ras). Cellular
lysates were immunoprecipitated with anti-phosphotyrosine antibody (p-Tyr IP), and gel separated total lysates (Total) were
immunoblotted for the detection of total PKC
levels with the
anti-PKC
antibody. A similar result was observed in one additional
experiment.
Figure 2:
Enzymatic activity of
tyrosine-phosphorylated PKC. Primary keratinocytes were cultured
for 4 days in medium containing 10 ng/ml TGF
, and the lysates (n = 3) were immunoprecipitated sequentially with an
anti-phosphotyrosine antibody followed by an anti-PKC
antibody.
PKC activity was assayed in the tyrosine-phosphorylated and
nonphosphorylated fractions in the presence or the absence of 1
µM TPA. For calculating PKC
-specific activities, the
amount of PKC
in each assay tube was normalized by immunoblotting
with an anti-PKC
antibody. Similar results were obtained in two
additional experiments.
Tyrosine phosphorylation of
PKC was also assessed in keratinocytes isolated from mice
harboring a disrupted EGFR gene(21) . The immunoblots in Fig. 3B show that PKC
was tyrosine-phosphorylated
to a similar extent in both EGFR +/+ and -/-
keratinocytes (1200 and 1700% increase, respectively) in response to
acute TPA treatment. In contrast, v-ras
transduction was less effective at inducing PKC
tyrosine
phosphorylation in the EGFR -/- keratinocytes than in the
+/+ cells of the same genetic background (60 and 250%
increase, respectively). There was no difference in the amount of total
PKC
between the EGFR +/+ and -/-
keratinocytes. These genetic data strongly support the involvement of
the EGFR in the v-ras
-induced PKC
tyrosine
phosphorylation.
Figure 4:
Tyrosine phosphorylation of PKC by
Src family kinases. In A, recombinant PKC
from
baculovirus-infected Sf-9 insect cells was incubated alone or with
purified EGFR, c-Src, c-Fyn, or a membrane fraction from untreated
normal keratinocytes in tyrosine kinase assay buffer as indicated.
Total reaction mixtures and PKC
immunoprecipitated from the
reactions were analyzed for phosphotyrosine by immunoblotting with
anti-phosphotyrosine antibody. In B, 10 µg of membrane
protein from untreated keratinocytes were incubated with the indicated
clearing antibody followed by protein A-Sepharose, and the cleared
lysate was added to recombinant PKC
for the tyrosine kinase assay.
The reaction mixtures were immunoprecipitated for phosphotyrosine and
stained with an anti-PKC
antibody. In C, primary
keratinocytes were treated with 10 ng/ml TGF
for 5 min (5M) or 5 days (5D) as indicated, and cellular
lysates immunoprecipitated with nonspecific rabbit IgG, c-Src, or c-Fyn
antibodies. The immunoprecipitates were incubated with tyrosine kinase
assay buffer (``Experimental Procedures''), and the
phosphorylated proteins were analyzed by immunoblotting with an
anti-phosphotyrosine antibody. Kinase refers to the band
corresponding to either Src or Fyn kinase. IP,
immunoprecipitation.
Figure 5:
Subcellular distribution of PKC after
TGF
or TPA treatment. In A, keratinocytes were treated
with 10 ng/ml TGF
for either 2 min or 4 days and either
fractionated into soluble and particulate fractions or lysates prepared
and immunoprecipitated with phosphotyrosine antibody. The fractionated
and immunoprecipitated samples were immunoblotted with anti-PKC
antibody. In B, keratinocytes were treated with 100 nM TPA for 10 min, and PKC
translocation and tyrosine
phosphorylation were assessed as described for A. IP,
immunoprecipitation.
To determine
if either c-Src or c-Fyn was present in the keratinocyte membranes and
was phosphorylating PKC, we immunoprecipitated c-Src or c-Fyn from
a solubilized membrane fraction and tested if this cleared lysate could
phosphorylate PKC
on tyrosine. Fig. 4B shows that
removal of c-Src or c-Fyn from the membrane lysate significantly
decreased the PKC
tyrosine kinase activity compared with the
nonspecific rabbit IgG cleared lysate. The tyrosine kinase activity of
c-Src and c-Fyn immunoprecipitated from keratinocytes treated with
TGF
for 5 min or 5 days is shown in Fig. 4C. The
autophosphorylation and heterophosphorylation of multiple proteins by
both c-Src and c-Fyn was increased by acute and chronic TGF
treatment. Kinase activity was determined by densitometry of the
phosphotyrosine immunoblots. C-Src kinase activity increased 1.42
± 0.35-fold and 1.66 ± 0.34-fold (mean ± standard
deviation, n = 2) after acute and chronic TGF
treatment. The effect of TGF
on c-Fyn kinase activity was greater,
increasing 2.05 ± 0.35-fold and 3.26 ± 0.50-fold after
acute and chronic TGF
(n = 3). Immunoprecipitation
with nonspecific IgG recovered no detectable tyrosine kinase activity (Fig. 4C). In addition, the recovery of tyrosine kinase
activity in c-Src and c-Fyn immunoprecipitates was completely blocked
by adding an excess of the peptide that the antibodies were raised
against (data not shown). PKC
was not present in c-Src and c-Fyn
immunoprecipitates when assayed by immunoblot (data not shown). These
results indicate that keratinocyte c-Src and c-Fyn can phosphorylate
PKC
and that both of these tyrosine kinases are activated in
response to TGF
treatment of keratinocytes.
Figure 6:
In vitro tyrosine phosphorylation
of PKC isozymes. In A, recombinant PKC isozymes from Sf-9
cells were incubated with either c-Src or c-Fyn kinase as described
under ``Experimental Procedures,'' and the total reaction
mixtures were analyzed for phosphotyrosine by immunoblotting. To
identify the location of each PKC isozyme, the blot was stripped and
restained for individual PKC isoforms. Similar results were obtained in
two additional experiments. In B, total tyrosine kinase
reaction mixtures of the indicated PKC isozyme either alone or with
c-Src or c-Fyn were immunoprecipitated with an anti-phosphotyrosine
antibody. The immunoprecipitates were immunoblotted with PKC antibodies
specific for PKC,
,
, or
as
indicated.
In this report, we demonstrate that TGF treatment
induces tyrosine phosphorylation of PKC
and inhibits its activity.
In view of the high level of TGF
produced by transformed mouse
keratinocytes (15, 16) , these results provide a
mechanism for the tyrosine phosphorylation of PKC
induced by
oncogenic ras(5) . Studies using keratinocytes from mice with
mutant alleles of EGFR revealed that the EGFR is required for
TGF
-induced tyrosine phosphorylation of PKC
, but biochemical
analysis indicates that the EGFR does not phosphorylate PKC
directly. We demonstrate that c-Src and c-Fyn kinases become activated
after TGF
treatment, and PKC
, as well as PKC
,
,
and
, are substrates for c-Src and c-Fyn kinases in
vitro.
Activation of Src family kinases in response to
EGFR-ligand interaction has been reported previously in other cell
types(34, 35) . Several possible mechanisms have been
proposed for the activation of Src family tyrosine kinase by EGFR. The
Src SH2 domain can bind to activated, tyrosine-phosphorylated
EGFR(36, 37) , and Src itself has been shown to
phosphorylate the EGFR and create a Src SH2 docking site(35) .
C-Src can also bind to tyrosine-phosphorylated Neu, which can be
transphosphorylated by activated EGFR(36, 38) .
Finally, signaling molecules downstream from the EGFR such as
p21Gap and Raf-1 associate with Src family members,
illustrating that direct association with the EGFR is not necessary for
Src activation(39, 40) .
The mutant mouse strain waved-2 harbors a point mutation in the EGFR, which decreases
its tyrosine kinase activity >90%(22) , consistent with the
lack of PKC tyrosine phosphorylation in waved-2 keratinocytes after acute EGF treatment. In contrast,
v-ras
keratinocytes from waved-2 mice have
tyrosine-phosphorylated PKC
, suggesting that EGFR ligands secreted
by transformed keratinocytes are responsible for the PKC
tyrosine
phosphorylation, just as chronic (4 days) treatment with EGF is capable
of inducing PKC
phosphorylation in waved-2 keratinocytes.
The role of EGFR ligands in v-ras-stimulated
PKC
tyrosine phosphorylation was strongly supported by experiments
using EGFR deficient keratinocytes (Fig. 3B). The
increase in PKC
tyrosine phosphorylation in the EGFR
-/- v-ras
keratinocytes was only 35%
± 17% (mean ± standard deviation, n = 2)
of that induced in the EGFR +/+ v-ras
keratinocytes of the same strain. In the same experiments, the
induction of PKC
tyrosine phosphorylation by TPA was almost
identical in the EGFR +/+ and -/- keratinocytes
(18.9-fold and 18.4-fold, respectively), indicating that the EGFR
kinase is not essential for PKC
tyrosine phosphorylation, that the
EGFR null keratinocytes are competent to respond to other external
stimuli, and that TPA targets PKC
through an independent pathway.
These studies establish the critical role for EGFR signal transduction
in altering PKC
tyrosine phosphorylation and subsequent enzymatic
activity in Ras-transformed keratinocytes. Nevertheless, transduction
of EGFR -/- keratinocytes with v-ras
induced a small increase in tyrosine phosphorylation of PKC
,
indicating that other pathways may contribute to Ras-induced PKC
tyrosine phosphorylation. For example, stimulation of
phosphatidylinositol turnover by AlF
slightly elevated PKC
tyrosine phosphorylation in normal
keratinocytes (Fig. 1A), and inositol phosphate
metabolism is up-regulated in v-ras
-transformed
keratinocytes(9, 41) .
Tyrosine phosphorylation of
PKC has been reported in the promyeloid cell line 32D and NIH-3T3
fibroblasts treated with the tumor promoter TPA or
PDGF(27, 29) . PKC
tyrosine phosphorylation may
also play a role in the IgE receptor signaling pathway of mast cells (32) and in saliva production by parotid acinar cells in
response to substance P or the muscarinic agonist
carbachol(31) . However, the published effects of tyrosine
phosphorylation on PKC
activity are ambiguous.
Tyrosine-phosphorylated PKC
isolated from ras
-transformed (5) and TGF
-treated
keratinocytes (Fig. 2) has reduced activity. Li et al. observed increased PKC activity in the membrane fraction of
32D/PDGF-
R/PKC-
or NIH-3T3/PKC-
cells treated with
either TPA or PDGF to increase the level of membrane-associated
tyrosine-phosphorylated PKC
(29) . However, this activity
may be due to nontyrosine-phosphorylated PKC
or other PKC isozymes
that translocate to the membrane fraction after TPA treatment. In
vitro tyrosine phosphorylation of PKC
by Fyn, insulin
receptor, or PDGF receptor also resulted in a <2-fold increase in
PKC activity (27) , but the relevance to tyrosine
phosphorylation in intact cells is unknown because differences in
substrates or assay conditions can influence the activity of PKC. For
example, the activity of tyrosine-phosphorylated PKC
in response
to activation of the IgE receptor showed decreased activity toward its
physiological substrate, the Fc
RI
chain, and increased
activity toward myelin basic protein(32) . Li et al. have also generated a mutant PKC
that was constitutively
phosphorylated on tyrosine and catalytically inactive, further
supporting an association between tyrosine phosphorylation and
inhibition of PKC
activity(42) . PKC
can be
tyrosine-phosphorylated on more than one site, and distinct
phosphorylation sites may regulate activity differently (43) . (
)To date, most experiments where the
tyrosine-phosphorylated PKC
is isolated from intact cells support
a role for tyrosine phosphorylation in the inhibition of PKC
activity (5, 32, 42) .
Translocation of
PKC in response to EGF is observed in some cell
types(44) , but not in others ( Fig. 5and (31) ), and our results indicate that translocation is not a
prerequisite for tyrosine phosphorylation. In unstimulated mouse
keratinocytes, 30-50% of the PKC
is localized to the
particulate fraction(11, 33) . Furthermore, as shown
in Fig. 4(A and B), c-Src and c-Fyn
constitute the major PKC
tyrosine kinase activity in the membrane
fraction of keratinocytes where tyrosine-phosphorylated PKC
is
localized(5, 29) . Thus, a pool of
particulate-associated PKC
exists in keratinocytes that can become
tyrosine-phosphorylated upon activation of the appropriate kinase.
Specialized functions for individual PKC isozymes have been
identified in several cell types(45) . PKC is involved in
myeloid differentiation (46) and secretion in basophilic
RBL-2H3 cells(47) . PKC
also regulates cell cycle
progression in CHO cells (48) and growth arrest in
fibroblasts(49, 50) . In primary mouse keratinocytes,
PKC
translocates in response to Ca
-induced
differentiation(11) . Moreover, TPA-induced keratinocyte
differentiation is inhibited by concentrations of bryostatin 1
(10-1000 nM) that protect PKC
from down-regulation (33) . Thus, activation of PKC
may be important for
commitment to keratinocyte terminal differentiation. Both
v-ras
and EGFR ligands modify keratinocyte
differentiation in vitro, and EGFR activation reproduces a
subset of phenotypic alterations characteristic of neoplastic
v-ras
keratinocytes(9, 14, 17) . Therefore,
the common target of PKC
tyrosine phosphorylation for
v-ras
and EGFR ligands may be relevant to the
phenotypic alterations in epidermal neoplasia.
In addition to
PKC, c-Src and c-Fyn phosphorylate other PKC isoforms (
,
, and
) in vitro, but we have not detected tyrosine
phosphorylation of any isoforms except PKC
in intact cells. The in vivo substrate specificity of c-Src and c-Fyn may be
determined by the subcellular distribution of the kinases and
substrates as well as direct physical associations. Thus, PKC isoforms
such as PKC
and
, which are readily phosphorylated in
vitro by c-Src and c-Fyn, may not be accessible to the appropriate
tyrosine kinase in living cells. As can be seen from Fig. 4C, c-Src and c-Fyn have a different pattern of
associated proteins, and these could influence their substrate
specificity.
This report defines a novel connection between tyrosine kinase signaling and the PKC family of enzymes, which may have important functional consequences for epithelial growth, differentiation, and carcinogenesis. The EGFR and PKC pathways are two major signaling systems for epidermal keratinocytes. A better understanding of these signal transduction pathways and cross-talk between the different kinase cascades provides new insight for the design of drugs to treat diseases involving this cell type.