(Received for publication, November 21, 1994; and in revised form, May 10, 1995)
From the
Rapid and long term effects of protein kinase C activation
on receptor tyrosine kinase signaling parameters were investigated in
human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes
of phorbol 12-myristate 13-acetate treatment, epidermal growth factor
receptor and HER2 tyrosine phosphorylation was decreased, while
platelet-derived growth factor receptor and insulin receptor
autophosphorylation was up-regulated. These effects are not mediated by
protein kinase C-dependent receptor tyrosine kinase phosphorylation but
apparently by activation or inactivation of receptor tyrosine
kinase-specific phosphatases, as indicated by neutralization of these
phenomena upon treatment of cells with sodium orthovanadate. In
contrast to these short term effects, sustained activation of protein
kinase C
by phorbol 12-myristate 13-acetate results in
translocation of protein kinase C from the cytosol to the membrane
fraction where it forms stable complexes with all receptor tyrosine
kinases investigated. Ligand-induced receptor tyrosine kinase/protein
kinase C association in NIH 3T3 fibroblasts is accompanied by a
mobility shift of the receptor, indicating phosphorylation by activated
protein kinase C. This phenomenon correlates with the disappearance of
receptor tyrosine kinases from the cell surface, implying that this
interaction plays a role in the process of receptor internalization and
degradation. Interestingly, ligand-stimulated receptor down-regulation
is also enhanced by overexpression of phospholipase C
, which
strongly indicates a role for this common receptor tyrosine kinase
substrate in negative regulation of growth factor signals.
Reversible protein phosphorylation on tyrosine residues
represents a regulatory mechanism that governs a wide spectrum of
fundamental processes in cells of higher eukaryotes. Among the cellular
enzymes that catalyze these reactions are tyrosine kinases with a
covalently connected extracellular ligand binding function, which play
a key role in the intercellular communication network that is crucial
for the regulation of growth, development, and metabolic homeostasis of
multicellular organisms. The tyrosine-specific phosphorylation function
of these receptor tyrosine kinases (RTKs) ()is indispensable
for the activation of signaling pathways that promote cellular
responses such as cell division, differentiation, and migration.
Engagement of such cellular effector systems occurs through
phosphorylated tyrosine residues of RTKs, which function as high
affinity binding sites for proteins containing Src homology 2 (SH2)
domains. These occur either in combination with a catalytic function
such as phospholipase C
(PLC
), the GTPase-activating protein
(GAP), the Src tyrosine kinase, the tyrosine phosphatase PTP1D/Syp, and
the GDP/GTP exchanger protein Vav or as adapter proteins such as the
p85 subunit of phosphatidylinositol 3`-kinase (PI 3`-kinase), CRK, NCK,
SHC, and GRB2 (reviewed in (1, 2, 3) ).
Despite extensive information concerning molecular interactions in the signaling cascade triggered by receptor-ligand interaction on the cell surface, the events that eventually activate specific gene transcription and define the ligand-characteristic response of the cell remain poorly understood. It is clear, however, that downstream from tyrosine-phosphorylated primary substrates the activation of serine/threonine kinases is of major significance for signal transmission and definition. These include the Raf-1 kinase(4, 5) , the S6 kinases(6, 7, 8) , mitogen-activated protein kinase kinase(9) , mitogen-activated protein kinase(10, 11, 12) , casein-2 kinases(13) , and the members of the protein kinase C (PKC) family.
The PKC serine/threonine kinases, a family of at least 12
isoenzymes with distinct tissue distribution characteristics, have been
subdivided on the basis of different primary structures and enzymatic
properties into Ca-dependent or conventional PKCs and
the Ca
-independent or novel PKCs (reviewed in ref.
14), and extensive evidence is available for their involvement in
control of cell proliferation, differentiation, and
motility(15) . While certain PKC isotypes are activated by
phorbol esters such as phorbol 12-myristate 13-acetate (PMA), their
physiological ligand is the second messenger diacylglycerol
(DAG)(16, 17) , a product of the
phosphoinositide-specific PLC, whose
subtype was shown to be
activated by mitogens such as epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF) through receptor-mediated
tyrosine
phosphorylation(18, 19, 20, 21, 22) .
In addition to DAG, PLC
-catalyzed hydrolysis of
phosphatidylinositol 4,5-biphosphate yields inositol
1,4,5-triphosphate, a regulator of intracellular
[Ca
]
, which together
with DAG activates PKC.
Among its diverse effects on the physiology
of cells (reviewed in (16) ), PKC was shown to phosphorylate
and activate the serine/threonine kinase
Raf-1(23, 24) , which triggers the mitogen-activated
protein kinase signaling pathway and ultimately leads to
transcriptional activation of specific genes(25) . In addition
to this positive influence on growth factor signals, PKC activation is
known to down-modulate the signaling potential of the receptors for EGF
and insulin. Phosphorylation of the EGF-R at Thr-654 attenuates high
affinity EGF binding (26) and causes a decrease in
EGF-stimulated tyrosine kinase activity and DNA
synthesis(27, 28) . Similarly, the signaling activity
of the insulin receptor, which appears to be primarily phosphorylated
on carboxyl-terminal serine and threonine
residues(29, 30) , is impaired after PMA treatment of
some cell types due to a reduction of its tyrosine kinase
activity(31, 32, 33) , while in other cells
an activation of RTK activity was
observed(34, 35, 36) . Moreover, as recently
shown, phorbol ester treatment of Chinese hamster ovary cells
overexpressing PKC and the insulin receptor resulted in
phosphorylation of receptor threonine and serine residues without an
effect on tyrosine phosphorylation but caused inhibition of
insulin-stimulated PI 3`-kinase activity(37) . Interestingly,
although the EGF-R and PDGF-R share common signaling pathways, they
differ with respect to PKC-dependent negative feedback mechanisms.
While PMA treatment of A431 cells blocks EGF-induced PI
turnover(38, 39) , it does not inhibit that induced by
PDGF(40, 41) , suggesting differential regulation of
RTKs by the PKC system within the same cell.
In addition to its
differential effects on receptor-ligand interactions and signal
transduction, PKC has been implicated in receptor internalization and
degradation. Chen et al.(42) identified a distinct
region within the EGF-R COOH terminus that appears to be required for
ligand-dependent down-regulation and degradation. Interestingly, the
high affinity binding site for PLC, Tyr-992, is located within
this region(43) , which suggests that PKC activation via
PLC
-generated second messengers is involved in receptor
down-regulation. Nevertheless, the molecular signals that are involved
are still poorly understood, and their interpretation is a matter of
controversy.
To further elucidate the complex role of PKC in the
cellular signaling network, we investigated its short and long term
effects on RTK function. While short term stimulation of PKC affects
the EGF and HER1-2 receptors differently than those for PDGF and
insulin, the functional consequences for these receptors are identical
after prolonged PKC activation. PKC-mediated signal modulation involves
ligand-induced, high affinity complex formation with receptors.
Moreover, our findings strongly support a role for PLC in
PKC-mediated down-regulation of RTK signals.
The following
monoclonal antibodies and polyclonal antisera were used: 108.1 (49) mouse monoclonal antibody against the extracellular domain
of the EGF-R; 5E2 mouse monoclonal antibody against phosphotyrosine (50) ; CT-PLC, rabbit polyclonal antiserum against a
peptide corresponding to amino acid residues 1255-1274 of human
PLC
(DFRISQEHLADHFDSRERR) (provided by Reiner Lammers); and
PKC-specific polyclonal antibodies (RIOS) (provided by Etta Livneh).
To analyze the functional relationship of PKC and various
RTKs in intact cells, we employed chimeric receptors consisting of the
human EGF-R extracellular domain fused to the cytoplasmic domains of
the murine PDGF--R (EP-R), human insulin receptor (EI-R), and the
EGF-R isozyme HER2 (HER1-2). As previously shown, all of these
chimeric receptors are functional and generate a cytoplasmic
domain-characteristic cellular signal upon stimulation with EGF (44, 45, 46) . The chimeric receptor cDNAs
and those of EGF-R, PLC
, GAP, p85 subunit of PI 3`-kinase, and
PKC
were used in the form of cytomegalovirus early promoter-driven
expression vector (CMV) constructs for transient expression experiments
in human 293 embryonic fibroblasts. To confirm results obtained in
transient overexpression experiments, we employed NIH 3T3 cell lines,
which had been transfected with RTK cDNA expression plasmids and were
established as stable lines.
Figure 1:
Effect of EGF, PMA, and sodium
orthovanadate on receptor tyrosine phosphorylation. 293 cells
overexpressing the receptor cDNA alone (lanes1, 2, 5, 6, 9, 11) or
together with PKC (lanes3, 4, 7, 8, 10, 12) were treated with
vehicle, 50 ng/ml EGF, EGF + PMA (1 µM), sodium
orthovanadate (1 mM), or EGF + PMA + orthovanadate
for 10 min as indicated. Receptors were immunoprecipitated using mAb
108.1, separated by SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with antiphosphotyrosine antibody 5E2. TPA, phorbol
12-myristate 13-acetate.
Transfected 293 cells transiently overexpressing either
RTKs alone or with PKC were metabolically labeled with
[
S]methionine for 18 h and treated with PMA for
60 min. As shown in Fig.2, in the absence of PKC
overexpression (lanes1, 2, 4, 5, 7, 8, 10, 11), PMA
treatment (lanes2, 5, 8, 11) caused no mobility shift of RTKs in SDS-PAGE, suggesting
that the differential effects on RTK phosphorylation shown in Fig.1were not due to covalent, PMA-inducible modifications. In
contrast, in cells coexpressing PKC
, PMA treatment resulted in a
mobility shift of all RTKs. Interestingly, under these conditions
immunoprecipitation with the EGF-R extracellular domain-specific mAb
108.1 led to coprecipitation of PKC
, suggesting that even in the
absence of ligand, the RTKs used in this experiment bound PMA-activated
PKC, but only when both proteins were overexpressed. This observation
was confirmed with PKC-specific antibodies, which coimmunoprecipitated
all receptors from samples that contained overexpressed PKC
(data
not shown). Moreover, PMA-activated PKC associated with the
kinase-deficient EGF-R mutant HERK721A (data not shown), further
supporting our observation that receptor autophosphorylation is not
required for PKC binding.
Figure 2:
Immunoprecipitation of radiolabeled
receptor tyrosine kinases after PMA treatment. 293 cells were
transfected with EP-R, EGF-R, HER1-2, EI-R expression plasmid
alone (lanes1, 2, 4, 5, 7, 8, 10, 11) or together with
PKC expression plasmid (lanes3, 6, 9, 12). Cells were metabolically labeled with
[
S]methionine and treated with PMA (lanes2, 3, 5, 6, 8, 9, 11, 12) for 60 min. Subsequently, the
receptors were immunoprecipitated using mAb 108.1 (
EGF-R) and
analyzed by SDS-PAGE.
To investigate whether complex formation
of RTKs with PKC was an artifact of overexpression, we examined NIH 3T3
cell lines expressing the various receptors at levels of 1.5-2.5
10
per cell. As shown in Fig.3,
immunoprecipitation of EP-R, EGF-R, HER1-2, and EI-R led to
coimmunoprecipitation of endogenous PKC already in the absence of EGF.
The amounts of RTK-bound PKC in starved cells varied, especially in the
case of HER1-2, between experiments from barely detectable to
somewhat further inducible by EGF treatment. Coimmunoprecipitation was
further increased by ligand addition for all cell lines, indicating
that receptor activation leads to enhanced PKC binding.
Figure 3:
Coimmunoprecipitation of PKC after
immunoprecipitation of RTKs stably expressed in NIH 3T3 cells. NIH 3T3
cells stably expressing the various receptors were cultured in low
serum overnight and subsequently treated with EGF (100 ng/ml) for 30
min as indicated. Cells were lysed and receptors were
immunoprecipitated using mAb 108.1 (EGF-R) and separated by
SDS-PAGE. After transfer to nitrocellulose, RTKs were detected using a
polyclonal antiserum against the human EGF-R extracellular domain
(antisera DIII, upperpanel), and
coimmunoprecipitation of endogenous PKC
was determined by using
PKC-specific antisera (lowerpanel).
Having
demonstrated that PKC associates with the receptors for PDGF, EGF,
HER2, and insulin in a ligand-dependent manner, we next investigated
the growth factor effect on coimmunoprecipitating PKC activity.
HER1-2-expressing NIH 3T3 cells were cultured overnight in low
serum medium and then stimulated with EGF or vehicle prior to lysis and
immunoprecipitation with mAb 108.1. The immunoprecipitates were washed
and subsequently incubated in the presence of [-
P]ATP, lipids, Ca
, and AC-MBP, a
PKC-specific substrate peptide (see ``Materials and
Methods''). As shown in Fig.4, PKC that was coupled to
unstimulated HER1-2 was activated by Ca
and
lipids and further enhanced by EGF addition in a time-dependent manner.
These data confirm the results shown in Fig.2and Fig. 3and indicate that receptor autophosphorylation, which is
completed seconds after ligand addition, does not directly lead to PKC
binding but that receptor-activated signaling pathway(s) indirectly
promote PKC activation, translocation to the membrane, and binding to
RTKs.
Figure 4:
Determination of receptor-bound PKC
activity. HER1-2-expressing cells were left untreated or treated
with EGF as indicated. HER1-2 receptor immunoprecipitates were
assayed for PKC activity as described under ``Materials and
Methods.'' The data given are the mean values from three
independent experiments and are expressed relative to the protein
kinase activity obtained in the presence of Ca and
phospholipids from untreated cells. 100% activity approximates to the
incorporation of 30 fmol
-
PO
min
mg
cell lysate
, under the standard assay
conditions.
To further investigate the time course of EGF- versus PMA-induced RTKPKC complex formation, HER1-2 NIH 3T3
cells were cultured for 24 h under low serum conditions and treated for
various times with EGF or PMA. Coimmunoprecipitation of PKC with
HER1-2 was then investigated by immunoblot analysis using
PKC
-specific antibodies. EGF treatment led to a transient increase
of PKC binding to HER1-2, reaching its maximum level after 30
min, while PMA treatment led to a more rapid and pronounced binding of
PKC to the receptor, which continued to increase for 3 h (data not
shown). Apparently, the experimental PKC activator PMA induces
constitutive association with RTKs, while endogenous, presumably RTK
activation-dependent signals are transient and subject to negative
feedback mechanisms.
As indicated by the amount of
PIP formed (Fig.5), almost no PI 3`-kinase activity
coimmunoprecipitated with the endogenous receptors in untransfected
cells under any of the stimulation conditions (lanes1-3). EGF stimulation of HER1-2-expressing
cells (lane5), however, led to a strong increase in
PIP levels, which was reduced by simultaneous addition of PMA (lane6), as measured by Cerenkov counting of the radiolabeled
phospholipids. When PKC was co-overexpressed with HER1-2,
EGF stimulation resulted in PI 3`-kinase activity association with the
receptor that was about 20% of that measured in the absence of PKC
overexpression (lane5). PMA addition further reduced
this effect (lane9), indicating that PKC activation
or overexpression alone results in uncoupling of receptor-regulated
signal transduction pathways.
Figure 5:
HER1-2-bound PI 3-kinase activity.
Untransfected (lanes1-3), HER1-2 (lanes4-6), and HER1-2 and PKC (lanes7-9) expressing cells were metabolically
labeled with [
S]methionine overnight and
subsequently left untreated, treated with EGF (50 ng/ml), or
simultaneously with EGF and PMA (1 µM) as indicated. After
immunoprecipitation with mAb 108.1 (
EGF-R), the samples were
divided into three aliquots. One aliquot was used to determine
receptor-bound PI 3`-kinase activity (lanes1-9), the second aliquot to determine receptor
expression and PKC coimmunoprecipitation (lanes10-12), and the third to determine receptor
autophosphorylation (not shown). Formation of PIP (lanes1-9) was determined by separation on TLC plates and
exposure to film. The PIP spots were subsequently scraped from the
plate and quantitated by Cerenkov counting. Expression of HER1-2
and coimmunoprecipitation of PKC was determined after receptor
immunoprecipitation with 108.1 antibodies, separation by SDS-PAGE, and
exposure to film (lanes10-12). TPA, phorbol
12-myristate 13-acetate.
To support this observation, we
investigated the interaction of HER1-2 with additional
substrates, including PLC, GAP, and p85, in the absence and
presence of PKC
overexpression. Without PKC
cotransfection,
EGF treatment resulted in coimmunoprecipitation of PLC
, GAP, and
p85 from transiently overexpressing 293 cells. Coincubation with PMA
reduced receptor tyrosine phosphorylation similar to the effect shown
in Fig.1(lane9) and concomitantly
coimmunoprecipitation of all substrates investigated. Co-overexpression
of PKC
reduced HER1-2 substrate interaction already in the
absence of PMA, while activation of overexpressed PKC
by PMA
almost completely abolished coprecipitation of PLC
, GAP, and p85
(data not shown). These results demonstrate that PKC
is very
potent in regulating the interaction of HER1-2 with subsequent
signaling mediators.
Figure 6:
EGF- and PMA-induced degradation of
radiolabeled HER1-2. 293 cells were transfected with HER1-2
expression plasmid alone (lanes1-3), together
with PKC expression plasmid (lanes4-6),
together with PLC expression plasmid (lanes7-9), and together with PKC and PLC
expression
plasmids (lanes10-12). Cells were
metabolically labeled with [
S]methionine
overnight, and after washing away the radiolabel, HER1-2
degradation was determined in the absence or presence of EGF or PMA.
After 4 h of incubation, cells were lysed, and HER1-2 was
immunoprecipitated using mAb 108.1. After separation by SDS-PAGE, the
gel was dried and exposed to film. TPA, phorbol 12-myristate
13-acetate.
The difference between PMA- and PLC-mediated receptor
degradation in the absence of overexpressed PKC indicates that both DAG
and elevated Ca
levels are required to fully activate
PKC isotypes involved in receptor down-regulation. PMA, which acts like
DAG, may not be sufficient under low intracellular Ca
concentrations to fully activate the respective isotypes, a
deficiency that is compensated under PKC overexpression conditions.
To further investigate whether the time course of RTKPKC
complex formation correlated with the disappearance of radiolabeled
receptors, 293 cells overexpressing HER1-2 and PKC were treated
for various times with EGF, PMA, EGF together with PMA, or left
untreated. The receptors were immunoprecipitated, separated by
SDS-PAGE, and exposed to film. Fig.7shows that for 6 h, EGF
had no effect on receptor degradation (compare untreated and
EGF-treated lanes) and did not lead to enhanced binding of PKC to the
receptor. Treatment with PMA alone or in combination with EGF induced,
with increasing incubation time, the formation of a
HER1-2
PKC complex and at the same time HER1-2
degradation. While the kinetics observed are influenced by de novo synthesis transport of receptors and PKC to the cell surface in
the overexpression cell system, the information obtained by this
analysis was not significantly affected. Identical results were
obtained with EP-R, EGF-R, and EI-R (not shown), indicating that
binding of activated PKC, leading to elimination of receptors from the
cell surface by internalization and subsequent degradation, represents
a general mechanism.
Figure 7:
Time course of HER1-2PKC
complex formation and HER1-2 degradation.
HER1-2-PKC
-overexpressing 293 cells were metabolically
labeled for 18 h using [
S]methionine. The
labeling medium was replaced by normal DMEM, and the cells were then
treated for various times with EGF, PMA, EGF + PMA, or left
untreated as indicated. After immunoprecipitation of HER1-2 and
separation by SDS-PAGE, the radiolabeled receptors and
coimmunoprecipitated PKC
were visualized by exposure to x-ray
film. TPA, phorbol 12-myristate 13-acetate.
In this study, we provide evidence that PKC may mediate rapid and delayed effects on RTKs. While the immediate consequences that influence receptor phosphorylation on tyrosine residues are diverse and RTK-specific, the delayed effects involve direct RTK-PKC interaction and appear to be common for different RTK subtypes.
EGF activates
autophosphorylation of EP-R, EGF-R, HER1-2, and EI-R
overexpressed in 293 fibroblasts. Simultaneous treatment with EGF and
PMA reduced the autophosphorylation of EGF-R and HER1-2 but
clearly enhanced EP-R and EI-R autophosphorylation. The PMA effect was
very rapid and could be detected within a few minutes. Phorbol ester
treatment of these cells did not lead to a mobility shift of RTKs in
the absence of cotransfected PKC, suggesting that the effects on
RTK phosphorylation on tyrosine residues were not due to endogenous
PKC-mediated RTK phosphorylation on serine and threonine residues. How
phosphorylation of overexpressed RTKs is regulated by PMA is not clear,
but one likely possibility is the involvement of
phosphotyrosine-specific phosphatases. This is supported by the
observation that orthovanadate, a potent inhibitor of this type of
enzyme, is able to neutralize the PMA-mediated effects in the absence
of overexpressed PKC
. Additionally, the PMA-mediated effects on
EGF-R and HER1-2 tyrosine phosphorylation were dominant in
experiments in which EGF and PMA were added simultaneously in cells
that were treated first with EGF for 5 min and subsequently with PMA
for 5 min or vice versa. (
)These data indicate that
PMA-induced receptor dephosphorylation can be reversed by orthovanadate
treatment and is not due to covalent receptor modifications that, by
inducing conformational changes, inhibit the intrinsic tyrosine kinase
activity, as speculated previously. This effect might be regulated by
PMA-inducible endogenous PKC isotypes, which phosphorylate and thereby
activate certain tyrosine-specific phosphatases. Such PMA- and protein
kinase C-dependent phosphorylation of tyrosine-specific phosphatases
has been demonstrated in vivo and in vitro; however,
no up-regulation of the enzymatic activity was detected (reviewed in (57) ). Alternatively, PMA might directly activate PTPases, but
up to now, no data exist to support this possibility.
In the
presence of overexpressed PKC, all investigated RTKs formed a
stable complex with PKC
upon PMA treatment, which at the same time
resulted in a mobility shift of the receptors. Under these conditions,
the PMA-inducible effects on tyrosine kinase activity were not
reversible by orthovanadate, indicating that receptor phosphorylation
on tyrosine residues is regulated by PKC-dependent phosphorylation on
serine and threonine residues. Alternatively, binding of activated PKC
to the receptor directly results in receptor inactivation. An EGF-R
composed of the extracellular ligand binding domain, the transmembrane
region, and 8 adjacent cytoplasmic amino acids (Na8) failed to interact
with activated PKC
, while deletion of the entire carboxyl-terminal
tail had no influence on the interaction, suggesting that the tyrosine
kinase or juxtamembrane domains contain the binding site for this SH2
domain-less signal regulator protein. In contrast to the EGF-R,
HER1-2, and EI-R, the PDGF-R kinase was only slightly affected by
PKC binding, which might be due to the unique kinase structure of type
III receptors.
Whether the inhibition of EGF-R, HER1-2, and EI-R autophosphorylation is indeed due to PKC-dependent transmodulation remains controversial. EGF-R phosphorylation at Thr-654 by activated PKC has been suggested to account for inhibition of tyrosine activity. This is supported by the observation that PMA fails to modulate receptor autophosphorylation in cells expressing EGF-receptors lacking Thr-654 (58, 59, 60, 61) . Activators of PKC also stimulate the phosphorylation of Thr-669 in vivo(62, 63) ; however, there is no evidence to date that phosphorylation of Thr-669 is required for inhibition of receptor autophosphorylation. In contrast, Morrison et al.(64) showed phorbol ester-stimulated EGF-R tyrosine kinase inhibition of a Thr-654 mutant expressed in Chinese hamster ovary cells and concluded that this residue is not sufficient to negatively regulate the EGF-R.
A second pathway of regulation of the EGF-R
tyrosine kinase activity is represented by the phosphorylation of the
receptor at Ser-1046/1047 in response to PMA treatment. Increased
phosphorylation at this site is associated with an inhibition of RTK
activity, and site-directed mutagenesis at Ser-1046/1047 has indicated
that these residues are also important for receptor
down-regulation(59, 65) . PKC itself is not able to
phosphorylate Ser-1046/1047; however, CAM kinase II has been shown in vitro to phosphorylate these residues and cause an
inhibition of protein kinase activity(65) . Thr-654 is
conserved within the closely related RTK HER2 (Thr-686), as is Ser-1047
(Ser-1113 in HER2). If these residues are indeed important for
regulating receptor tyrosine kinase activity in vivo, this
could explain why HER2 is similarly affected by PMA and overexpressed
PKC.
While PKC-dependent transmodulation of the PDGF-R has not
yet been described, it is well established that the insulin receptor is
a PKC substrate. PKC-mediated insulin receptor phosphorylation can lead
to a marked increase in the phosphorylation of the -subunit (34, 35, 36, 66) but can also
inhibit autophosphorylation in certain cell types (31, 32, 33) . Additionally, it was recently
demonstrated that in response to phorbol esters, Chinese hamster ovary
cells overexpressing PKC isotypes
,
1, and
, but not
, exhibit about 4-fold higher levels of insulin receptor
phosphorylation. This increased phosphorylation occurred exclusively on
serine and threonine (serine 1305/1306, threonine 1348, and other
unidentified sites) without inhibition of receptor tyrosine kinase
activity. However, activated PKC
inhibited the insulin-stimulated
increase of phosphatidyl 3-kinase activity(37) .
Simultaneous treatment of HER1-2-expressing cells with EGF and
PMA inhibited not only receptor autophosphorylation but also substrate
binding, as investigated by coimmunoprecipitation of coexpressed
PLC, GAP, and p85. In cells that also overexpress PKC
, the
inhibitory effect on receptor autophosphorylation and substrate
coimmunoprecipitation was even more dramatic. Activated PKC also
abolished receptor-bound PI 3-kinase activity. These data are in
agreement with the observation that phosphorylated tyrosine residues
within the receptor are binding sites for SH2 domain-containing
proteins such as PLC
, GAP, and p85 PI 3-kinase subunit. Previously
reported evidence indicates that PKC inhibits EGF-R-dependent tyrosine
phosphorylation of PLC
and activation of phosphoinositide
hydrolysis(67) . Moreover, a link between GAP and PKC-mediated
signaling pathways has been reported by several
laboratories(68, 69, 70, 71) . As
recently demonstrated, PMA treatment of T cells increases the amount of
active GTP-bound Ras in cell membranes(68) , and furthermore,
this increase is caused by PKC-mediated reduction of GAP activity,
resulting in a positive response of the cell. In this context, it is
important to note that direct activation of PKC by phorbol esters or
indirect activation by mitogens such as PDGF, thrombin, and bombesin
stimulates the proliferation of many cell
types(72, 73, 74, 75) . In addition,
PKC has been implicated in mediating crucial events in neoplastic
transformation (76) .
The data indicate that the short term effect of activated PKC uncouples the EGF-R and HER2 receptors (subclass II RTKs) from their signal transduction pathways by inducing a reduction of receptor autophosphorylation, resulting in inhibition of EGF-induced mitogenicity. On the other hand, these data suggest that activated PKC, which enhances autophosphorylation of PDGF-R and insulin receptor in these cells, couples these receptors more tightly to their signal transduction pathways. However, whether PDGF and PMA, and insulin and PMA are additive in these cells with respect to mitogenicity remains to be investigated.
RTKPKC complex
formation was time dependent and must be regarded as a long term
effect. EGF-induced complex formation in NIH 3T3 cells stably
expressing HER1-2 reached its maximum after about 30 min and was
undetectable after 180 min, while PMA induced a more rapid complex
formation, which was still increasing after 180 min. This difference
between EGF- and PMA-induced complex formation might be explained by
the finding that growth factors transiently activate PLC
(reviewed
in (77) ), which then leads to transient activation of PKC,
while PMA directly and constitutively activates PKC.
The time course of PKC binding to the receptors correlated extremely well with the time course of translocation of PKC from the cytosolic to the membrane fraction upon PMA treatment (data not shown). An involvement of PKC in receptor internalization and degradation has been proposed for several years. Exposing cells to phorbol esters results in decreased EGF binding and can be observed in most cell types. Immunofluorescent and electron microscopic localization of the EGF-R after phorbol ester treatment showed that a significant percentage of receptors become internalized(78) .
Deletion mutations within the EGF-R COOH
terminus have been shown to cause an inhibition of EGF-R
endocytosis(79) . The importance of PKC-mediated endocytosis
and degradation is supported by the observation that Tyr-992, the major
PLC interaction site, is located within this subdomain, suggesting
that EGF receptors lacking this region are unable to activate PKC via
PLC
-activated polyphosphoinositide hydrolysis.
PKC mediation of
EP-R, EGF-R, HER1-2, and EI-R internalization and degradation
could be clearly demonstrated in 293 cells. Overexpression of the RTK
alone and subsequent treatment with EGF or PMA had no effect on
receptor degradation, while coexpression of PKC resulted in a
marked loss of radiolabeled receptors upon PMA treatment. Determination
of the time course of receptor degradation in 293 cells overexpressing
HER1-2 and PKC showed that EGF was not able to induce receptor
degradation and PKC complex formation, whereas PMA induced receptor/PKC
complex formation, receptor phosphorylation, and, simultaneously,
receptor degradation. It is interesting to note that increased PKC
binding clearly correlated with the loss of receptors. As mentioned
earlier, EGF and PMA do not mediate receptor down-regulation when the
receptor is overexpressed alone, suggesting that the endogenous
down-regulating pathways are not sufficiently stimulated to activate
degradation of these elevated amounts of receptors. Coexpression of PKC
and PMA treatment can restore this pathway, whereas EGF still has no
effect. If the assumption is correct that the ligand-activated receptor
activates PLC
, which leads to activation of PKC, then PLC
should be the limiting factor in the latter case. Simultaneous
overexpression of the receptor, PLC
, and PKC should therefore
restore the entire pathway and render it EGF-inducible. This is indeed
the case, indicating that PLC
is an important regulator of the
receptor down-regulation pathway and supports the assumption that
receptor internalization and degradation are dependent on PKC-mediated
receptor phosphorylation.
In conclusion, the studies reported here show that PKC mediates short and long term effects on RTKs. The immediate effects, which appear to involve PMA-responsive tyrosine-specific phosphatases, are RTK-specific and regulate the phosphorylation state of the receptor on tyrosine residues, i.e. its ability to interact with SH2 domain-containing cellular signal transducers. In contrast, the PKC long term effects seem to be general in nature and are induced by translocation from the cytosol to the plasma membrane and formation of stable complexes with the receptor, concomitant with phosphorylation followed by internalization and degradation. To fully understand these mechanisms, characterization of specific phosphatases and an evaluation of the importance of PKC-mediated RTK phosphorylation in the triggering of receptor degradation are important goals for future research.