Molecular and Cellular Biology, October 1998, p. 5888-5898, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Is an Important Signaling Molecule in
Insulin-Like Growth Factor I Receptor-Mediated Cell
Transformation
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892,1 and Department of Microbiology, The Mount Sinai Medical Center, New York, New York 100292
Received 23 January 1998/Returned for modification 2 March 1998/Accepted 20 July 1998
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ABSTRACT |
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To investigate the potential role of protein kinase C-
(PKC-
)
in insulin-like growth factor I receptor (IGF-IR)-mediated cell
transformation, an oncogenic gag-IGF-IR
-fusion receptor lacking the entire extracellular domain, which was designated NM1, and
a full-length IGF-IR were coexpressed with either wild-type PKC-
(PKC-
WT) or an ATP-binding mutant of PKC-
(PKC-
K376R) in NIH
3T3 fibroblasts. While overexpression of PKC-
WT did not affect NM1-
and IGF-IR-induced focus and colony formation of NIH 3T3 cells,
expression of PKC-
K376R severely impaired these events. In contrast,
NM1-mediated cell growth in monolayer was not affected by coexpressing
PKC-
K376R. PKC-
WT and PKC-
K376R were constitutively phosphorylated on a tyrosine residue(s) in the NM1- and
IGF-IR-expressing cells and were associated with them in an
IGF-I-independent manner. Activated IGF-IR was able to phosphorylate
purified PKC-
in vitro and stimulated its kinase activity.
Furthermore, the level of endogenous PKC-
protein was up-regulated
through transcriptional activation in response to long-term IGF-IR
activation. Taken together, our results demonstrate that PKC-
plays
an important role in IGF-IR-mediated cell transformation, probably via
association of the receptor with PKC-
and its activation through
protein up-regulation and tyrosine phosphorylation. Competition with
endogenous PKC-
for NM1 and IGF-IR association by PKC-
K376R is
probably an important mechanism underlying the PKC-
K376R-mediated
inhibition of cell transformation by NM1 and IGF-IR.
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INTRODUCTION |
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Insulin-like growth factor I
receptor (IGF-IR) is a type II tyrosine kinase receptor which is
composed of two extracellular
subunits and two membrane-spanning
subunits linked by disulfide bonds (49, 50). IGF-I
stimulation of IGF-IR results in receptor autophosphorylation and
phosphorylation of certain signaling molecules such as Shc,
phosphatidylinositol 3' kinase (PI 3'K), Grb2, Grb10, insulin receptor
substrate 1 (IRS-1), and interleukin 4-phosphorylated substrate
(4PS)/IRS-2 (5). Activation of the IGF-IR signaling pathway
leads to proliferation, differentiation, and inhibition of apoptosis in
different model systems (5). The importance of IGF-IR in
cell growth and development has been demonstrated by the targeted
disruption of the IGF-IR gene (2, 29). The size of newborn
mice lacking the IGF-IR was reduced by 70% in comparison to that of
wild-type littermates. Moreover, IGF-IR activation has been
demonstrated to play an important role in transformation of cultured
cells and in tumor progression in syngeneic animals and nude mice
(3). Embryonic fibroblasts established from IGF-IR
/
mice
(R
cells) are resistant to transformation induced by a
variety of oncogenes, growth factor receptors, and viral proteins,
including v-ras, raf, platelet-derived growth
factor
receptor (PDGF-
R), epidermal growth factor receptor
(EGFR), simian virus 40 (SV40) T antigen, and the E5 protein of bovine
papillomavirus (6, 7, 32, 33, 41, 42). Reconstitution of
R
cells with wild-type IGF-IR restored the susceptibility
of transformation by those oncogenes and growth factor receptors.
Inhibition of the IGF-IR signaling pathway by expression of anti-sense
IGF-I (48) or IGF-IR (17, 34, 36, 40, 43), by
expression of dominant-negative IGF-IR (18, 39), or by
microinjection of neutralizing antibody against IGF-IR (1,
15) has been shown to abolish or delay the progression of a
variety of tumors in animal models. For example, down-regulation of the
IGF-IR by antisense oligonucleotide blocking has been demonstrated to
reduce the tumorigenicity of human glioblastoma T98G, rat glioblastoma C6, human breast carcinoma MC-F7, and mouse melanoma B16-F10 cells (5).
Protein kinase C-
(PKC-
) is a serine/threonine kinase whose
activation has been tightly linked to monocytic differentiation of the
32D myeloid progenitor cell line in response to
12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation
(31). PKC-
has also been demonstrated to be
phosphorylated on a tyrosine residue(s) both in vitro and in vivo in
response to a variety of stimuli (9, 22). Recently, PKC-
has been identified as an important downstream signaling molecule of
the PDGF-
R (23, 24). Autophosphorylation, membrane translocation, and membrane-associated kinase activity of PKC-
increased in response to PDGF stimulation in NIH 3T3 fibroblasts overexpressing PKC-
and in 32D cells coexpressing PDGF-
R and PKC-
. PKC-
was tyrosine phosphorylated both in vitro and in vivo
by the activated PDGF-
R (20, 22, 24). Coexpression of an
ATP binding mutant of PKC-
(PKC-
K376R) (25) with the sis oncogene which encodes the PDGF-B chain significantly
inhibited sis/PDGF-
R-mediated cell transformation of NIH
3T3 fibroblasts, strongly suggesting that PKC-
is a physiological
substrate involved in PDGF-
R-mediated cell transformation
(21).
We have previously constructed a gag-IGF-IR
fusion
receptor by deleting the entire extracellular domain of human IGF-IR and fusing the remaining sequence to the avian sarcoma virus UR2 gag and have designated it NM1 (14, 27, 28).
Expression of NM1 in chicken embryo fibroblasts (CEF) resulted in
constitutive receptor autophosphorylation and cell transformation, as
reflected in morphological alteration and colony formation in soft
agar. NM1 also induced tumors in chicken efficiently. In this study, we
investigated the potential role of PKC-
in the native and fusion
IGF-IR-induced transformation of NIH 3T3 cells. Our results demonstrate
that transformation of NIH 3T3 cells by NM1 is severely impaired by
coexpression of PKC-
K376R. The PKC-
K376R mutant is also capable
of blocking full-length IGF-IR-mediated focus formation in response to
exogenous IGF-I. Furthermore, we show that PKC-
is tyrosine
phosphorylated in NM1- and IGF-IR-expressing cells and is associated
with these receptor tyrosine kinases in vivo. In addition, purified
PKC-
is tyrosine phosphorylated in vitro by NM1 and IGF-IR, and this
phosphorylation results in increased PKC-
activity. Finally, we
present evidence that endogenous PKC-
is up-regulated in protein and
RNA levels upon long-term NM1 and IGF-IR activation, thus providing a
potential link between IGF-IR and PKC-
in the NM1- and
IGF-IR-mediated cell transforming pathway.
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MATERIALS AND METHODS |
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cDNA construction.
The construction of oncogenic NM1
gag-IGF-IR and T6 gag-IR has been described
elsewhere (27, 28). The NM1 mutant F1136, containing the
Y1136-to-F1136 mutation of NM1, has been described elsewhere
(14). Construction of the mutant dS2, from which 19 amino
acids in the subtransmembrane region of NM1 were deleted, will be
described elsewhere. All of these fusion receptor genes have been
subcloned into a Moloney murine leukemia virus long terminal
repeat-based expression vector pMEXneo as described
previously (14, 27, 28). The full-length IGF-IR in
pMEXneo was obtained from William Rutter. Cloning of
PKC-
WT and PKC-
K376R into the pLTRgpt vector was
reported before (25).
Transfection and focus formation assay.
Transfection of NIH
3T3 cells was performed by the calcium phosphate precipitation, as
previously described (21). Briefly, 1.5 × 105 cells were plated on 10-cm tissue culture plates 1 day
before transfection. Equal amounts of precipitated DNA were added to two separate plates for each transfecting sample. One of these plates
was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, BRL)
containing 5% of calf serum for the focus-forming assay, while the
other plate was selected in medium containing geneticin and/or
mycophenolic acid, depending on the selectable marker gene present in
the expression vectors. The number of drug-resistant colonies formed
per plate was determined in order to ensure that equivalent amounts of
DNA were utilized and that comparable transfection efficiencies were
obtained. Media were changed twice a week following the transfection.
Three weeks after transfection, the nonselected plates were fixed,
stained with Giemsa dye (Fisher Scientific), and photographed for
assessing focus formation. The selected plates were enumerated for the
drug-resistant colonies, and cells of combined colonies from
drug-resistant plates were used for further biochemical studies and for
colony-forming assays (see below). When the full-length IGF-IR (coding
for both the
and
subunits) cloned in pMEXneo was
utilized in the focus formation assay, media containing 1% of calf
serum instead of 5% were included for the nonselective plates either
in the absence or in the presence of 50 ng of human IGF-I per ml for
focus induction. The electroporation method for 32D cell transfection
has been reported before (31).
Soft agar colony formation assay. The soft agar assay measuring anchorage-independent growth has been reported elsewhere (21). Briefly, 105 NIH 3T3 stable transfectants were suspended in 4 ml of DMEM supplemented with 10% calf serum and 0.4% Seaplaque agarose in 6-cm tissue culture plates containing 4 ml of 0.8% agarose containing DMEM underlay. Cultures were fed with 0.2 ml of DMEM containing 10% of calf serum twice a week for 2 weeks. The colonies were stained with p-iodonitrotetrazolium violet (Sigma) after 2 weeks and photographed on an inverted light.
Monolayer cell growth determination.
Each NM1 transfectant
coexpressing the various PKC-
constructs was plated in a six-well
Coaster plate at 1 × 104 to 5 × 104
cells/well with DMEM containing 10% calf serum. On the following day,
the cells were washed once with DMEM and maintained in DMEM containing
either 1% or 10% calf serum. Cell numbers were counted from one of
these wells on day 0 and were continuously counted every other day from
each of these wells until day 8 by using an automatic cell counter
(Coulter Corporation). Two wells from each sample were counted at each
time, and the mean values were calculated and expressed in the
corresponding figure.
Protein extraction, immunoprecipitation, and immunoblot
analysis.
Overnight serum starvation, growth factor stimulation,
and lysis of cells have been described elsewhere (24, 25).
For direct anti-PKC-
and anti-IGF-IR immunoblot analysis, equivalent amounts of total cellular proteins (100 µg per sample) extracted with
the lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophosphate, 50 mM sodium fluoride,
1 mM Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10-µg/ml aprotinin, and 10-µg/ml leupeptin were
immunoblotted with anti-PKC-
serum (1 to 1,000 dilution [R&D
Aba.]) or with anti-IGF-IR serum (1 to 1,000 dilution
[27]). For measuring IGF-IR tyrosine phosphorylation
and PKC-
tyrosine phosphorylation and for coimmunoprecipitation of
IGF-IR with PKC-
, cells were serum starved overnight and were either
untreated or stimulated with human IGF-I (10 ng/ml) for 10 min at
37°C and lysed with the same lysis buffer described above. Equivalent
amounts of cell lysates (2 to 5 mg per sample) were immunoprecipitated
with either anti-IGF-IR or anti-PKC-
. Proteins were fractionated and
transferred to Immobilon membranes (Millipore) and immunoblotted with
antiphosphotyrosine (anti-pTyr [UBI; 2 µg/ml]), anti-PKC-
, or
anti-IGF-IR antibody as described in the corresponding figures.
Immune complex assay for IGF-IR autophosphorylation.
Cells
were either untreated or stimulated with IGF-I for 10 min after
overnight serum starvation and lysed in the lysis buffer described
above. Equal amounts of protein from cell lysates (2 mg per sample)
were immunoprecipitated with anti-IGF-IR serum. The immunoprecipitates
were washed with the lysis buffer and incubated in a reaction solution
containing 25 mM HEPES (pH 7.5), 0.1% Nonidet P-40 (NP-40), 10 mM
MgCl2, 3 mM MnCl2, and 30 µM
Na3VO4 and 10 µCi of
[
-32P]ATP at room temperature for 10 min. The reaction
was stopped by adding an equal volume of 2× sample loading buffer, and
the mixture was boiled and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dried gel
was autoradiographed.
In vitro PKC-
phosphorylation by IGF-IR and PKC-
activity
assay.
IGF-IR from NIH 3T3 cells and various transfectants was
immunoprecipitated with anti-IGF-IR serum. The immunoprecipitates were
incubated with the baculovirus-derived PKC-
for 20 min at room
temperature in cold-ATP-containing buffer as reported elsewhere (22). After reaction, 2 µl of each reaction mixture was
used as a PKC-
source to measure its activity by using PKC-
pseudosubstrate region-derived peptide in the presence of
[
-32P]ATP (22). Briefly, purified PKC-
before and after tyrosine phosphorylation was incubated at room
temperature in 40 µl of reaction buffer containing 10 µM PKC-
substrate derived from the PKC-
pseudosubstrate region, 20 mM
Tris-HCl (pH 7.5), 1 mM CaCl2, 10 µM magnesium acetate, 1 µM TPA, 50-µg/ml phosphatidylserine (Sigma), 30 µM cold ATP, and
30 µCi of [
-32P]ATP for 20 min. The reaction tube
was centrifuged, and 20 µl of the supernatant was spotted on
phosphocellulase disks (Life Technologies, Inc./BRL). The disks were
washed twice with 1% phosphoric acid and twice with distilled water,
and samples were analyzed by liquid scintillation. The remaining
reaction mixture was subjected to SDS-PAGE and immunoblotted with
anti-pTyr or anti-PKC-
.
Northern blot analysis.
The method with full-length mouse
PKC-
cDNA as the probe in Northern blot analysis has been described
before (24).
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RESULTS |
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Expression of PKC-
K376R mutant severely impairs NM1-, IGF-IR-,
and T6-induced focus formation of NIH 3T3 fibroblasts.
To
investigate the potential role played by PKC-
in IGF-IR-mediated
cell transformation of NIH 3T3 fibroblasts, we cotransfected expression
vectors containing various PKC-
cDNAs together with NM1 plasmid.
Consistent with its transforming activity in CEF (27, 28),
NM1 was able to induce focus formation of NIH 3T3 fibroblasts with an
efficiency of 6 × 102 foci/µg of DNA (Fig.
1A). Coexpression of PKC-
WT with NM1
did not affect the focus-forming activity induced by NM1 (Fig. 1A). In
striking contrast, coexpression of PKC-
K376R with NM1 reduced its
focus-forming activity by 90%. dS2 contains a 19-amino-acid deletion
in the juxtamembrane region of NM1, and its transforming activity in
CEF was dramatically reduced compared to that of NM1 (14a).
Similarly, the focus-forming activity of dS2-transfected NIH 3T3 cells
was only one-fifth of that of NM1-transfected cells (Fig. 1A, panel 2).
Tyrosine 1136 of NM1 or IGF-IR has been demonstrated to be important
for their transforming activity, as measured by colony formation of
CEF, NIH 3T3, and mouse embryonic cells in soft agar (12, 14,
18). However, expression of the NM1 F1136 mutant in NIH 3T3 cells
showed that it still retained about 50 to 60% of the focus-forming
activity of NM1 (Fig. 1A, panel 3). Both dS2- and F1136-induced
focus-forming activities were also severely inhibited by coexpression
of PKC-
K376R. T6 is a gag-insulin receptor
(gag-IR) fusion protein that is similar in structure to NM1
(38). Although T6 has a transforming activity comparable to
that of NM1 in CEF (38), its focus-forming activity was
remarkably reduced in comparison with that of NM1 in NIH 3T3 cells
(Fig. 1A, panel 4). The focus-forming activity of T6 was nearly
abolished in the presence of PKC-
K376R, which was a result
consistent with that of NM1.
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K376R. NIH 3T3
cells were cotransfected with full-length IGF-IR and the PKC-
WT or
PKC-
K376R plasmid. The transfected cells were maintained in DMEM
containing 1% calf serum in the absence or presence of 50 ng of human
IGF-1 per ml. As shown in Fig. 1B, fewer than 30 foci were observed in
cells cotransfected with IGF-IR and pLTR in the absence of exogenous
IGF-I. IGF-I stimulation of the same transfectant for 3 weeks resulted
in more than 200 foci. Similar numbers of foci were observed in the
IGF-IR and pLTR-
WT cotransfectant upon IGF-I stimulation. In
contrast, expression of PKC-
K376R totally abolished focus formation
by spontaneous and IGF-I-stimulated IGF-IR activation.
Anchorage-independent growth of NIH 3T3 cells induced by NM1 is
inhibited by PKC-
K376R.
To test whether expression of
PKC-
K376R also affected NM1-mediated anchorage-independent growth,
we generated stable NIH 3T3 transfectants coexpressing NM1 and
PKC-
K376R. As shown in Fig. 2,
parental NIH 3T3 cells formed only a few spontaneous colonies in media
containing 10% calf serum. NM1-expressing NIH 3T3 cells formed more
than 200 colonies. In striking contrast, expression of PKC-
K376R
significantly reduced the number of NM1-induced colonies in soft agar.
Consistent with the results of the focus formation assay, expression of
PKC-
WT had no effect on the colony-inducing activity of NM1 compared
to that of NM1/pLTR-cotransfected cells. Taken together, these results
demonstrate that PKC-
plays a pivotal role in NM1-, IGF-IR-, and
T6-mediated transformation of NIH 3T3 fibroblasts.
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PKC-
K376R expression does not affect the growth rate of
NM1-expressing cells in monolayer.
The growth rates of cells
stably cotransfected with NM1 and PKC-
WT or PKC-
K376R in
monolayer culture were measured in the presence of two serum
concentrations. Transfectants cultured in 10% serum reached confluence
in about 6 days, whereas those in 1% serum grew much more slowly, even
though they were transformed by NM1 (Fig.
3). Nevertheless, neither coexpression of
PKC-
WT nor that of PKC-
K376R affected the growth rate of
NM1-expressing cells at either serum concentration. Since the same
transfectants were utilized for both the soft agar colony formation
assay and the monolayer growth assay, these data provide the evidence
that PKC-
-mediated signaling is important for IGF-IR-induced
anchorage-independent growth and escape from contact inhibition, but
not for proliferation of cells in monolayer culture. Segregation of
signaling pathways leading to cell growth on monolayer versus those for
focus and colony formation has been observed previously in other
oncogene systems, including the differential effect exerted by various Ros mutants (52). However, the result here represents the
initial observation that PKC-
plays a differential role in distinct
biological pathways mediated by IGF-IR activation.
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IGF-IR expression and kinase activity are not affected by
coexpression of the PKC-
K376R mutant.
PKC-
expression levels
among the various transfectants were measured by direct immunoblot
analysis with anti-PKC-
serum. The expression levels of PKC-
WT
and PKC-
K376R were increased by five- and threefold, respectively,
over that of the endogenous PKC-
(Fig.
4A). The protein level for the
PKC-
K376R mutant was lower than that of PKC-
WT in the different
transfectants, despite the use of the same expression vector. This was
also observed in our previous studies, in which we attempted to express
this mutant in 32D myeloid progenitor cells and to coexpress it with the sis oncogene in NIH 3T3 cells (21, 25). The
expression levels of the 53-kDa NM1 protein in the control and PKC-
transfectants were very similar (Fig. 4B). IGF-IR transfectants
displayed a fivefold increase in IGF-IR expression over that of the
endogenous IGF-IR, as judged by the expression of the 97-kDa IGF-IR
subunit (Fig. 4C). Again, no differences in IGF-IR protein levels were detected among the various PKC-
transfectants.
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WT or PKC-
K376R would
affect NM1 and IGF-IR tyrosine kinase activities, the extents of
receptor tyrosine phosphorylation and in vitro kinase activities of NM1
and IGF-IR were measured. Expression of NM1 resulted in a
constitutively tyrosine-phosphorylated protein, which migrated as a
broad band of 53 to 60 kDa (Fig. 5A). We
did not observe any significant changes in the level of NM1 protein
tyrosine phosphorylation upon coexpression of either PKC-
WT or
PKC-
K376R. Overexpression of the full-length IGF-IR resulted in
basal phosphorylation of the receptor. Ligand stimulation greatly
increased tyrosine phosphorylation of the receptor (Fig. 5A). Again,
ligand-dependent phosphorylation of IGF-IR was not affected by
coexpression of PKC-
K376R.
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K376R on NM1 and IGF-IR kinase activities. As seen in Fig.
5B, the autophosphorylation activities in all NM1 transfectants were
indistinguishable. An 80-kDa phosphorylated protein was coprecipitated
by anti-IGF-IR only in lysates from the NM1/pLTR-
WT cotransfectant
(Fig. 5B [indicated by the asterisk]), but not from the NM1/pLTR or
NM1/pLTR-
K376R cotransfectant. The ligand-dependent activation of
the endogenous IGF-IR in various PKC-
single transfectants was also
measured by the kinase assay (Fig. 5B). Expression of PKC-
K376R did
not affect activation of the endogenous IGF-IR. These results indicate
that the inhibitory effect of PKC-
K376R on NM1 and IGF-IR
transformation is not due to its effect on receptor activation and
expression.
Tyrosine phosphorylation of cellular proteins in response to IGF-I
stimulation was also examined by anti-pTyr immunoblot analysis. As seen
in Fig. 5C, a relatively high level of IGF-IR
subunit tyrosine
phosphorylation was observed in all three IGF-IR-transfected lines, but
not in NIH 3T3 cells. Ligand stimulation resulted in increased tyrosine
phosphorylation of the overexpressed IGF-IR to a similar extent in
PKC-
WT and PKC-
K376R cotransfectants. Interestingly, a 180-kDa
tyrosine phosphorylated protein, which may represent endogenous IRS-1,
was detected in response to IGF-I stimulation in all the lines tested.
Again, no obvious difference was observed in tyrosine phosphorylation
of cellular proteins among all of the PKC-
cotransfectants.
Overexpressed PKC-
is constitutively phosphorylated on a
tyrosine residue(s) in NM1- or IGF-IR-cotransfected NIH 3T3 cells.
PKC-
has been previously demonstrated by our laboratory and several
others to be phosphorylated on a tyrosine residue(s) in vivo and in
vitro (8, 9, 11, 16, 20, 22, 24, 44, 45, 51). Tyrosine
phosphorylation of PKC-
was considered an indicator of its
activation, since only the membrane-associated PKC-
was found to be
phosphorylated (24). We were interested to know whether
activation of IGF-IR was able to induce tyrosine phosphorylation of
PKC-
. As shown in Fig. 6A,
coexpression of NM1 with PKC-
WT resulted in constitutive tyrosine
phosphorylation of PKC-
WT. Phosphorylation of the PKC-
K376R
mutant protein by NM1 was much higher than that of PKC-
WT, even
though mutant protein expression was two- to threefold lower (Fig. 4A).
This is consistent with our previous finding that PKC-
K376R protein
was constitutively and highly phosphorylated on tyrosine, which may be
due to its exclusive localization in the membrane fraction of the cell.
Surprisingly, PKC-
WT overexpression in an IGF-IR/pLTR-
WT
cotransfectant resulted in constitutive tyrosine phosphorylation of
PKC-
independent of IGF-I, although IGF-IR tyrosine phosphorylation
was greatly increased upon IGF-I stimulation (Fig. 5A). Likewise,
PKC-
K376R was constitutively phosphorylated on tyrosine at a level
higher than that of PKC-
WT in the IGF-IR/pLTR-
K376R
cotransfectant.
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WT by overexpressed IGF-IR
was in sharp contrast to PKC-
activation by the PDGF-
R, in which
PKC-
was tyrosine phosphorylated in a PDGF-dependent manner
(24). IGF-IR overexpression led to a relatively high level
of autophosphorylation in the absence of addition of exogenous ligand
(Fig. 5A and C). Furthermore, we have previously shown that
overexpression of PKC-
resulted in its partial localization in the
membrane fraction of the cell before stimulation (25). To
test whether constitutive tyrosine phosphorylation of PKC-
WT prior
to IGF-I stimulation was due to its overexpression, we chose to
stimulate 32D cells with IGF-I or TPA. 32D cells express functional IGF-IR (data not shown) and higher endogenous PKC-
than NIH 3T3 cells whose tyrosine phosphorylation in response to PDGF
(24) or TPA (see below) was easily detected in vivo. As
shown in Fig. 6B, tyrosine phosphorylation of endogenous PKC-
from
32D cells in response to IGF-I stimulation for 5 and 30 min was
increased by at least two- to threefold, strongly suggesting that
PKC-
is a physiological substrate of IGF-IR. Overexpression of
IGF-IR in 32D cells led to some constitutive PKC-
tyrosine
phosphorylation, although a onefold increase of PKC-
tyrosine
phosphorylation was still observed in response to IGF-I stimulation for
5 min. These data clearly indicate that overexpression of IGF-IR can cause constitutive tyrosine phosphorylation of endogenous PKC-
, which may be contributed by the leaky IGF-IR due to its overexpression. Tyrosine phosphorylation of PKC-
WT became fully independent of IGF-I
when both IGF-IR and PKC-
were overexpressed in 32D cells. This
result mimics the phenomenon observed with NIH 3T3 cells coexpressing
IGF-IR with PKC-
(Fig. 6A). As reported elsewhere (22,
24), endogenous PKC-
and overexpressed PKC-
were tyrosine phosphorylated in response to TPA stimulation (Fig. 6B). We conclude that overexpression of PKC-
with NM1 or IGF-IR overexpressed in NIH
3T3 cells results in constitutive tyrosine phosphorylation of PKC-
.
Based on the data obtained from the 32D cell system, it is speculated
that endogenous PKC-
of NIH 3T3 cells may also be tyrosine
phosphorylated and activated by IGF-IR in vivo in a ligand-dependent
fashion when it reaches a certain expression level during the IGF-IR
transformation process. This hypothesis is further supported by the
up-regulation of endogenous PKC-
through long-term IGF-IR activation
in NIH 3T3 cell system (see Fig. 9).
Purified PKC-
can be phosphorylated by NM1 and IGF-IR with
increased activity in vitro.
Having demonstrated that PKC-
was
tyrosine phosphorylated in the NM1 or IGF-IR cotransfectant, we were
interested to know whether activated IGF-IR was able to phosphorylate
PKC-
directly and affect PKC-
kinase activity. For this purpose,
IGF-IR from the NM1 or IGF-IR transfectant and NIH 3T3 parental line
was immunoprecipitated and subjected to an in vitro kinase assay in the
presence of purified PKC-
derived from baculovirus and cold ATP
(22). After reaction, some of the supernatant containing the
purified PKC-
was assayed for PKC-
activity, while the remaining
reaction mixture was subjected to immunoblot analysis with anti-pTyr.
As shown in Fig. 7A, coincubation of the
purified PKC-
in vitro with immunoprecipitated endogenous IGF-IR
resulted in PKC-
tyrosine phosphorylation. The level of PKC-
tyrosine phosphorylation was increased with immunoprecipitates from
NM1- or IGF-IR-overexpressed cells (Fig. 7A; compare lanes 1 to 2 and
3). The same amount of purified PKC-
was used for the in vitro
tyrosine phosphorylation reaction in all of the samples, as determined
by reprobing the same membrane shown in Fig. 7A with anti-PKC-
(Fig.
7B). Subsequent analysis of PKC-
activity demonstrated that its
activity was increased by 1.2- to 1.4-fold after it was tyrosine
phosphorylated, compared to the non-tyrosine-phosphorylated PKC-
(Fig. 7C; compare lanes 1 to 3 to 4). These results suggest that
PKC-
is phosphorylated by NM1 and IGF-IR in vitro and that this
phosphorylation increases PKC-
activity.
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PKC-
associates with NM1 and IGF-IR intracellularly.
Association of tyrosine kinase receptors with their substrates in vivo
occurs frequently via Src homology 2 (SH2) or phosphotyrosine binding
(PTB) domains of these substrates (37). Although PKC-
does not possess an SH2 or PTB domain, it has been shown to be tyrosine
phosphorylated by various protein tyrosine kinase receptors, including
PDGF-
R, IR, and EGFR (8, 22, 24), and to associate with
p85 subunit of PI 3'K (10) and v-src
(51) in vivo. An 80-kDa phosphoprotein from the
NM1/pLTR-
WT cotransfectant was reproducibly detected in the in vitro
IGF-IR kinase assay (Fig. 5B). Since PKC-
possesses
autophosphorylation capacity and is known to migrate as an 80-kDa
protein by SDS-PAGE, we suspected that the 80-kDa phosphoprotein could
be PKC-
. Failure to detect the same 80-kDa protein from the
NM1/pLTR-
K376R cotransfectant further suggested its identity as
PKC-
, since PKC-
K376R is not able to undergo autophosphorylation
(25). To test our hypothesis, we performed reciprocal
immunoprecipitation and immunoblotting analyses using anti-IGF-IR and
anti-PKC-
sera. As shown in Fig. 8A,
both PKC-
WT and PKC-
K376R proteins were detected in anti-IGF-IR immunoprecipitates from lysates of NM1-cotransfected cells. The amounts
of PKC-
associated with NM1 protein were proportional to the
expression levels of PKC-
WT and PKC-
K376R in the respective cotransfectants (Fig. 4A). PKC-
WT and PKC-
K376R
coimmunoprecipitated by anti-IGF-IR migrated as doublets or triplets on
SDS-PAGE gels, which may be due to existence of multiple phosphorylated
forms of PKC-
as reported elsewhere (25). Endogenous
PKC-
was not detected under such condition. PKC-
WT in the
IGF-IR/pLTR-
WT cotransfectant was constitutively associated with
IGF-IR in this assay (Fig. 8A). PKC-
K376R was also detected from
anti-IGF-IR immunoprecipitates in the IGF-IR/pLTR-
K376R
cotransfectant, albeit at a much lower level (data not shown). To
exclude any possibility of nonspecific coimmunoprecipitation of
overexpressed PKC-
from anti-IGF-IR immunoprecipitates, we
immunoprecipitated IGF-IR/pLTR-
WT transfectant with preimmune serum.
As seen in Fig. 8B, preimmune serum did not precipitate PKC-
from
the transfectant, suggesting that detection of PKC-
from anti-IGF-IR
immunoprecipitates is due to the specific interaction of PKC-
with
the IGF-IR.
|
immunoprecipitates from the IGF-IR/pLTR, IGF-IR/pLTR-
WT, or IGF-IR/pLTR-
K376R cotransfectant, with the most abundant association detected from the IGF-IR/pLTR-
WT
cotransfectant (Fig. 8C). Like tyrosine phosphorylation of PKC-
by
IGF-IR, its association with IGF-IR appeared to be mainly ligand
independent. These results demonstrate that PKC-
WT and PKC-
K376R
are associated with both IGF-IR and NM1 in a ligand-independent manner
in NIH 3T3 cells overexpressing these proteins.
Endogenous PKC-
protein and RNA levels are increased upon
constitutive or long-term IGF-IR activation.
To further explore
the role of endogenous PKC-
in NM1- and IGF-IR-mediated cell
transformation, we examined the endogenous PKC-
protein levels in
NM1- and IGF-IR-overexpressing NIH 3T3 cells. As shown in Fig.
9A, when the cells were cultured in media containing 10% calf serum, overexpression of NM1 or IGF-IR resulted in
a twofold increase in the PKC-
protein level compared to that of the
parental NIH 3T3 cells. When NM1 and IGF-IR transfectants were serum
starved for 8 h and then treated with IGF-I for another 16 h,
endogenous PKC-
from IGF-I-treated IGF-IR transfectant was
up-regulated by twofold (Fig. 9B). As expected, TPA treatment for
16 h completely degraded endogenous PKC-
protein. The level of
PKC-
in the NM1 transfectant was equivalent to that of the IGF-I-treated IGF-IR transfectant and was independent of IGF-I stimulation, indicating that constitutively activated NM1 was able to
up-regulate the PKC-
protein level even in the absence of serum.
|
messages by one- to twofold in NM1 and IGF-IR transfectants compared to that of NIH 3T3 cells was clearly observed (Fig. 9C). Taken
together, our results suggest that association with and tyrosine
phosphorylation of PKC-
by IGF-IR and up-regulation of the
expression of PKC-
by long-term IGF-IR overexpression and activation
play an important role in NM1- and IGF-IR-mediated cell transformation.
| |
DISCUSSION |
|---|
|
|
|---|
Although activation of IGF-IR due to its mutations was not
reported in samples from tumor patients, overexpression of functional IGF-IR has been repeatedly documented in different cancers
(3-5). In the present study, we provide evidence that
overexpression of native and oncogenic IGF-IR can lead to NIH 3T3 cell
transformation, clearly indicating the causal role of overexpressed
IGF-IR in cell transformation. The IGF-IR- and oncogenic IR-mediated
transformation is inhibited by coexpression of an ATP binding mutant of
PKC-
(PKC-
K376R). Since PKC-
WT overexpression did not enhance
IGF-IR-mediated transformation, it is likely that the level of
endogenous PKC-
is not limiting for relaying IGF-IR transformation
signals. It is also possible that downstream signaling molecules of
PKC-
are limiting. Therefore, transformation would not be enhanced when PKC-
WT is overexpressed together with NM1 or IGF-IR. To date,
we have shown that c-Sis (PDGF-B)-, IGF-IR-, and IR-induced, but not
v-H-Ras- and v-Raf-induced, transformation of NIH 3T3 cells can be
blocked by coexpressing PKC-
K376R, indicating specificity in the
dominant inhibitory effect of this mutant on oncogene-mediated cell
transformation. PKC-
K376R expression did not affect NM1 or IGF-IR
tyrosine kinase activities, indicating that PKC-
K376R must exert its
inhibitory effect downstream of receptor activation.
Our data show association of IGF-IR with PKC-
and tyrosine
phosphorylation of PKC-
in NM1- and IGF-IR-transfected cells coexpressing PKC-
. The activated IGF-IR was also demonstrated to
phosphorylate purified PKC-
in vitro, leading to increased PKC-
enzymatic activity. In addition, PKC-
tyrosine phosphorylation correlated with its association with NM1 or IGF-IR in vivo. All of
these results strongly suggest that PKC-
is a direct in vivo substrate of IGF-IR. That the endogenous PKC-
was tyrosine
phosphorylated in response to short-term IGF-I stimulation in 32D cells
further substantiates the role of PKC-
as a physiological substrate
of IGF-IR in vivo. Our previous study also demonstrated that the IR was
able to phosphorylate purified PKC-
in vitro and that tyrosine
phosphorylation of PKC-
by IR increased PKC-
kinase activity
(22). Inhibition of oncogenic IR-induced transformation by
the PKC-
K376R mutant correlates with these observations.
Although our data did not provide evidence that PKC-
is directly
activated by short-term IGF-I stimulation in the NIH 3T3 cell system,
it has been reported that insulin stimulation leads to diacylglycerol
production and subsequent PKC activation (46, 47). In
addition, long-term stimulation by NM1 or IGF-I treatment of IGF-IR in
NIH 3T3 transfectants results in up-regulation of PKC-
protein
levels. In addition, endogenous PKC-
of 32D cells is tyrosine
phosphorylated by the activated IGF-IR (Fig. 6B), an indicator of
PKC-
activation (24). Thus, endogenous PKC-
may be
regulated in an IGF-I-dependent manner during the transformation process. Inhibition of NM1 and IGF-IR transformation by the
PKC-
K376R mutant and its association with these receptors further
substantiate the specific role of endogenous PKC-
in
IGF-IR-mediated cell transformation. The exact mechanism
underlying PKC-
K376R inhibition of NM1 and IGF-IR
transformation is still unclear. However, the association of
PKC-
K376R with NM1 and IGF-IR in vivo strongly suggests that
PKC-
K376R might compete with endogenous PKC-
for IGF-IR binding.
Thus, PKC-
K376R competition may block the PKC-
-mediated signal transduction pathway utilized by IGF-IR by sequestering important substrates whose activation requires phosphorylation by
endogenous PKC-
.
Our results utilizing the PKC-
ATP binding mutant indicate that
IGF-IR-mediated cell proliferation can be segregated from focus and
colony formation. It appears that PKC-
activation is involved in
cell transformation but not in matrix-attached cell growth induced by
IGF-IR. Systematic deletion and point mutation of the cytoplasmic
domain of IGF-IR have also suggested that IGF-IR-mediated soft agar
growth, mitogenicity, and inhibition of apoptosis are separable
(35). A cluster of serine residues at the COOH terminus of
IGF-IR has been identified as important for IGF-IR-induced transformation, but not for its mitogenicity (19). Whether
these serines are phosphorylated by PKC-
remains to be determined.
Recently, conflicting results concerning the role played by PKC-
in
cellular transformation have been observed in different cell systems.
PKC-
was suggested to be a tumor suppressor gene in
c-Src-transfected 3Y1 fibroblasts (30). c-Src transformed 3Y1 cells only when TPA was present. It was proposed that the synergistic effect between TPA and c-Src on 3Y1 cell transformation was
due to the down-regulation of functional PKC-
. Expression of a
dominant-negative mutant of PKC-
(13), similar to that generated in our laboratory and used in our present study, enhanced the
colony-forming activity of c-Src-expressing cells. In contrast to this
study, a positive role for PKC-
in malignant transformation was
demonstrated in a separate report (26). When rat embryo fibroblasts were transformed by SV40 T antigen, endogenous PKC-
levels were increased by more than threefold. When clones capable of
growing in soft agar were analyzed, their endogenous PKC-
levels
were found to be further increased compared to those of the original
SV40-transformed cells. Expression of the NH2 terminus of
PKC-
, which appeared to act in a dominant inhibitory fashion, completely suppressed soft agar colony formation by SV40-transformed cells (26). More recently, this group further demonstrated
that PKC-
may play an important role in determining the metastatic property of SV40-transformed cells (13a). In our hands, both PDGF and IGF-I, two important mitogens for cell proliferation, are able
to up-regulate endogenous PKC-
levels (Fig. 9) (24). Whether the role of PKC-
in cellular transformation is cell type specific or oncogene specific remains to be determined.
In summary, our results provide the first evidence that PKC-
is a
direct tyrosine substrate of IGF-IR and plays a pivotal role in
IGF-IR-mediated transformation in NIH 3T3 cells. Association of the
PKC-
dominant-negative mutant with NM1 or IGF-IR may block IGF-IR-mediated signal transduction by competing with endogenous PKC-
for receptor association and activation. The identification of
PKC-
as a substrate of tyrosine kinase receptors, such as the IGF-IR
and PDGF-
R, will further allow us to evaluate novel mechanisms of
receptor-substrate interaction and activation. Since PKC-
does not
possess SH2, PTB, or pleckstrin homology domains which have been
documented as the important modules in protein-protein interactions
(37), it will be interesting to determine whether association of PKC-
with NM1 or IGF-IR in vivo is direct, and, if
so, which regions of PKC-
and IGF-IR are required for this association. We are also very interested to determine if endogenous PKC-
is critical in tumorigenesis, in which the up-regulation of
IGF-IR and activation of its signaling pathway are tightly involved.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported in part by NIH grant CA55054.
We thank Nelson Ellmore for excellent technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Laboratory of Cellular and Molecular Biology, Building 37, Room 1E24, NCI, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-1347. Fax: (301) 496-8479. E-mail: Liwe{at}dc37a.nci.nih.gov.
| |
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