(Received for publication, May 4, 1995; and in revised form, July 13, 1995)
From the
Stimulation of the activity of protein kinase C by pretreatment
of cells with phorbol esters was tested for its ability to inhibit
signaling by four members of the insulin receptor family, including the
human insulin and insulin-like growth factor-I receptors, the human
insulin receptor-related receptor, and the Drosophila insulin
receptor. Activation of overexpressed protein kinase C resulted in
a subsequent inhibition of the ligand-stimulated increase in
antiphosphotyrosine-precipitable phosphatidylinositol 3-kinase mediated
by the kinase domains of all four receptors. This inhibition varied
from 97% for the insulin receptor-related receptor to 65% for the Drosophila insulin receptor. In addition, the activation of
protein kinase C
inhibited the in situ ligand-stimulated
increase in tyrosine phosphorylation of the GTPase-activating
protein-associated p60 protein as well as Shc mediated by these
receptors. The mechanism for this inhibition was further studied in the
case of the insulin-like growth factor-I receptor. Although the in
situ phosphorylation of insulin-receptor substrate-1 and p60 by
this receptor was inhibited by prior stimulation of protein kinase
C
, the in vitro tyrosine phosphorylation of these two
substrates by this receptor was not decreased by prior stimulation of
the protein kinase C
in the cells that served as a source of the
substrates. Finally, the insulin-like growth factor-I-stimulated
increase in cell proliferation was found to be inhibited by prior
activation of protein kinase C
. These results indicate that the
ability of activated protein kinase C
to antagonize signaling by
the human insulin receptor is shared by the other members of the
insulin receptor family despite their considerable differences in amino
acid sequence. Moreover, the present study shows that this antagonism
is exerted at a very early step, the initial tyrosine phosphorylation
of three distinct endogenous substrates. Finally, the present study
indicates that this inhibition is not caused by an increased Ser/Thr
phosphorylation of these two substrates.
Most studies have documented a critical role for the intrinsic
tyrosine kinase activity of the insulin receptor (IR) ()in
mediating subsequent biological responses(1, 2) .
After binding insulin, the receptor autophosphorylates on several
specific tyrosines and then tyrosine-phosphorylates several endogenous
substrates including insulin-receptor substrate (IRS)-1, an
SH2-containing protein called Shc, and various 60-kDa proteins
including one that is tightly bound by the GTPase-activating protein of
Ras(3, 4, 5, 6, 7, 8, 9, 10) .
The tyrosine phosphorylation of IRS-1 results in its being bound by
several SH2-containing proteins including the phosphatidylinositol (PI)
3-kinase, a tyrosine phosphatase, and two SH2 linker proteins called
Grb-2 and Nck(3, 11) . A number of studies utilizing a
variety of approaches have implicated the tyrosine phosphorylation of
IRS-1 as being important in initiating several biological responses
including stimulation of growth responses as well as stimulation of
glucose
uptake(12, 13, 14, 15, 16, 17) .
Because of the critical role of the receptor tyrosine kinase in initiating subsequent biological responses, a major question is how this receptor kinase activity may be regulated. Interest in this question is also stimulated by the finding that the IR kinase may be negatively regulated in patients with non-insulin-dependent mellitus, possibly contributing to the insulin resistance observed in this condition(18, 19, 20, 21, 22, 23, 24, 25) . Negative regulation of the IR kinase in several cell systems by a variety of different types of prior treatments has also been reported; for example, treatment of cells with high concentrations of insulin for long periods of time, with tumor necrosis factor, with activators of protein kinase C or cyclic AMP, or with inhibitors of Ser/Thr phosphatases and even incubation of the cells with high concentrations of glucose have all been reported to decrease the ability of insulin to stimulate the IR kinase activity (26, 27, 28, 29, 30, 31, 32, 33) . In most of these cases, the detailed biochemical mechanism whereby these agents elicit the inhibition is still not known. In one of the best understood systems, pretreatment of adipocytes with the Ser/Thr phosphatase inhibitor, okadaic acid, an increase in the Ser/Thr phosphorylation of IRS-1 was observed, which inhibited its subsequent ability to be tyrosine-phosphorylated in vitro by the IR and to be bound by the PI 3-kinase(32) . However, it is not clear which kinase is responsible for this hyperphosphorylation of IRS-1 and which specific Ser/Thr residues in IRS-1 are responsible for its decreased ability to be tyrosine-phosphorylated.
Another system that
has been extensively studied is the ability of activated protein kinase
C (PKC) to antagonize IR signaling. This system is particularly
attractive, since several reports have indicated that hyperglycemia can
result in activation of PKC(34, 35) . Although some
studies have indicated that activation of PKC increases the Ser/Thr
phosphorylation of the IR and this phosphorylation inhibits its ability
to subsequently tyrosine-phosphorylate in vitro various
exogenous substrates(29, 36) , other studies do not
confirm this
finding(30, 37, 38, 39, 40) .
Activation of overexpressed PKC was observed to stimulate the
Ser/Thr phosphorylation of the IR and to inhibit the in situ insulin-stimulated increase in tyrosine phosphorylation of the
endogenous substrate IRS-1 and its subsequent binding by PI 3-kinase
even under conditions in which no decrease in receptor kinase activity
could be detected in in vitro assays utilizing exogenous
substrates(30, 38) . In addition, a number of specific
Ser/Thr residues in the IR have been identified as phosphorylation
sites both in vitro and in vivo. These include two
serines (Ser 1305/1306 and Ser 1327) and one threonine (Thr 1348) in
the carboxyl tail of the IR, serines in the juxtamembrane region (Ser
967/968), and serines in the kinase domain of the IR (Ser
1035/1037)(41, 42, 43, 44, 45, 46, 47, 48, 49) .
The role of these Ser/Thr phosphorylation sites in the IR in mediating
the PKC-induced inhibition is not clear since mutant IR lacking these
Ser/Thr phosphorylation sites still exhibited decreased signaling
abilities after activation of
PKC(46, 47, 50) .
In order to further
elucidate the mechanism whereby activation of PKC inhibits subsequent
signaling by the IR, we set out in the present study to examine the
ability of overexpressed PKC to antagonize signaling by the other
members of the IR family, including the human receptor for insulin-like
growth factor I (hIGFR), the human insulin receptor-related receptor
(hIRR), and the Drosophila IR (dIR). These three receptors
differ considerably in their amino acid sequences from the human
IR(51, 52, 53) . For example, the carboxyl
tail of IRR lacks the two serine and one threonine phosphorylation
sites present in human IR(52) , whereas the dIR juxtamembrane
and kinase domains are only 29 and 64% identical to the respective
regions of the human IR(53) . Despite these considerable
differences in amino acid sequence, in the present study we show that
the ligand-stimulated increase in PI 3-kinase mediated by all four
receptors is inhibited by the activation of PKC
, although the
extent of inhibition did differ to some extent. Moreover, in the
present report we show that the ligand-stimulated increase in tyrosine
phosphorylation of the GAP-associated p60 as well as the adaptor
protein Shc mediated by these receptors is also inhibited by activation
of PKC. Finally, the inhibition of IRS-1 and p60 tyrosine
phosphorylation observed in situ could not be replicated in vitro by the use of IRS-1 or p60 from PKC-activated cells.
These results support the hypothesis that the inhibition observed with
PKC activation is important in modulating signaling by all members of
the IR family and indicates that this inhibition is not caused by an
increased Ser/Thr phosphorylation of these substrates.
To study the interactions of PKC with the different
members of the IR family, we isolated stably transfected CHO cell lines
overexpressing PKC
and these four different receptors. For hIRR
and dIR, chimeric receptors were utilized that contained the
extracellular domain of the human IR and the cytoplasmic domains of
these two receptors(56, 57) . These different cell
lines (called CHO-PKC-hIR, CHO-PKC-hIGFR, CHO-PKC-hIR/hIRR, and
CHO-PKC-hIR/dIR for the cells overexpressing the hIR, the hIGFR, the
chimeric hIR/hIRR, and the chimeric hIR/dIR, respectively) all
expressed comparably elevated levels of PKC
in comparison with
CHO-hIR cells (data not shown) and elevated levels of the transfected
receptors (the relative amounts of these four receptors could not be
directly compared since different antibodies were required to visualize
the different receptors). Activation of the overexpressed PKC
by a
20-min pretreatment with the phorbol ester PMA was found to inhibit the
subsequent ligand-stimulated increase in
anti-phosphotyrosine-precipitable PI 3-kinase mediated by all four
receptors, whereas this treatment had no significant effect on CHO-hIR
cells, which do not overexpress PKC
(Fig. 1). The
inhibitions observed were 70, 80, 97, and 65% (average of four
different experiments) for the cells overexpressing PKC
and the
human IR, IGFR, IRR, and Drosophila IR, respectively. For each
receptor, the results were confirmed with a second independent clone of
cells. Again, the cells overexpressing the hIRR exhibited a greater
inhibition than the hIR, whereas the cells overexpressing the Drosophila IR exhibited slightly less inhibition than the hIR.
Figure 1:
Effect of PKC activation on the
ligand-induced increase in anti-phosphotyrosine-precipitable PI
3-kinase mediated by the four members of the IR family. CHO cells
overexpressing only the human IR (CHO-hIR) or PKC
and the
human IR (CHO-PKC-hIR), the human IGFR (CHO-PKC-hIGFR), the chimeric hIR/hIRR (CHO-PKC-hIR/hIRR), or the chimeric human and Drosophila IR (CHO-PKC-hIR/dIR) were prestimulated with PMA. Then
ligand was added, and after an additional 10 min the cells were lysed.
The amount of anti-phosphotyrosine-precipitable PI 3-kinase was
measured. Results are the means ± S.E. for four or five
experiments, where the amount of PI 3-kinase activity present in the
PMA-treated cells is expressed as a percentage of the response in the
same line of cells treated with ligand
alone.
The above described differences in inhibition between the various
members of the IR family suggested that the PKC effects may be mediated
via an effect of this Ser/Thr kinase on the receptors themselves. If
this were true, one might expect that activation of the PKC would
also inhibit the receptor-mediated tyrosine phosphorylation of other
substrates. To test this, the various cell lines were again pretreated
with PMA and then stimulated with ligand, and the extent of tyrosine
phosphorylation of another endogenous substrate was measured, the p60
GAP-associated protein(9) . PMA pretreatment was found to
inhibit the ligand-stimulated increase in tyrosine phosphorylation of
this substrate in the CHO cells overexpressing all four members of the
IR receptor family and PKC
but not the control cells, which
overexpressed the hIR but not PKC
(Fig. 2A). The
tyrosine phosphorylation of p60 was likely to be mediated by the
overexpressed IR family member in each of these cell lines since the
extent of tyrosine phosphorylation of this protein was in all cases
increased over that observed in cells overexpressing only PKC
(Fig. 2A). In these cells, unlike another previously
described cell line(60) , PMA treatment alone did not stimulate
the tyrosine phosphorylation of p60 (data not shown).
Figure 2:
Effect of PKC activation on the
ligand-induced tyrosine phosphorylation of p60 in situ (A) and in vitro (B) and on the in
situ tyrosine phosphorylation of Shc (C). A, in situ tyrosine phosphorylation of p60. CHO cells were the
same as in Fig. 1with the addition of cells only overexpressing
PKC
(CHO-PKC). Cells were pretreated with PMA and then
stimulated with ligand. After an additional 10 min they were lysed, and
the p60 was immunoprecipitated and analyzed by SDS gel electrophoresis
and immunoblotting with an anti-phosphotyrosine antibody. B, in vitro tyrosine phosphorylation of p60. The p60 was
immunoprecipitated from either control or PMA-treated CHO-PKC-IGFR
cells and then incubated with either activated IGFR or buffer, as
indicated. Control precipitations were performed with normal Ig (NM) and also incubated with IGFR. The samples were then
analyzed by SDS gel electrophoresis and immunoblotting with
anti-phosphotyrosine antibodies. C, in situ tyrosine
phosphorylation of Shc. The different CHO cells were treated as in A and lysed, and the Shc was immunoprecipitated and analyzed
by SDS gel electrophoresis and immunoblotting with an
anti-phosphotyrosine antibody. The blot was then stripped and probed
with the anti-Shc antibodies. The position of the major Shc band is
indicated.
The
ligand-stimulated increase in tyrosine phosphorylation of a third
endogenous substrate, Shc, (5, 8) was also examined in
these cells. The tyrosine phosphorylation of Shc was increased in the
cells overexpressing the hIR, hIGFR, and hIR/hIRR (but not in the cells
expressing hIR/dIR) over that observed in the parental cells and in the
cells overexpressing only PKC (Fig. 2C). In each
of these cells, activation of the overexpressed PKC
was found to
inhibit the ligand-stimulated increase in tyrosine phosphorylation of
Shc (Fig. 2C). As with the other two substrates, the
insulin-stimulated increase in tyrosine phosphorylation of Shc was not
inhibited in the cells containing only the endogenous levels of PKC (Fig. 2C).
To further study the mechanism whereby
PKC activation inhibits signaling, the ability of the isolated IGFR was
examined for its capacity to phosphorylate in vitro IRS-1 that
had been isolated from either control cells or cells in which PKC
had been previously activated (Fig. 3). No significant
difference was observed in the extent of IRS-1 phosphorylation in
vitro by isolated IGFR (in three experiments, the ratio of
tyrosine phosphorylation of IRS-1 isolated from PMA-treated cells to
phosphorylation of IRS-1 isolated from control cells was 1.1 ±
0.25). In contrast, in parallel experiments, the IGF-I-stimulated
tyrosine phosphorylation of IRS-1 that occurred in situ was
decreased by about 70% after PMA pretreatment (Fig. 3) (the
ratio of IRS-1 tyrosine phosphorylation in PMA-treated cells/control
cells was 0.28 ± 0.18), a value that is in good agreement with
the 80% decrease in the IGF-I-stimulated increase in
anti-phosphotyrosine-precipitable PI 3-kinase (Fig. 1).
Figure 3: In situ versus in vitro phosphorylation of IRS-1. Parallel plates of CHO-PKC-IGFR cells were pretreated with PMA or buffer and then either utilized for the in situ or in vitro phosphorylations. For the in situ phosphorylations, the cells were stimulated with ligand, and the IRS-1 was immunoprecipitated and analyzed by immunobloting with anti-phosphotyrosine antibodies. For the in vitro phosphorylations, the IRS-1 was immunoprecipitated from cells and then incubated with either activated IGFR or buffer, as indicated. The samples were then analyzed by SDS gel electrophoresis and immunoblotting with anti-phosphotyrosine antibodies. The total amount of IRS-1 immunoprecipitated from the different cells was also analyzed and found to be the same by stripping the blot and reprobing it with a polyclonal anti-IRS-1 antibody. Ppt ab, precipitating antibody.
We
also compared the ability of the IGFR to phosphorylate in vitro p60 from either PMA-treated or control cells. The extent of p60
phosphorylation in vitro was not significantly decreased if
the p60 was isolated from PMA-treated cells versus control
cells (Fig. 2B). Again, in contrast, in parallel plates
the in situ tyrosine phosphorylation of p60 was greatly
inhibited by prior activation of the PKC (Fig. 2A).
To determine whether activation of
PKC would affect the ability of the IGFR to mediate a biological
response, CHO-PKC-IGFR cells were treated with or without PMA, and then
the ability of IGF-I to stimulate proliferation was measured. PMA
pretreatment was found to greatly inhibit the ability of IGF-I to
stimulate the proliferation of these cells (Fig. 4).
Figure 4: Effect of PMA-treatment on IGF-I-stimulated proliferation of CHO-PKC-IGFR cells. Cells were treated with the indicated concentrations of IGF-I in the presence or absence of 1 µM PMA. Proliferation was assessed by the use of the phenazine methosulfate assay. Results shown are means ± S.E. for five experiments, and the results have been expressed as a percentage of the proliferative response observed in the presence of 1 nM IGF-I.
In the present studies, activation of overexpressed PKC
was found to antagonize the signaling abilities of four distinct
members of the IR family including the human IR and IGFR, the orphan
receptor called IRR and the Drosophila IR (Fig. 1). The
sequences of these four receptors differ
considerably(51, 52, 53) , although their
signaling abilities all appear to be quite similar ( (55, 56, 57) and the present study). The
inhibition occurred at one of the earliest steps mediated by these
receptors, the ligand-stimulated increase in
anti-phosphotyrosine-precipitable PI 3-kinase, a monitor of IRS-1
tyrosine phosphorylation(3) . In addition, activation of the
overexpressed PKC
was found to inhibit the ligand-stimulated
increase in tyrosine phosphorylation of two other endogenous
substrates, the GAP-associated p60 (9) (Fig. 2A) as well as the adaptor protein
Shc (4, 5) (Fig. 2C). The tyrosine
phosphorylation of IRS-1 and its subsequent association and activation
of the PI 3-kinase has been found to be critical for subsequent
signaling by the IR and presumably by the other members of this family (12, 13, 14, 15, 16, 17) .
This effect of the activated PKC
could therefore explain its
ability to inhibit the IGF-I-induced proliferative response in these
cells (Fig. 4). Alternatively, PKC
could also be acting
through an effect on a downstream molecule in the cascade, which leads
to the proliferative response(61) .
In the present studies, activation of the endogenous PKC was found to be insufficient to inhibit signaling by the overexpressed human IR in the CHO cells ( Fig. 1and Fig. 2). In contrast, in several prior reports activation of the endogenous levels of PKC have been reported to inhibit signaling via the IR, both in cells with and without overexpressed human IR(29, 33, 50) . Some of these differences could be due to different levels of endogenous PKC in the various cell types studied as well as differences in the various isoenzymes of PKC present in these different cells. The ratio of PKC to IR may be critical in determining whether the inhibition of IR signaling is observed. In this regard, it would be important to determine whether activation of endogenous PKC in normal target tissues of insulin action is sufficient to inhibit insulin-stimulated increases in IRS-1-associated PI 3-kinase.
Activation of overexpressed
PKC has previously been found to stimulate to a limited extent the
Ser/Thr phosphorylation of IRS-1 (38) . This increased
phosphorylation of IRS-1 could have explained its decreased ability to
be tyrosine-phosphorylated by the IR and the other members of this
family. However, no decrease in the in vitro phosphorylation
of IRS-1 by the IGFR was observed when the IRS-1 was isolated from
cells in which the PKC
had been previously activated (Fig. 3). These findings differ from another cellular system of
insulin resistance, okadaic acid-treated adipocytes(32) . In
this system, the Ser/Thr phosphatase inhibitor okadaic acid was
observed to cause a hyperphosphorylation of IRS-1, which was evident
from a shift in its mobility on SDS gel electrophoresis, and this
hyperphosphorylation was reported to inhibit the subsequent in
vitro phosphorylation of IRS-1 by the IR. Such a shift in IRS-1
has not been observed after the activation of PKC
(38) ,
consistent with the lack of effect in the in vitro phosphorylation (Fig. 3). In addition, activation of the
PKC
inhibited the in situ phosphorylation of the second
endogenous substrate, the GAP-associated p60 protein, without effecting
the in vitro phosphorylation of this substrate (Fig. 2). These results indicate that the PKC
is not
causing its effect by stimulating the Ser/Thr phosphorylation of these
two endogenous substrates.
This hypothesis is also supported by the
finding that the extent of inhibition by PKC varied to some extent
for the four different receptors. Most notable was the greater
inhibition of the signaling by hIRR (Fig. 1). This receptor
differs from the hIR in part by lacking 48 amino acids in the carboxyl
tail of the
chain(52) , including two previously
identified serine phosphorylation sites as well as a
threonine-phosphorylated residue in this
region(41, 42, 43, 48) . These
results, therefore, indicate that phosphorylation of these residues in
the carboxyl tail of the IR may not be important in mediating the
negative regulation of the IR kinase; this conclusion is consistent
with prior studies that have indicated that Ser/Thr phosphorylations in
the carboxyl tail region have little effect on the IR
kinase(49, 50) . A comparison of the amino acid
sequences of the cytoplasmic domains of these four receptors indicates
that there are 11 conserved serines in all four molecules (Fig. 5). Only one of these serines (Ser-1190) is conserved in
other receptor tyrosine kinases such as the epidermal growth factor
receptor(62) . The one conserved serine in the juxtamembrane
region of these four receptors (Ser-974) has been previously found by
site-directed mutagenesis not to play a major role in mediating the
PKC-induced inhibition of the IR kinase(46) . It is therefore
possible that the phosphorylation of one or a combination of these
serine residues in the kinase domains of these receptors is responsible
for the observed inhibitions.
Figure 5: Comparison of the amino acid sequences of the cytoplasmic domains of the four members of the IR family. Residues that are conserved in all four proteins are capitalized, and the 11 conserved serines are in boldface type.
The inhibition of the in situ tyrosine phosphorylation of IRS-1 and GAP-associated p60 caused by
activation of PKC is not reflected in a decrease in receptor
kinase activity in in vitro assays(30, 38) .
Several hypotheses could explain these data. First, it is possible that
the in vitro conditions do not replicate the conditions of the in situ situation. Second, it is possible that a third protein
is involved in the intact cells. This protein may preferentially
recognize the Ser/Thr-phosphorylated IR and inhibit its kinase activity in situ; such a protein would function like arrestin, a
protein that preferentially recognizes the serine-phosphorylated
rhodopsin(63) . Alternatively, it is possible that under
nonstimulated conditions, a third protein tethers the IR together with
its various substrates. After PMA-stimulated Ser/Thr phosphorylation,
the IR may release this protein, thereby resulting in the decreased
tyrosine phosphorylation of IRS-1 and other substrates. Such a protein
would be similar to the yeast protein Ste5, which has been hypothesized
to make a complex with various components of the yeast MAP kinase
cascade, thereby facilitating their interactions(64) .