Insulin Stimulates PKC
-mediated Phosphorylation of Insulin
Receptor Substrate-1 (IRS-1)
A SELF-ATTENUATED MECHANISM TO NEGATIVELY REGULATE THE FUNCTION
OF IRS PROTEINS*
Yan-Fang
Liu,
Keren
Paz,
Avia
Herschkovitz,
Addy
Alt
,
Tamar
Tennenbaum
,
Sanford R.
Sampson
,
Motoi
Ohba§,
Toshio
Kuroki§,
Derek
LeRoith¶, and
Yehiel
Zick
From the Department of Molecular Cell Biology, The Weizmann
Institute of Science, Rehovot 76100, Israel, the
Faculty of Life Sciences, Gonda-Goldschmied Center,
Bar-Ilan University, Ramat-Gan 52900, Israel, the
§ Institute of Molecular Oncology, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan, and the
¶ Molecular and Cellular Endocrinology Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, August 10, 2000, and in revised form, January 2, 2001
 |
ABSTRACT |
Incubation of rat hepatoma Fao cells with
insulin leads to a transient rise in Tyr phosphorylation of insulin
receptor substrate (IRS) proteins. This is followed by elevation in
their P-Ser/Thr content, and their dissociation from the insulin
receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K)
inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1,
its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases
that negatively regulate IRS-1 function are downstream effectors of
PI3K. PKC
fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKC
in Fao cells, by
infection of the cells with adenovirus-based PKC
construct, had no
effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR·IRS-1 complexes and the rate of Tyr
dephosphorylation of IRS-1. The insulin-stimulated negative regulatory
role of PKC
was specific and could not be mimic by infecting Fao
cells with adenoviral constructs encoding for PKC
,
, or
.
Because the reduction in P-Tyr content of IRS-1 was accompanied by a
reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKC
, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKC
inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKC
itself. These findings implicate PKC
as a key element in a multistep negative feedback control mechanism of IRS-1 functions.
 |
INTRODUCTION |
The insulin receptor mediates insulin action through the
phosphorylation of substrate proteins on Tyr residues (1-3). The major
substrates of the insulin receptor kinase are Shc (4) and the
IRS1 family of proteins, IRS-1
(5), IRS-2 (6), IRS-3 (7), and IRS-4 (8). IRS proteins contain a
conserved PH (pleckstrin homology) domain (9,
10) located at the amino terminus, adjacent to a phosphotyrosine
binding (PTB) domain. The PTB domain is present in a number of
signaling molecules (11) and shares 75% sequence identity between
IRS-1 and IRS-2 (12). This domain interacts with the NPXY
motif of the juxtamembrane region of IR and promotes IR-IRS-1
interaction (13-15). The carboxyl-terminal region of IRS proteins is
poorly conserved. It contains multiple tyrosine phosphorylation motifs
that serve as docking sites for SH2 (Src homology-2) domain-containing proteins like the p85
regulatory subunit of PI3K, Grb2, Nck, Crk,
Fyn, and SHP-2, which mediate the metabolic and growth-promoting functions of insulin (1-3).
IRS proteins contain more than 70 potential Ser/Thr phosphorylation
sites for kinases like PKA (cAMP-dependent protein kinase), PKC, and MAPK (5, 6, 16). The phosphorylation of Ser/Thr residues of
IRS proteins has a dual function, serving either for a positive or
negative modulation of insulin signal transduction. Phosphorylation of
Ser residues within the PTB domain of IRS-1 by insulin-stimulated PKB
protects IRS proteins from the rapid action of protein tyrosine
phosphatases and enables the Ser-phosphorylated IRS proteins to
maintain their Tyr-phosphorylated active conformation, implicating PKB
as a positive regulator of IRS-1 functions (17). In contrast, a
wortmannin-sensitive Ser/Thr kinase, different from PKB, phosphorylates
IRS proteins and acts as a negative feedback control regulator that
turns off insulin signals by inducing the dissociation of IRS proteins
from IR (17). These observations raise the question as to which kinases
act as negative modulators of IRS proteins function.
Several Ser/Thr kinases located downstream of PI3K are potential
candidates to fulfill this role. These include the mammalian target of
rapamycin (mTOR) (18) and p70S6 kinase (19), which are activated by
phosphoinositide-dependent kinase-1 (PDK-1) (20). Other
candidates are members of the PKC family. Indeed,
12-O-tetradecanoylphorbol 13-acetate, a potent activator of
various PKC isoforms, effectively inhibits both IRS-1 interactions with
the juxtamembrane region of the insulin receptor and insulin's ability
to phosphorylate IRS proteins, thus implicating
diacylglycerol-activated PKCs as potential regulators of IR-IRS
interactions (21-23).
Atypical PKCs, exemplified by PKC
, are downstream effectors of PI3K
(24) and PDKs (25) and act as mediators of insulin action. These
proteins do not contain PH domains, and the exact mechanism of their
activation is unknown. Atypical PKCs bind phosphatidylinositol 1,4,5-trisphosphate with reasonably high affinities, which
results in their activation (26). Hence, phosphatidylinositol
1,4,5-trisphosphate may promote PKC signaling by colocalizing the
enzyme in close proximity to its substrates. PKC
can be activated by
insulin, and this activation is blocked by inhibitors of PI3K and the
expression of dominant negative p85 (27, 28). Phosphorylation of PKC
is required for its full activation; two of the regulatory
phosphorylation sites on PKC
are targets for phosphorylation by
PDK-1 and PDK-2 (25), thus representing another mechanism by which
activation of PI3K affects the activity of PKC
.
In the present study we provide evidence that overexpression of PKC
,
but not other PKC isoforms potentiates the insulin-stimulated dissociation of IRS-1 from the insulin receptor, accelerates the rate
of Tyr dephosphorylation of IRS-1, and as a result, reduces complex
formation between IRS-1 and PI3K. These findings implicate PKC
as an
insulin-stimulated and a PI3K-dependent kinase that down-regulates IRS-1 functions by a tightly regulated process. Agents
that induce insulin resistance, such as TNF, a known activator of
PKC
(29, 30) could take advantage of this mechanism. Activation of
PKC
by TNF in an insulin-independent manner (29, 30) could account
for the enhanced Ser phosphorylation of IRS proteins and their
dissociation from IR, which takes place when cells are exposed to TNF
(21), thus providing us with a possible molecular mechanism for the
induction of an insulin-resistant state.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human insulin was a gift
from Novo-Nordisc (Copenhagen, Denmark). Wortmannin, wheat-germ
agglutinin coupled to agarose beads, protease inhibitor mixture,
protein A-Sepharose CL-4B, and monoclonal anti-phospho-MAPK antibodies
were purchased from Sigma. Rapamycin, PD98059, Go6983,
bisindolylmaleimide I (GF109203X), and
12-(2-cyanoethyl)-6,7,2,13,-terahydro-13-methyl-5-oxo-(5H)-indolo[2,3-
]-pyrrolo[3,4-c]carbazole (Go6976) were purchased from Calbiochem. Monoclonal PY-20 antibodies and polyclonal anti-IR
-subunit antibodies were obtained from Transduction Laboratories (Lexington, KY). Polyclonal IRS-1 antibody (YR-1) was prepared as described (31). Polyclonal anti-PKC
/
was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Polyclonal anti-p85 antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-phospho-PKB
(Ser473) was purchased from New England Bio-Labs, Inc.
(Beverly, MA).
Treatment of Cells--
Rat hepatoma Fao cells were grown in
RPMI medium supplemented with 10% fetal calf serum as described (21,
32). H-35 rat hepatoma cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. At 80%
confluence, the cells were deprived of serum for 16 h before each
experiment. The medium was aspirated, and the cells were incubated with
the indicated inhibitors in serum-free medium for different time
periods at 37 °C. Cells were then incubated with or without 100 nM insulin for 1 or 60 min at 37 °C. Cells were washed
three times with ice-cold phosphate-buffered saline and harvested in
buffer A (25 mM Tris-HCl, 2 mM sodium
orthovanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM
-glycerophosphate, 25 mM NaCl, protease inhibitor
mixture 1:1000, pH 7.4). Following three cycles of freezing and
thawing, the cell extracts were centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatants were
collected. Samples were resolved by means of SDS-PAGE and immunoblotted
with the indicated antibodies.
Adenovirus Constructs--
Recombinant adenoviruses were
constructed in three steps essentially as described (33). The cDNA
encoding mouse wild-type PKC
, a kinase-inactive form of this enzyme
(PKC
-KD, generated by replacing K282 (AAG) at the ATP binding site
by R(AGG)), as well as cDNA encoding PKC
,
, and
were
inserted into a cassette cosmid. The cassette cosmid for constructing
recombinant Ad of the E1 substitution type, pAdex1, was an 11-kilobase
pair charomid vector bearing an Ad5 genome spanning 0 to 99.3 map units with deletions of E1 (map units 1.3-9.3) and E3 (map units
79.6-84.8), in which a unique SwaI site was created by
linker insertion at the E1 deletion. The expression unit was excised
with the appropriate restriction enzymes, blunt-ended with Klenow
fragment of DNA polymerase I, and purified by gel electrophoresis.
Thereafter the fragment was ligated with SwaI-linearized
pAxCAwt (33). After overnight ligation, the DNA sample was digested
with SwaI to exclude empty religated cosmids lacking a
coding sequence, and an aliquot was packaged in vitro using
Gigapack (Stratagene, La Jolla, CA). Colonies were obtained after
plating the transduced Escherichia coli DH5
; the majority of the clones contained the desired insert. Ad5-dIX, which
has an E3 deletion (map units 79.6-84.8) was used as the parent virus
for recombinant Ad construction. The DNA-terminal protein complex of
the parent Ad was prepared and purified utilizing a CsCl density
gradient with guanidine hydrochloride. The EcoT22I-digested adenovirus DNA-terminal protein complex was mixed with the cassette cosmid bearing the desired expression unit, and human embryonic kidney
293 cells were transfected with the mixed DNA by the calcium phosphate
method using a CellPhect Transfection kit (Amersham Pharmacia Biotech).
One day later, the cells were dispensed in 96-well plates in 10-fold
serial dilutions and mixed with untransfected 293 cells. After being
maintained in culture for 10-15 days, virus-containing supernatants
were isolated and propagated further to assess restriction analysis and
expression of inserted genes.
Infection of Fao Cells with Adenovirus-PKC--
Fao cells in
90-mm plates were cultured in RPMI medium supplemented with 10% fetal
calf serum till 60% confluence. The medium was aspirated, and 150-µl
aliquots of PKC
,
,
, or
recombinant adenoviruses were
added to the plate in 3 ml of RPMI medium containing 10% fetal calf
serum for 2 h at 37 °C. The cultures were supplemented with 8 ml of RPMI medium containing 10% fetal calf serum and were further
incubated with the adenoviral constructs for 16 h. The cultures
were then washed twice with RPMI, trypsinized, and divided into three
samples. 48 h post-infection, cells were starved for 16 h,
further treated as indicated above, and then harvested in buffer A.
Binding of IRS-1 to Immobilized IR--
Insulin receptors were
extracted from Fao cells at 70% confluence with buffer B (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM NaF, 150 mM NaCl, and protease inhibitor
mixture 1:1000, pH 7.4). Cell extracts were centrifuged at 12,000 × g for 20 min, and the supernatant, which contained the
soluble IR, was collected. Immobilization of IR on wheat germ
agglutinin-coupled agarose beads was carried out as described
previously (34). In brief, wheat germ agglutinin-coupled beads were
equilibrated in buffer B, and aliquots (1 mg) of the solubilized
IR were incubated with 30 µl of packed wheat germ agglutinin-beads
for 1 h at 4 °C. The immobilized IR was washed with buffer A
prior to use. Fao cells at 80% confluence grown in 90-mm dishes were
treated as indicated, washed three times with phosphate-buffered
saline, and harvested in 400 µl of buffer A. Following three cycles
of freezing and thawing, the cell extracts were centrifuged at
12,000 × g for 30 min at 4 °C, and the supernatants were collected. Aliquots (1.0 mg) were incubated for 2 h with 30 µl of immobilized IR while shaking at 4 °C. The beads were washed
three times with buffer A and boiled in 50 µl of Laemmli "sample
buffer" (35). Samples were resolved by means of SDS-PAGE and
immunoblotted with anti-IRS-1 or anti-IR
subunit antibodies.
Immunoprecipitation--
Fao cells were solubilized at 4 °C
in buffer A. Cell extracts were then centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatants were
collected. Aliquots (1.0 mg) were incubated for 2 h at 4 °C
with polyclonal IRS-1 antibody coupled to 15 µl of packed protein
A-Sepharose beads. Immunocomplexes were washed three times with buffer
A, resolved by means of SDS-PAGE, and immunoblotted with the indicated antibodies.
 |
RESULTS |
IRS-1 Serves as a Substrate for an Insulin-stimulated
Wortmannin-sensitive Ser/Thr Kinase--
Consistent with our previous
studies (17), incubation of Fao cells with 10
7
M insulin rapidly stimulated Tyr phosphorylation of the IRS
proteins (Fig. 1, A, a and
B, e). Phosphorylation was maximal 1 min
following insulin treatment and declined by 60 min incubation with the
hormone, accompanied by a decrease in the electrophoretic mobility of
IRS-1 and a marked (>40%) reduction in its ability to interact
in vitro with the insulin receptor (Fig. 1B,
f). Preincubation of the cells with wortmannin, a potent
inhibitor of PI3K, eliminated both the mobility shift and the reduction
in the P-Tyr content of IRS-1, whereas preincubation of the cells with
rapamycin, which inhibits the activation of p70 S6 kinase (36),
partially reversed the long-term effects of insulin. Similarly,
wortmannin effectively prevented the dissociation of IR·IRS-1
complexes that occurs following a 60-min treatment with insulin. In
contrast, PD-98059, a specific MEK (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase) inhibitor (37),
had no such protective effect, although it effectively inhibited
insulin-stimulated activation of MAPK (Fig. 1A,
d). Notably, both wortmannin and, to a lesser extent,
rapamycin prevented insulin-stimulated Ser phosphorylation of IRS-1,
but only wortmannin inhibited the activation of PKB, indicating that
the insulin-stimulated IRS-1 kinases that inhibit its Tyr
phosphorylation in response to insulin differ from PKB, the positive
regulator of IRS proteins function (17).

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Fig. 1.
Effects of different inhibitors on Tyr
phosphorylation of IRS-1 and its interaction with IR. Fao cells
were incubated with or without the indicated inhibitors for 60 min at
37 °C. Cells were then further incubated with 100 nM
insulin for the indicated times. A, cytosolic extracts were
prepared, and samples (1 mg) were subjected to immunoprecipitation
(IP) with IRS-1 antibodies. The immunocomplexes were
resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY
antibody (a). In parallel, samples (100 µg) of total cell
extracts were resolved by means of 7.5% SDS-PAGE and
immunoblotted (IB) with anti-IRS-1 (b),
anti-phospho-PKB (c), and anti-phospho-MAPK
(d) antibodies. B, cell extracts (1 mg) were
bound to immobilized IR as described under "Experimental
Procedures." Bound IRS-1 was detected following immunoblotting with
anti-IRS-1 antibodies (f). In parallel, cell extracts (100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted with
anti-P-Tyr (e) or anti-IRS-1 antibodies (g).
The bar graphs represent quantitation of the results
(mean ± S.D., n = 3-5). *, significantly
different from the measurement at 1 min (p < 0.001);
**, significantly different from the measurements taken at 1 min
(p < 0.05) and at 60 min (p < 0.01);
***, significantly different from the measurement at 1 min
(p < 0.025); #, significantly different from the
measurement at 1 min (p < 0.001). The p
values are based upon a paired t test.
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|
Insulin-induced Ser Phosphorylation of IRS-1 Impairs Its
Interaction with the p85 Regulatory Subunit with PI3K--
To
determine whether insulin-stimulated Ser phosphorylation of IRS-1 also
affects its ability to couple with downstream effectors, binding of
IRS-1 to p85
, the regulatory subunit of PI3K, was studied.
Consistent with numerous studies (cf. Refs. 1-3),
insulin-stimulated Tyr phosphorylation of IRS-1 resulted in the
effective coupling of IRS-1 to p85
(Fig.
2). Furthermore, the reduced level of Tyr phosphorylated IRS-1 following 60 min of insulin treatment was accompanied by a significant reduction in the amount of p85-associated IRS-1. Accordingly, pretreatment with wortmannin increased both the
P-Tyr content of IRS-1 and its ability to interact with p85. In
contrast, the PKC inhibitors Go6976 (inhibitor of PKC
,
I, and
µ) and GF109203X (inhibitor of PKC
,
I,
II,
,
, and
) failed to exert a protective effect. The PKC inhibitor Go6983 (inhibitor of PKC
,
,
,
, and
) exerted a partial
inhibitory effect, indicating that PKC
, which is inhibited only by
this inhibitor, could mediate insulin-stimulated Ser phosphorylation of
IRS-1.

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Fig. 2.
Effects of PKC inhibitors on Tyr
phosphorylation of IRS-1 and its association with PI3K. Fao
cells were incubated with the indicated inhibitors for 60 min. Cells
were then further incubated with 100 nM insulin for the
indicated time at 37 °C, and cytosolic extracts were prepared.
Samples (1 mg) were subjected to immunoprecipitation (IP)
with IRS-1 antibodies (a and b). Immunocomplexes
were resolved by means of 7.5% SDS-PAGE and immunoblotted
(IB) with anti-PY (a) or anti-p85 antibodies
(b). In parallel, samples (100 µg) of total cell extracts
were resolved by means of 7.5% SDS-PAGE and immunoblotted with
anti-IRS-1 (lane c), anti-phospho-PKB
(d), or anti-phospho-MAPK (lane e)
antibodies. The bar graphs represent quantitation of
the results mean ± S.D., n = 3-5). *,
significantly different from both the measurement taken at 1 min
(p < 0.050 and the measurement taken at 60 min
(p < 0.003); **, significantly different from the
measurement at 1 min (p < 0.05); #, significantly
different from the measurement at 1 min (p < 0.01);
##, different from the measurement 60 min (p = 0.07).
The p values are based upon a paired t
test.
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|
PKC
Mediates Ser Phosphorylation of IRS-1--
To determine
whether PKC
is directly involved in the negative regulation of IRS-1
protein functions, PKC
was overexpressed in Fao cells by infection
with an adenoviruses vector. As shown in Fig.
3 (inset) PKC
is endogenously
expressed in Fao cells, whereas infection with an adenovirus-based
PKC
construct markedly elevated the cellular content of PKC
,
assayed 48 h post-infection. Infection of Fao cells with
adenoviral constructs bearing
- galactosidase (Fig.
4B) or PKC
(Fig. 3b)
did not affect the cellular content of IRS-1 nor did it affect the
extent of Tyr phosphorylation of IRS-1 following a 1-min insulin
treatment (Figs. 3 and 4). However, the P-Tyr content of IRS-1, assayed
60 min following insulin stimulation, was significantly reduced in
cells overexpressing PKC
when compared with noninfected cells. The
infection of Fao cells with an adenoviral construct expressing a
kinase-inactive form of PKC
(K282R) failed to mimic the inhibitory
effects of the wild-type PKC
on the insulin-stimulated Tyr
phosphorylation of IRS-1 following 60 min of insulin treatment (Fig.
4A), indicating that only an active PKC
mediates the
inhibitory effects. Wortmannin effectively inhibited the reduction in
P-Tyr content of IRS-1 following 60 min of insulin treatment both in the nontreated as well as the PKC
-overexpressing cells, indicating that activation of the overexpressed PKC
is controlled by the endogenous PI3K activity and is independent of MAPK activation. Similarly, overexpression of PKC
did not affect the
insulin-stimulated activation of PKB (Fig. 3c),
indicating that PKB is not a downstream effector of PKC
.
Overexpression of PKC
significantly potentiated insulin-stimulated
MAPK activation (Fig. 3e), but MAPK could not be considered
an insulin-stimulated IRS-1 kinase because its inhibition by PD98059
did not affect the P-Tyr content of IRS-1, assayed 60 min following
insulin stimulation (Fig. 1).

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Fig. 3.
Effects of overexpression of
PKC on insulin-stimulated Tyr phosphorylation
of IRS-1. Fao cells remained uninfected (None) or were
transiently infected with Ad-PKC . 48 h post-infection, cells
were incubated with or without 100 nM wortmannin for 60 min
and were then further incubated with 100 nM insulin for the
indicated time at 37 °C. Cell extracts (1 mg) were subjected
to immunoprecipitation (IP) with IRS-1 antibody
(a and b). Immunocomplexes were resolved by means
of 7.5% SDS-PAGE and immunoblotted with anti-PY (a) or
anti-IRS-1 (b) antibodies. In parallel, total cell extract
(100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted
with anti-PKC (inset), anti-phospho-PKB (c),
or anti-phospho-MAPK ( ) antibodies. The bar graphs
represent quantitation of the results (mean ± S.D.,
n = 3-5). *, significantly different from the
measurement taken at 1 min (p < 0.001) and the
measurement taken at 60 min (p < 0.001) in noninfected
cells. The p values are based upon paired t test.
WT, wild type.
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Fig. 4.
Effects of overexpression of a
kinase-inactive PKC (K282R) on
insulin-stimulated Tyr phosphorylation of IRS-1. Fao cells were
remained uninfected (None) or were transiently infected with
Ad- -galactosidase overexpressing -galactosidase (B) or
Ad-PKC KD overexpressing a kinase-inactive PKC (K282R)
(A). 48 h post-infection, cells were incubated with 100 nM insulin for the indicated time at 37 °C. Cell
extracts (1 mg) were subjected to immunoprecipitation (IP)
with IRS-1 antibody (a, b, and d).
Immunocomplexes were resolved by means of 7.5% SDS-PAGE and
immunoblotted with anti-PY (a and d) or
anti-IRS-1 (b) antibodies. In parallel, total cell extract
(100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted
with anti-PKC (c and f) or anti-IRS-1
(e) antibodies. The results of a representative experiment
carried out six times (B) or two times (A) are
shown.
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|
Overexpression of PKC
Inhibits Complex Formation between IR and
IRS-1 and the Subsequent Association of IRS-1 with PI3K--
The
consequences of the reduction in P-Tyr content of IRS-1 as a result of
PKC
overexpression were evaluated next. As shown in Fig.
5a overexpression of PKC
,
accelerated the dissociation of IR·IRS-1 complexes observed following
60 min of insulin treatment (Fig. 5a, lane 3 versus 9). The marked reduction in complex
formation was evident when we compared the amount of the
Ser/Thr-phosphorylated IRS-1 bound to IR (the upper IRS-1
band in Fig. 5a, lanes 3 and 9) with
the total Ser/Thr-phosphorylated IRS-1 protein (Fig. 5c, lanes 3 and 9). Notably, the nonphosphorylated
(faster migrating) form of IRS-1, which is practically
undetected in the total IRS-1 blots of cells treated with
insulin for 60 min (compare Fig. 5c, lane 2 versus 3 or lane 8 versus 9) is
concentrated upon binding to IR, and becomes visible when IRS-1-bound
to IR is analyzed (Fig. 5a, upper versus lower
bands in lanes 3, 5, 6, 9, 11, and 12). Here again, pretreatment with the PKC inhibitors Go6976
and GF109203X was ineffective, whereas Go6983, which inhibits several PKC isoforms including PKC
, partially restored complex formation (Fig. 5a, lane 3 versus 5)
but only in the noninfected cells. The reduction in the IR·IRS-1
complex formation and the subsequent reduction in the P-Tyr content of
IRS-1 (at 60 min; Figs. 3 and 6), resulted in reduced association
between IRS-1 and its downstream effector, PI3K (Fig.
6); wortmannin but not PD98059 could
reverse this effect. Of note, insulin-stimulated activation of IRS-1
kinases in cells overexpressing PKC
was also regulated by mTOR or
its downstream effectors, because rapamycin partially reversed the inhibitory effects of the overexpressed PKC
on Tyr phosphorylation of IRS-1 and its association of p85 (Fig. 6).

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Fig. 5.
Effects of overexpression of
PKC on the interaction of IRS-1 with IR.
Fao cells remained uninfected or were transiently infected with
Ad-PKC . 48 h post-infection, cells were incubated with the
indicated inhibitors for 60 min and were then further incubated with
100 nM insulin for the indicated time at 37 °C. Cell
extracts were prepared in buffer A, and samples (1 mg) were bound to
immobilized IR as described under "Experimental Procedures."
IRS·1-IR complexes were resolved by means of 7.5% SDS-PAGE and
immunoblotted with anti-IRS-1 (a) and anti IR (b)
antibodies. In parallel, samples of total cell extracts (100 µg) were
resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1
(c) or anti-PKC (d) antibodies, respectively.
The bar graphs represent quantitation of the results
(mean ± S.D., n = 3-5). *, significantly
different (p < 0.001) from the measurement taken at 1 min (lanes 2 and 8) and the measurements taken at
60 min (lane 3) in noninfected cells (p < 0.002); **, significantly different (p < 0.02) from
the measurement taken at 60 min in noninfected cells (lane
3); ***, not significantly different (p = 0.21)
from the measurement taken at 60 min in PKC -infected cells
(lane 9). The p values are based upon a paired
t test.
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Fig. 6.
Effects of overexpression of
PKC on insulin-stimulated association of IRS-1
with PI3K. Fao cells remained uninfected or were
transiently infected with Ad-PKC . 48 h post-infection, cells
were incubated with the indicated inhibitors for 60 min and were then
further incubated with 100 nM insulin for the indicated
time at 37 °C. Cell extracts were prepared in buffer A, and samples
(1 mg) were subjected to immunoprecipitation (IP) with IRS-1
antibodies (a-c). Immunocomplexes were resolved by means of
7.5% SDS-PAGE and immunoblotted (IB) with anti-PY
(a), anti-IRS-1 (b), or anti p85 antibodies
(c). In parallel, samples (100 µg) of total cell extracts
were resolved by means of 7.5% SDS-PAGE and immunoblotted anti p85
antibodies (d). The bar graphs represent
quantitation of the results (mean ± S.D., n = 3-5). *, significantly different (p < 0.03) from the
measurement taken at 60 min (lane 9) in PKC -infected
cells; **, significantly different (p < 0.004) from
the measurement taken at 60 min in noninfected cells (lane
3); ***, significantly different (p < 0.04) from
the measurement taken at 1 min (lane 8) and the measurements
taken at 60 min (lane 9) in PKC -infected cells
(p < 0.08); # significantly different
(p < 0.006) from the measurement taken at 1 min
(lane 2) and the measurement taken at 60 min (lane
3) (p < 0.04) in noninfected cells. The
p values are based upon a paired t test.
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The Specificity of the Effects of PKC
--
The specificity of
the effects of PKC
was evaluated next. As shown in Fig.
7, overexpression of PKC
, PKC
, or
PKC
in Fao cells, introduced by infection with an adenoviral vector,
failed to reproduce the effects of PKC
and did not accelerate the
rate of Tyr dephosphorylation of IRS-1 following 60 min of insulin treatment. These PKC isoforms failed to induce MAPK activation in
response to insulin. These results indicate that the negative feedback
control on IRS protein function is selectively mediated by PKC isoforms
that are downstream effectors of PI3K along the insulin signaling
pathway.

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|
Fig. 7.
Effects of overexpression of different PKC
isoforms on insulin-stimulated Tyr phosphorylation of IRS-1. Fao
cells were remained uninfected or were infected with Ad-PKC
(A), (B), or (C) as described
under "Experimental Procedures." 48 h post-infection, cells
were incubated for 60 min with or without 100 nM wortmannin
as indicated. Cells were then further incubated with 100 nM
insulin for the indicated time at 37 °C. Cytosolic extracts were
prepared, and samples (1 mg) were subjected to immunoprecipitation
(IP) with IRS-1 antibody (a, b, e, f, i, and
j). Immunocomplexes were resolved by means of 7.5% SDS-PAGE
and immunoblotted with anti-PY (a, e, and i) or
anti-IRS-1 (b, f, and j) antibodies. In
parallel, samples (100 µg) of total cell extracts were resolved by
means of 7.5% SDS-PAGE and immunoblotted with anti-PKC
(c), anti-PKC (g), or anti-phospho-MAPK
(d and h) antibodies.
|
|
 |
DISCUSSION |
Atypical PKC isotypes PKC
and PKC
are important elements in
insulin signal transduction. They are activated by insulin through a
PI3K-dependent mechanism (27, 38), and insulin-stimulated glucose transport depends upon the activation of one or both of these
enzymes (27, 28, 38, 39). In the present study we provide evidence that
PKC
also plays a major regulatory role in a negative feedback
control mechanism induced by insulin to terminate its own signaling
pathways. This mechanism involves PKC
-mediated Ser/Thr
phosphorylation of IRS-1 that leads to its dissociation from the
insulin receptor. The dissociated IRS protein fails to undergo further
Tyr phosphorylation by the insulin receptor kinase, while being
subjected to the action of protein Tyr phosphatases that reduce its
P-Tyr content. The resulting Tyr-dephosphorylated IRS-1 is unable to
recruit downstream effectors like PI3K, and the insulin signal is
therefore terminated.
Several lines of evidence support such a mechanism. First, we could
demonstrate that of several inhibitors tested, wortmannin, a PI3K
inhibitor, effectively inhibits the dissociation of IRS-1 from the
insulin receptor and the subsequent reduction in its P-Tyr content
observed following a 60-min insulin treatment. Hence, a
wortmannin-sensitive Ser/Thr kinase presumably acts as the feedback control regulator that turns off insulin signals. PKC
, an
insulin-stimulated Ser/Thr kinase downstream of PI3K, could fulfill
this role, and indeed Go6983, an inhibitor of several PKC isoforms
including PKC
, partially prevented the reduction in P-Tyr content of
IRS-1 following a 60-min insulin treatment. Second, overexpression of PKC
by infection of Fao cells with an adenoviral-based expression vector markedly potentiated the dissociation of IR·IRS-1 complexes and the resulting reduction in P-Tyr content of IRS-1. The
overexpressed PKC
, like its endogenous counterpart, remained under
the control of the insulin signaling pathway, since its effects were
inhibited in the presence of wortmannin. Furthermore, overexpression of PKC
did not impair the acute (1 min) effects of insulin on Tyr phosphorylation of IRS-1, indicating that the basal activity of the
overexpressed PKC
is rather low.
The effects of PKC
on Ser phosphorylation of IRS-1 seems to be quite
unique in the sense that other PKC isoforms such as PKC
,
, and
, when overexpressed in Fao cells, fail to mimic the effects of
PKC
following insulin stimulation. Similarly, a kinase-inactive form
of PKC
fails to mimic the inhibitory effects of its wild-type
counterpart when transiently overexpressed in Fao or H-35 cells. Still,
these observations do not exclude the possibility that other PKC
isoforms could phosphorylate the IRS proteins in an insulin-independent
manner. In fact, we have shown (21) that
12-O-tetradecanoylphorbol 13-acetate, a potent activator of
conventional and novel PKC isoforms, effectively inhibits both IRS-1
interactions with the juxtamembrane region of the insulin receptor and
insulin's ability to phosphorylate IRS proteins (21). Similarly, the
mutation of Ser612 of IRS-1, a potential MAPK
phosphorylation site, eliminates the ability of
12-O-tetradecanoylphorbol 13-acetate to induce IR-IRS dissociation, indicating that diacylglycerol-activated PKCs (40) could
act as potential regulators of IR-IRS interactions (22, 23). It should
be noted, however, that although overexpression of PKC
potentiates
insulin-mediated activation of MAPK, the latter does not seem to
mediate PKC
effects on IRS-1 phosphorylation because PD98059, which
effectively inhibits activation of MAPK does not inhibit Ser/Thr
phosphorylation of IRS-1 in response to insulin treatment.
Although IRS proteins contain several PKC phosphorylation sites, it is
presently unclear whether PKC
phosphorylates IRS-1 directly or
whether its effects are mediated by a downstream effector of PKC
.
Recent studies suggest that IRS-1 serves as an in vitro substrate for PKC
(41). Furthermore, endogenous IRS-1 coprecipitates with endogenous PKC
, and this association is increased 2-fold upon
insulin stimulation (41). These findings suggest that PKC
could
function as a direct IRS-1 kinase. However, several Ser/Thr kinases
that are downstream effectors of PKC
could also fulfill this role. A
potential candidate is the IkB kinase
(IKK
), which binds PKC
as well as the atypical PKC
both in vitro and in
vivo, and serves as an in vitro substrate for PKC
(42). Overexpression of PKC
positively modulates IKK
activity,
whereas the transfection of a dominant negative mutant of PKC
severely impairs the activation of IKK
in TNF-stimulated cells
(42).
The p70 S6 kinase (19) is also a potential candidate in view of the
fact that rapamycin, which inhibits the insulin-stimulated activation
of p70S6K (43), partially prevents the reduction in P-Tyr content of
IRS-1 following 60 min of insulin treatment. Indeed,
mTOR-mediated phosphorylation on Ser632,
Ser662, and Ser731 of IRS-1 was shown to
inhibit its insulin-stimulated Tyr phosphorylation and its ability to
bind PI3K (23). Furthermore, p70 S6 kinase is activated by PKC
and
participates in a PI3K-regulated signaling complex in some (44) but not
all cellular models (30). Recently, the c-Jun NH2-terminal
kinase (JNK) was shown to promote insulin resistance during association
with IRS-1 and phosphorylation of Ser307. However it still
needs to be determined whether JNK is a downstream effector of PI3K.
Although earlier studies suggest that it is (45), recent findings
indicate that phosphatidylinositol 3-kinase is not part of
the insulin signaling pathway that leads to JNK activation (46).
Our current work and previous studies (17, 21) indicate that Ser/Thr
phosphorylation of IRS protein following insulin stimulation has a dual
role, either to enhance or to terminate the insulin signal. Ser
residues of the PTB domain of IRS-1, located within consensus PKB
phosphorylation sites, presumably function as positive effectors of
insulin signaling (17). Once phosphorylated by PKB
, they serve to
protect IRS proteins from the rapid action of protein Tyr
phosphatases. In such a way, PKB
acts to propagate and
accelerate insulin signaling by phosphorylating downstream effectors
and phosphorylating IRS proteins, thus generating a positive feedback
loop for insulin action. Insulin also activates PKC
, which mediates
phosphorylation of yet unidentified Ser/Thr residues within the IRS
protein. Phosphorylation of these sites is part of the negative
feedback control mechanism induced by insulin, which leads to the
dissociation of the IR·IRS complexes and results in the
termination of insulin signal. Agents that induce insulin resistance,
such as TNF, take advantage of this mechanism by stimulating Ser
phosphorylation of IRS proteins and the dissociation of IR·IRS
complexes (21). Interestingly, TNF induces the activation of PKC
in
an insulin-independent manner (29, 30), implicating PKC
itself or
its downstream Ser/Thr kinases (i.e. IKK
) as potential
TNF-stimulated IRS-1 kinases. Both Ser/Thr kinases, which phosphorylate
IRS-1, PKB the positive regulator, and PKC
the negative regulator,
are downstream effectors of PI3K. This finding suggests that
their action should be orchestrated in a way that enables sustained
activation of IRS-1, as a result of its phosphorylation by PKB, prior
to the activation of PKC
, the action of which is expected to
terminate insulin signal transduction. Our studies further indicate
that the negative feedback control mechanism induced by PKC
has a
built-in self-attenuation signal. Accordingly, insulin-stimulated and
PI3K-mediated activation of PKC
inhibits IRS-1 functions and reduces
complex formation between IRS-1 and PI3K, which then inhibits further
activation of PKC
. It is presently unclear how PKC
promotes
insulin action and glucose transport (27, 28, 38, 39) while acting as a
negative feedback regulator of insulin signal transduction. Most
likely, the positive and negative regulatory roles of PKC
are
subjected to a tight spatio-temporal control along the insulin signal
transduction pathway, such that the positive effects are turned on and
terminated before the negative regulatory functions are being
activated. Further studies are required to unravel the mechanisms that
govern this intricate regulatory process.
 |
ACKNOWLEDGEMENT |
We thank Dr. Ronit Sagi-Eisenberg for helpful
comments and discussions.
 |
FOOTNOTES |
*
This work was supported by research grants from the Israel
Ministry of Health, the Juvenile Diabetes Foundation International (1-1998-228), the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the Minerva Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Incumbent of the Marte R. Gomez professorial chair. To whom
correspondence should be addressed. Tel: 972-8-9342-380; Fax: 972-8-9344-125; E-mail: yehiel.zick@weizmann.ac.il.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M007281200
 |
ABBREVIATIONS |
The abbreviations used are:
IRS, insulin
receptor substrate;
IR, insulin receptor;
PTB, phosphotyrosine binding;
PKB, protein kinase B;
PKC, protein kinase C;
PI3K, phosphatidylinositol 3-kinase;
PDK, phosphoinositide-dependent
kinase;
PKC
-KD, a kinase-inactive PKC
;
MAPK, mitogen-activated
protein kinase;
mTOR, mammalian target of rapamycin;
TNF, tumor
necrosis factor
;
PAGE, polyacrylamide gel electrophoresis;
JNK, c-Jun NH2-terminal kinase;
Ad, adenovirus.
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