Insulin Induces Specific Interaction between Insulin Receptor and Protein Kinase C
in Primary Cultured Skeletal Muscle
Liora Braiman,
Addy Alt,
Toshio Kuroki,
Motoi Ohba,
Asia Bak,
Tamar Tennenbaum and
Sanford R. Sampson
Faculty of Life Sciences (L.B., A.A., A.B., T.T., S.R.S.)
Gonda-Goldschmied Center Bar-Ilan University Ramat-Gan 52900,
Israel
Institute of Molecular Oncology (T.K.) and
Department of Microbiology (M.O.) Showa University Tokyo
142-8555, Japan
 |
ABSTRACT
|
---|
Certain protein kinase C (PKC) isoforms, in
particular PKCs ßII,
, and
, are activated by insulin
stimulation. In primary cultures of skeletal muscle, PKCs ßII and
, but not PKC
, are activated via a phosphatidylinositol 3-kinase
(PI3K)-dependent pathway. The purpose of this study was to investigate
the possibility that PKC
may be activated upstream of PI3K by direct
interaction with insulin receptor (IR). Experiments were done on
primary cultures of newborn rat skeletal muscle, age 56 days in
vitro. The time course of insulin-induced activation of PKC
closely paralleled that of IR. Insulin stimulation caused a selective
coprecipitation of PKC
with IR, and these IR immunoprecipitates from
insulin-stimulated cells displayed a striking induction of PKC activity
due specifically to PKC
. To examine the involvement of PKC
in the
IR signaling cascade, we used recombinant adenovirus constructs of
wild-type (W.T.) or dominant negative (D.N.) PKC
.
Overexpression of W.T.PKC
induced PKC
activity and
coassociation of PKC
and IR without addition of insulin.
Overexpression of D.N.PKC
abrogated insulin- induced
coassociation of PKC
and IR. Insulin-induced tyrosine
phosphorylation of IR was greatly attenuated in cells overexpressing
W.T.PKC
, whereas in myotubes overexpressing D.N.PKC
, tyrosine
phosphorylation occurred without addition of insulin and was sustained
longer than that in control myotubes. In control myotubes IR displayed
a low level of serine phosphorylation, which was increased by insulin
stimulation. In cells overexpressing W.T.PKC
, serine phosphorylation
was strikingly high under basal conditions and did not increase after
insulin stimulation. In contrast, in cells overexpressing D.N.PKC
,
the level of serine phosphorylation was lower than that in
nonoverexpressing cells and did not change notably after addition of
insulin. Overexpression of W.T.PKC
caused IR to localize mainly in
the internal membrane fractions, and blockade of PKC
abrogated
insulin-induced IR internalization. We conclude that PKC
is involved
in regulation of IR activity and routing, and this regulation may be
important in subsequent steps in the IR signaling cascade.
 |
INTRODUCTION
|
---|
In the cascade of events leading to the multiple effects of
insulin, this hormone activates its receptor tyrosine kinase by
autophosphorylation of the ß-subunits. This leads to stimulation of a
number of downstream signaling factors, among which are several insulin
receptor substrates (IRS1, IRS2, IRS3), phosphatidylinositol 3-kinase
(PI3K), mitogen-activated protein (MAP) kinase and other protein
kinases (1). Whereas activation of the insulin receptor (IR) depends on
tyrosine phosphorylation, recent studies have shown that IR also
undergoes serine phosphorylation. The mechanisms and importance of
these serine phosphorylations are presently unknown.
We and others have reported that among the proteins activated by
insulin stimulation of skeletal muscle and fat cells are certain
members of the protein kinase C (PKC) family of serine-threonine
kinases (2, 3, 4, 5, 6, 7, 8), in particular PKCs ßII,
, and
. PKCs ßII and
are activated via products of PI3K activity (7). While the pathway
for insulin stimulation of PKC
in skeletal muscle is not known, it
is clearly independent of PI3K activity (6). These PKC isoforms, being
activated by insulin, are possible candidates for phosphorylating the
receptor on serine residues and affecting its routing. In fact, several
studies have been directed toward investigating this possibility, but
the findings are inconclusive (9, 10).
We have reported that insulin stimulation of PKC
is associated with
phosphorylation on tyrosine residue (7). It is known that tyrosine
phosphorylation of PKC
occurs only in the presence of an activator
and appears to be restricted to the activated form of the enzyme (11).
Thus, tyrosine phosphorylation sites may be exposed only upon an
activator-induced conformational change of PKC
. This phosphorylation
modifies the activity of PKC
toward certain substrates. Among the
proteins that have been shown to tyrosine phosphorylate PKC
in vitro is IR tyrosine kinase (12).
In an earlier study on insulin action in primary cultures of rat
skeletal muscle (7), we reported that insulin-induced activation and
tyrosine phosphorylation of PKC
were readily detectable within 1
min. This, together with the lack of PI3K involvement in
insulin-induced PKC
activation, suggested to us that tyrosine
phosphorylation of PKC
occurs upstream in the insulin signaling
pathway, perhaps at the level of IR itself. Accordingly, we have
investigated the relation between PKC
and IR in primary cultures of
rat skeletal muscle. These cells, plated initially as individual
myoblasts, align and fuse into multinucleated muscle fibers by day 34
in vitro. The mature fibers display resting membrane and
action potentials and other membrane properties that are nearly
identical to those seen in vivo (13, 14, 15, 16). The results show
that insulin specifically induces PKC
to associate with IR and that
this IR-PKC
association plays an important role in early IR
signaling. Our results further indicate that PKC
induces serine
phosphorylation of IR and may be involved in the routing of the
receptor.
 |
RESULTS
|
---|
We have recently shown that in cultured skeletal muscle, insulin
induces activation of PKC
as well as PKCs
and ßII, and that
activation and tyrosine phosphorylation of the latter two isoforms
occur via a wortmannin-sensitive pathway (7). The pathway for
activation of PKC
is not yet known. We found, however, that
activation as well as tyrosine phosphorylation of PKC
by insulin
occurred sooner than PKCs ßII and
, i.e. 1 min as
compared with 10 min, respectively, after insulin stimulation. These
results suggested that insulin-induced activation and tyrosine
phosphorylation of PKC
could be occurring upstream and independent
of PI3 kinase. Therefore, we initially attempted to clarify the pathway
by which resident PKC
could be related to the initial steps of the
insulin-induced signaling pathway in intact muscle cells.
Insulin Induces Association between IR and PKC
One possible mechanism for insulin-induced activation and tyrosine
phosphorylation of PKC
, suggested both by the rapidity of the effect
and the close association with IR tyrosine phosphorylation, might
involve a direct physical linkage of this isoform with IR (12). Several
studies on cells transfected either with IR or PKC
have shown that
the two proteins can associate in vitro (12, 17). To examine
this, we immunoprecipitated IR with anti-IRß antibody from control
and insulin-stimulated muscle cultures and probed the blots with
specific antibodies to PKC isoforms. Before treatment with insulin, no
PKC isoform could be detected in IR immunoprecipitates. By 1 min
following insulin stimulation, PKC
was found to coprecipitate with
IR; the amount of IR-PKC
complex decreased by 10 min, and by 30 min
PKC
could no longer be detected in association with IR (Fig. 1A
). This rapid induction of
insulin-induced IR-PKC
coprecipitation was essentially parallel to
insulin-induced PKC
tyrosine phosphorylation and activation (6).
Moreover, neither PKC
nor PKCßII, which were activated and
tyrosine phosphorylated by insulin, nor PKC
, which was not activated
or tyrosine phosphorylated by insulin, was found to coprecipitate with
IR. Similar results were obtained in studies in which PKCs ßII,
,
and
were immunoprecipitated and the blots were probed with anti-IR
antibody (not shown). Thus, insulin induced IR to selectively associate
with PKC
.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 1. Insulin Induces Selective Association of PKC
with IR
A, Western blot of insulin-induced coprecipitation of IR with PKC .
Studies were performed on 6-day-old cultured myotubes, which were
transferred to serum-free, low glucose minimal essential medium
(EM), 24 h before experiments were conducted. Protein extracts
from untreated cultures (C), or insulin-stimulated cultures treated for
different time periods (1, 10, or 30 min) were immunoprecipitated with
specific anti-IR antibodies. Immunoprecipitates were run on SDS-PAGE,
transferred to filters and immunoblotted with specific anti-PKC ,
anti-PKC , or anti-PKC antibodies. Coprecipitation of PKC with
IR was induced within 1 min following insulin stimulation, while PKC
and PKC did not coprecipitate with IR. The data presented are
representative of four separate experiments. B. Western blot of
tyrosine phosphorylation of IR induced by IGF-I or insulin (IN).
Studies were done on cells as described in panel A. Protein extracts
from untreated cultures (C), or cultures stimulated with IGF-I for 5 or
15 min, or with insulin for 5 min, were immunoprecipitated with
specific anti-IR antibodies. Immunoprecipitates were run on SDS-PAGE,
transferred to filters, and immunoblotted with specific
antiphosphotyrosine (p-ty) or anti-IR antibodies. The data presented
are representative of three separate experiments. C, Western blot of
insulin-induced coprecipitation of IR with PKC . Protein extracts
from untreated cells (C) or cells stimulated with insulin or IGF-I for
different time periods (1, 10, or 30 min) were immunoprecipitated with
specific anti-IR antibodies. Immunoprecipitates were run on SDS-PAGE,
transferred to filters, and immunoblotted with specific anti-PKC ,
anti-IR, or anti-IGFR antibodies. Insulin but not IGF-I induced
coprecipitation of IR with PKC . D, Western blot of IGF-I-induced
coprecipitation of IGFR with PKC . Protein extracts from untreated
cells (C) or cells stimulated with insulin or IGF-I for different time
periods (1, 10, or 30 min) were immunoprecipitated with specific
anti-IR antibodies. Immunoprecipitates were run on SDS-PAGE,
transferred to filters, and immunoblotted with specific anti- PKC ,
anti-IR, or anti-IGFR antibodies. IGF-I but not insulin induced
coprecipitation of IGFR with PKC by 10 min after stimulation.
|
|
To further demonstrate the specificity of insulin induction of
IR-PKC
association, we treated cells with insulin-like growth factor
I (IGF-I), which also activates IR (18, 19, 20, 21). Figure 1B
shows that IGF-I
induces tyrosine phosphorylation of IR in this preparation of cultured
myotubes. In spite of this stimulation of IR, IGF-I did not induce
PKC
to associate with IR (Fig. 1C
). Figure 1D
shows that IGF-I
induced coimmunoprecipitation of PKC
with IGFR within 10 min.
Insulin also induced tyrosine phosphorylation of IGFR but did not
induce association between IGFR and PKC
. Finally, insulin did not
induce PKC
to associate with other receptor kinases such as
epidermal growth factor receptor (EGFR) or nerve growth factor receptor
(NGFR) (data not shown).
Insulin Induces PKC Activity in IR Immunoprecipitates
We next examined whether the PKC
that is physically associated
with IR after insulin stimulation is indeed active. This can be
determined by measuring PKC activity in IR immunoprecipitates from
nonstimulated and insulin-stimulated cells. Accordingly, we
immunoprecipitated IR from untreated and insulin-treated cultures and
performed a PKC activity assay on those immunoprecipitates. Figure 2
shows that after 1 min of insulin
treatment, IR immunoprecipitates displayed a striking level of PKC
activity that was sustained for at least 5 min. In contrast, IR
immunoprecipitates from untreated cultures had no detectable PKC
activity. To determine that the increased PKC activity of
insulin-stimulated IR was exclusively associated with PKC
induced by
insulin stimulation, we immunoprecipitated different PKC isoforms from
nonstimulated and insulin-stimulated cell lysates and then
immunoprecipitated IR from the remaining
supernatant. We subjected these IR immunoprecipitates to PKC activity
assay. The results of these experiments are also shown in Fig. 2
.
Removal of PKC
resulted in a loss of PKC activity in IR
immunoprecipitates. In contrast, removal of PKC
(or of PKC
or of
PKCßIInot shown) did not reduce PKC activity of the IR
immunoprecipitates. This further confirms that the PKC activity
detected in IR immunoprecipitates was specifically due to PKC
.
Overexpressed W.T.PKC
Associates with IR
The insulin-induced association of PKC
and IR raises the
possibility that PKC
may be involved in some aspects of the IR
signaling cascade. To examine this possibility, we have used
recombinant adenovirus constructs of PKC
to overexpress either
W.T.PKC
or a kinase-inactive PKC
(D.N.PKC
) in mature myotubes,
and then to investigate the effects of PKC
overexpression and
blockade on the initial steps in the insulin-induced signaling cascade.
Cells infected with W.T.PKC
or with D.N.PKC
expressed higher
protein levels of these isoforms, and overexpressed W.T.PKC was found
in an activated state (6). We initially examined the effects of
overexpression of W.T.PKC
on insulin-induced association of PKC
and IR. Interestingly, overexpression of W.T.PKC
resulted in an
association of PKC
and IR without addition of insulin (Fig. 3
). Treatment of the cultures with
insulin could not further increase this association. Indeed, insulin
stimulation appeared to decrease IR-PKC
association. In contrast,
the inactive mutant form of PKC
did not associate with IR. Moreover,
overexpression of D.N.PKC
prevented insulin-induced association of
IR with the native PKC
. We have confirmed our earlier report (6)
that expression of D.N.PKC
prevents insulin-induced activation of
PKC
. These findings thus suggest that activation of PKC
is
necessary and sufficient for the interaction between the IR and
PKC
.
PKC
Regulates Serine Phosphorylation of IR
The findings so far demonstrate not only that PKC
is induced by
insulin to associate with IR but also that the association between the
two proteins may regulate the state of IR phosphorylation itself. The
regulation of IR tyrosine phosphorylation and activation is also
reported to be associated with changes in phosphorylation on serine
residues of the IR (see Ref. 22). Indeed, serine phosphorylation of IR
is considered one of the possible mechanisms for regulation of IR
function (9). In view of our results, one possible mechanism for
regulation of IR activation and distribution associated with serine
phosphorylation of IR could be via activation of PKC
. Therefore, we
investigated effects of overexpression of W.T.PKC
and D.N.PKC
on
serine phosphorylation of IR and effects of insulin stimulation
thereon. Figure 4
shows serine
phosphorylation of IR in cultured myotubes under different conditions.
In control myotubes, IR displayed a low level of serine
phosphorylation, and on stimulation by insulin, serine phosphorylation
began to increase by 5 min and reached maximum by 10 min, similar to
findings reported for other cell types (10, 23). In cells
overexpressing W.T.PKC
, serine phosphorylation was strikingly high
under basal conditions (without insulin stimulation) and was decreased
by insulin stimulation (Fig. 4
, lower panel). In contrast,
in cells overexpressing D.N.PKC
, the level of serine phosphorylation
was lower than that in control, and insulin-induced serine
phosphorylation was completely abrogated. These findings suggest that
PKC
may be involved in insulin-induced serine phosphorylation of
IR.
PKC
Alters Insulin-Induced Changes in IR Distribution
Serine phosphorylation of IR is associated with changes in
IR receptor distribution between the plasma and internal membrane
components. To determine whether PKC
may also play a role in IR
distribution, we followed the expression of IR in different cellular
compartments after insulin treatment of control skeletal myotubes. This
was done indirectly by probing plasma and internal membrane fractions
with anti-IR antibody at different times after insulin stimulation.
Before insulin stimulation, IR was detected almost exclusively in the
plasma membrane, where it remained for at least 5 min after addition of
insulin (Fig. 5A
). By some 10 min after
stimulation, IR had largely left the plasma membrane and could be
detected in increased amounts in the internal membrane fraction.
Interestingly, in myotubes overexpressing W.T.PKC
(Fig. 5B
),
only a small amount of IR was detected in the plasma membrane before
insulin stimulation; most of the IR was found in the internal membrane
fractions. Thus, the activated overexpressed PKC
, which associates
with IR, appeared to transfer IR out of the plasma membrane. Moreover,
insulin had no effect on amount of IR in the plasma membrane and
appeared to decrease the amount of IR in the internal membrane
fraction. In contrast, in cells overexpressing D.N.PKC
, IR was
enriched in the plasma membrane before addition of insulin and remained
in the plasma membrane for at least 30 min after insulin stimulation.
At the same time, under all experimental conditions, there was no
significant change in total IR expression as measured by Western blot
analysis of total cell lysates. In other words, W.T.PKC
appeared to
induce redistribution of IR, and D.N.PKC
prevented insulin-induced
changes in IR distribution between plasma and internal membrane
fractions. This indicates that insulin-activated PKC
is required for
insulin-induced changes in IR distribution.
We also examined effects of short-term blockade of PKC
on
insulin-induced IR distribution with the use of Rottlerin, which in low
concentrations is a selective inhibitor of PKC
. We have shown that
this substance at a concentration of 5 µM inhibits
insulin-induced activation of PKC
but not that of PKCßII or
(6). In the current study, cells were pretreated with 5
µM Rottlerin for 10 min before addition of insulin.
Effects of selective inhibition of PKC
by Rottlerin were similar to
those of overexpression of dominant negative PKC
(Fig. 5C
). In other
words, in Rottlerin-treated cells, IR did not move to the intracellular
compartment after insulin stimulation. Instead, IR remained in the
plasma membrane for at least 30 min after addition of insulin.
PKC
Regulates the Tyrosine Phosphorylation State of IR
Activation of IR is characterized by tyrosine autophosphorylation.
As we have shown that PKC
is involved in serine phosphorylation and
distribution of IR, we considered it important to examine effects of
PKC
overexpression and blockade on insulin-induced tyrosine
phosphorylation of IR. To accomplish this, we analyzed insulin-induced
changes in tyrosine phosphorylation of IR in plasma membrane and
internal membrane fractions prepared from noninfected cells and cells
overexpressing either W.T. or D.N.PKC
. The time course of
insulin-induced tyrosine phosphorylation of IR in these fractions from
noninfected cells is shown in the upper blot in Fig. 6A
. In confirmation of our earlier
findings, tyrosine phosphorylation of IR in control myotubes reached a
maximum 1 min after addition of insulin and returned to near basal
levels by 10 min (6). In cells overexpressing W.T.PKC
(Fig. 6B
), the
basal level of tyrosine phosphorylation of IR in the plasma membrane
was unchanged from control, uninfected cells. However, tyrosine
phosphorylated IR was now detected in immunoprecipitates from the
internal membrane fraction, even in the absence of insulin stimulation.
Insulin had no additional effect on the level of phosphorylation. In
myotubes expressing the D.N.PKC
mutant (Fig. 6B
), basal tyrosine
phosphorylation of IR in the plasma membrane fraction was higher than
that in control, noninfected cells. On stimulation by insulin, IR
tyrosine phosphorylation in D.N.PKC
-expressing cells was higher than
that in control uninfected cells treated with insulin. In addition, the
high level of tyrosine phosphorylation was sustained for at least 30
min. Moreover, no tyrosine-phosphorylated IR could be detected in IR
immunoprecipitates from internal membrane fractions of D.N.PKC
expressing cells with or without insulin stimulation.
 |
DISCUSSION
|
---|
In this report we have shown for the first time that insulin
induces an immediate physical linkage between IR and PKC
, and that
this occurs with the same time course as tyrosine phosphorylation of
both IR and PKC
. In contrast, PKC isoforms ßII and
, while also
activated and phosphorylated on tyrosine by insulin, were not induced
to coprecipitate with IR. We have already shown that tyrosine
phosphorylation and translocation of PKCs ßII and
occur via a
wortmannin-sensitive pathway, probably involving PI3 kinase, while
PKC
activation and tyrosine phosphorylation were independent of PI3
kinase. Our findings thus indicate that the change in activation state
of PKC
may be accomplished by direct action of IR tyrosine kinase or
another, as yet unidentified, tyrosine kinase upstream in the IR
signaling pathway to phosphorylate this isoform on tyrosine residues.
Our results on intact muscle cells in primary culture are entirely
consistent with the in vitro studies of Li et al.
(12), who reported that coincubation of IR with PKC
resulted in
phosphorylation on tyrosine of this PKC isoform associated with an
increase in PKC activity. It is important to emphasize, however, that,
in contrast to the results of Li et al. (12), our findings
were obtained on PKC
and IR proteins resident in intact skeletal
muscle cells in primary culture. This indicates for the first time that
the phenomenon may be an important step in vivo in the
insulin-signaling pathway. Numerous other studies on various cell types
have shown that PKC
is phosphorylated on tyrosine sites in response
to activators and growth factors, and that this is sometimes associated
with an increase in the serine-threonine kinase activity of PKC
(12, 24, 25, 26).
Insulin-induced coimmunoprecipitation of PKC and IR was also specific
for IR. In other words, insulin did not cause association between
PKC
and other receptor tyrosine kinases such as IGF receptor (IGFR),
EGFR, and NGFR. Although IGF-I did induce PKC
to associate with
IGFR, this effect occurred only after 10 min. This is in contrast to
the rapid induction of IR-PKC
association in response to
insulin.
We also showed that the IR-PKC
complex contained considerable PKC
activity, and that this was due specifically to PKC
. Thus, removal
of PKC
by immunoprecipitation of this protein with specific
anti-PKC
antibodies abrogated the insulin-induced PKC activity of
immunoprecipitated IR, whereas immunoprecipitation of other PKC
isoforms did not. Results similar to these have not heretofore been
reported and indicate that tyrosine phosphorylation of PKC
may occur
as a result of the direct and immediate interaction of PKC
with
IR.
This is the first demonstration of insulin or other growth
factor-induced coprecipitation of PKC
with IR involving resident
proteins in intact cells. A recent study by Formisano et al.
(17) demonstrated that in NIH-3T3 cells expressing human IR
(3T3hIR), insulin could induce coprecipitation of
IR with PKCs
,
, and
. There are certain other important
differences between the studies of Formisano et al. and
those reported here. First, the insulin-induced coprecipitation in our
studies was specific for PKC
; neither PKC
nor PKC
was induced
by insulin to coprecipitate with IR. This specificity for
insulin-induced coprecipitation of PKC
and IR was also observed in
skeletal muscle cultures in which PKCs
and
were transiently
overexpressed. Second, insulin-induced association of PKC
with IR in
skeletal muscle occurred within 1 min, whereas that in
3T3hIR cells was observed after 30 min. There was
no indication if cells were examined earlier than 30 min after insulin
stimulation. Nonetheless, the more rapid time course of insulin effects
on natively expressed PKC
and IR may be a reflection of
physiological as opposed to in vitro conditions.
We reported previously that insulin-induced activation of PKC
occurs via a pathway independent and possibly upstream of PI3 kinase
(7). In this study, we found a strong relation among the time courses
of insulin-induced tyrosine phosphorylation of PKC
, PKC
activation, and the time course of PKC
-IR coprecipitation. Indeed,
insulin-induced tyrosine phosphorylation of PKC
and IR-PKC
coprecipitation paralleled the time course of insulin-induced
autophosphorylation of IR as well. This indicates that IR tyrosine
kinase may be responsible for tyrosine phosphorylation of PKC
, as
shown by Li et al. (12) in vitro. This is
supported by the additional finding that the PKC
in these
coprecipitates was found to be activated by insulin stimulation. The
physical association between IR and PKC
may indicate that IR is an
endogenous substrate for PKC
in skeletal muscle. As pointed out by
Gschwendt (27), tyrosine phosphorylation of PKC
, possibly by IR
tyrosine kinase, is important for the determination of PKC
substrate
specificity. Thus, after the insulin-induced association of IR with
PKC
and its tyrosine phosphorylation, PKC
proceeds to serine
phosphorylate the IR. We are unable, however, to rule out the
possibility that either or both tyrosine phosphorylation and activation
of PKC
may occur via some other tyrosine kinase upstream in the IR
signaling cascade.
Also of interest are the results implicating PKC
in serine
phosphorylation of IR. Serine phosphorylation has been shown to be an
important initial step in IR routing (28, 29, 30, 31, 32, 33, 34, 35, 36, 37). We found that IR was
phosphorylated on serine residues in cells overexpressing W.T.PKC
without insulin stimulation, and that IR in these cells was localized
primarily in the internal membrane fractions. In addition, when
D.N.PKC
was expressed in the muscle cells, insulin-induced serine
phosphorylation was blocked and IR remained in the plasma membrane for
at least 30 min after insulin stimulation. These findings, in agreement
with earlier in vitro studies on several cell types
transfected with IR and different PKC isoforms (17, 29, 38, 39),
suggest that PKC
induces serine phosphorylation of IR after insulin
stimulation and that this PKC isoform plays a major role in IR
routing.
Our results regarding effects of overexpression of W.T. and D.N. PKC
on tyrosine phosphorylation of IR in plasma and internal membrane
fractions indicate that phosphorylation state and routing are very
closely coupled. Moreover, our findings support the concept that IR
remains tyrosine phosphorylated until after it is routed to internal
membranes, as suggested by Di Guglielmo et al.(22). We found
that PKC
appeared to act in a manner similar to insulin. Thus,
overexpression of PKC
resulted in tyrosine phosphorylation of IR and
routing of phosphorylated IR to the internal membrane fraction, whereas
overexpression of inactive PKC
prevented the internalization of IR
where it remained tyrosine phosphorylated.
In conclusion, we propose that PKC
is essential for IR
internalization. We further suggest that serine phosphorylation occurs
via PKC
activity on IR in the plasma membrane, and that serine
phosphorylation per se does not prevent tyrosine
phosphorylation of IR. Rather, phosphorylation of IR on serine residues
plays a role in the internalization of tyrosine-phosphor-ylated IR
to internal membranes, where IR is dephosphorylated. The results
presented in this report, together with our previous study implicating
PKC
in insulin-induced glucose transport (6), demonstrate a cardinal
role of this PKC isoform in the mediation of a multitude of
insulin-induced effects in skeletal muscle.
 |
MATERIALS AND METHODS
|
---|
Materials
Tissue culture media and serum were purchased from Biological
Industries (Beit HaEmek, Israel). Enhanced chemical luminescence (ECL)
kit was purchased from Bio-Rad Laboratories, Inc. (Rishon
le Zion, Israel). Antibodies for various proteins were obtained
from the following sources: monoclonal antibodies to IRß were
purchased from Transduction Laboratories, Inc. (Lexington,
KY). Polyclonal antiphosphoserine was obtained from Zymed Laboratories, Inc. (South San Francisco, CA), and monoclonal
antiphosphotyrosine was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-PKC antibodies were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA;
polyclonal) and from Transduction Laboratories, Inc.
(monoclonal). Horseradish peroxidase, and antirabbit and antimouse IgG
were obtained from Bio-Rad Laboratories, Inc. Leupeptin,
aprotinin, phenylmethylsulfonylfluoride (PMSF), orthovanadate, and
pepstatin were purchased from Sigma (St. Louis, MO).
Preparation of Rat Muscle Cell Cultures
Skeletal muscle cultures were prepared from thigh muscles
obtained from 1- to 2-day-old neonatal rats as described previously
(7). On day 5 in culture, myotubes were transferred to low-glucose (4.5
mM), serum-free DMEM containing 1% BSA for 24 h
before study. On the day of study, cells were transferred to PBS (pH
7.36) containing 2 mM glucose for 10 min before addition of
insulin (70100 nM).
Immunoprecipitation
Culture dishes (90 mm; Nunc, Rothskilde, Denmark) containing the
muscle cells were washed with
Ca2+/Mg2+- free PBS and
then mechanically detached in radioimmunoprecipitation assay
buffer containing a cocktail of protease inhibitors. After scraping,
the preparation was centrifuged at 20,000 x g for 20
min at 4 C. The supernatant was used for immunoprecipitation as
described previously (6, 7).
Cell Fractionation
Crude membrane preparations were isolated from muscle cell
cultures as described (6, 7). Culture dishes (90 mm; Nunc) containing
the muscle cells were washed with
Ca2+/Mg2+-free PBS and then
mechanically detached in
Ca2+/Mg2+- free PBS
containing 2 mM EDTA with a rubber policeman. The cells
were pelleted by centrifugation at 500 x g for 10 min
at 4 C. The cells were resuspended in sonication buffer (50
mM Tris HCl, pH 7.4; 150 mM
NaCl; 2 mM EDTA; 1 mM EGTA;
sucrose, 25 mM sucrose) containing leupeptin, 20
µg/ml; aprotinin, 10 µ g/ml; 0.1 mM PMSF; 1
mM dithiothreitol (DTT);
200 µM orthovanadate; and pepstatin, 2 µg/ml.
The suspension was homogenized in a Dounce glass homogenizer (30
strokes) and centrifuged at 1,100 x g for 5 min. The
supernatant was centrifuged at 31,000 x g for 60 min.
The supernatant from this centrifugation was centrifuged at
190,000 x g for 60 min to collect the light microsome
fraction. The 31,000 x g pellet was resuspended in
homogenization buffer to a final volume of 500 µl and placed on a
discontinuous sucrose gradient of 500 µl each of 32% (wt/wt), 40%
(wt/wt), and 50% (wt/wt) sucrose solution in 5
mM Tris, pH 7.5. This gradient was centrifuged at
210,000 x g for 50 min. The plasma membranes banded
above the 32% layer, and the 32/40% and 40/50% interfaces were
collected by puncture with a syringe. These fractions were diluted in
homogenization buffer containing 1% Triton X100, freeze-thawed four
times, centrifuged at 30,000 x g for 30 min, and the
supernatant was designated as the membrane protein. All membrane
fractions were stored at -70 C until use.
Western Blot Analysis
Crude and fractionated lysates of control and insulin-stimulated
cultures were subjected to SDS-PAGE and electrophoretic transfer to
Immobilon-P (Millipore Corp., Bedford, MA) membranes. The
membranes were subjected to standard blocking procedures and were
incubated with monoclonal antibodies against specific PKC isoforms or
phosphotyrosine, and with polyclonal antibodies against glucose
transporters as described previously (6, 7).
PKC Recombinant Adenoviruses and Viral Infection of
Cultures
The recombinant adenoviruses were constructed as described (40).
The dominant negative mutant of mouse PKC
was generated by
substitution of the lysine residue at the ATP binding site with alanine
(41). The mutant
cDNA was cut from SRD expression vector with
EcoRI and ligated into the pAxCA1w cosmid cassette to
construct Ax vector. Its kinase-negative nature was demonstrated by
abrogation of autophosphorylation activity (41).
After differentiation of cultured rat myoblasts into myotubes, the
culture medium was aspirated and cultures were infected with medium
containing PKC
or PKC
recombinant adenoviruses as recently
described (6).
PKC Activity
Specific PKC activity was determined in freshly prepared
immunoprecipitates from mature muscle cultures after appropriate
treatments as described (6, 7). These lysates were prepared in RIPA
buffer without NaF. Activity was measured using the SignaTECT Protein
Kinase C Assay System (Promega Corp., Madison, WI).
 |
FOOTNOTES
|
---|
Address requests for reprints to: S. R. Sampson, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail:
sampsos{at}mail.biu.ac.il
Supported in part by the Sorrell Foundation, the Ben and Effie Raber
Research Fund, The Harvett-Aviv Neuroscience Research Fund, and grants
from The Israel Science Foundation founded by the Israel Academy of
Sciences and Humanities, and from the Chief Scientists Office of the
Israel Ministry of Health. S.R.S. is the incumbent of the Louis Fisher
Chair in Cellular Pathology.
Received for publication August 3, 2000.
Revision received November 22, 2000.
Accepted for publication December 13, 2000.
 |
REFERENCES
|
---|
-
White MF, Kahn CR 1994 The insulin signaling system.
J Biol Chem 269:14[Free Full Text]
-
Acevedo-Duncan M, Cooper DR, Standaert ML, Farese RV 1989 Immunological evidence that insulin activates protein kinase C in
BC3H-1 myocytes. FEBS Lett 244:174176[CrossRef][Medline]
-
Bandyopadhyay G, Standaert ML, Galloway L, Moscat J, Farese
RV 1997 Evidence for involvement of protein kinase C (PKC)-
and
noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated
glucose transport in L6 myotubes. Endocrinology 138:47214731[Abstract/Free Full Text]
-
Standaert ML, Galloway L, Karnam P, Bandyopadhyay G, Moscat
J, Farese RV 1997 Protein kinase C-
as a downstream effector of
phosphatidylinositol 3-kinase during insulin stimulation in rat
adipocytes. Potential role in glucose transport. J Biol Chem 272:3007530082[Abstract/Free Full Text]
-
Bandyopadhyay G, Standaert ML, Zhao LYB, Yu B, Avignon A,
Galloway L, Karnam P, Moscat J, Farese RV 1997 Activation of protein
kinase C (
, ß, and
) by insulin in 3T3/L1 cells. Transfection
studies suggest a role for PKC-
in glucose transport. J Biol
Chem 272:25512558[Abstract/Free Full Text]
-
Braiman L, Alt A, Kuroki T, Ohba M, Bak A, Tennenbaum T,
Sampson SR 1999 Protein kinase C
mediates insulin-induced glucose
transport in primary cultures of rat skeletal muscle. Mol Endocrinol 13:20022012[Abstract/Free Full Text]
-
Braiman L, Sheffi-Friedman L, Bak A, Tennenbaum T, Sampson SR 1999 Tyrosine phosphorylation of specific protein kinase C isoenzymes
participates in insulin stimulation of glucose transport in primary
cultures of rat skeletal muscle. Diabetes 48:19221929[Abstract]
-
Chalfant CEKY, Ohno S, Konno Y, Fisher AA, Bisnauth LD,
Watson JE, Cooper DR 1996 A carboxy-terminal deletion mutant of protein
kinase C ß II inhibits insulin-stimulated 2-deoxyglucose uptake in L6
rat skeletal muscle cells. Mol Endocrinol 10:12731281[Abstract]
-
Kellerer M, Mushack J, Seffer E, Mischak H, Ullrich A, Haring
HU, 1998 Protein kinase C isoforms
,
and
require insulin
receptor substrate-1 to inhibit the tyrosine kinase activity of the
insulin receptor in human kidney embryonic cells (HEK 293 cells).
Diabetologia 41:833838[CrossRef][Medline]
-
Strack V, Stoyanov B, Bossenmaier B, Mosthaf L, Kellerer M,
Haring HU 1997 Impact of mutations at different serine residues on the
tyrosine kinase activity of the insulin receptor. Biochem Biophys Res
Commun 239:235239[CrossRef][Medline]
-
Gschwendt M 1999 Protein kinase C
. Eur J Biochem 259:555564[Abstract/Free Full Text]
-
Li W, Mischak H, Yu JC, Wang LM, Mushinski JF, Heidaran MA,
Pierce JH 1994 Tyrosine phosphorylation of protein kinase C-
in
response to its activation. J Biol Chem 269:23492352[Abstract/Free Full Text]
-
Brodie C 1990 Regulation by thyroid hormones of glucose
transport in cultured rat myotubes. J Neurochem 55:186191[Medline]
-
Brodie C, Bak A, Sampson SR 1985 Dependence of
Na+ K+ ATPase and
electrogenic component of Em in cultured myotubes
on cell fusion. Brain Res 336:384386[CrossRef][Medline]
-
Brodie C, Bak A, Shainberg A, Sampson SR 1987 Role of Na-K
ATPase in regulation of resting membrane potential of cultured rat
skeletal myotubes. J Cell Physiol 130:191198[Medline]
-
Brodie C, Brody M, Sampson SR 1989 Characterization of the
relation between sodium channels and electrical activity in cultured
rat skeletal myotubes: regulatory aspects. Brain Res 488:186194[CrossRef][Medline]
-
Formisano P, Oriente F, Miele C, Caruso M, Auricchio R,
Vigliotta G, Condorelli G, Beguinot F 1998 In NIH-3T3 fibroblasts,
insulin receptor interaction with specific protein kinase C isoforms
controls receptor intracellular routing. J Biol Chem 273:1319713202[Abstract/Free Full Text]
-
De Meyts P, Wallach B, Christoffersen CT, Urso B, Gronskov K,
Latus LJ, Yakushiji F, Ilondo MM, Shymko RM 1994 The insulin-like
growth factor-I receptor. Structure, ligand-binding mechanism and
signal transduction. Horm Res 42:152169[Medline]
-
Dealy CN, Kosher RA 1995 Studies on insulin-like growth
factor-I and insulin in chick limb morphogenesis. Dev Dyn 202:6779[Medline]
-
Bailyes EM, Nave BT, Soos MA, Orr SR, Hayward AC, Siddle K 1997 Insulin receptor/IGF-I receptor hybrids are widely distributed in
mammalian tissues: quantification of individual receptor species by
selective immunoprecipitation and immunoblotting. Biochem J 327:209215[Medline]
-
Wertheimer E, Trebicz M, Eldar T, Gartsbein M, Nofeh-Mozes S,
Tennenbaum T 2000 Differential roles of insulin receptor and
insulin-like growth factor-1 receptor in Differentiation of murine skin
keratinocytes. J Invest Dermatol 115:2429[Abstract/Free Full Text]
-
Di Guglielmo GM, Drake PG, Baass PC, Authier F, Posner BI,
Bergeron JJ 1998 Insulin receptor internalization and signalling. Mol
Cell Biochem 182:5963[CrossRef][Medline]
-
De Fea K, Roth RA 1997 Protein kinase C modulation of insulin
receptor substrate-1 tyrosine phosphorylation requires serine 612.
Biochemistry 36:1293912947[CrossRef][Medline]
-
Stempka L, Girod A, Muller HJ, Rincke G, Marks F, Gschwendt M,
Bossemeyer D 1997 Phosphorylation of protein kinase C
(PKC
) at
threonine 505 is not a prerequisite for enzymatic activity. Expression
of rat PKC
and an alanine 505 mutant in bacteria in a functional
form. J Biol Chem 272:68056811[Abstract/Free Full Text]
-
Soltoff SP, Toker A 1995 Carbachol, substance P, and phorbol
ester promote the tyrosine phosphorylation of protein kinase C d in
salivary gland epithelial cells. J Biol Chem 270:1349013495[Abstract/Free Full Text]
-
Smith H, Chang EY, Szallasi Z, Blumberg PM, Rivera J 1995 Tyrosine phosphorylation of protein kinase C-d in response to the
activation of the high affinity receptor for immunoglobulin E modifies
its substrate recognition. Proc Natl Acad Sci USA 92:91129116[Abstract]
-
Gschwendt M 1999 Protein kinase C
. Eur J Biochem 259:555564[Abstract/Free Full Text]
-
Liu F, Roth RA 1994 Identification of serines-1035/1037 in the
kinase domain of the insulin receptor as protein kinase C
mediated
phosphorylation sites. FEBS Lett 352:389392[CrossRef][Medline]
-
Kellerer M, Haring HU 1995 Pathogenesis of insulin resistance:
modulation of the insulin signal at receptor level. Diabetes Res Clin
Pract 28[Suppl]:S173S177
-
Nakashima S, Morinaka K, Koyama S, Ikeda M, Kishida M, Okawa
K, Iwamatsu A, Kishida S, Kikuchi A 1999 Small G protein Ral and its
downstream molecules regulate endocytosis of EGF and insulin receptors.
EMBO J 18:36293642[Abstract/Free Full Text]
-
Smith RM, Harada S, Smith JA, Zhang S, Jarett L 1998 Insulin-induced protein tyrosine phosphorylation cascade and signalling
molecules are localized in a caveolin-enriched cell membrane domain.
Cell Signal 10:355362[CrossRef][Medline]
-
Ceresa BP, Kao AW, Santeler SR, Pessin JE 1998 Inhibition of
clathrin-mediated endocytosis selectively attenuates specific insulin
receptor signal transduction pathways. Mol Cell Biol 18:38623870[Abstract/Free Full Text]
-
Chow JC, Condorelli G, Smith RJ 1998 Insulin-like growth
factor-I receptor internalization regulates signaling via the
Shc/mitogen-activated protein kinase pathway, but not the insulin
receptor substrate-1 pathway. J Biol Chem 273:46724680[Abstract/Free Full Text]
-
Giorgetti-Peraldi S, Ottinger E, Wolf G, Ye B, Burke TRJ,
Shoelson SE 1997 Cellular effects of phosphotyrosine-binding domain
inhibitors on insulin receptor signaling and trafficking. Mol Cell Biol 17:11801188[Abstract]
-
Eriksson JW, Lonnroth P, Wesslau C, Smith U 1997 Insulin
promotes and cyclic adenosine 3',5'-monophosphate impairs functional
insertion of insulin receptors in the plasma membrane of rat
adipocytes: evidence for opposing effects of tyrosine and
serine/threonine phosphorylation. Endocrinology 138:607612[Abstract/Free Full Text]
-
Smith RM, Harada S, Jarett L 1997 Insulin internalization and
other signaling pathways in the pleiotropic effects of insulin. Int Rev
Cytol 173:243280[Medline]
-
Carpentier JL, Hamer I, Gilbert A, Paccaud JP 1996 Molecular
and cellular mechanisms governing the ligand-specific and non-specific
steps of insulin receptor internalization. Z Gastroenterol 34 [Suppl
3]:7375
-
Bollag GE, Roth RA, Beaudoin J, Mochly-Rosen D, Koshland
DEJ 1986 Protein kinase C directly phosphorylates the insulin receptor
in vitro and reduces its protein-tyrosine kinase activity.
Proc Natl Acad Sci USA 83:58225824[Abstract]
-
Bossenmaier B, Mosthaf L, Mischak H, Ullrich A, Haring HU 1997 Protein kinase C isoforms ß 1 and ß 2 inhibit the tyrosine kinase
activity of the insulin receptor. Diabetologia 40:863866[CrossRef][Medline]
-
Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K,
Tokuda C, Saito I 1996 Efficient generation of recombinant adenoviruses
using adenovirus DNA- terminal protein complex and a cosmid bearing
the full-length virus genome. Proc Natl Acad Sci USA 93:13201324[Abstract/Free Full Text]
-
Ohno S, Konno Y, Akita Y, Yano A, Suzuki K 1990 A point
mutation at the putative ATP-binding site of protein kinase C
abolishes the kinase activity and renders it
down-regulation-insensitive. A molecular link between
autophosphorylation and down-regulation. J Biol Chem 265:62966300[Abstract/Free Full Text]