From the Dipartimento di Biologia e Patologia Cellulare e
Molecolare "L. Califano" and Centro di Endocrinologia ed Oncolgia
Sperimentale del Consiglio Nazionale delle Ricerche (CNR), "Federico
II" University of Naples Medical School, Naples, Italy
Insulin increased protein kinase C (PKC) activity
by 2-fold in both membrane preparations and insulin receptor (IR)
antibody precipitates from NIH-3T3 cells expressing human IRs
(3T3hIR). PKC-
, -
, and -
were barely
detectable in IR antibody precipitates of unstimulated cells, while
increasing by 7-, 3.5-, and 3-fold, respectively, after insulin
addition. Preexposure of 3T3hIR cells to staurosporine
reduced insulin-induced receptor coprecipitation with PKC-
, -
,
and -
by 3-, 4-, and 10-fold, respectively, accompanied by a
1.5-fold decrease in insulin degradation and a similar increase in
insulin retroendocytosis. Selective depletion of cellular PKC-
and
-
, by 24 h of 12-O-tetradecanoylphorbol-13-acetate
(TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-
. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs
(3T3Mut), insulin-induced coprecipitation of PKC-
, -
,
and -
with the IR was reduced by 10-, 7-, and 3-fold, respectively.
Similar to the 3T3hIR cells chronically exposed to TPA,
untreated 3T3Mut featured a 3-fold decrease in insulin
degradation, with a 3-fold increase in intact insulin retroendocytosis.
Thus, in NIH-3T3 cells, insulin elicits receptor interaction with
multiple PKC isoforms. Interaction of PKC-
and/or -
with the IR
appears to control its intracellular routing.
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INTRODUCTION |
The turn on of the insulin signaling mechanism by the insulin
receptor (IR)1 involves a
complex network of protein-protein interactions (1). Insulin-bound
receptors phosphorylate a variety of docking proteins which include the
IRS and Shc systems. Once phosphorylated, the docking proteins recruit
and activate multiple insulin effectors (1). By employing the IRSs to
engage Src homology 2 domain proteins, the IR avoids the stoichiometric
constraints encountered by receptors which directly recruit these
signaling molecules to their autophosphorylation sites (1, 2). The IRSs
widen the connection and the tuning opportunities of the insulin
signaling (3). There is evidence, however, that certain insulin
bioeffects also follow the direct interaction of the IR with major
insulin effectors. These effectors include phosphatidylinositol
3-kinase (4, 5) and, possibly, protein kinase C (PKC) (6, 7).
PKCs represent a family of structurally and functionally related
serine/threonine kinases derived from multiple genes as well as from
alternative splicing of single mRNA transcripts (8, 9). The
individual isoforms differ in their regulatory domains and in their
dependence on Ca2+, as well as in their tissue distribution
and intracellular localization (10, 11). PKCs appear to play a dual
role in the insulin signaling network. First, PKCs control
insulin-dependent receptor kinase activation (12-15) and
may regulate IRS-1 signaling as well (12, 13). Second, at least in
certain cells and tissues, insulin activation of PKCs is required to
evoke insulin effects on glucose transport and its intracellular
metabolism (16, 17). Current evidence (6) indicates that chimeric
receptors consisting of the EGF receptor extracellular domain fused to
the cytoplasmic domain of the IR form stable complexes with PKC-
following EGF binding. It has also been reported that insulin increases
PKC activity in Tyr(P) Ab precipitates from KB cells (22). However, the
molecular mechanisms of PKC activation in response to insulin as well
as the role of each individual PKC isoform in insulin signal
transduction is still unsettled.
In previous reports (23) we demonstrated that Arg1152 and
Lys1153 in the regulatory domain of the IR kinase are
crucial for enabling IR phosphorylation by PKC. A peptide encoding the
receptor sequence surrounding these residues inhibited phosphorylation
of IR by PKC. In contrast, a mutant peptide in which the Arg and Lys
were substituted by neutral amino acids exhibited no inhibitory
effects, suggesting that IR phosphorylation by PKC follows direct
IR-PKC interaction (23). In the present work we have shown that insulin controls IR association with PKC-
, -
, and -
. In turn, in the NIH-3T3 cells, PKC-
and/or -
association with IR appears crucial for enabling proper intracellular sorting of the receptor to the insulin degradative route.
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EXPERIMENTAL PROCEDURES |
Materials--
Rabbit polyclonal antibodies toward specific PKC
isoforms were purchased from Life Technologies, Inc. Ab50 and B10
receptor antibodies were a generous gift from Drs. D. Accili and P. Gorden (National Institutes of Health, Bethesda, MD). Protein
electrophoresis reagents were from Bio-Rad. Western blotting and ECL
reagents were from Amersham Corp. Media and sera for cell culture were from Life Technologies Inc. All other reagents were from Sigma.
Mutant Cell Clones, Extract Preparations, and PKC
Assays--
The NIH-3T3 cell clones expressing the QA mutant insulin
receptors have been previously reported (23). For determination of PKC
activity, the cells were solubilized with 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton
X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin (extraction buffer)
and then clarified by centrifugation at 10,000 × g for
15 min at 4 °C. Upon protein quantitation, equal aliquots of the
extract were added to the lipid activators (10 mM phorbol
12-myristate 13-acetate, 0.28 mg/ml phosphatidyl serine, and 4 mg/ml
dioleine, final concentrations), and the 32P/substrate
solution (50 mM Ac-MBP(4-14), 20 mM ATP, 1 mM CaCl2, 20 mM MgCl2,
4 mM Tris, pH 7.5, and 10 mCi/ml (3,000 Ci/mM)
[
-32P]ATP), in the presence or the absence of 25 µM PKC pseudosubstrate inhibitor peptide PKC(19-36)
(24). The samples were incubated for 20 min at room temperature and
rapidly cooled on ice, and 20-µl aliquots were spotted onto
phosphocellulose disc papers (Life Technologies, Inc.). Discs were
washed twice with 1% H3PO4, followed by two
more washes with water, and disc-bound radioactivity was quantitated by
liquid scintillation counting.
Co-precipitation Studies--
Cell extracts were prepared as
described above. Samples were precipitated with protein
A-Sepharose-bound Ab50 or B10 insulin receptor antibodies, and the
immunocomplexes were resuspended either in extraction buffer, for
quantitation of PKC activity, or in Laemmli buffer (25), for
SDS-polyacrylamide gel electrophoresis protein separation. These
proteins were then blotted on nitrocellulose filters, probed with
isoform-specific PKC antibodies, and detected by the ECL method as
described by the manufacturer. Alternatively, the immunoprecipitations
were performed using isoform-specific PKC Abs, followed by
immunoblotting and detection with insulin receptor antibodies.
Quantitation of the autoradiographs was obtained by laser
densitometry.
Insulin Binding, Internalization, and Intracellular
Processing--
125I-Labeled insulin binding and
internalization were analyzed as described previously (26).
Internalization rates were calculated according to Lund et
al. (27). Degraded and intact 125I-insulin in the
incubation media or the cell lysates were determined by trichloroacetic
acid precipitation as described in Formisano et al.
(26).
 |
RESULTS |
PKC Co-precipitation with the Insulin Receptor--
NIH-3T3 cells
expressing human wild-type insulin receptors (3T3hIR cells)
were exposed for 30 min to 100 nM insulin or 1 µM TPA. This treatment increased the ability of plasma
membrane preparations from the cells to phosphorylate the Ac-MBP(4-14)
substrate for PKC by 2- and 10-fold, respectively (Fig.
1, top panel). Insulin effect
was accompanied by a similarly sized increase in phosphorylation of the
PKC substrate by specific IR Ab precipitates from these same cells
(Fig. 1, bottom panel). At variance, substrate
phosphorylation by the IR Ab precipitates from TPA-stimulated cells was
increased by only 35 ± 3% (value ± S.D.) above the basal
levels. Simultaneous preincubation of the cells with both insulin and
TPA did not significantly enhanced the effect of insulin alone.
Ac-MBP(4-14) phosphorylation by the immunoprecipitates from both basal
and insulin and/or TPA-stimulated cells were blocked by the specific
PKC inhibitory peptide PKC(19-36), indicating co-precipitation of PKC
with the insulin receptor.

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Fig. 1.
PKC activity in membrane preparations and IR
Ab precipitates from 3T3hIR cells. Top panel,
PKC activity was assayed by incubating the Ac-MBP(4-14) substrate with
membrane preparations from basal and insulin- or TPA-stimulated cells,
as described under "Experimental Procedures." Bottom
panel, cell lysates were immunoprecipitated with Sepharose-bound
IR Ab, and PKC activity was assayed in the absence or in the presence
of excess PKC(19-36) inhibitory peptide. Data represent the means ± S.D. of four triplicate experiments.
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To further investigate the PKC-IR co-precipitation, we immunoblotted
the IR Ab precipitates with isoform-specific PKC Abs. In precipitates
from insulin-unstimulated cells, PKC-
and -
were detectable at
low levels at the expected Mr 80,000 (Fig. 2). A faint band was also revealed by
PKC-
Abs, corresponding to the predicted Mr
65,000 (Fig. 2C). PKC-
was undetectable in these IR Ab
precipitates although clearly recognizable in total lysates of the
cells. In contrast PKC-
, -
1, and -
2
could not be demonstrated in either the cell lysates or the IR
precipitates (not shown). Upon stimulation of the cells with 100 nM insulin for 30 min, the levels of PKC-
, -
, and
-
detected on blots increased by 7-, 3.5-, and 3-fold, respectively,
while PKC-
remained undetectable. Almost identical results were
obtained by precipitating the cell lysates with isoform-specific PKC
Abs followed by blotting and detection with IR Abs (data not shown). As
shown in Fig. 3, the effect of insulin on
PKC-
, -
, and -
co-precipitation with the IR exhibited very
similar dose responses. Insulin effects were well detectable at 10
10 M, half-maximal at 2-5 × 10
9, and reached a plateau at 10
7
M. Differing from dose responses, time courses of the
insulin effect on IR-PKC co-precipitation were specific for each PKC
isoform. In the case of PKC-
, the insulin effect was maximal after
30 min of exposure, remaining unchanged for up to 2 h, and then
vanishing in 16 h (Fig. 4, top
panel), while, in the case of PKC-
, the effect reached a
maximum after 15 min and disappeared by 60 min (Fig. 4, middle
panel). For PKC-
, the insulin effect was diphasic, with an
early spike (maximum in 5 min), which returned to basal levels in 15 min and was followed by a more sustained increase lasting for up to
16 h (Fig. 4, bottom panel).

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Fig. 2.
Co-precipitation of IR with individual PKC
isoforms. 3T3hIR cells were exposed to 100 nM insulin for 30 min as indicated. Lysates from the cells
were then precipitated with Sepharose-bound IR Ab ( -IR
precip.) separated by SDS-polyacrylamide gel electrophoresis, and
immunoblotted with specific PKC- , - , - , and - Abs. Aliquots
of the cell lysates were directly immunoblotted with no previous
precipitation (total lysates). The experiment shown is
representative of four independent experiments.
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Fig. 3.
Insulin dose-response curve of IR-PKCs
co-precipitation. 3T3hIR cells were incubated with the
indicated concentrations of insulin. Cell extracts were then
precipitated with Sepharose-bound IR Ab and blotted with PKC-
(filled circles), PKC- (open triangles), and
PKC- Abs (open circles) as described under
"Experimental Procedures." Detection was achieved by ECL and
autoradiography, and the intensity of the bands was quantitated by
laser densitometry. Data represent the means of three independent
experiments with each individual PKC isoform.
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Fig. 4.
Time course of IR-PKCs co-precipitation.
3T3hIR cells were exposed to 100 nM insulin for
the indicated times. Cell extracts were then precipitated with
Sepharose-bound IR Ab and blotted with PKC- (top panel),
PKC- (middle panel), and PKC- Abs (bottom
panel). After ECL and autoradiography (see "Experimental
Procedures"), the intensity of the bands was quantitated by laser
densitometry. Each data point represents the mean ± S.D. of three
independent experiments.
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Effects of PKC Inhibition and Cellular Depletion on IR
Routing--
Evidence is available that PKC has an important role in
regulating the internalization and degradation of several tyrosine kinase receptors following binding with their specific ligand (6). We
therefore addressed the questions whether this is also the case for the
IR kinase, and whether specific PKC isoforms are involved in this
regulation. To this end, we analyzed insulin-induced IR internalization
and intracellular routing following either simultaneous inhibition of
PKC-
, -
, and -
activities with staurosporine or cell depletion
of TPA-sensitive PKCs (PKC-
and -
) by a 24-h preincubation with 1 µM TPA. Preincubation of the cells with 6 µM staurosporine before insulin stimulation inhibited PKC
activity in total cell lysates by almost 55%. Concomitantly, as
revealed by immunoblot studies, the levels of PKC-
, -
, and -
in IR Ab precipitates were reduced by 3-, 4-, and 10-fold, respectively (Fig. 5, top panel),
suggesting that PKC activation is necessary for its association with
the insulin receptor. Chronic treatment of the cells with TPA decreased
PKC activity by >80% and also reduced recovery of PKC-
and -
in
the IR Ab precipitates by 6- and 5-fold, respectively. At variance from
the staurosporine however, similar amounts of the PKC-
isoform were
evidenced in the immunoprecipitates from TPA-treated cells and in the
precipitates from untreated cells. Upon entering cells, normal
insulin-bound IRs return to the plasma membrane mainly through an
intracellular compartment where insulin is detached from the receptor
and degraded (26, 28). Small amounts of internalized receptors are also rapidly recycled through a distinct retroendocytotic mechanism (26,
29). Following TPA and staurosporine preincubation, however, the amount
of trichloroacetic acid-soluble (degraded) 125I-insulin
released by the cells decreased by 66 ± 7 and 32 ± 3%, respectively (Fig. 5, middle panel). These changes were
accompanied by 60 ± 5 and 22 ± 4% respective increases in
the amount of trichloroacetic acid-precipitable (intact)
125I-insulin released into the medium by TPA- and
staurosporine-treated cells, respectively (Fig. 5, bottom
panel). No significant change on insulin-induced IR
internalization by the cells occurred in either the TPA- or the
staurosporine-treated cells (data not shown). Thus, preserved
association of the IR with PKC-
and -
but not PKC-
correlated
with normal receptor intracellular sorting.

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Fig. 5.
Effect of PKC inhibition on IR-PKC
coprecipitation and insulin degradation and retroendocytosis. Top
panel, 3T3hIR cells were preincubated for either 30 min with 6 µM staurosporine or 24 h with 1 µM TPA and further stimulated with insulin, as indicated.
Cell extracts were then precipitated with IR Ab, blotted with PKC- ,
- or - Abs and revealed by ECL. A representative autoradiograph
is shown. Middle and bottom panels, cells treated
with either staurosporine for 30 min or TPA for 24 h were
incubated with 125I-insulin, acid-washed to remove
extracellular insulin, and warmed at 37 °C for 30 further min.
Aliquots of media were then precipitated with trichloroacetic acid.
trichloroacetic acid-soluble radioactivity represents insulin
degradation (middle panel), while trichloroacetic
acid-precipitable radioactivity represents the intact insulin
(bottom panel). Bars represent the means ± S.D. of four triplicate experiments.
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IRMut-PKC Interaction and Intracellular
Routing--
To further explore the potential for PKC-IR interaction
to regulate IR intracellular routing, we have studied the
internalization and intracellular sorting of the R1152Q/K1153A double
mutant insulin receptor (IRMut). In previous studies, we
have shown that this receptor is not phosphorylated by PKC, but
responds to insulin with auto- and substrate phosphorylation, and
transduction of metabolic and mitogenic responses (23). At variance
with the 3T3hIR cells, insulin exposure did not increase
PKC activity in plasma membranes from cells expressing the mutant
receptors (3T3Mut; Fig. 6,
top panel). Based on IR-PKC co-precipitation, the amounts of
PKC-
, -
, and -
that associated with the insulin-activated IRs
were, respectively, 10-, 7-, and 3-fold less in NIH-3T3 cells expressing IRMut than in the 3T3hIR cells (Fig.
6, bottom left). The levels of the each individual PKC
isoform in total cell lysates were identical in the two cell types,
however (Fig. 6, bottom right), indicating that
IRMut was unable to properly associate with and activate
PKC following insulin binding, despite normal levels of these kinases
in the cells. Based on the appearance of trypsin-resistant insulin
binding (intracellular receptors), the IRMut underwent
rapid and time-dependent internalization in response to
insulin, superimposable to that of the wild-type IR (data not shown).
Preincubation with 100 nM insulin for 0.5, 1, and 16 h
also reduced subsequent 125I-insulin binding by 20, 28, and
31% in the 3T3Mut cells and by 19, 25, and 28% in the
3T3hIR cells, indicating that the mutant receptor undergoes
normal insulin-dependent down-regulation as well (Fig.
7, top panel). However,
insulin degradation levels were reduced by 3-fold in the
3T3Mut cells as compared with 3T3hIR cells
(Fig. 7, middle panel). Conversely, intact
125I-insulin release into the medium was 3-fold greater in
the 3T3Mut than in the 3T3HIR cells (Fig. 7,
bottom panel). Thus, similar to wild-type IR in PKC-depleted
cells with IRMut, lack of interaction with PKCs is
accompanied by a preferential receptor convey to the cell surface
through the retroendocytotic rather than the insulin-degradative
route.

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Fig. 6.
Membrane PKC activity and insulin-induced
IR-PKC co-precipitation in 3T3Mut cells. Top,
PKC activity was assayed in membrane preparations from basal and
insulin-stimulated 3T3Mut cells as described in the legend
to Fig. 1. Bottom, 3T3hIR and 3T3Mut
cells were exposed to 100 nM insulin as described under
"Experimental Procedures." Cell extracts were either precipitated
with IR Ab and blotted with PKC- , - and - Abs ( -IR
precip.) or directly blotted with the specific PKC Abs
(total lysates) as described in Fig. 2. A representative
experiment is shown.
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Fig. 7.
Receptor down-regulation, insulin
degradation, and retroendocytosis in 3T3Mut cells.
Top panel, 3T3hIR and 3T3Mut cells were
preincubated with 100 nM insulin for the indicated times
and then thoroughly washed to remove all free and cell-surface bound
insulin. 125I-Insulin binding was subsequently determined
as described under "Experimental Procedures." Data represent the
means ± S.D. of at least three triplicate experiments. Insulin
degradation (middle panel) and retroendocytosis
(bottom panel) were measured in 3T3hIR and
3T3Mut cells, as described in Fig. 5. Bars
represent the means ± S.D. of four triplicate experiments.
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DISCUSSION |
PKC activation in response to insulin has been reported in several
tissues and cell types (18, 30). However, neither the molecular
mechanisms by which the activation occurs nor the functional role of
PKC in the insulin signaling system have been completely elucidated. In
the present work, we have shown that, in NIH-3T3 cells expressing human
IRs, insulin induces membrane recruitment of PKC-
, -
, and -
,
but not of the -
isoforms. PKC-
, -
, and -
recruitment in
response to insulin linearly correlated with their appearance in the IR
Ab precipitates. This observation is consistent with a previous report
by Liu and Roth (22) in which the recovery of an insulin-stimulated PKC
activity in phosphotyrosine Ab precipitates from insulin-stimulated
cells was described. While eliciting a 10-fold greater PKC
translocation to the plasma membrane, TPA was less effective than
insulin in promoting co-precipitation of PKC-
and -
with the IR
and was unable to promote that of PKC-
. Neither EGF or
platelet-derived growth factor elicited any significant IR-PKC
co-precipitation (not shown). It appears therefore that, in NIH-3T3
cells, a specific association of PKC-
, -
, and -
with IR occurs
in response to insulin.
We have further observed that, in NIH-3T3 cells, inhibition of PKC-
,
-
, and -
activity by staurosporine also inhibited insulin-induced
PKC-IR association, suggesting that the insulin-induced activation of
these PKC isoforms is necessary to allow their subsequent association
with the receptor. Staurosporine pretreatment of the cells also
increased the routing of the insulin-receptor complexes through the
retroendocytotic pathway, thus shifting the internalized receptors from
the degradative to the retroendocytotic compartment. This change in
receptor sorting was not accompanied by alterations in insulin-induced
receptor internalization or down-regulation, indicating that PKC-IR
association is crucial in specifically controlling the intracellular
sorting of the receptor following insulin-dependent
internalization. Consistent with these data and with the relevance of a
direct PKC-IR interaction for proper sorting of the receptor, the
IRQA mutant, which is unable to interact with PKC-
,
-
, and -
, but is normally responsive to insulin in term of auto-
and substrate phosphorylation, kinase activation, and signaling (23),
exhibited identical abnormalities in intracellular cell sorting as the
wild-type human IR in staurosporine-treated cells. It is possible that
the abnormal routing of IRQA is caused by decreased
phosphorylation by one or more PKC isoforms because the QA double
mutation impairs receptor phosphorylation by these kinases both
in vitro and in intact cells (23). The altered intracellular
routing of IRQA might also be directly caused by its
decreased ability to activate PKC. This is less likely, however, because TPA activation of PKC in IRQA-expressing cells does
not restore normal receptor routing (data not shown). A different mutant (IRQK) also exhibits similar abnormalities in the
intracellular sorting as the IRQA (26). Similar to
IRQA, IRQK is unable to interact with PKC-
,
-
, and -
upon insulin exposure (not shown). At variance with the
IRQA, however, IRQK does not respond to insulin in terms of autophosphorylation and kinase activation (31), indicating
that receptor phosphorylation and kinase activation are not sufficient
for enabling receptor-PKC association.
Pretreatment of NIH-3T3 cells with TPA before insulin stimulation
depleted the cells of PKC-
and -
, but had no effect on PKC-
levels. Accordingly, the levels of PKC-
co-precipitating with IRs
following insulin stimulation were identical in cells treated with TPA
or not, while the levels of PKC-
and -
were greatly reduced.
Nevertheless, in TPA-pretreated cells, we observed a shift of the
internalized insulin-IR complexes from the degradative toward the
retroendocytotic route that was almost 2-fold more pronounced than that
caused by staurosporine. We suggest, therefore, that PKC-
and/or
-
but not PKC-
are responsible for PKC control of IR routing in
these cells. Based on the findings reported in the present work,
PKC-
appears the most likely candidate for this regulatory role. In
fact, (i) the time course of insulin-induced IR engagement in the
degradative route better correlates with the time course of
insulin-dependent PKC-
-IR co-precipitation than with
that of PKC-
-IR co-precipitation, and (ii) chronic exposure of the
cells to TPA, to which the IR sorting mechanism is extremely sensitive,
determines a 3-fold greater depletion of PKC-
than -
from the
cells. In addition, consistent with this possibility, Seedorf et
al. (6) have recently shown that chimeric receptors engineered
with the EGF receptor extracellular domain fused to the cytoplasmic
domain of several different tyrosine kinase receptors, including IR,
form stable complexes with PKC-
upon EGF binding and promote
receptor tyrosine kinase internalization and degradation. TPA-sensitive
PKCs are known to bind and phosphorylate cytoskeletal proteins such as
F-actin (32, 33), talin (34), and other cytoskeleton-associated
proteins (35, 36), as well as proteins regulating vesicle formation and
trafficking such as dynamin I (37). It is tempting therefore to
speculate that, once bound to the IR, PKC-
might control the IR
intracellular routing by interacting with specific cytoskeletal
elements.
Evidence is available that PKCs are required for insulin-induced
regulation of gene expression (18, 38), protein synthesis (30), glucose
uptake (18, 20, 21), and pyruvate dehydrogenase activity (19). Previous
studies have also demonstrated an important role of the PKC system in
controlling the IR kinase and signaling in several physiological (39)
and pathological (40) conditions. By describing the role of PKC in
controlling the IR intracellular sorting, the findings in the present
work indicate the existence of an additional step where PKCs may
control the function of the insulin receptor and thus insulin
action.
We are grateful to Drs. E. Consiglio and G. Vecchio for their continuous support and advice during the course
of this work, to Drs. P. Gorden and D. Accili (National Institutes of
Health, Bethesda, MD) for generously donating anti-insulin receptor
antibodies. We also thank Dr. M. Bifulco for helpful discussions and
Dr. D. Liguoro for the technical help.
The authors dedicate this paper to the late Professor Gaetano
Salvatore.