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Endocrinology 149(4):1718 –1727
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2007-1572
Insulin Increases Nuclear Protein Kinase C␦ in L6
Skeletal Muscle Cells
Miriam Horovitz-Fried,* Tamar Brutman-Barazani,* Dov Kesten, and Sanford R. Sampson
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel 52900
Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of
cell growth, differentiation, apoptosis, and other cellular
functions. PKC proteins reside in the cytoplasm in an inactive
state translocate to various membranes to become fully activated in the presence of specific cofactors. Recent evidence
indicates that PKC isoforms have an important role in the
nucleus. We recently showed that insulin rapidly increases
PKC␦ RNA and protein. In this study we initially found that
insulin induces an increase in PKC␦ protein in the nuclear
fraction. We therefore attempted to elucidate the mechanism
of the insulin-induced increase in nuclear PKC␦. Studies were
performed on L6 skeletal myoblasts and myotubes. The increase in nuclear PKC␦ appeared to be unique to insulin be-
P
ROTEIN KINASE C (PKC) isozymes are a family of
serine/threonine kinases involved in the transduction
of number signals important for the regulation of cell growth,
differentiation, apoptosis, and many other functions (1– 4).
At present, 11 isoforms have been cloned and identified.
They have been classified into three main groups that share
a common requirement for phospholipids for their activity
but differ in structure and their dependence on other activators. Conventional PKCs (␣, ␤I, ␤II,␥) require phosphatidylserine (PS), Ca2⫹, and diacylglycerol or phorbol esters for
activation. Novel PKCs (␦, ␧, ␯, ␪) require diacylglycerol and
PS for activation. Atypical PKCs (␨, ␫, ␭) require only PS for
activation (5– 8). The biological functions of PKC have mostly
been linked with events occurring at the plasma membrane
or other membrane components in the cytoplasm. This is
because PKCs proteins are believed to remain in an inactive,
cytoskeletal-associated state in the cytoplasm; after phosphorylation, they translocate to the plasma membrane or
membranes of cytoplasmic organelles to become fully activated in the presence of specific cofactors (1, 9, 10). Recent
studies, however, indicate that PKC proteins may also have
a role in the nucleus. Thus, it has been suggested that nuclear
PKCs may participate in cascades that communicate signals
that are generated at the plasma membrane and are transmitted to the nucleus (11–13). Studies have identified several
First Published Online December 27, 2007
* M.H.-F. and T.B.-B. contributed equally to this study.
Abbreviations: AD, Actinomycin D; CH, cycloheximide; DRB, 5,6dichlorobenzimidazole riboside; IR, insulin receptor; LMB, leptomycin
B; PKC, protein kinase C; PS, phosphatidylserine; WGA, wheat germ
agglutinin.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
cause it was not induced by other growth factors or rosiglitazone. Inhibition of transcription or translation blocked the
insulin-induced increase in nuclear PKC␦, whereas inhibition
of protein import did not. Inhibition of protein export from the
nucleus reduced the insulin-induced increase in PKC␦ in the
cytoplasm and increased it in the nucleus. The increase in
nuclear PKC␦ induced by insulin was reduced but not abrogated by treatment of isolated nuclei by trypsin digestion.
Finally, we showed that insulin induced incorporation of 35Smethionine into nuclear PKC␦ protein; this effect was not
blocked by inhibition of nuclear import. Thus, these results
suggest that insulin may induce nuclear-associated, or possibly nuclear, translation of PKC␦ protein. (Endocrinology 149:
1718 –1727, 2008)
proteins that can act as PKC substrates (11, 14, 15) and other
nuclear PKC-binding proteins (16 –19). Nuclear PKCs are
involved in the regulation of several important biological
process such as cell proliferation [PKC␤II (20, 21)], cell differentiations [PKC␣ (22)], neoplastic transformation [PKC␤
(23, 24)], and apoptosis [PKC␣ and PKC␦ (25, 26)].
We recently reported that insulin stimulation of skeletal
muscle rapidly increases total PKC␦ RNA and protein levels
on a time scale of within 5 min (27). These effects are accompanied by rapid activation of the PKC␦ promoter and are
abrogated by pretreatment with inhibitors of either translation [cycloheximide (CH)] or transcription [actinomycin D
(AD) and 5,6-dichlorobenzimidazole riboside (DRB)]. Thus,
insulin induces de novo PKC␦ protein synthesis as well as
transcription of new PKC␦ RNA. During the course of these
studies, we examined the possibility that elevations in total
cytosolic PKC␦ might result from translocation from other
subcellular fractions. To our surprise, our results indicated
that the ability of insulin to induce an increase in PKC␦
protein occurred with the same rapid time course in the
nucleus as it does in the cytoplasmic and cytoskeletal fractions, i.e. within 5 min of insulin stimulation. These observations suggested that insulin may induce rapid translocation of nascent PKC␦ into the nucleus or de novo synthesis of
the PKC␦ protein itself within or associated with the nucleus.
According to accepted paradigm, polymerase II transcribes DNA to mRNA; the RNA then translocates to the
cytoplasm in which it is translated to protein by the ribosomes. Synthesis of protein in the nucleus is considered
unlikely, despite occasional reports that suggest the existence
of this phenomenon (28 –31). These studies performed on
HeLa cells showed that whereas most of protein translation
occurs in the cytoplasm, some (10 –15%) protein translation
may occur in the nucleus. Nuclear translation has been re-
1718
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
futed by other studies (32–34), in which it was concluded that
if nuclear translation occurs, it would be limited to less than
1% of the total protein.
In the current study, we examined the mechanisms underlying the rapid increase in nuclear PKC␦ in response to
insulin of skeletal muscle. We found that the effect appeared
to be limited to insulin and was not blocked by inhibitors of
nuclear import or export. Our data suggest that after insulin
stimulation, PKC␦ may be both transcribed and translated
within the cell nucleus.
A
IB:: α-tubulin
α
IB: actin
Cell culture
L6 cells were grown in ␣MEM supplemented with 10% fetal calf
serum for 4 d after confluence, with media changed daily; cells were
allowed to differentiate spontaneously or induced by changing the media to one supplemented with 2% fetal calf serum. In some experiments
(immunostaining), studies were done on L6 myoblasts, which are also
used as a model system for insulin signaling (35, 36).
Cytoplasmic extract
Dishes were washed with Ca2⫹/Mg2⫹-free PBS and then mechanically detached with cell scraper in radioimmunoprecipitation assay
buffer [50 mm Tris-HCl (pH 7.4); 150 mm NaCl; 1 nm EDTA; 10 mm NaF;
1% Triton X-100; 0.1% sodium dodecyl sulfate; 1% Na deoxycholate]
containing a cocktail of antiproteases and antiphosphatases (Sigma).
After scraping, the preparation was centrifuged at 20,000 ⫻ g for 20 min
at 4 C. The supernatant from this step was designated the cytoplasmic
fraction.
Nuclear extracts
Nuclear extracts were prepared as described (35). Dishes were
washed with Ca2⫹/Mg2⫹-free PBS and then mechanically detached with
a cell scraper in PBS. Cells were transferred to Eppendorf tubes and
centrifuged at 200 ⫻ g (1500 rpm) for 15 min at 4 C. The pellet was
resuspended in buffer A [1 m Tris (pH 7.5), 2.5 m NaCl, 2 mm EDTA, 1
mm EGTA] containing a cocktail of protease and phosphatase inhibitors
(Sigma). The suspension was then homogenized in a Dounce glass
homogenizer (30 strokes) and centrifuged at 400 ⫻ g (2000 rpm) for 15
min at 4 C. The pellet was resuspended again in buffer A containing
cocktails of protease and phosphatase inhibitors and centrifuged at
400 ⫻ g for 15 min at 4 C. The pellet from this centrifugation was
suspended in Buffer 1 (0.5 m sucrose; 5 mm MgCl2; 0.1 mm EDTA; 10 mm
Tris, pH 8; 1 mm dithiothreitol) and centrifuged at 23,100 ⫻ g for 45 min
at 4 C. Purification of the nuclear preparation (Fig. 1) was verified by
scanning electron microscopy (SEM) at the step prior to disruption of the
nuclei and by Western blotting for tubulin (a cytoplasmic marker) and
as well as for actin (to control for equal loading of protein). The pellet
from this step was resuspended in buffer 2 (buffer 1 without EDTA, with
0.6 m KCl) and left in ice for 30 min. The suspension was then centrifuged
Cyto
Nuc
Cyto
Nuc
B
IB:H3
IB:actin
Materials and Methods
Tissue culture media and serum were purchased from Biological
Industries (Beit HaEmek, Israel). Enhanced chemical luminescence was
performed using antibodies purchased from Bio-Rad (Hercules, CA)
and other reagents from Sigma (St. Louis, MO). The following antibodies
were used: anti-PKC␦ and anti-PKC␣ (Santa Cruz Biotechnology, Santa
Cruz, CA); antiskeletal muscle ␤-actin (Sigma); horseradish peroxidaseconjugated antirabbit and antimouse IgG (Bio-Rad); Alexa fluor488 goat
antirabbit IgG was purchased from Invitrogen (Eugene, OR).
Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, dithiothreitol,
orthovanadate, pepstatin, cyclohexamide, DRB, AD, and wheat germ
agglutinin (WGA) were purchased from Sigma. Insulin (HumulinR,
recombinant human insulin) was purchased from Lilly France SA (Fergersheim, France). Leptomycin B (LMB) was purchased from Calbiochem
(La Jolla, CA). 35S-methionine was purchased from PerkinElmer (Boston,
MA).
1719
C
X400
FIG. 1. Western blot and photomicrograph showing purity of nuclear
preparations. Differentiated myotubes were subjected to cell fractionation, SDS-PAGE, and transfer, as described in Materials and
Methods. A, Transferred proteins were immunoblotted (IB) with antibodies to tubulin [cytoplasmic marker (Cyto)] and actin (to verify
equal loading). The Western blots are from two separate experiments.
Tubulin is undetectable in the nuclear fraction (Nuc). B, Transferred
proteins were immunoblotted with antibodies to histone H3 (nuclear
marker) and actin (to verify equal loading). The Western blots are
from two separate experiments. Histone is undetectable in the cytoplasmic fraction. C, Photographs (SEM) of nuclei isolated from L6
myotubes. No cellular debris is detectable.
at 75,500 ⫻ g for 60 min at 4 C. The supernatant from this step was
designated the nuclear fraction.
Western blot analysis
Western blots were performed as described (38 – 41). Equal protein
loading of Western blots was confirmed by immunoblotting for skeletal
muscle ␤-actin.
RT-PCR
Total RNA was obtained using RNeasy minikit (QIAGEN, Valencia,
CA) from control and insulin stimulated cells. Reverse transcription was
performed on 1 ␮g total RNA using One Tube RT-PCR system (Roche,
Mannheim, Germany) and 10 ␮m specific primers for PKC␦ and S12
control (rRNA, housekeeping gene). The reverse transcription reaction
was amplified for 40 cycles (94 C, 1 min; 60 C, 1 min; 72 C, 1 min; 70 C,
10 min). Finally, 50% of the amplified products were resolved on a 1%
agarose gel. Specific primers for PKC␦ and S12 were designed based on
reported sequences as described (27).
Cell preparation for immunofluorescence imaging
Cells were seeded on coverslips at 3 ⫻ 105 cells/well in six-well plates.
After appropriate treatment, cells were fixed with 4% paraformaldehyde
and dried. Coverslips were washed three times, permeabilized (isotonic
PBS buffer, 10% BSA, 1% Triton X-100), and incubated with PKC␦
primary antibody for 1 h. After further wash steps, cells were stained
with a fluorescent-conjugated secondary antibody, washed, mounted,
and imaged under a fluorescent microscope at ⫻400 magnification.
1720
35
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
sized PKC␦ might occur in, or be targeted to, a particular cell
fraction. Accordingly, we performed studies in which PKC␦
levels were examined in the membrane cytosolic and nuclear
fractions in control and insulin-stimulated cells. As shown in
Fig. 2A, insulin induced a rapid increase, within 5 min, in
PKC␦ protein in each of the fractions tested, membrane,
cytosolic, and nuclear. The increase in nuclear PKC␦ was also
verified by confocal microscopy. As shown in Fig. 2B, PKC␦
levels increased dramatically in both the nuclei and surrounding cytoplasm after insulin stimulation for 5 and 15
min. In contrast, insulin induced an increase in cytosolic, but
a decrease in nuclear, PKC␣ protein levels (Fig. 2C).
S-methionine uptake
Culture dishes (90 mm; Nunc, Glostrup, Denmark) containing the
muscle cells were transferred to methionine-free medium for 3 h. The
medium was then changed to medium containing 35S-methionine (40
␮Ci/ml) for 5–15 min after treatment with insulin for 5–15 min. Cells
were washed three times with PBS, scraped, and lysates prepared for
immunoprecipitation with specific antibody to PKC␦, as described below. The immunoprecipitates were then subjected to SDS-PAGE. The
resulting gel containing immunoprecipitated PKC␦ was exposed to xray film at ⫺70 C for 2 wk.
Immunoprecipitation
Specific antibody to PKC␦ (dilution 1:100) was added to 400 ␮g
protein from the nuclear extract and was rotated continuously for 60 min
at 4 C. After the samples were rotated for 60 min, 30 ␮l of A/G Sepharose
was added and rotated overnight at 4 C. The samples were then centrifuged at 2000 ⫻ g for 10 min at 4 C, and the pellet was washed three
times with buffer 2 by centrifugation at 2000 ⫻ g for 2 min at 4 C. The
pellet was then resuspended in 25 ␮l of sample buffer [0.5 m Tris HCl
(pH 6.8); 10% sodium dodecyl sulfate; 10% glycerol; 4% 2-␤-mercaptoethanol; 0.05% bromophenol blue]. The suspension was again centrifuged at 500 ⫻ g (at 4 C for 10 min), boiled for 5 min, and then subjected
to SDS-PAGE.
The ability to increase nuclear PKC␦ is unique to insulin
The question arises as to whether the effect to increase
nuclear PKC␦ might be induced by other growth factors and
agents with insulin-like actions or whether the increase in
nuclear PKC␦ might be unique to insulin. To accomplish this,
we examined effects of agents known to produce insulin-like
responses or activate common enzymes in the insulin signaling pathway in skeletal muscle. We studied two categories of agents: 1) growth factors, such as IGF-I, epithelial
growth factor, and nerve growth factor, all activators of receptor tyrosine kinases. Indeed, IGF-I is known to signal via
shared elements in the insulin receptor (IR) signaling cascade
and activate the IR ␤-subunit tyrosine kinase; and 2) rosiglitazone (RG), a member of the thiazolidinedione family of oral
antidiabetic agents, which improves insulin sensitivity and
glucose homeostasis in type 2 diabetic patients as well as
various animal models of diabetes and obesity. Neither IGF-I
Results
Insulin increases PKC␦ protein levels in the nucleus of L6
skeletal muscle cells
We recently reported that insulin induces an increase in
total PKC␦ protein levels by a process involving both transcription of RNA and translation into new protein (27). These
experiments were done on whole-cell lysates of a variety of
skeletal muscle cell preparations. In the current study, we
initially attempted to determine whether the newly synthe-
A
Mem
Nuc
Cyto
IB:PKCδ
δ
IB:actin
FIG. 2. Insulin increases PKC␦ protein in the cytoplasmic (Cyto), membrane (Mem), and nuclear (Nuc)
fractions of L6 skeletal muscle. Differentiated myotubes were treated with insulin (10⫺7 M) for 5 min.
After cell fractionation, SDS-PAGE, and transfer, lysates were probed with specific antibodies against
PKC␦. A, Western blots of insulin (Ins)-induced increase PKC␦ protein in all three fractions. IB, Immunoblot. B, Photographs of immunostained L6 myoblasts showing effect of insulin on PKC␦ protein levels
in the cytoplasm and nucleus. C, Western blot showing that insulin-induced an increase in cytoplasmic
PKC␣ and a decrease in nuclear PKC␣. Differentiated
myotubes were treated with insulin (10⫺7 M) for 5
min. After cell fractionation, SDS-PAGE, and transfer, proteins were probed with specific antibodies
against PKC␣.
Ins (min)
0
5
15
0
5
15
0
B
Ins
-
+
C
Nuc
Cyto
IB:PKCα
α
IB:actin
Ins (min)
0
5
15
0
5
15
5
15
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
(27) nor any of the other growth factors increased PKC␦
protein levels, in either total cell lysates or any subcellular
fractions (not shown). On the other hand, RG increased total
PKC␦ levels within 5 min, similar to the effects of insulin (Fig.
3A). To investigate further the mechanism by which RG
increases PKC␦ protein, we pretreated cells with inhibitors of
transcription or translation. Interestingly, pretreatment of
cells with CH abrogated the RG-induced increase in PKC␦
protein, whereas the effect of RG was not altered by pretreatment with AD (Fig. 3B), an inhibitor of transcription.
These findings indicated that RG increases translation of
PKC␦ RNA into protein but does not induce an increase in
PKC␦ RNA. Furthermore, this increase in PKC␦ protein occurred only in the cytoplasm (Fig. 3B) and not in the nucleus
(Fig. 3C). This was further confirmed in studies on PKC␦
RNA in which we found that RG, in contrast to insulin, did
not increase PKC␦ RNA levels (Fig. 3D).
the insulin-induced increase in nuclear PKC␦ protein. Cells
were treated with CH, AD, or DRB before stimulation with
insulin. Nuclear fractions were then prepared and subjected
to SDS-PAGE and Western blotting. In addition, cells were
fixed on coverslips for fluorescence microscopy (as described
in Materials and Methods). Figure 4 shows results obtained in
these experiments. Pretreatment of L6 cells with CH almost
completely abrogated the insulin-induced increase in nuclear
PKC␦ protein, thus indicating that increased PKC␦ protein in
the nucleus derives from a newly synthesized pool. These
results are further strengthened by studies in which cells
were treated with inhibitors of transcription (AD or DRB),
which also blocked the insulin-induced increase in nuclear
PKC␦ (Fig. 5).
Inhibition of nuclear import does not abrogate the insulininduced increase in PKC␦ protein levels in the nucleus
The results so far are consistent with the possibility that
the insulin-induced increase in nuclear PKC␦ protein may
result from rapid synthesis followed by translocation into
the nucleus. To investigate this possibility, we treated cells
with WGA (100 mg/ml, 3 h), an inhibitor of nuclear import
(42– 44) before insulin stimulation. Treatment with WGA
caused an increase in PKC␦ in the absence of insulin treatment, but to our surprise, inhibition of nuclear import did
not have any effect on the insulin-induced increase in
nuclear PKC␦ (Fig. 6, A and C). In contrast, this treatment
did inhibit insulin-induced translocation of specificity
protein-1 from the cytoplasm to the nucleus (Fig. 6B; see
The insulin-induced increase in nuclear PKC␦ protein is
abrogated by inhibitors of transcription and translation
Our findings indicate that the ability to induce a rapid
increase in nuclear PKC␦ protein is unique to insulin and is
closely linked in time to PKC␦ RNA transcription. One possible mechanism for the increase in nuclear PKC␦ is its translocation to the nucleus either from an existing pool or newly
synthesized PKC␦ protein in the cytoplasm. Because we have
previously shown that insulin induces rapid de novo synthesis of PKC␦ protein (27), we first investigated the effects of
inhibition of translation (CH) or transcription (AD, DRB) on
A
Cyto PKCδ
δ
RG
200
OD % of control
INS
IB: PKCδ
δ
FIG. 3. RG increases PKC␦ protein in the cytoplasm
but not nuclear PKC␦ or PKC␦ RNA in skeletal muscle. Differentiated myotubes were treated with RG
(10⫺6 M) for the times indicated. Cell fractionation,
SDS-PAGE, and transfer were performed as in Fig.
2. A, Western blots showing effects of insulin (Ins)
and RG on cytosolic PKC␦ protein levels. The graph
to the right shows densitometry measurements of the
Western blots. IB, Immunoblot. B, Western blot of
cytosolic (Cyto) PKC␦ in control (Con) and RG-stimulated cells. The blot shows that CH (1 mM, 12 h) but
not AD (1 ␮g/ml, 3 h) abrogates RG-induced increase
in cytosolic PKC␦ protein. C, Nuclear PKC␦ protein
is not increased by RG. Nuclear fractions were prepared from control and RG-stimulated cells as described in Materials and Methods. After cell fractionation, SDS-PAGE, and transfer, nuclear proteins
were probed with specific antibodies against PKC␦.
D, PKC␦ RNA is not increased by RG. RNA was
extracted from control and RG-stimulated cells, and
RT-PCR was performed as described in Materials
and Methods.
IB: actin
150
100
50
0
C
Time ( min)
B
1721
0
5
15
5
INS 5
INS 15
RG 5
RG 15
15
Cyto PKCδ
δ
IB:PKCδ
δ
IB:actin
Rg (min)
0
5
15
0
Con
C
5
15
0
5
CH
Nuclear PKCδ
δ
15
AD
D
PKCδ
δ RNA
δ
IB: PKCδ
δ
S12
IB: actin
Rg ( min )
0
5
15
Rg (min)
0
5
15
1722
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
B
FIG. 4. Inhibition of translation blocks the insulin-induced increase in nuclear PKC␦. Differentiated myotubes were pretreated with CH (3 h, 1
mM) and then with insulin (Ins; 10⫺7 M) for 5 min.
Nuclear extracts were prepared as described in
Materials and Methods. After SDS-PAGE and
transfer, nuclear proteins were probed with specific antibodies against PKC␦. A, Western blots
showing the ability of CH to block insulin-induced increase in nuclear PKC␦ protein. IB, Immunoblot. B, Graph displaying results of densitometry measurements made on Western blots;
each bar represents the mean ⫾ SE of the measurements of the ratio of PKC␦ to actin in three
separate experiments (*, P ⬍ 0.005 vs. control).
C, Photographs of immunostained L6 myoblasts
showing the blocking by CH of the insulin-induced increase in PKC␦ protein levels in the nucleus.
IB: PKCδ
δ
IB: actin
Ins
CH
-
+
-
+
-
150
100
50
0
0
+
-
5
OD(% of control)
IB:PKCδ
δ
IB:actin
+
+
+
+
+
+
possible that PKC␦ might be translated on the nuclear
envelope in which it remains bound. To examine this
possibility, we isolated nuclei from insulin-stimulated and
unstimulated cells and treated the isolated nuclei with
B
A
+
-
CONTROL
CH
200
C
Ref. 42). This results in a slight reduction in the insulininduced increase in cytosolic PKC␦ (42). The insulin-induced decrease in level of nuclear PKC␣ was essentially
unaffected by treatment with WGA (Fig. 6A). It remains
-
+
+
250
Ins (min)
Ins
CH
Ins
AD
OD(% of control)
A
300
CONTROL
AD
250
200
150
100
50
0
0
5
Ins(min)
C
Ins
DRB
-
+
-
+
+
+
FIG. 5. Inhibition of transcription blocks insulin-induced increase in nuclear and cytoplasmic PKC␦ protein. Differentiated myotubes were
pretreated with AD (3 h, 1 ␮g/ml) or DRB (1 h, 50 ␮g/ml) and then with insulin (Ins; 10⫺7 M) for 5 min. Nuclear extracts were prepared as described
in Materials and Methods. After SDS-PAGE and transfer, proteins were probed with specific antibodies against PKC␦. A, Western blots showing
the ability of AD to block insulin-induced increase in nuclear PKC␦ protein. IB, Immunoblot. B, Graph displaying results of densitometry
measurements made on Western blots; each bar represents the mean ⫾ SE of the measurements of the ratio of PKC␦ to actin in three separate
experiments (*, P ⬍ 0.005 vs. control). C, Photographs of immunostained L6 myoblasts showing effect of DRB to inhibit insulin-induced increase
in PKC␦ protein levels in the nucleus.
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
A
Nucleus
300
OD % of control
IB: PKCδ
250
150
100
IB: actin
Ins
WGA
B
control
WGA
200
IB: PKCα
FIG. 6. Inhibition of protein import does not reduce and
slightly increases the induction by insulin of PKC␦ protein in the nuclear fraction. Differentiated myotubes were
pretreated with import inhibitor (WGA, 1 h, 100 ␮g/ml)
and then with insulin (Ins; 10⫺7 M) for 5 min. Nuclear
extracts were prepared as described in Materials and
Methods. After SDS-PAGE and transfer, proteins were
probed with specific antibodies against PKC␦ or PKC␣. A,
Western blots showing that after treatment with WGA,
basal and insulin-induced increase in nuclear PKC␦ was
increased. In contrast, WGA did not alter the effect of
insulin to decrease nuclear PKC␣ levels. The graph displays results of densitometry measurements made on
Western blots; each bar represents the mean ⫾ SE of the
measurements of the ratio of PKC␦ to actin in three separate experiments (*, P ⬍ 0.005 vs. control). IB, Immunoblot. B, Western blots showing that after treatment
with WGA, the level of specificity protein-1 (SP-1) in basal
and insulin-stimulated cells was reduced in the nuclear
fraction. C, Photographs of immunostained L6 myoblasts
showing effects of inhibition of nuclear import on insulininduced increase in nuclear PKC␦ protein. D, Trypsin
digestion of proteins from the nuclear envelope does not
abrogate the insulin-induced increase in nuclear PKC␦
protein. Differentiated myotubes were treated with insulin (10⫺7 M) for 5 min. After cell fractionation nuclei
were treated or not with trypsin (T.V) as described in
Materials and Methods. After SDS-PAGE and transfer,
proteins were probed with specific antibodies against
PKC␦.
1723
-
+
-
+
+
+
50
0
0
Ins (min)
5
IB: SP-1
IB: actin
Ins
WGA
-
+
-
+
+
+
C
Ins
WGA
-
+
-
+
+
+
D
IB: PKCδ
IB: actin
Ins
T.V
trypsin (1 mg/ml) to dislodge any PKC␦ bound to the
nuclear envelope. As can be seen in Fig. 6D, the insulininduced increase in PKC␦ was maintained but reduced
slightly in trypsin-treated isolated nuclei.
Inhibition of nuclear export increases the insulin-induced
increase in PKC␦ protein levels in the nucleus and reduces the
insulin-induced increase in PKC␦ protein levels in the cytoplasm
The results so far show that the insulin-induced increase in
nuclear PKC␦ protein does not appear to be explained by translocation from the cytoplasm to the nucleus. This leads to the
possibility that PKC␦ may be translated in or associated with
the nucleus in response to insulin. We attempted to further rule
out the possibility of nuclear translation by inhibition of protein
export. We reasoned that, after insulin induction of PKC␦ RNA
transcription, PKC␦ RNA may migrate from the nucleus to the
-
+
-
+
+
+
cytoplasm in which it is translated into PKC␦ protein, which is
then rapidly translocated back into the nucleus. Blockade of
nuclear export would inhibit the export of any PKC␦ protein (to
the extent that it is synthesized or preexists in the nucleus) but
allow the newly synthesized PKC␦ RNA to migrate to the
cytoplasm and undergo translation into new PKC␦ protein.
Assuming that insulin induces an increase in PKC␦ synthesis in
the cytoplasm and that the increase in nuclear PKC␦ results
from its translocation from the cytoplasm to the nucleus, inhibition of nuclear export should not affect the insulin-induced
increase in cytoplasmic PKC␦. We studied this possibility by
treating unstimulated and insulin-stimulated cells with LMB
(20 ng/ml, 2 h before addition of insulin), an inhibitor of nuclear
export (45, 46). The results of these studies are shown in Fig. 7.
In fact, inhibition of nuclear export reduced the insulin-induced
increase in cytoplasmic PKC␦ protein and further increased the
1724
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
A
Cytosol
IB:PKCδ
δ
IB:actin
Ins
LMB
B
+
+
450
IB:PKCδ
δ
IB:actin
INS
LMB
C
-
+
-
+
+
+
400
CONTROL
350
LMB
300
250
200
150
100
50
0
0
Ins (min)
5
IB:PKCα
α
IB:actin
Ins
LMB
-
+
-
+
+
+
D
insulin-induced PKC␦ protein level in the nucleus (Fig. 7, A, B,
and D). In contrast, the insulin-induced decrease in PKC␣ was
eliminated by treatment with LMB (Fig. 7C).
35
+
Nucleus
Ins
LMB
Insulin increases
PKC␦ protein
+
-
O.D. (% of control)
FIG. 7. Inhibition of nuclear export reduces the insulin-induced increase in cytosolic PKC␦ levels and
increases the insulin-induced increase in nuclear
PKC␦ protein. Differentiated myotubes were pretreated with the nuclear export inhibitor, LMB (2 h,
20 ng/ml) and then with insulin (Ins; 10⫺7 M) for 5
min. Nuclear and cytosolic extracts were prepared
as described in Materials and Methods. After SDSPAGE and transfer, proteins were probed with specific antibodies against PKC␦. A, Blockade of nuclear export reduces insulin-induced increase in
cytosolic PKC␦. IB, Immunoblot. B, Western blots
showing that inhibition of nuclear export increases
PKC␦ protein and slightly elevates insulin-induced
increase in nuclear PKC␦ protein. The graph to the
right displays results of densitometry measurements made on Western blots; each bar represents
the mean ⫾ SE of the measurements of the ratio of
PKC␦ to actin in three separate experiments (*, P ⬍
0.005 vs. control). C, Western blots showing that
inhibition of nuclear export blocks insulin-induced
decrease in PKC␣ protein. D, Photographs of immunostained L6 myoblasts showing effect of nuclear export inhibition on insulin-induced increase
in nuclear PKC␦ protein levels.
-
S-methionine incorporation into nuclear
The results so far appear to indicate that PKC␦ protein,
once rapidly elevated in the nucleus, is closely time linked
to translocation from the nucleus to the cytoplasm. As we
have just shown, inhibition of nuclear export reduced insulin-induced increase in cytosolic PKC␦ and increased
insulin-induced nuclear PKC␦. These results indicate the
possibility of nuclear-associated or nuclear translation of
PKC␦ protein. To further investigate this last hypothesis,
we labeled the cells with radioactive 35S-methionine (40
mCi/ml) and examined the uptake of 35S-methionine into
nuclear PKC␦ of control and insulin-stimulated cells. As
can be seen in Fig. 8A, insulin induced an increase in
35
S-methionine incorporation into PKC␦ protein, which
rapidly increases in the nucleus. These data appear to
suggest the possibility that PKC␦ protein may indeed be
translated in or associated with the nucleus. Treatment
-
+
-
+
+
+
with WGA (nuclear import inhibitor) did not prevent the
effect of insulin on35S-methionine incorporation into nuclear PKC␦ protein (Fig. 8B).
Insulin increases PK␦ protein in isolated, live nuclei in vitro
The results indicate that insulin increases nuclear PKC␦
protein via a mechanism independent of nuclear import or
export. This suggests that the effect may occur directly in the
nucleus or on nuclear-associated elements (e.g. nuclear envelope associated proteins). To examine this further, we performed studies on isolated nuclei stimulated in vitro. Nuclei
were prepared as described in Materials and Methods. Results
are shown in Fig. 9, A and B. Figure 9A is a Western blot
showing that insulin administered directly to the nuclei
caused an increase in nuclear PKC␦. These results were confirmed by confocal microscopy, as shown in Fig. 9B.
Discussion
In this study we report that insulin stimulation of skeletal
muscle induces a rapid increase in PKC␦ protein in the nuclear
fraction of cell lysates. In contrast, insulin decreases PKC␣ pro-
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
A
Endocrinology, April 2008, 149(4):1718 –1727
IP:PKCδ
δ 35S-methionine
Ins
-
+
B
Ins
WGA
-
-
+
+
+
-
+
FIG. 8. Insulin increases 35S-methionine incorporation into nuclear
PKC␦ protein. Differentiated myotubes were labeled with 35S-methionine (40 ␮Ci/ml) as described in Materials and Methods. The cells were
treated with insulin (Ins; 10⫺7 M) for 5 min, after which a nuclear extract
was prepared. PKC␦ was immunoprecipitated (IP) from nuclear extracts
with anti-PKC␦ antibodies as described in Materials and Methods and
then subjected to SDS-PAGE and autoradiography. B, Insulin-induced
increase 35S-methionine incorporation into nuclear PKC␦ is not blocked
by inhibition of nuclear import. Differentiated myotubes were pretreated
with import inhibitor (WGA, 1 h, 100 ␮g/ml) and then with insulin (10⫺7
M) for 5 min. Nuclear extracts were prepared as described in Materials
and Methods. PKC␦ was immunoprecipitated from nuclear extracts with
anti-PKC␦ antibodies as described in Materials and Methods and then
subjected to SDS-PAGE and autoradiography.
tein levels in the nucleus. RG, which does increase total PKC␦,
does not increase PKC␦ RNA or nuclear PKC␦ protein. These
results are consistent with our recent report that showed that
insulin induces a rapid increase in total PKC␦ protein and RNA
(27). Recently evidence has accumulated to show that some
PKC isoforms are translocated to the nucleus in response to
certain stimuli (11, 13). Several reports suggest that nuclear
PKCs have a role in cascades that communicate signals generated at the plasma membrane and transmitted to the nucleus
(47). Most PKC isoforms have been found to contain a nuclear
localization sequence, and studies have shown that the PKC␦
nuclear localization sequence is highly conserved in this kinase
family (12). We showed previously that inhibition of translation
and transcription of the cells blocked the insulin-induced increase in PKC␦ RNA and protein (27).
We attempted to elucidate the mechanism by which insulin
induces a rapid increase in nuclear PKC␦ protein. We first
examined the effect of translation inhibition on the insulininduced increase in nuclear PKC␦ protein levels. An inhibitor
of translation (CH) blocked the insulin-induced increase in nuclear PKC␦ protein levels. This blockade might occur as a result
of inhibition of PKC␦ translation in the cytoplasm. After treatment with cycloheximide, less PKC␦ is found in the cytoplasmic
fraction, and therefore, less protein would be translocated to the
nucleus. As expected, inhibition of transcription (AD) also
blocked the insulin-induced increase in nuclear PKC␦ protein
levels. Because of the inhibition of transcription, less PKC␦ is
transcribed; less mRNA of PKC␦ is translated into protein; and,
therefore, less PKC␦ would be translocated to the nucleus.
To demonstrate that PKC␦ protein is indeed translocated
from the cytoplasm fraction to the nuclear fraction, we performed studies in which protein import into the nucleus was
inhibited by treatment with a protein import inhibitor. We
found that inhibition of protein import into the nucleus did not
1725
block or even reduce the insulin-induced increase in nuclear
PKC␦ protein. Interestingly, blockade of nuclear import induced an increase in nuclear PKC␦, even in the absence of
insulin stimulation. We can only speculate as to the mechanism
of this effect. WGA would be expected to inhibit import of many
proteins into the nucleus. This could include factors that may
down-regulate PKC␦ (as well as other proteins) synthesis or
degradation. In any case, our data are strongly suggestive that
insulin induces PKC␦ synthesis associated with (or within) the
nucleus. Alternatively, the effect might be related to the ability
of WGA to mimic certain effects of insulin stimulation (48).
Clearly, additional studies are necessary to clarify this effect
further.
In current studies (Attali, V., M. Horovitz-Fried, T. BrutmanBarazani, and S. Sampson, studies in progress) in which we are
investigating effects of thiazolidinediones (rosiglitazone and
troglitazone) on PKC␦ expression in skeletal muscle, we have
observed that these compounds caused an increase in protein
levels of PKC␦ but not other PKC isoforms. Several relatively
recent studies have shown that these compounds have effects
that, whereas in a sense positively up-regulate IR signaling,
may appear to be independent of peroxisomal proliferatoractivated receptor-␥-related actions (36, 49 –51). In this study,
we also examined the possibility that RG might also increase
nuclear PKC␦ protein. We found, however, that the effect of RG
appeared to occur only in the cytoplasm. Thus, the effect of
insulin to increase nuclear PKC␦ would appear to be selective
for this hormone. It is also important to emphasize that the effect
of insulin also appears to be selective for PKC␦ because levels
of PKC␣ were decreased by insulin; we have not observed
effects of insulin on any other PKC isoforms.
The traditional view of transcription/translation in eukaryotes is that polymerase II transcribes the DNA to mRNA
in the nucleus; the mRNA is translocated to the cytoplasm, in
which it is translated to protein by the ribosomes. Synthesis of
protein in the nucleus has been considered unlikely, despite
occasional reports that appear to demonstrate the existence of
this phenomenon (28 –31). Most reports agree that, whereas
most of the cellular protein is translated in the cytoplasm, it has
been estimated that at most as much as 1% of the proteins in the
cell may be translated in the nucleus (32–34). There is both direct
and indirect evidence for translation of proteins in the nucleus
occurring immediately after transcription, such as occurs in
bacteria. Studies with labeled amino acids have shown that,
indeed, most of the labeled residues are incorporated into cytoplasmic proteins, but a small fraction of the labeled amino
acids may be incorporated into polypeptides in the nucleus (30).
Labeling the cells with florescent lysine showed that the accumulation of the florescence in the nucleus was time dependent
and sensitive to eukaryotic but not to bacterial translation inhibitors. The translation sites that were labeled by the lysine
were not scattered in the nucleus but overlapped with the
transcription sites. All the factors necessary for the translation
process have been found in the nucleus. These findings provide
indirect evidence for the possibility of nuclear translation. On
the other hand, there are several studies that refute this possibility based mainly on the objection that the nuclear preparations could have been contaminated with cytoplasmic components (32–34). It should be pointed out, however, that even these
studies left open the possibility that some cellular protein trans-
1726
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
A
nuclei
Nuclei in vitro
IB:PKCδ
δ
IB:actin
Ins
FIG. 9. Insulin increases PKC␦ protein levels in isolated,
live nuclei in vitro. Nuclear fraction and fraction of live
nuclei were prepared as described in Materials and Methods. A, Western blots showing that insulin (Ins) induces
an increase in PKC␦ protein levels from isolated live
nuclei in vitro as well as in nuclear fraction. IB, Immunoblot. B, Photographs of immunostained isolated nuclei
showing the effect of insulin on PKC␦ protein levels. The
green (left lane) represents the levels of PKC␦ and the red
(middle lane) is immunostaining of the nuclei with
propidium iodide. In the right lane are shown merged
photographs demonstrating PKC␦ within the nucleus
(orange).
-
+
-
+
B
Control
Ins 5 min
Ins 15 min
lation might occur in the nucleus. We did not find evidence for
contamination of the nuclear preparations in this study with
cytoplasmic components. SEM examination indicated that the
fraction was comprised only of nuclei; no other cellular debris
was detected, and Western blotting studies showed that tubulin, a cytoplasmic protein, was undetectable in the nuclear
fraction.
We attempted to further rule out the possibility of nuclear
translation by inhibition of protein export. We reasoned that
after insulin induction of PKC␦ transcription, PKC␦ RNA
would migrate from the nucleus to the cytoplasm in which it is
translated into PKC␦ protein, which is then translocated into the
nucleus. Blockade of nuclear protein export would inhibit the
export of any PKC␦ protein (to the extent that it is synthesized
or preexists in the nucleus) but allow the newly synthesized
PKC␦ RNA to migrate to the cytoplasm and undergo translation into new PKC␦ protein. Assuming that insulin induces an
increase in PKC␦ synthesis in the cytoplasm and that the increase in nuclear PKC␦ results from its translocation from the
cytoplasm to the nucleus, inhibition of nuclear export was not
expected to affect the insulin-induced increase in cytoplasmic
PKC␦. In fact, inhibition of nuclear export reduced the insulininduced increase in cytoplasmic PKC␦ protein and increased
even more the insulin-induced PKC␦ protein level in the nucleus. Similarly, inhibition of nuclear import did not reduce the
ability of insulin to increase nuclear PKC␦ protein. These findings would appear to be consistent with a nucleus-associated
origin of the newly translated protein.
These results indicate that it is possible that PKC␦ protein
may belong to a group of proteins that represent a very small
percentage of total cell proteins whose translation takes place in
close proximity to, or possibly within, the nucleus immediately
after transcription. To directly examine this possibility, we labeled cells with 35S-methionine and showed that insulin induced a rapid (within 5 min) increase in 35S-methionine incorporation into nuclear PKC␦ protein. This effect persisted despite
pretreatment of the cells with WGA, which inhibits nuclear
import. Thus, all these results indicate that insulin induces
nuclear-associated translation of PKC␦ protein.
In conclusion, this study suggests the possibility that PKC␦
protein may be translated in skeletal muscle cells in response to
insulin, closely time linked to its transcription in the nucleus.
This possible mechanism might explain the rapid increase in
PKC␦ protein in the nuclear fraction. The idea of rapid transcription and translation of proteins is highly unconventional
and runs counter to current concepts of protein synthesis, as has
been discussed (32). However, this mechanism may be unique
for insulin and PKC␦ as the data in our study appear to indicate.
Insulin is released from pancreatic ␤-cells in a pulsatile, periodic
manner, sometimes as frequently as every 20 –30 min, depending on fluctuations in blood sugar levels. Studies have shown
that certain PKC isoforms, PKC␦ in particular, play a key role
in the initial steps of IR signaling and may participate in IR
internalization and tyrosine phosphorylation (39). Thus, it
should not be surprising that expression of PKC isoforms, such
as PKC␦, may be regulated in a rapid manner at multiple levels
to sustain these kinases at effective concentrations during the
course of insulin action in insulin target tissues. One of these
mechanisms might include rapid and closely linked nuclear
transcription and translation.
Horovitz-Fried et al. • Insulin Increases Nuclear PKC␦
Endocrinology, April 2008, 149(4):1718 –1727
Acknowledgments
Received November 15, 2007. Accepted December 20, 2007.
Address all correspondence and requests for reprints to: S. R. Sampson, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900,
Israel. E-mail: [email protected].
This work was supported in part by the Russell Berrie Foundation
and D-Cure, Diabetes Care in Israel, grants from the Chief Scientist’s
Office of the Israel Ministry of Health, and the Sorrell Foundation.
Disclosure Statement: The authors have nothing to disclose.
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