From the
Insulin regulates a diverse array of cellular signaling
processes involved in the control of growth, differentiation, and
cellular metabolism. Insulin increases glucose transport via a protein
kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function
of specific PKC isozymes in insulin action has not been elucidated. Two
isoforms of PKC
Protein kinase C (PKC)
Insulin activates and translocates PKC
The
PKC
To our knowledge, this is the first report of a peptide
hormone regulating alternative splicing of mRNA within minutes.
Furthermore, the protein product of the alternatively spliced mRNA
turns out to be a relevant intracellular signaling component for the
hormone, insulin. This data sheds new light on the details of insulin
signaling as well as PKC isozyme function. The alternatively spliced
PKC
The regulation of alternative splicing
involves a switch in mRNA processing
(11) . Here we demonstrated
a change in alternatively spliced products using a probe that overlaps
the C4 or catalytic region and the alternative splice site. By
selecting a region of PKC spanning the area of the mRNA, which is
alternatively spliced, both PKC
One explanation for the decrease in PKC
These studies further demonstrate the
expression of PKC
Our PKC
In our studies, we are able
to demonstrate that increasing levels of PKC
Functional differences between
PKC
The present studies
indicate that the ability of cells to respond to signals from insulin
may depend upon the presence of one PKC
Heterogeneous pools of stably transfected cells were
grown in 24-well plates until >80% confluent. NIH-
We thank Dr. Walter Kolch, Institute for Clinical
Molecular Biology and Tumor Genetics, GSF, Munich, Germany; Dr. J. F.
Mushinski, National Cancer Institute, National Institutes of Health,
Bethesda, MD; and Dr. Duane Eichler, University of South Florida,
Tampa, FL for thoughtful and critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
result via alternative splicing of precursor mRNA.
As now shown, both isoforms are present in BC3H-1 myocytes, and insulin
induces alternative splicing of the PKC
mRNA thereby switching
expression from PKC
I to PKC
II mRNA. This effect occurs
rapidly (15 min after insulin treatment) and is dose-dependent. The
switch in mRNA is reflected by increases in the protein levels of
PKC
II. High levels of
12-0-tetradecanoylphorbol-13-acetate, which are commonly used
to deplete or down-regulate PKC in cells, also induce the switch to
PKC
II mRNA following overnight treatment, and protein levels of
PKC
II reflected mRNA increases. To investigate the functional
importance of the shift in PKC
isoform expression, stable
transfectants of NIH-3T3 fibroblasts overexpressing PKC
I and
PKC
II were established. The overexpression of PKC
II but not
PKC
I in NIH-3T3 cells significantly enhanced insulin effects on
glucose transport. This suggests that PKC
II may be more selective
than PKC
I for enhancing the glucose transport effects of insulin
in at least certain cells and, furthermore, that insulin can regulate
the expression of PKC
II by alternative mRNA splicing.
(
)
mediates cellular
responses elicited by hormones, neurotransmitters, and growth factors.
Molecular cloning has revealed that PKC represents a multigene family,
to date consisting of 11 different isozymes encoded by 10 distinct
genes. Depending on the cofactor requirements, PKCs can be subdivided
into three groups: classical PKCs (
,
I,
II,
),
which require diacylglycerol, phospholipids, and Ca
for full activity; novel PKCs (
,
,
,
),
which are phospholipid and diacylglycerol-dependent but
Ca
-independent; and atypical PKCs (
,
(
)
, µ), which require only phospholipids (for review, see
Refs. 1-3). Activation of classical and novel PKCs involves their
translocation from the cytoplasm to various cell membranes and
cytoskeletal structures after binding of diacylglycerol, which is
generated by agonist-induced hydrolysis of phosphatidylinositol
bisphosphate, phosphatidylcholine, a phosphatidylinositol-containing
glycan, or from the de novo synthesis of phosphatidic
acid
(4) .
in
BC3H-1 myocytes, a nonfusing murine cell line with many phenotypic
characteristics of muscle
cells
(5, 6, 7, 8) . Insulin stimulation
of glucose uptake in myocytes is inhibited by PKC
inhibitors
(9) . Treatment of BC3H-1 myocytes overnight with TPA
failed to down-regulate insulin-stimulated glucose
uptake
(6, 7, 8, 10) . The retention of
PKC-dependent vinculin phosphorylation following TPA treatment
suggested that the remaining apparent PKC
(as per hydroxyapatite
column purification and immunoreactivity) could explain the continued
response of these cells to insulin
(6, 7) .
gene encodes two mRNAs that originate from alternative
splicing of the C-terminal exons
(11) . The resulting
polypeptides, PKC
I and PKC
II, diverge in the sequence of
their C-terminal 50 or 52 amino acids,
respectively
(11, 12) . Fig. 1provides a schematic
of the functional organization of PKC
. The expression of different
polypeptides and prediction they will occur from the same gene by
alternative splicing has been observed for a growing number of genes
including fibronectin
(13) , the insulin receptor
(14) ,
and a nontransmembrane phosphotyrosine phosphatase
(15) . In
these cases, alternative splicing patterns are influenced by stages of
development, differentiation, growth factors, or disease. The
functional significance of alternatively spliced peptides is not fully
understood.
Figure 1:
Structure of PKCI
and -
II gene products. A schematic representation of PKC
as
deduced from cDNA sequence analysis (40) is shown. PKC
I and
-
II are designated as the 671- and 673-amino acid products,
respectively. Coding sequences are represented by blocks.
C1-C4 represent the conserved regions.
V1-V5 are the variable domains. C1 and
C2 form the regulatory domain (shaded). Cysteine-rich
zinc finger domains are indicated by ovals. The V5,
variable region is shaded. Regions corresponding to the
PKC
I probe used for the RNase protection assay and location of the
primers used for RT-PCR analysis are indicated below the
scheme. The numbering of nucleotides corresponds to that of Knopf
et al. (41).
Differential expression of both PKC messages has
been demonstrated in brain, hemopoietic cells, B lymphoblastoid cells,
and cardiac myocytes
(11, 12, 16, 17) .
Immunochemical methods with specific PKC
I, -
II, and
pan-specific antibodies demonstrate that both isoforms are present in
these cells and tissues as well as in rat soleus muscle, rat
adipocytes, and BC3H-1 murine
myocytes
(6, 18, 19) . These studies did not,
however, evaluate whether expression is regulated in vitro and
in vivo. We found that the levels of mRNA for PKC
I
declined quickly with insulin treatment, and PKC
II mRNA levels
increased, concomitantly, along with protein levels of PKC
II in
BC3H-1 myocytes. High levels of TPA mimicked insulin, and PKC
II
was the major isoform of PKC
retained in BC3H-1 myocytes following
chronic TPA treatment. To determine if PKC
I and -
II function
differently in response to insulin, both were stably overexpressed in
NIH-3T3 fibroblasts, which normally express PKC
and only
negligible levels of PKC
. PKC
II overexpression enhanced
insulin-stimulated 2-DOG uptake significantly above empty vector
control cells or stable transfectants overexpressing PKC
I. Thus,
one alternatively spliced form may be more specific than the other for
increasing glucose transport responses in certain cell types.
Cell Culture
BC3H-1 myocytes were grown in low
glucose Dulbecco's modified Eagle's medium with 10%
controlled process serum replacement (Sigma) for 11-14 days on
collagen-coated 100-mm plates. Glucose (25 mM) was added to
culture media for 24-48 h prior to treatment with varying
concentrations of insulin or TPA (Sigma) as indicated prior to
isolation of total RNA or cell lysates.
Isolation of RNA, Ribonuclease Protection Analysis (RPA),
and RNA Half-life
Total cellular RNA was obtained using a
single-step method
(20) . For determining mRNA half-life, cells
were treated with actinomycin D (5 µg/ml, Sigma) alone or
actinomycin D and insulin. For RPA, the PstI fragment of
PKCI (BIV5) was subcloned into pGEM-4Z (Fig. 1). Based on
predicted splice events (16), PKC
I-specific transcripts yield a
716-base pair protected band, while PKC
II encoded mRNA is detected
as a 566-base pair band. Antisense PKC
I was transcribed with T7
polymerase and labeled with [
-
P]UTP using
the Promega RNA transcription kit. Total RNA (10 µg) was hybridized
with the
P-labeled BIV5 cRNA probe (1.2-5
10
cpm/hybridization) using the RPA II ribonuclease
protection assay kit as described (Ambion). Protected RNA fragments
were detected following electrophoresis on denaturing polyacrylamide
gel (5% polyacrylamide, 8 M urea). The sizes of the probe and
protected fragments were verified by 5`-labeled
X174
DNA/HinfI ladder. Hybridized fragments were detected by
autoradiography.
Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
Generation of single-stranded cDNA templates for RT-PCR
was carried out on BC3H-1 myocyte total RNA using SuperScript
preamplification kit (Life Technologies, Inc.). The cDNA was aliquoted,
and three reactions were amplified: (a) sense primers
corresponding to the V3 region of PKC
(ATGAAACTGACCGATTTTAACTTCCTG) and antisense primers corresponding to
the V5 region of PKC
I (AAGAGTTTGTCAGTGGGAGTCAGTTCC), (b)
sense primers to V3 region and antisense primers corresponding to the
V5 region of PKC
II (CGGAGGTCTACAGATCTACTTAGCTCT), and (c)
sense and antisense primers for
-actin (Clontech). For all primer
pairs, cycling conditions were an initial 96 °C melt, 5 min, and
then 95 °C, 1 min; 58 °C, 1 min; 72 °C, 3 min. This cycle
was repeated 34 times ending with 95 °C, 1 min; 58 °C, 1 min;
and 72 °C, 10 min. Specificity of the primers was verified with
cDNA for PKC
I and PKC
II (from Dr. John Knopf, Genetics
Institute, Cambridge, MA).
Western Blot Analysis
Cell lysates (50 µg)
from BC3H-1 myocytes and NIH-3T3 cells overexpressing PKCI and
II were analyzed as described previously
(21, 22) .
PKC
I antibodies were obtained from Research and Diagnostic
Antibodies, from Drs. Susan Jaken and Susan Kiley (W. Alton Jones Cell
Science Center, Lake Placid, NY), or from Dr. Yusaf Hannun (Duke
University Medical Center, Durham, NC). PKC
II antibodies were
provided by Dr. D. Kirk Ways (East Carolina University, Greenville, NC)
or Drs. Jaken and Kiley. Following incubation with primary antibody,
blots were developed as described previously
(23) or with the
ECL kit (Amersham Corp.). The PKC antisera were characterized
previously
(16, 17) . Specificity of PKC
II antisera
obtained from Dr. D. Kirk Ways for PKC
II in BC3H-1 myocytes was
performed by preabsorption of antisera with peptides.
Overexpression of PKC
NIH-3T3 cells were stably transfected as described with
MTHI and -
II in NIH 3T3
Cells
II
(24) or MV7
I
(25) using Lipofectin (Life
Technologies, Inc.). Stable transfectants were selected in the presence
of 750 µg/ml G418 (Life Technologies, Inc.), and the more than 100
clones obtained were cultured in bulk cultures. In the case of
PKC
II, data from one stable clone is also shown with the stable
bulk transfected cells. Stable transfectants are grown in high glucose
Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. Overexpression was evaluated by Western blot analysis and by PKC
activity toward histone III-S (Sigma) as described
previously
(21) . 2-[1,2-
H]Deoxy-D-glucose
uptake-NIH-3T3 cells overexpressing PKC
I and -
II were
grown in high glucose Dulbecco's modified Eagle's medium
with 10% fetal bovine serum (Sigma) in 24-well plates until >80%
confluent. Prior to 2-[
H]DOG uptake, cells are
switched to Dulbecco's modified Eagle's medium without
serum overnight. 2-[
H]DOG uptake is assayed as
described previously
(21) . 2-Deoxyglucose uptake refers to
transport across the plasma membrane operating in tandem with
phosphorylation by hexokinase.
PKC
To simultaneously examine PKCI mRNA Switches to PKC
II mRNA in the
Presence of Insulin
I and
-
II mRNA in BC3H-1 myocytes, we used a cRNA probe spanning the
alternative splice site for PKC
I and -
II for RPA
(Fig. 1). When BC3H-1 myocytes were treated overnight with
increasing concentrations of insulin, cellular expression switched
PKC
mRNA from the PKC
I to PKC
II mRNA as demonstrated by
the appearance of the shorter, 566-base pair, protected fragment
(Fig. 2A). This suggested that insulin regulated
alternative splicing of PKC
mRNA. This phenomenon was
dose-dependent and was apparent at 0.2 nM insulin. Initially,
PKC
I was the primary mRNA detected by this assay. In the presence
of insulin, PKC
II mRNA became the primary splice product. A time
course showed that the PKC
I to PKC
II mRNA shift was complete
after 15 min (Fig. 2B).
Figure 2:
RNase protection analysis of PKCI and
II mRNA in BC3H-1 myocytes after insulin treatment. PanelA, RNase protection analysis of BC3H-1 myocytes following
treatment with insulin. Glucose (25 mM) was added to culture
media prior to treatment with varying concentrations of insulin
overnight prior to isolation and analysis of total RNA (10 µg) as
described under ``Materials and Methods.'' Y1 and
Y2 refer to the undigested and digested probe. Panel
B, cells were treated with insulin (200 nM) for 15 min to
1 h prior to isolation of total RNA. The data shown are representative
of more than five separate experiments.
Demonstration of PKC
To establish that insulin regulated the expression of
PKCI and PKC
II mRNA Switch by
RT-PCR
I and -
II mRNA using another method, the alternative
splice products were examined by RT-PCR (Fig. 1). This assay
allows for the relative evaluation of PKC
I and -
II mRNA, but
it is not quantitative. The amplification spans several splice sites in
mature PKC
mRNA and eliminates amplification of any precursor mRNA
forms. As observed with the RPA, the switch from PKC
I to
PKC
II mRNA was complete after 15 min following insulin treatment
(Fig. 3). With this assay, both PKC
I and -
II mRNA were
detected in unstimulated BC3H-1 myocytes, but only minor traces of
PKC
I mRNA could be detected in cells that had been incubated with
insulin for up to 60 min.
Figure 3:
RT-PCR analysis of BC3H-1 myocyte
PKCI and PKC
II mRNA. Total RNA (1 µg) was subjected to
RT-PCR. Polymerase chain reaction fragments were fractionated on
agarose gels and transferred to nylon for verification of products by
Southern blot using a
P-labeled full-length PKC
I/II
cDNA probe as shown. The data shown are representative of three
separate experiments.
Determination of Insulin Effect on PKC
The rapid disappearance of the PKCI mRNA
Stability
I mRNA in the
presence of insulin suggests that another event accompanying the switch
to PKC
II mRNA is the destabilization of PKC
I mRNA. To
evaluate this, cells were treated with actinomycin D alone or with
insulin for varying times. In the presence of insulin, PKC
I mRNA
was decreased by 75% after 15 min, demonstrating that the effect of
insulin on PKC
I mRNA was to destabilize the message
(Fig. 4). In the absence of insulin, PKC
I message was only
detected for up to 1 h in cells treated with actinomycin D. This
indicates that message stability may play some role in regulating
differential expression of PKC
I mRNA.
Figure 4:
Message stability of PKCI mRNA in
BC3H-1 myocytes. Total RNA from BC3H-1 myocytes previously treated with
5 µg/ml actinomycin D or actinomycin D with insulin (200
nM) for 15 min was analyzed by RPA with the BIV5 probe cut
with BbuI. Only PKC
I is evaluated with this probe.
Autoradiograms were analyzed by
densitometry.
Phorbol Ester Mimicked Insulin in Switching the Splice
Product at Concentrations Exceeding 1 µM
To
determine whether the activation and subsequent down-regulation of PKC
was involved in PKC alternative splicing, PKC was directly
activated with TPA. Under these conditions, we have shown that BC3H-1
myocytes appear to retain PKC
immunoreactive
material
(7, 8) . TPA treatment increased PKC
I mRNA
levels by 50% at lower concentrations, which is not surprising, as TPA
induces the basal promoter of PKC
(26) . However, at higher
concentrations of TPA, 1-5 µM, alternative splicing
of PKC
I to PKC
II was induced (Fig. 5).
Figure 5:
Effect of TPA on PKCI and PKC
II
mRNA. Total RNA from BC3H-1 myocytes cultured overnight in the presence
of 1 nM to 5 µM TPA or 0.01% dimethyl sulfoxide
(control vehicle) was assayed by RPA using the BIV5 probe. The effect
of TPA was noted in three separate
experiments.
Demonstration of Increased PKC
To determine whether the shift from
PKCII Protein Levels with
Insulin and TPA Treatment
I to PKC
II mRNA following insulin treatment was reflected
at the protein level, lysates from insulin-treated BC3H-1 myocytes were
analyzed. Very low levels of PKC
II protein (a single 82 kDa band)
were detected in untreated cells, but levels increased more than 5-fold
after 30 min of treatment and more than 12-fold following 24 h of
insulin treatment (Fig. 6). The induction of PKC
II by
insulin was apparent using antibodies from Drs. Jaken and Kiley and Dr.
Ways. PKC
I, the predominant PKC
mRNA and protein form in
resting BC3H-1 myocytes, diminished to half of its initial level with
insulin treatment. PKC
I was detected as a doublet at 74-76
kDa and sometimes as another doublet at 80-82 kDa in whole cell
lysates with antibodies from Drs. Jaken and Kiley and Dr. Hannun, Life
Technologies, Inc. (affinity purified), and Research and Diagnostic
Antibodies. A PKC
II immunoreactive band (at 80-82 kDa) was
demonstrated in BC3H-1 myocytes following overnight treatment with 5
µM TPA with antisera from Dr. Ways (Fig. 7) and Drs.
Jaken and Kiley (data not shown). PKC
I levels (at 74-76 kDa)
were diminished by more than 80% following TPA treatment as detected
with antibody from Life Technologies, Inc. that recognizes the (V3)
region.
(
)
The greater relative diminution of
PKC
I following treatment with TPA versus insulin may
reflect the ability of TPA to activate PKC
I while insulin may
preferentially stimulate PKC
II under these conditions, and insulin
may not enhance the proteolytic turnover of PKC
I to the same
extent as TPA. Thus, the switch from PKC
I to PKC
II mRNA is
paralleled by the increased expression of PKC
II.
Figure 6:
Effect of insulin on PKCI and
PKC
II protein expression in BC3H-1 myocytes. BC3H-1 myocytes were
treated with insulin (200 nM) as indicated prior to the
addition of Laemmli lysis buffer to washed cell monolayers. Results of
densitometric scans are shown with insets of the Western blot
data using PKC
I (lower band analyzed) and PKC
II antibodies
from Drs. Susan Jaken and Sue Kiley as described under ``Materials
and Methods.'' Similar patterns for PKC
I (a doublet) and
PKC
II were noted in three separate passages of cells with three
different antibodies to PKC
I and two antibodies to
PKC
II.
Figure 7:
Effect of phorbol esters on PKCI and
PKC
II protein expression in BC3H-1 myocytes. BC3H-1 myocytes were
treated with vehicle (A) or 5 µM TPA (B)
overnight. Cytosolic (75 and 60 µg of protein for PKC
I and
PKC
II, respectively) and membrane (150 and 40 µg of protein
for PKC
I and PKC
II, respectively) fractions were prepared for
Western blot analysis using alkaline phosphatase detection as described
previously (21). Antibody against PKC
hinge region (Life
Technologies, Inc.) was used here to detect PKC
I as the lower, 74
kDa, band. (This antibody does not detect PKC
II unless
chemiluminesence is used for detection.) PKC
II antibody was from
Dr. D. Kirk Ways. Similar decreases in PKC
I and increases in the
upper PKC
II band were detectable using the Life Technologies, Inc.
PKC
antibody with ECL detection. The 80 kDa marker is indicated by
.
The increase
in PKCII protein with insulin occurs rapidly, and its induction
may play an important role in glucose transport. The functional
significance of this finding is difficult to address in the BC3H-1
myocytes due to the expression of PKC
and other PKC isoforms (6).
There is also the potential for counter-regulation of these PKC
isoforms and glucose transporter levels with insulin. We found that the
insulin effect on glucose uptake was enhanced in TPA-treated BC3H-1
myocytes even though acute TPA stimulatory effects were no longer
observed. For example, we found 789 ± 20 cpm of
2-[
H]DOG uptake/well for basal uptake versus 1749 ± 35 cpm for insulin (200 nM), and, following
1 µM TPA treatment overnight, we found 1161 ± 69
cpm for basal versus 3609 ± 139 cpm for insulin-treated
cells.
Potential Functional Significance of the Switch from
PKC
The observation that PKCI to PKC
II
is an
insulin responsive PKC isozyme infers that the switch from PKC
I to
PKC
II expression induced by insulin or high concentrations of
phorbol ester may be of functional significance for glucose uptake. If
PKC
II protein expression reflects an increase in the alternative
expression of PKC
II mRNA, one might predict that
insulin-stimulated glucose transport will be increased when PKC
II
is increased. Although we found that insulin effects on glucose uptake
were retained and enhanced by chronic TPA treatment, it was also
evident that another PKC isozyme, PKC
, was down-regulated in
myocytes
(6, 7) . To avoid the effects of down-regulating
PKC
and other isozymes in BC3H-1 myocytes, we used NIH-3T3 cells
overexpressing PKC
I and PKC
II to examine the effects of
increased PKC
II. PKC
and negligible amounts of PKC
are
expressed in parent NIH-3T3 cells used here
(27) , thus allowing
us to assess the role of PKC
isoforms without encountering
significant endogenous PKC
as detected in BC3H-1 myocytes. Western
blot analysis of transfected NIH-3T3 cells demonstrated the stable
overexpression of PKC
I and PKC
II (Fig. 8). PKC activity
as determined with histone III-S as substrate was increased 2-fold in
cytosol and 3-4-fold in membranes compared with empty vector
control cell lines for both isozymes.
Figure 8:
Characterization of PKCI and
PKC
II protein overexpression in stably transfected NIH-3T3 cells
by Western blot analysis. Cell lysates (50 µg) from NIH-3T3 cells
overexpressing PKC
I and PKC
II were prepared as described
previously (7). Antisera to PKC
I was from Research and
Diagnostics, and antiserum for PKC
II was from Dr. D. Kirk Ways.
LanesA, C, and E denote the empty
vector control cell lines; laneB represents
transfected cells overexpressing PKC
I; laneD is
from transfected cells overexpressing PKC
II; and laneF is a clonal line (clone 10) selected from the batch
transfection of PKC
II.
We previously found in
adipocytes and BC3H-1 myocytes that PKC contributes to
insulin-stimulated glucose transport
(28, 29) . In
NIH-3T3 cells, we found that 1 and 10 nM insulin increases
glucose uptake by 10 and 30%, respectively, in parental or control
cells (). This effect of insulin is statistically
significant and may reflect a ``PKC
component'' of
glucose transport in NIH-3T3 cells. Cells overexpressing PKC
I did
not respond differently than control cells in that insulin-stimulated
2-DOG uptake remained at 10 and 30% above basal uptake. Cells
overexpressing PKC
II demonstrated enhanced, dose-dependent
(1-10 nM), insulin-stimulated 2-DOG uptake of 60 and
100%, respectively, above basal uptake. This enhanced response was also
observed in the clonal line overexpressing PKC
II as well as in
stable bulk cultures of PKC
II transfectants. The insulin effect is
PKC-dependent as indicated by inhibition of 2-DOG uptake with GF
109203X (Calbiochem) and CGP 41251 (Ciba Giegy), specific PKC
inhibitors
(30, 31) . These results indicate that
PKC
II but not PKC
I is capable of further enhancing
insulin-stimulated glucose transport in NIH-3T3 cells and further
suggests a physiological significance in the regulation of PKC
alternative splicing.
exon detected here corresponds to the C-terminal variable
region, V5, which is believed to determine substrate
specificity
(1) .
I and PKC
II mRNA are detected
in the same sample following RNase digestion. Probes directed solely to
the V5 region or downstream untranslated region can hybridize with
precursor PKC
I mRNA in addition to alternatively spliced mRNA, and
the detection of a splicing event is not obvious. We further supported
our observation by amplifying mRNA by RT-PCR. The ability to detect
PKC
II mRNA in untreated cells using RT-PCR is attributed to the
superior sensitivity of the RT-PCR method to amplify low message
levels. Using both methods, we found that PKC
I mRNA levels quickly
diminish with insulin treatment and, in contrast, PKC
II mRNA
levels increase.
I mRNA
may be that insulin diminishes message stability. Although this was
found to be the case in experiments where cells were treated with
actinomycin D, message destabilization does not explain why PKC
II
mRNA levels are then increased, and it is clear that alternative
splicing to the default product, PKC
II, is occurring. One scheme
of alternative splicing for PKC
is that retention of the
PKC
II exon results in PKC
II mRNA. When this exon is excised,
PKC
I is encoded, and the PKC
I polyadenylation site was used
by both PKC
I and PKC
II transcripts
(11) . A likely role
for insulin might involve regulation of splicing factors required for
the mRNA splicing complex, and the factors involved in alternative
pre-mRNA splicing are possibly induced by insulin
(32) . One such
factor was isolated from regenerating liver (33). It is of interest
that insulin regulates alternative splicing of insulin receptor
isoforms in hepatic FAO cells similar to the time course we see in the
BC3H-1 myocyte for PKC
splicing
(34) . Changes in
alternative splicing of the insulin receptor is also reported in a
diabetic patient
(14) .
I and PKC
II by BC3H-1 myocytes. Using two
techniques for detecting mRNA and six different antibodies, we found
mRNA and protein PKC
levels are regulated by insulin and phorbol
ester. Recent studies from other labs failed to detect PKC
I or
PKC
II in BC3H-1 myocytes
(35) . Moreover, as will be
reported later, PKC
I was not only fully down-regulated by
overnight TPA treatment, but it was acutely translocated by TPA (as
well as by insulin). The failure of Stumpo et al.(35) to detect PKC
I in untreated BC3H-1 myocytes may be due
to differences in antisera, detection techniques or cell culture. The
chemiluminesence method used here may be more sensitive for detecting
PKC
I. In our studies, PKC
I appears at a lower apparent
molecular mass, 74-76 kDa, than PKC
II, which migrates at
80-82 kDa. PKC
I and
II isoforms were often detected as
doublets. The difference in apparent molecular masses between the two
isozymes may reflect a number of cellular phosphorylations that PKC may
undergo in the processing of newly translated protein or limited
proteolysis of PKC
I as suggested
(36) . In addition, we
frequently change our culture stocks, and cells are serum-starved prior
to experiments.
protein analyses of myocyte lysates are
similar to studies with IM-9 and BJA-B cell lines, in which PKC
II
migrated as an upper 77 kDa and lower 74 kDa band
(16) . PKC
I migrated at a lower molecular mass in that study. The upper band
did not down-regulate with long term phorbol ester
treatment
(16) . In our studies, a similar upper band was induced
with TPA and insulin in BC3H-1 myocytes and is consistent with a switch
from PKC
I to PKC
II. In these studies it was not possible to
compare the amounts of PKC
I with PKC
II. However, the relative
changes in PKC
I and PKC
II with insulin and phorbol ester
reflect changes related to mRNA levels.
II protein accompany
the switch of mRNA products. In this case, we have linked the
functional significance of alternative mRNA splicing of PKC
I and
II to glucose transport. Glucose transport is stimulated by
insulin in a number of tissues and cells
(37) . There appear to
be PKC
and PKC
-dependent components in rat adipocytes
demonstrated with antisense oligonucleotides to down-regulate these
isozymes
(29) . In other studies, insulin-dependent glucose
transport was restored by introducing PKC
and PKC
enzymes
into PKC-depleted adipocytes (28). For the present studies, we stably
transfected NIH-3T3 cells to overexpress PKC
I and PKC
II.
Overexpression of PKC
II but not PKC
I further enhanced
insulin-dependent increases in glucose transport. Whether this same
relationship between PKC
I and PKC
II is relevant to other cell
types must await further studies. It may be of interest that both
BC3H-1 myocytes and NIH-3T3 cells, murine cell lines, express
GLUT1-type glucose transporters.
I and -
II have also been demonstrated in other systems.
PKC
I was found to be more efficient in inactivating glycogen
synthase kinase-3
by phosphorylation than PKC
II
(38) .
In cardiac myocytes the redistribution of PKC
I from the
perinuclear membrane to the nucleolus while PKC
II translocated
from the cytoplasm to the perinuclear and plasma membrane following TPA
and norepinephrine treatment was shown
(39) . These studies
demonstrate that not only do the catalytic potentials of PKC
I
and-
II differ, but their ability to translocate to distinct
intracellular compartments is also distinct.
isoform versus the other, and their expression is regulated by insulin. Insulin
stimulation of cells leads to increases in diacylglycerol and, hence,
activates several PKC isozymes. Our data suggest that this rather
nonspecific activation of PKC isozymes is further fine tuned by the
control of PKC isozyme expression via alternative splicing of PKC
.
The rapidity of the switching observed in BC3H-1 myocytes infers that
this process has functional consequences under varying physiological
states. Although it is recognized that developmental stages or cell
types can express different PKC
transcripts
(11, 12, 16) , this study indicates
that a cell can alter expression of one splice product versus another in response to a peptide hormone to alter metabolic
pathways.
Table:
Effect of insulin on 2-deoxyglucose uptake in
NIH-3T3 cells stably transfected to overexpress PKCI and
PKC
II
II-clone 10
denotes one clone from the heterogeneous pool of transfectants. Prior
to 2-[
H]DOG uptake, cells are switched to
Dulbecco's modified Eagle's medium without serum overnight
(21). Data are the mean and S.E. of picomoles uptake for the number of
separate cell passages assayed (n). Results of two-way ANOVA
at p < 0.05 are indicated by * for the effect of insulin on
2-DOG uptake and ** for the effect of the PKC
II vector on the
insulin effect compared with vector control.
, presumably PKC
I, when
blots were developed and visualized using alkaline phosphatase. This
antibody detected higher molecular mass forms of PKC
II as well as
lower molecular mass PKC
I when Amersham ECL was used for
visualization.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.