From the The protein kinase C Protein kinase C The regulation of PKC Cell Culture--
L6 rat skeletal myoblasts (obtained from Dr.
Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown
in Reverse Transcriptase-Polymerase Chain Reaction--
Total RNA
was isolated from L6 myotubes (13) and 1 µg was used to synthesize
first strand cDNA using an oligo(dT) primer and Superscript II
reverse transcriptase (Life Technologies pre-amplification kit). For
quantitative RT-PCR, 5% of the cDNA was amplified in the presence
of 10 Departments of Biochemistry and Molecular
Biology and
Internal Medicine,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(PKC
) gene encodes two
isoforms, PKC
I and PKC
II, as a result of alternative splicing.
The unique mechanism that underlies insulin-induced alternative
splicing of PKC
pre-mRNA was examined in L6 myotubes. Mature
PKC
II mRNA and protein rapidly increased >3-fold following
acute insulin treatment, while PKC
I mRNA and protein levels
remained unchanged. Mature PKC
II mRNA resulted from inclusion of
the PKC
II-specific exon rather than from selection of an alternative
polyadenylation site. Increased PKC
II expression was also not likely
accounted for by transcriptional activation of the gene or increased
stabilization of the PKC
II mRNA, and suggest that PKC
II
expression is regulated primarily at the level of alternative splicing.
Insulin effects on exon inclusion were observed as early as 15 min
after insulin treatment; by 20 min, a new 5
-splice site variant of
PKC
II was also observed. After 30 min, the longer 5
-splice site
variant became the predominate species through activation of a
downstream 5
splice site. Similar results were obtained using IGF-I.
Although the role of this new PKC
II mRNA species is presently
unknown, inclusion of either PKC
II-specific exon results in the same
PKC
II protein.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(PKC
)1 is a member of the
PKC family, which is a serine/threonine kinase family that mediates
cellular responses elicited by hormones, neurotransmitters, and growth
factors (1-3). The PKC
gene encodes two mRNAs that originate
from alternative splicing of exons encoding the carboxyl terminus (see
Fig. 1A) (4). The resulting polypeptides, PKC
I and
PKC
II, diverge in the sequence of their COOH-terminal 50 (PKC
I)
or 52 (PKC
II) amino acids, respectively (4, 5). PKC
is involved
in insulin-stimulated glucose transport based on studies in the BC3H-1
myocytes, rat soleus muscle, adipocytes, L6 myotubes, and vascular
smooth muscle cells (6-11). Our work demonstrated that PKC
I and
-
II have different and distinct functions in response to insulin in
stable transfectants of NIH-3T3 fibroblasts (12). PKC
II
overexpression enhanced insulin-stimulated 2-deoxyglucose uptake
significantly above control cells or stable transfectants
overexpressing PKC
I (12). The contribution of PKC
II activity to
insulin-stimulated [3H]2-deoxyglucose uptake was also
examined in rat L6 myotubes, a cell line phenotypically similar
to skeletal muscle expressing GLUT4 and GLUT1 type glucose transporters
(8). Transient expression of a PKC
II-specific dominant negative
blocked insulin-stimulated 2-deoxyglucose uptake (8). CG53353, a
PKC
II-specific inhibitor at 1 µM, also inhibited
insulin-stimulated 2-deoxyglucose uptake (8). Thus, one alternative
splice variant of the PKC
gene is more effective than the other as a
positive transducer for glucose transport responses.
gene expression has not been extensively
studied. Our laboratory was the first to demonstrate that expression of
these two messages was regulated acutely by insulin (12). In BC3H-1
myocytes, insulin induced alternative splicing of the PKC
mRNA,
thereby switching expression from PKC
I to PKC
II mRNA (12).
The switch in mRNA was reflected by increased protein levels of
PKC
II (12). However, the mechanism of how insulin regulated the
post-transcriptional processing of PKC
pre-mRNA was unclear.
There were several options. For example, insulin could affect
polyadenylation site selection to define the carboxyl terminus, and, in
another case, insulin could affect the inclusion of the
PKC
II-specific exon into the mature message since the PKC
II-specific exon includes a translation stop codon. Increased transcription or changes in PKC
II mRNA stability may also affect post-transcriptional processing. In this report, each of these possibilities were considered in L6 skeletal muscle cells, which, unlike the BC3H-1 myocytes, express the GLUT4 type glucose transporter and are a fully differentiating cell model for insulin action. We
demonstrate that insulin specifically enhances the inclusion of an exon
encoding the last 52 COOH-terminal amino acids of PKC
II. Insulin
also activated two 5
splice sites, a previously known site and a
second newly identified 5
splice site, to include the
PKC
II-specific exon, which contains a stop codon, thereby encoding
only the PKC
II protein. This is the first report of a hormone
affecting the 5
splice site selection in pre-mRNA. Since our
knowledge of alternative splicing is derived primarily from in
vitro biochemical approaches, this study expands our understanding of how splicing is regulated in vivo in eukaryotes and
demonstrates L6 skeletal muscle cells as a model for hormone-induced
alternative splicing.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-minimum essential medium (
-MEM) supplemented with 10% fetal
bovine serum (Sigma) to confluence. Myoblasts were fused into myotubes
by changing media to
-MEM supplemented with 2% fetal bovine serum
for 2-4 days after confluence. Cells were incubated in
-MEM + 0.1%
bovine serum albumin for 6 h and placed in phosphate-buffered
saline (PBS) + 0.1% bovine serum albumin just prior to treatment with insulin (200 nM) or IGF-I (Long R3 IGF-I,
GroPep) (20 ng/ml). For mRNA stability studies, actinomycin D (10 µg/ml) was added 5 min prior to addition of insulin or PBS sham
control.
2 amol of mimic DNA using primers specific for
PKC
I, PKC
II, or
-actin and Taq DNA polymerase from
Perkin-Elmer. The PKC
II-specific primers correspond to Primer A, a
sense primer to the V3 region of PKC
(5
-ATGAAACTGACCGATTTTAACTTCCTG-3
), and Primer B, an antisense primer
corresponding to the V5 region of PKC
II
(5
-CGGAGGTCTACAGATCTACTTAGCTCT-3
) (Fig. 1B). The
PKC
I-specific primers were Primer C, a sense primer corresponding to
the C3 region (5
-CCGCCTCTACTTTGTGATGGA-3
), and Primer D, an antisense
primer corresponding to the V5 region of PKC
I
(5
-TGCCTGGTGAACTCTTTGTCG-3
) (Fig. 1B). Sense and antisense primers for
-actin (CLONTECH) were used to
normalize for total RNA. Mimics (competitors) for PKC
I and PKC
II
were constructed using the CLONTECH MIMIC
construction kit (CLONTECH). The mimic is a neutral
piece of DNA containing primer sequences for either PKC
I or PKC
II
on its 5
and 3
ends. The mimic will specifically compete for primer
binding sites with the target PKC
I or PKC
II cDNA. Extension
rates of competitor mimics are within 5% of the target cDNA
extension rate. Following 30 cycles of amplification in a Biometra
Trioblock thermocycler (
-actin and PKC
II: 94 °C, 1 min;
58 °C, 1 min; and 72 °C, 3 min; PKC
I: 94 °C, 30 s;
58 °C, 30 s; and 72 °C, 1 min), 20% of the PCR reaction was
resolved on a 1% agarose gel. The photograph of the ethidium
bromide-stained PCR products was quantified by scanning densitometry.
II-specific exon inclusion, Primer E, an upstream
sense primer corresponding to the C4 kinase domain common to both
PKC
I and -
II (5
-GTTGTGGGCCTGAAGGGGAACG-3
), and Primer D
(described above) were used. This RT-PCR assay allows for relative comparison of PKC
II versus PKC
I mRNA levels.
Western Blotting--
L6 myotube cell lysates (40 µg) were
subjected to 9% SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(14). Proteins were electrophoretically transferred to nitrocellulose
membranes, blocked with Tris-buffered saline, 0.1% Tween 20 containing
5% nonfat dried milk, washed, and incubated with a polyclonal antibody
against either the COOH terminus of PKCII or PKC
I (Santa Cruz
Technologies and Dr. Yoshiko Akita, Tokyo Metropolitan Institute of
Medical Science, Tokyo, Japan). Detection was performed using enhanced chemiluminescence (ECL, Amersham).
Random Prime Labeling--
PKCII cDNA (25 ng) was labeled
with [
-32P]CTP (3000 Ci/mmol) using random prime
labeling (14). Labeled cDNA was purified from unincorporated
nucleotides using G50 Sephadex chromatography (14).
Southern Blot Analysis--
DNA was transferred to Hybond N
nylon membrane (Amersham) and baked at 80 °C for 2 h. Blots
were prehybridized for 1 h in 5 × SSC, 5 × Denhardt's
solution, 0.5% sodium dodecyl sulfate (SDS), and 50% formamide at
43 °C. Random prime labeled PKCII cDNA (1 × 106 cpm) was added, and blots were hybridized for 16 h
at 43 °C and washed for 15 min at room temperature with 1 × SSC, 0.1% SDS and 0.1 × SSC, 0.1% SDS. Blots were exposed to an
imaging screen and quantitated using the Molecular Dynamics
PhosphorImager system.
Transient Transfections and Reporter Gene Assays--
PKC
promotor (
2200 to +43) constructs were cloned into a luciferase
reporter plasmid as described previously (15). L6 rat skeletal muscle
myotubes were grown in 35-mm tissue culture dishes and co-transfected
with 3.5 µg of the PKC
promotor/luciferase construct and 1 µg of
SV40
-galactosidase vector using the calcium phosphate method (14).
Cells were incubated in calcium phosphate/DNA precipitate for 16 h, washed twice with phosphate-buffered saline, and placed in medium
(2% fetal bovine serum) for 8 h (14). Serum was removed for
6 h prior to insulin (200 nM) treatment. Cells were
lysed with 1 × lysis buffer (Promega Corp.) and assayed for luciferase and
-galactosidase activity following the manufacturer's protocol (Promega Corp.).
Sequencing-- All PCR products were verified by sequencing at the University of South Florida Genome Center.
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RESULTS |
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Insulin Increased PKCII Mature mRNA and Had No Effect on
PKC
I Mature mRNA Levels--
Although insulin regulation of
alternative splicing of PKC
II pre-mRNA has been reported
previously in BC3H-1 myocytes, the mechanism by which insulin affected
splicing was not examined. Since the mechanism is likely to be relevant
in insulin action, we used L6 myotubes in this study. These cells
represent a model of fully differentiated skeletal muscle myotubes that
express GLUT4 type glucose transporters, in contrast to the BC3H-1
myocytes. Using competitive RT-PCR, the expression of PKC
II mature
mRNA was examined in response to acute insulin treatment in
differentiated L6 myotubes (Fig.
1B). Upon treatment with
insulin (200 nM) for 15 min, a 3-fold increase in PKC
II
mRNA was observed (Fig. 2). The
up-regulation of PKC
II mRNA increased 3.5-4-fold at 1 h
after insulin treatment. This extended our earlier findings in BC3H-1 myocytes to L6 myotubes for examining the molecular basis of
hormone-induced alternative splicing.
|
|
The PKCII mRNA Increase Is Reflected at the Protein
Level--
To examine whether insulin effects on PKC
I and -
II
mRNA were reflected at the protein level, polyclonal antibodies
specific for the COOH terminus of either PKC
I or PKC
II proteins
were used for detection on Western blots. Insulin treatment increased PKC
II immunoreactive protein levels greater than 3.5-fold (350%), while PKC
I immunoreactive protein levels remained unchanged (Fig. 3). The data shown are composites of five
separate experiments with each time point examined for an individual
effect. Thus, the increase in PKC
II mRNA expression was directly
reflected at the protein level between 15 min and 24 h as with the
BC3H-1 myocytes. Differences in the amount of basal PKC
II
immunoreactive protein between experiments are attributed to variables
in cell culture and sensitivity of chemiluminescence detection. The
overall increase in PKC
II immunoreactive protein following insulin
treatment was consistent.
|
The Rapid Up-regulation in PKCII mRNA Was Not Reflected by
Increased Transcriptional Activity--
If the alternative splicing
that leads to mature PKC
II mRNA is under negative regulation by
a limited amount of RNA transactivating factors, a rapid increase in
PKC
pre-mRNA via transcriptional activation may account for
increased PKC
II expression. To examine whether the up-regulation of
PKC
II mRNA was related to an increase in transcriptional
activity, several PKC
promotor constructs of varying lengths
including a (
2200 to +43) construct were examined for insulin
responsiveness. The PKC
promotor (
511 to +43)/luciferase construct
was the only construct activated in response to insulin following
transient transfection (data not shown). This construct contains an
AP2, an E-box, and two Sp1 sites. With this construct, no change in
PKC
promotor/luciferase activity was observed within 30 min and only
modest increases were noted after 16 h. Based on these data and
the observation that PKC
I mRNA is not increased in response to
insulin, the early increase in PKC
II mRNA was not likely due to
an increase in transcriptional activity of the PKC
gene. Treatment
with tetradecanoyl phorbol acetate to activate the promotor and
transfection with a fully active SV40/luciferase construct were used as
positive controls to demonstrate promotor activation (15). Transfection
efficiency was normalized using a ratio of the co-transfected
pSV-
-galactosidase activity.
The Early Up-regulation in PKCII mRNA Was Not Likely Due to
Increased Message Stability--
The possibility of
post-transcriptional regulation of PKC
II expression at the level of
message stability was also evaluated. To demonstrate whether PKC
II
mRNA was up-regulated via increases in message stability, insulin
effects on PKC
I and PKC
II mRNA levels were determined after
treatment with the transcription inhibitor, actinomycin D, after 1 h of acute insulin treatment. Using competitive RT-PCR, no differences
in mRNA levels were detected between control and insulin-treated L6
myotubes (data not shown). This suggests that insulin effects on
PKC
II message were not likely due to rapid increases in
mRNA stabilization. Also of note is the observation that insulin
did not destabilize PKC
I message as in the BC3H-1 myocytes,
demonstrating either the lack of a hormone-induced destabilization
system or a requirement for PKC
I in insulin-activated metabolic or
mitogenic processes in L6 myotubes.
Alternative Polyadenylation Site Selection Did Not Occur in
Response to Insulin--
Since insulin treatment did not result in
transcriptional activation or increased message stability, the
possibility that PKCII expression was regulated primarily at the
level of alternative splicing was likely. Early studies by Nishizuka
and co-workers (4) suggested PKC
II mRNA is encoded via inclusion
of the PKC
II exon into the mature mRNA transcript in brain
tissue. In B lymphoblastoid cells, however, Hannun and co-workers (16)
demonstrated that polyadenylation site selection might govern PKC
II
splicing. Based on the information available about the arrangement of
terminal exons in the PKC
gene, it was possible that the
alternatively spliced PKC
II mRNA resulted from one of these two
mechanisms, inclusion of the PKC
II-specific exon or alternative
polyadenylation site selection, in L6 myotubes. Regulation of either
mechanism by a hormone had not been demonstrated. In the case of
polyadenylation site selection, insulin would activate a
polyadenylation site located downstream of the PKC
II exon, thereby
producing mature PKC
II mRNA. In the case of an exon inclusion
mechanism, the PKC
I polyadenylation site would be used, but a stop
codon in the PKC
II exon terminates translation and results in a
protein with the PKC
II COOH terminus. If the PKC
II exon were
skipped, then PKC
I would be encoded. To determine if the increase in
PKC
II mRNA was due to regulation of PKC
II polyadenylation
site selection, an RT-PCR-based assay was designed. Using primer E, a
sense primer to the C4 region, and primer G, an antisense primer to a
sequence downstream of the PKC
II exon and near the PKC
II
polyadenylation site, only a polyadenylated form of PKC
II mRNA
could be amplified. Neither control L6 myotubes nor insulin-treated L6
myotubes expressed a polyadenylated form of PKC
II even after 55 PCR cycles (Fig. 4). Therefore, the
increase in PKC
II mRNA in response to insulin was not likely due
to regulation of PKC
II polyadenylation site selection. As a positive
control, 10
3 amol of a PKC
II cDNA containing the
3
-untranslated region of the polyadenylated form of PKC
II was
amplified (obtained from Dr. S. Ohno, Yokohama City University School
of Medicine, Yokohama, Japan).
|
Insulin Enhanced the Inclusion of the PKCII Exon into the Mature
mRNA Transcript--
Since alternative polyadenylation site
selection did not likely account for the effect of insulin on PKC
II
expression, the possibility that insulin affected inclusion of the
PKC
II-specific exon in L6 myotubes was a likely alternative. An
RT-PCR assay that allowed for the simultaneous detection of PKC
II
and PKC
I mRNA utilizing primer E, a sense primer to the upstream
PKC
common exon (C4 kinase domain), and primer D, an antisense
downstream primer specific for PKC
I (V5 domain) was designed (Fig.
5, A and B). In
response to acute insulin treatment, PKC
II message (374-bp PCR
product) increased to levels that exceeded PKC
I message (153-bp PCR
product) (Fig. 6). The amount of mature
PKC
II mRNA increased from 15% exon-included message to 60% by
15 min to a maximum of 80% exon-included message at 30 min after
treatment with insulin. This demonstrated that insulin rapidly enhanced inclusion of the PKC
II-specific exon.
|
|
Identification of a Previously Unidentified 5 Splice Site for the
PKC
II Exon Encoding an Additional Splice Variant of
PKC
II--
An additional, longer PCR product was also observed
following exon inclusion analysis, which did not correspond to the
anticipated PKC
II amplification product (Figs. 6) (4). The new
fragment hybridized with a labeled PKC
cDNA probe upon Southern
blot analysis suggesting that the amplified fragment was not
artifactual. Sequence analysis revealed it was a new splice variant of
PKC
II, which included approximately 136 nucleotides of additional
extended 3
-untranslated sequence (Fig. 5C). After comparing
this sequence to the published sequence of the PKC
II exon and
surrounding intronic sequences, the activation of a second 5
splice
site, CAG/GTGGCAT, was demonstrated (Fig.
7A) (4). Insulin action
culminated in the activation of both 5
splice sites that resulted in
exon inclusion (Fig. 7, A and B).
|
Insulin Sequentially Activates a Second 5 Splice Site in the
PKC
II Exon--
To further investigate the effects of insulin on
exon inclusion and the activation of the two 5
splice sites, we
amplified the region between the PKC
II exon and the PKC
I terminal
exon (Fig. 5, A and C). This RT-PCR-based assay
examines specifically exon-included PKC
II mRNA and therefore
increases our detection sensitivity for PKC
II message. We found that
insulin activated the second downstream 5
splice site in a
time-dependent manner. After a 20 min insulin treatment,
activation of the second splice site was detected by RT-PCR followed by
Southern blot analysis. By 30 min after insulin treatment, the second
splice site was preferentially activated since the longer transcript
"exceeded" transcripts using the conventional 5
splice site by a
ratio of 2 to 1 (Fig. 8). Acute IGF-I
treatment also activated the second 5
splice site at 30 min,
demonstrating that other related peptide hormones have the capability
of affecting PKC
II exon inclusion and 5
splice site selection in L6
myotubes (Fig. 8). This is the first report of a hormone affecting 5
splice site selection and the first report of IGF-I affecting
alternative splicing.
|
Insulin Effects on mRNA Splicing Are Specific for PKCII
mRNA in L6 Myotubes--
The effect of insulin on alternative
splicing of the insulin receptor was also reported in FAO cells, but
the mechanism was neither specified nor elucidated. To examine if the
effects of insulin on exon inclusion reflect a more general effect on
splicing, the potential regulation of insulin receptor exon 11 inclusion was examined in L6 myotubes. Primers that amplified from exon 10 to exon 12 of the insulin receptor gene were used to observe the
inclusion of the insulin receptor exon 11 (17). In control and
insulin-treated L6 myotubes, both type A (exon 11 excluded) and type B
(exon 11 included) insulin receptor mRNA were detected with type B
receptor mRNA predominating (85%). As shown in Fig. 9, insulin, however, had no affect on the
ratio of type A to type B insulin receptor mRNA after 30 min. These
results suggest that insulin's effects on exon inclusion are probably
not a general phenomenon, but specifically targeted to certain
pre-mRNAs.
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DISCUSSION |
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Our earlier studies in the BC3H-1 myocytes demonstrated that
PKCII expression was regulated by insulin (12). L6 myotubes were
used to determine the mechanism responsible for insulin effects on
PKC
II expression. The BC3H-1 myocytes used in previous studies were
a non-fusing cell line and only expressed GLUT1 transporters. A fully
differentiated cell line such as the L6 cell line that more closely
resembles skeletal muscle expressing both GLUT4 and GLUT1 type glucose
transporters provides a more physiologically relevant model for
studying splicing (18). To examine PKC
I and PKC
II mRNA
levels, a highly sensitive method to quantitate mRNA levels by
RT-PCR was developed. In L6 myotubes, as in the BC3H-1 myocytes, mature
PKC
II mRNA and protein increased more than 3.5-fold following
acute insulin treatment. Levels of PKC
I mature mRNA and
immunoreactive protein remained unchanged following insulin treatment.
Prior studies in the BC3H-1 myocytes showed that PKC
I mRNA and
protein levels decreased in response to insulin. This was due to
destabilization of PKC
I mRNA in response to insulin. The
different observations between the two cell types may be explained in
two ways. The lack of a insulin-induced destabilization system in the
L6 myotubes or a role for PKC
I in insulin action in the L6 myotubes.
Other findings from our laboratory suggest a role for PKC
I in
insulin action. We found that overexpression of PKC
I in L6 myotubes
overcomes the inhibition of insulin-stimulated 2-deoxyglucose uptake by
wortmannin, a phosphatidylinositol 3-kinase inhibitor (19).
Overexpression of PKC
I in L6 myotubes also increases
insulin-stimulated glycogen synthase
activation.2 These data
suggest that PKC
I as well as PKC
II have distinct roles in the
metabolic effects of insulin. However, the mechanism by which insulin
affected PKC
pre-mRNA splicing had not been elucidated, and
whether PKC
pre-mRNA preferentially processed to PKC
I or
PKC
II mature mRNA was not known. It also remained to be
elucidated whether PKC
pre-mRNA splicing was positively or
negatively regulated by insulin and whether RNA transactivating factors
were involved. Transactivating factors are known to be involved in
regulating post-transcriptional as well as transcriptional events
(20).
A possible mechanism to preferentially increase PKCII mRNA
expression could be through increased transcriptional activation in
response to insulin. In this case, the RNA splicing of PKC
pre-mRNA to mature PKC
II mRNA may be negatively regulated in the basal state. For example, in the absence of insulin RNA
transactivating factors may inhibit the inclusion of the
PKC
II-specific exon or recognition of the PKC
II polyadenylation
site, thereby leading to the production of mature PKC
I mRNA. A
rapid increase in the transcription of the PKC
gene in response to
insulin may increase the levels of PKC
pre-mRNA. If the
inhibitory RNA transactivating factors are limiting, the newly
transcribed PKC
pre-mRNA would be processed to mature PKC
II
mRNA. The PKC
message occurs at relatively low levels.
Therefore, to investigate the possibility of a transcriptional
mechanism of regulation, we initially evaluated the effect of insulin
on several PKC
promotor/luciferase constructs spanning
2200 to +43
bp (15). Only a
511/+43 PKC
promotor construct was activated by
insulin. All other constructs, including a
2200 to +43 bp construct,
exhibited no response to insulin. Insulin stimulation of the
511/+43
bp PKC
promotor construct occurred, however, at later times (16 h),
and induction was minimal. Hence, the increase in PKC
II mRNA was
not likely accounted for at the level of transcriptional activation,
although one could argue that the promotor constructs used required an
upstream enhancer element to respond to insulin.
An increase in PKCII mRNA stability could also increase PKC
II
mRNA levels in response to insulin. To determine if increases in
PKC
II mRNA were due to increased mRNA stability, relative decay rates of PKC
I and PKC
II mRNA were measured following
actinomycin D treatment. Relative decay rates did not change within
1 h of insulin treatment, and the increase in PKC
II mRNA
levels were not likely justified by increased PKC
II mRNA
stability.
Mature PKCII mRNA could also result from alternative exon
selection or alternative polyadenylation site selection (4, 12, 16).
The regulation of either nuclear mechanism by an external cell receptor
signaling cascade had not been reported. In the L6 myotubes, we found
that PKC
II mRNA was produced via inclusion of an exon that
encodes a stop codon defining the V5 region of PKC
II in response to
insulin treatment. This mechanism produces a message with the PKC
I
exon including a polyadenylation site spliced onto the 3
end of the
PKC
II exon (Fig. 7B). Insulin regulation of PKC
II exon
inclusion changed the ratio of PKC
I to PKC
II mRNA following
insulin treatment. The levels of newly processed PKC
II mRNA
exceeded the synthesis of PKC
I mRNA, demonstrating that insulin
enhanced PKC
II exon inclusion into the mature mRNA transcript.
The molecular mechanism through which insulin affects PKCII exon
inclusion has not been addressed. Our findings suggest that alterations
in splicing factors associated with the PKC
II exon may be occurring.
Several RNA cis-elements that are known to affect exon
inclusion and splice site selection were presently identified in the
PKC
II exon and its surrounding intronic sequences. They include a
binding site for phosphorylated SRp40, several purine-rich exon
splicing enhancers, and multiple pyrimidine tracts (Fig. 7A).2 Serine/arginine-rich (SR) proteins and
pyrimidine tract-binding protein are known RNA transactivating factors
that bind to these cis-elements and affect exon inclusion
and 5
splice selection (21). Since SR proteins can be regulated via
phosphorylation by SR protein kinases (22, 23), it is possible that
insulin may activate a protein kinase that modulates SR protein
phosphorylation and their RNA binding properties thereby affecting exon
inclusion.
An even more novel mechanism of insulin regulation of PKC
pre-mRNA splicing was the observation that insulin activated a second 5
splice site downstream of the previously reported 5
splice
site for the PKC
II exon. This additional downstream 5
splice site
was preferentially activated compared with the conventional 5
splice
site by 30 min after insulin treatment. This second splice variant for
PKC
II extended the 3
-untranslated region by approximately 136 nucleotides. The observation that insulin activated two 5
splice sites
for PKC
II exon inclusion is intriguing. The
time-dependent manner in which the downstream 5
splice
site is activated following insulin treatment suggests that the
additional untranslated region serves a functional purpose. This novel
mechanism may introduce features into the newly spliced PKC
II
mRNA that further regulate translation, long term stability, or
nuclear export of mature mRNA. Extending this observation, we found
that IGF-I also stimulated splicing via exon inclusion and activated the second 5
splice site. This is the first report of IGF-I affecting the alternative splicing of pre-mRNA and suggests that both
peptides stimulate similar signaling pathways culminating in nuclear
post-transcriptional processing of mRNA.
Insulin effects on exon inclusion appear to have some specificity,
since it did not further alter the inclusion of exon 11 into insulin
receptor mRNA, another gene whose pre-mRNA splicing has been
demonstrated to be affected rapidly by insulin (17). This, therefore,
argues against widespread activation of RNA trans-activating factors and nonspecific insulin effects on splicing. These data also
suggest a different insulin signaling pathway or RNA processing mechanism for regulation of PKC pre-mRNA splicing compared with insulin receptor mRNA splicing.
PKC is activated and translocated by insulin in L6 myotubes, and we
have previously reported that PKC
II is associated with enhanced
insulin-stimulated 2-deoxyglucose uptake, thus suggesting a
physiological significance for increasing PKC
II expression (8, 12,
24). A possible link of PKC
II to insulin-stimulated 2-deoxyglucose
uptake could be hypothesized by the fact that PKC
II specifically
encodes an F-actin binding site in its V5 region that PKC
I does not
encode. PKC
II specifically binds and is activated by F-actin (25).
The actin cytoskeleton undergoes rapid rearrangement in response to
acute insulin treatment in a phosphatidylinositol 3-kinase-dependent manner (26). Disassembly of the actin
network with cytochalasin D inhibits insulin-stimulated GLUT4
translocation in L6 muscle cells (27). Therefore, PKC
II may function
in the process of insulin-stimulated actin rearrangement leading to
activation or translocation of glucose transporters to the plasma
membrane in L6 myotubes.
In conclusion, PKCII expression is increased through enhanced exon
inclusion and additional 5
splice site activation, which introduces a
new COOH-terminal exon containing a stop codon that defines the V5
region of PKC
II. This is the first report of insulin altering
nuclear mRNA processing through 5
splice site regulation. Insulin
regulation of post-transcriptional splicing events demonstrates a novel
mechanism for hormonal responses to rapidly alter gene expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Harald Mischak for providing the
design of the PKCII primers used in the competitive RT-PCR assay and
Dr. Yoshiko Akita for the gracious gift of the PKC
I- and
PKC
II-specific antibodies.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Service of the Department of Veterans Affairs (to D. R. C.), by National Science Foundation Grant 9318124 (to D. R. C.), and by a grant from the American Heart Association, Florida Affiliate (to D. R. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: J. A. Haley Veteran's Hospital (VAR 151), 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-972-2000 (Ext. 7017); Fax: 813-974-7357; E-mail: dcooper{at}com1.med.usf.edu.
1
The abbreviations used are: PKC, protein kinase
C; PAGE, polyacrylamide gel electrophoresis; IGF-I, insulin-like growth
factor-I; bp, base pair(s); -MEM,
-minimal essential medium; PBS,
phosphate-buffered saline; PCR, polymerase chain reaction; RT-PCR,
reverse transcriptase-polymerase chain reaction; SR,
serine/arginine-rich.
2 D. R. Cooper, J. E. Watson, and C. E. Chalfant, unpublished observations.
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REFERENCES |
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