From the Department of Biochemistry and
Molecular Biology, University of South Florida and the
§ James A. Haley Veterans Hospital, Tampa, Florida 33612 and ¶ Isis Pharmaceuticals, Carlsbad, California 92008
Received for publication, February 8, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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Insulin regulates the inclusion of the exon
encoding protein kinase C (PKC) Insulin regulates levels of protein kinase C
(PKC)1 Pre-mRNA splicing occurs on nuclear spliceosomes, a macromolecular
complex consisting of small nuclear ribonucleoproteins, proteins
associated with heterogeneous nuclear RNA, and other splicing factors
including serine-arginine-rich (SR) proteins (14, 15). Exon splicing is
highly regulated, and numerous consensus sequences that bind specific
factors participate in the control of tissue-specific or
developmentally controlled splicing via SR protein-RNA and
protein-protein interactions (16). SR and SR-like proteins are
characterized by a modular composition with one or more RNA recognition
motifs and an arginine and serine domain (RS domain) in which the
serine residues can be highly phosphorylated. The RS domain is
responsible for protein-protein interactions and nuclear localization
(17-19). SR and SR-like proteins have been implicated in 5'-splice
site recognition and in the communication of splice sites caused by a
network of SR proteins (20). They can bind to exon enhancer motifs that
are often purine-rich sequences that promote the use of suboptimal
splice sites (21). Their interaction with exon enhancers results in a
concentration-dependent influence on alternative splicing
(22-25). Several SR protein kinases have been reported, including a U1
snRNP 70K-associated kinase, SR protein kinase (SRPK1), lamin B
receptor kinase, and a family of CDC2-like kinases (10, 26-28). Both
hyper- and hypophosphorylation of SR proteins has been shown to
influence splicing (29-31), and the interaction of SR protein kinases
with SR proteins can also influence their subcellular localization (32,
33). However, at this time the regulation of SR protein kinases by
peptide hormone-activated signal transduction pathways has not been
demonstrated to our knowledge.
The precise mechanisms by which SR proteins govern alternative splicing
are under investigation in many laboratories. One model proposes that
different concentrations of spliceosomal proteins in different cell
types cause alternative processing of pre-mRNAs. Evidence for this
mechanism is based on the variable expression levels of some SR
proteins in tissues as a function of cell growth or differentiation
(24, 34-36). Another model proposes the existence of cell and/or
developmental specific splicing factors that modulate splice site
selection. For example, the female-specific expression of
Drosophila transformer protein determines the sexual fate of the fruit fly by directing splicing decisions (37, 38). In addition,
our recent finding that insulin regulated 5'-splice site selection of
the PKC It is well known that insulin binding to its receptor activates at
least three kinase pathways that can signal to the nucleus (41, 42).
Insulin-induced mitogen-activated protein kinase (MAPK) activation is
associated with mitogenic signaling of insulin, and insulin-induced
signal transducers and activators of transcription or JAK/signal
transducers and activators of transcription pathways lead to nuclear
transcriptional activator and repressor activation involved in cell
differentiation (43, 44). In contrast, insulin activation of the
phosphatidylinositol 3-kinase pathway is associated with metabolic
signaling, consistent with the observation that insulin regulates
PKC Our studies focused initially on SRp40 for the following reasons. One,
it was first described as an early response gene (HRS/SRp40). Two, SRp40 concentrations were increased by insulin in the regenerating liver where it is induced as a delayed early gene. Three, SRp40 levels
are transcriptionally up-regulated by insulin. Four, SRp40 effects on
exon inclusion have been demonstrated previously for the alternative
splicing of fibronectin mRNA (45). Finally, increased SR protein
concentrations during development, cell differentiation, and cell
proliferation determine alternative splicing decisions (24, 25, 46). In
our case, however, insulin regulation of PKC In the present study, we provide evidence to support SRp40 involvement
in the regulation of PKC Cell Culture--
Rat L6 skeletal myoblasts (obtained from Dr.
Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown
on Preparation of Nuclear Extracts--
L6 myotube nuclear
extract was prepared from cells treated with or without insulin for 30 min as described by Dignam et al. (47).
Immunoprecipitation of SRp40--
L6 myotubes were collected by
centrifugation, and pellets were lysed in 20 volumes of 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%
SDS, and 50 mM Tris, pH 8.0, with protease inhibitors as
follows: benzamidine HCl, 16 µg/ml; aprotinin, 10 µg/ml; leupeptin,
10 µg/ml; phenylmethylsulfonyl fluoride, 1 mM. Cells were
placed on ice for 30 min, and insoluble material was pelleted at
12,000 × g for 10 min at 4 °C. An aliquot (500 µl) of lysate was incubated at a final concentration of 1 µg/ml
with anti-SRp40 polyclonal antibody followed by agitation at 4 °C
for 2 h. A 40-µl aliquot of protein A-Sepharose beads in a 1:1
suspension with the lysis buffer was added and incubated again for
1 h. After centrifugation at 10,000 × g, beads
were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl, followed by a wash in lysis buffer with no NaCl.
After adding 50 µl of SDS-PAGE sample buffer, the precipitate was
boiled for 5 min, centrifuged at 1000 rpm for 5 min before loading on
the gel, followed by Western blot analysis.
Western Blot Analysis--
L6 muscle cell lysates (40 µg) or
immunoprecipitates were subjected to 9% SDS-polyacrylamide gel
electrophoresis (PAGE) (48). 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 SRp40 or monoclonal
antibody against the phosphoepitope of SR proteins, mAb104 (49), or
anti-PKC Overexpression of SRp40 in L6 Rat Skeletal Muscle Cells--
L6
myoblasts were stably transfected with pCMV5 (empty vector control) or
HRS/SRp40 in pCMV5 (46), using a calcium phosphate/DNA precipitate for
16 h. Cells were then washed twice with phosphate-buffered saline
and placed in media for 18 h prior to selection of stable transfectants selected in the presence of 750 µg/ml G418. Cells were
selected and grown in bulk cultures. Overexpression was demonstrated by
Western blot analysis using anti-SRp40 and mAb104 antibodies, described
above. Transient overexpression was accomplished using LipofectinTM (Life Technologies, Inc.) and was normalized
by co-transfecting Transient Transfection of Antisense--
Antisense
2'-O-methoxyethyl oligonucleotides (20 mers) were designed
to bind to a putative SRp40-binding region (5'-TGGGAGCTTGGCTTGA-3') located 351 bases downstream from the first RT-PCR Analysis--
Total RNA (1 µg) was used to synthesize
first strand cDNA using an oligo(dT) primer and Superscript II
reverse transcriptase. Inclusion of the PKC 2-[1,2-3H]Deoxy-D-glucose
(2-Deoxyglucose) Uptake--
L6 myoblasts were grown and
differentiated as described above in 24-well plates. Prior to
2-[3H]deoxyglucose uptake, cells were switched to Materials--
The SRp40 cDNA construct was kindly provided
by Dr. Taub (46). Tissue culture media were purchased from Life
Technologies, Inc. Fetal bovine serum was from Atlanta Biologicals
(Norcross, GA). Porcine insulin was obtained from Sigma. Stat-60 was
from Molecular Research Center, Inc. (Cincinnati, OH). The reagents used for polyacrylamide gel electrophoresis were from GradiGels (North
Ryde, Australia). Antibody to the phosphoepitope of SR proteins
(mAb104) was obtained from hybridoma cells (CRL 2067, ATCC).
Anti-PKC Insulin Activation of PI 3-Kinase Results in Exon
Inclusion--
Since insulin regulates the alternative splicing of
PKC
To examine involvement of the insulin-stimulated PI 3-kinase pathway,
L6 myotubes were pretreated with LY294002, a specific PI 3-kinase
inhibitor (52), prior to insulin addition. RT-PCR analysis was used to
evaluate changes in PKC SR Proteins Are Expressed by L6 Myotubes--
Involvement of SR
proteins in regulating alternative mRNA splicing has been
well-established (20), and members of this family are characterized by
a C-terminal domain with extensive arginine/serine motifs that are
hyperphosphorylated. Since the phosphorylation state of SR proteins has
also been proposed to regulate their function (53), we examined which
SR proteins were expressed in L6 myotubes, using a monoclonal antibody
(mAb104) raised to the phosphodomain of SR proteins to analyze nuclear
extracts from control and insulin-treated cells. At least seven
different SR proteins showed increased phosphorylation in response to
insulin treatment, including SRp75, SRp55, SRp40, and SRp30a/b (Fig.
2). In control cells which were
serum-starved, and represent basal conditions for splicing of the
- SRp40 Was Phosphorylated by a PI 3-Kinase-dependent
Pathway in Response to Insulin--
Although the L6 cells were not
dividing and represented fully differentiated insulin-responsive
skeletal muscle cells, it was possible that insulin increased SRp40
phosphorylation state as well as SRp40 concentration. To examine this
possibility, SRp40 was immunoprecipitated in lysates from cells treated
with insulin and LY294002 using a polyclonal antibody developed for
HRS/SRp40 (46). The immunoprecipitates were analyzed by Western blot
analysis probed with mAb104. As shown in Fig.
3A, following insulin
treatment for 15 and 30 min, SRp40 phosphorylation increased.
Pretreatment of cells with LY294002 blocked insulin effects on SRp40
phosphorylation. The cell lysates were also analyzed directly by
Western analysis using anti-SRp40 antibody. In this, insulin treatment
for up to 30 min was shown to have no discernible effect on SRp40
protein levels (Fig. 3B). SRp40 protein levels were also
unaltered by LY294002 pretreatment.
These results indicated that in non-dividing L6 myotubes, insulin
affects SRp40 function via phosphorylation rather than increasing concentrations of the factor, and SRp40 phosphorylation was blocked by
an inhibitor of PI 3-kinase.
SRp40 Overexpression Mimicked Insulin Effects on Splice Site
Selection--
To establish further SRp40 involvement, SRp40
overexpression experiments were carried out (46). The co-transfection
of HRS/SRp40 and a fibronectin minigene in H35 cells was correlated to
HRS-mediated regulation of EIIIB exon inclusion, and this correlated to
induction of HRS protein and fibronectin EIIIB+ transcripts in
developing liver. By analogy, we stably overexpressed SRp40 cDNA in
L6 myoblasts to determine whether overexpression would mimic insulin
effects on splicing. As shown in Fig. 4,
A and B, increased SRp40 levels resulted in the
inclusion of the PKC
Protein levels of SRp40 and PKC Effect of Antisense Oligonucleotide Targeted to Putative
SRp40-binding Site in the Intron Spanning Effects of Cycloheximide, SRp40 Overexpression, and Antisense
Oligonucleotides on Cellular 2-Deoxyglucose Uptake--
To show that
the functional consequences of SRp40 overexpression correlated to
PKC
By serum-depriving L6 myotubes for longer periods (up to 18 h)
before measuring glucose uptake, PKC
As a control, L6 myotubes were pretreated for 2 h with LY379196,
which inhibits PKC Insulin is known to activate PI 3-kinase (41), and several
downstream kinases are activated in a PI 3-kinase-dependent
manner including Akt, PKC The C-terminal portion of SR proteins contains a domain rich in serine
and arginine residues that is highly phosphorylated. At least eight
members of the SR family, including SRp40 and SF2/ASF, contain
phosphoepitopes that are recognized by mAb104 (68). The finding that
insulin treatment increased the phosphorylation state of at least seven
proteins in nuclear extracts from skeletal muscle cells is consistent
with the observation that the RS domains are highly phosphorylated
in vivo (10, 69).
We focused on HRS/SRp40, a splicing factor with a molecular mass
of about 40 kDa that was originally shown to be induced by insulin in
rat hepatocytes (71). By using immunoprecipitation of SRp40 followed by
detection with an antibody to examine serine phosphorylation
specifically, it became evident that insulin treatment increased the
phosphorylation state of the SR protein rather than its concentration.
The phosphorylation was blocked by pretreatment with LY294002 as was
splicing. This indicated a role for PI 3-kinase in alternative
splicing, and to our knowledge, this is the first report of a hormone
signaling the phosphorylation of an SR protein.
Since the overexpression of trans-factors has
also been useful in establishing their role in splicing, SRp40 cDNA
was expressed in differentiated myotubes and resulted in exon
inclusion. This was used as evidence to link the regulation of SR
concentration to splice site selection in previous studies (46). Here,
the overexpression was linked to increased RS domain phosphorylation, increased PKC Two consensus sequences have been proposed for SRp40-binding sites (58,
73). Both are present in the introns flanking the The effect of the PI 3-kinase inhibitor, LY294002, to block glucose
uptake has been reported (57), and its effect is consistent with a role
for PI 3-kinase in insulin action (53) since this signaling pathway
directly links downstream kinases with the recruitment of glucose
carriers to the plasma membrane and results in increased glucose
uptake. Next, we evaluated the effect of newly synthesized PKC We demonstrated that overexpression of SRp40 mimicked insulin to
increase basal glucose uptake. This is consistent with the effects of
increased PKC Our studies in cells support in vitro observations for an
SRp40 role in splice site selection where addition of one or more SR
proteins to in vitro deficient splicing extracts restored
splice site selection in a concentration-dependent manner
(7, 70). The identification of SRp40 as a component of
insulin-regulated splicing was defined by the following criteria: its
ability to mimic insulin effects on PKCII mRNA. In this report, we show
that insulin regulates this exon inclusion (alternative splicing) via
the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway
through the phosphorylation state of SRp40, a factor required for
insulin-regulated splice site selection for PKC
II mRNA. By
taking advantage of a well known inhibitor of PI 3-kinase, LY294002,
we demonstrated that pretreatment of L6 myotubes with LY294002 blocked
insulin-induced PKC
II exon inclusion as well as phosphorylation of
SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells
mimicked insulin-induced exon inclusion. When antisense
oligonucleotides targeted to a putative SRp40-binding sequence in the
II-
I intron were transfected into L6 cells, insulin effects on
splicing and glucose uptake were blocked. Taken together, these results
demonstrate a role for SRp40 in insulin-mediated alternative splicing
independent of changes in SRp40 concentration but dependent on serine
phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This
switch in PKC isozyme expression is important for increases in the
glucose transport effect of insulin. Significantly, insulin regulation
of PKC
II exon inclusion occurred in the absence of cell growth and
differentiation demonstrating that insulin-induced alternative splicing
of PKC
II mRNA in L6 cells occurs in response to a metabolic change.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II mRNA in
skeletal muscle by a novel mechanism that signals the activation of
splice sites in the last intron of the pre-mRNA. Exon inclusion results in an mRNA that now encodes the C-terminal region of
PKC
II affecting subcellular localization and substrate specificity
of the kinase. The terminal PKC
I-specific exon with its
3'-untranslated region is spliced to the PKC
II-specific exon via
exon inclusion such that a stop codon is introduced at the splice site,
and as a result, the PKC
I exon becomes part of an extended
3'-untranslated region of PKC
II mRNA (1, 2). Therefore, PKC
II
and PKC
I differ only by their C-terminal 52-50 amino acids,
respectively. In contrast to PKC
I, increased expression of PKC
II
results in activation/inactivation of the mitogen-activated kinase
cascade (3), glycogen kinase synthase 3
(4), TLS/Fus (5), insulin receptor signaling (6), cyclin-dependent kinase
(CDK)-activating kinase,2 as
well as cell proliferation (8-10), protein trafficking (11), apoptosis, and glucose transport (12, 13).
II-specific exon within minutes after it binds to cell
surface receptors suggested a third possibility. SR proteins could
regulate alternative splicing via a receptor-linked signaling pathway
responding to metabolic change rather than to a change in growth or
development (39, 40).
II exon inclusion independent of cell growth and differentiation.
Therefore, the possibility that a PI 3-kinase-dependent signaling pathway could alter the phosphorylation of
post-transcriptional regulatory factors such as SR proteins as a step
in the regulation of PKC
II expression was examined.
II exon inclusion in
BC3H-1 myocytes and L6 myotubes occurs within 15 min, prior to SRp40
transcriptional up-regulation and increases in its concentration. This
suggested that if SRp40 was involved in the insulin-induced alternative
splicing that results in PKC
II mRNA, there must be another
mechanism that influences SRp40 activity, other than changes in concentration.
II exon inclusion by insulin via its
increased phosphorylation by a PI 3-kinase-dependent pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MEM supplemented with 10% fetal bovine serum to confluency in
100-mm or 6-well plates. Myoblasts were fused into myotubes by changing media to
-MEM supplemented with 2% fetal bovine serum for 4-days post-confluency. The extent of differentiation was established by
observation of multinucleation of 85-90% of cells. For experiments, myotubes were incubated in
-MEM with 0.1% bovine serum albumin (BSA) for 6 h and placed in phosphate-buffered saline with 0.1% BSA just prior to treatment with insulin.
II antibody. Following incubation with anti-rabbit IgG or
anti-mouse IgM-horseradish peroxidase, detection was performed using
enhanced chemiluminescence (Amersham Pharmacia Biotech).
-galactosidase (50).
II exon 5'-splice site.
The sequence of the antisense was 5'-ATTCAAGCCAAGCTCCCCAGC-3'. As a
control, 4 base mismatches were introduced as indicated, 5'-ATTCCAGGCAACCTCCAAGC-3'.
Antisense (50 and 100 nM) was introduced into cells using
LipofectinTM transfection for 3 h. Cells were then
placed in
-MEM with 2% fetal bovine serum overnight prior to
treatment with insulin. Total RNA was isolated using Stat-60, and
RT-PCR analysis was performed as described below. The transfectivity of
L6 myotubes was shown to be >60% (51).
II-specific exon was
detected using an upstream sense primer corresponding to the C4 kinase
domain, common to both PKC
I and -
II
(5'-GTTGTGGGCCTGAAGGGGAACG-3'), and an antisense primer to the -
IV5
exon common to both transcripts, (5'-TGCCTGGTGAACTCTTTGTCG-3'). The PCR
products would be 159 bp for PKC
I, 374 bp for PKC
II (where the
first splice site was activated, SSI), and 510 bp for PKC
II (where
the second splice site, SSII, was activated). We found that insulin
activated two 5'-splice sites in a time-dependent manner in
some experiments (39). This assay allows for relative comparison of
both PKC
II versus PKC
I mRNA levels in the same reaction. Following 35 cycles of a two-step PCR amplification program
(95 °C, 30 s; and 58 °C, 2 min) using
Taq-platinum DNA polymerase (PerkinElmer Life Sciences),
50% of the PCR was resolved by electrophoresis on 1.2% agarose gels
containing 0.05% ethidium bromide at 120 V for 60 min. An additional
set of primers was also used to evaluate only PKC
II mRNA in some
experiments. A sense primer corresponding to the coding region of the
II exon (5'-CACCCGCCATCCACCAGTCCT-3') was used with antisense
corresponding to -
IV5 as described above. The resulting products
would be 227 bp. This assay offers increased sensitivity for detecting
the
II exon since PKC
I mRNA was not amplified. For each
experiment,
-actin was determined to compare RNA levels between
samples. PCR products were visualized using a Kodak Digital Analysis
System 120. The assay was verified against a competitive RT-PCR system, and equivocal results were obtained with the three PCR methods (39).
-MEM
with 0.1% bovine serum albumin for 6 h.
2-[3H]Deoxyglucose uptake was assayed as described (51).
Cells were preincubated for 60 min with/without inhibitors in
Dulbecco's phosphate-buffered saline with 1% bovine serum albumin
(BSA), insulin (100 nM), or the vehicle, and Dulbecco's
phosphate-buffered saline + BSA was added, and cells were incubated an
additional 30 min at 37 °C prior to the addition of 10 nmol of
2-[3H]deoxyglucose (50-150 µCi/µmol) for 6 min at
37 °C. The uptake was terminated by aspiration of media; cells were
washed 3 times with cold Dulbecco's phosphate-buffered saline and
lysed in 1% SDS. Radioactivity was determined by liquid scintillation counting.
II (polyclonal antibody), anti-rabbit and anti-mouse IgG and
IgM antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-SRp40 (polyclonal antibody) was provided by Dr. Taub. LY294002 was
obtained from Calbiochem. LY379196 was provided by Eli Lilly
(Indianapolis, IN). ECL reagents were from Amersham Pharmacia Biotech
(Arlington Heights, IL). Antisense oligonucleotides were synthesized by
ISIS Pharmaceuticals (Carlsbad, CA). PCR primers were synthesized by
MSW BIOTECH, Inc. (High Point, NC). Primers for
-actin were obtained
from CLONTECH (Palo Alto, CA). Superscript II
reverse transcriptase was from Life Technologies, Inc.
Taq-platinum polymerase was from PerkinElmer Life Sciences.
LipofectinTM was from Promega. All other biochemicals and
reagents were purchased from the usual vendors.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II mRNA independent of growth and differentiation, we
proposed that an insulin-stimulated PI 3-kinase pathway was involved.
An insulin-sensitive p85/p110 PI 3-kinase is recruited to protein
phosphotyrosine residues in response to activation of the insulin
receptor tyrosine kinase producing phosphatidylinositol
3,4,5-trisphosphate which is necessary for metabolic and some mitogenic
actions of insulin (41, 53, 54), and several protein serine/threonine
kinases downstream of PI 3-kinase are implicated in the regulation of
glucose transporter recruitment, glycogen synthesis, protein synthesis,
and gene transcription, making this pathway a likely candidate for
regulating post-transcriptional events such as alternative splicing
(55).
pre-mRNA splicing and PKC
II mRNA
production (Fig. 1). Insulin stimulated
exon inclusion as evidenced by the presence of the
II-specific exon
in the mature mRNA as reported previously (39), and LY294002
inhibited this insulin effect. To verify that LY294002 blocked PI
3-kinase, 2-deoxyglucose uptake, also dependent upon the
insulin-induced PI 3-kinase pathway, was assessed and inhibited greater
than 60% (Table I).
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Fig. 1.
A PI 3-kinase inhibitor, LY294002, blocked
insulin effects on exon inclusion. A, schematic diagram
of the relevant portion of PKC pre-mRNA along with the positions
of the primers used for detection of PKC
II mRNA. C4
represents the last exon (indicated as a box) common to both
II and
I. Lines represent introns. B,
RT-PCR assay of L6 myotubes treated with or without insulin (100 nM, 30 min) and LY294002 (100 nM, 1 h
pretreatment); M indicates the 100-bp DNA ladder included
for size determination.
-Actin levels in corresponding samples are
shown in the lower panel.
-Actin levels in corresponding
samples are shown in the lower panel. Detection of PKC
II
mRNA was negligible in cells treated with LY294002. The experiment
was repeated on four occasions with the same results.
Insulin effects on 2-deoxyglucose uptake in L6 myotubes
II exon, only three major SR proteins, corresponding to SRp30a/b,
SRp55, and SRp75, were detected with mAb104. Thus, although the
phosphorylation states of seven SR proteins increased with insulin
treatment, a significant change in SRp40 was observed over basal
conditions and therefore was likely involved.
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Fig. 2.
Identification of SR proteins in nuclear
extracts from L6 myotubes treated with insulin. Cells were treated
with insulin (Ins; 100 nM, 30 min) prior to
isolation of nuclear extracts. Following SDS-PAGE, Western blot
analysis was performed using mAb104, which detects the phosphoepitope
of SR proteins. Bovine serum albumin (BSA) was included in
the 1st lane to demonstrate the specificity of the antibody.
The experiment was repeated to ensure reproducible results.
Con, control.
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Fig. 3.
Insulin increased the phosphorylation state
of SRp40 in L6 myotubes without altering concentrations of the
factor. A, cells were treated with or without insulin
(100 nM, 15 and 30 min) and LY294002 (10 µM,
1 h pretreatment). Lysates were immunoprecipitated (Ip)
with anti-SRp40 antibody, and blots were probed with mAb104.
B, lysates were analyzed by Western blot using anti-SRp40
antibody. Results were repeated on at least three occasions with
similar results.
II-specific exon as determined by RT-PCR
analysis, and the effect was analogous to that observed for insulin
treatment. Basal levels of PKC
II mRNA were negligible in
serum-starved L6 myotubes, but upon stimulation with insulin for 30 min, inclusion of the 216-bp PKC
II exon was evident. Transient overexpression of SRp40 also mimicked insulin-induced PKC
II exon inclusion, and the splicing observed in the presence of transient SRp40
expression was also blocked by LY294002 (Fig. 4C).
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Fig. 4.
Exon inclusion in
PKC II mRNA in the presence of insulin and
SRp40 cDNA. Cells were stably transfected with SRp40 cDNA
containing plasmids or the empty vector, pCMV. A, schematic
diagram of relevant portions of the PKC
II gene
and location of the primers designed to amplify insertion of the 216-bp
II exon. Predicted splice products and their lengths are indicated.
B, RT-PCR analysis of PKC
II exon inclusion in L6 myotubes
stably expressing either the empty vector or pCMV-SRp40. As a control,
-actin was amplified. C, RT-PCR analysis of PKC
II exon
inclusion in L6 myotubes transiently transfected with SRp40 cDNA.
The experiments were repeated to ensure reproducible results.
II were also analyzed to determine
that the transfected cDNA was expressed and to determine whether
the increase in PKC
II mRNA resulted in newly synthesized protein. The transfection of cells with SRp40 constructs increased levels of the protein, >5-fold, over endogenous SRp40 levels (Fig. 5A). The phosphorylation of
SRp40 in response to insulin also increased 5-fold over
non-insulin-stimulated levels as determined using mAb104 to detect
phosphorylated SRp40 (Fig. 5B). SRp40 overexpression increased levels of PKC
II protein expression in a manner consistent with insulin treatment (Fig. 5C).
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Fig. 5.
Immunoblot analysis with mAb104, anti-SRp40
and anti-PKC II, of L6 myotubes treated with
insulin or overexpressing SRp40 cDNA. A, whole cell
lysates from insulin-treated cells (100 nM, 30 min) or
cells stably overexpressing SRp40 cDNA were separated on SDS-PAGE
and blotted with anti-SRp40. B, analysis of blot developed
with mAb104 (detects the phosphoepitope of SR proteins). C,
analysis of blot developed with anti-PKC
II (detects an 81-kDa
protein). The experiment was repeated on at least three occasions with
similar results.
II-
I Exons--
Since
SRp40 mimicked insulin to enhance exon inclusion, the
cis-elements involved in the regulation are likely to occur
in the exon or intron sequence proximal to the splice site. An
SRp40-binding motif, TGGGAGCTTGGCTTGA, downstream from the PKC
II
exon 5'-splice site was identified from sequence analysis. This site is
similar to a cis-element predicted earlier,
TGGGAGCNNRGCTCGY, with a 2-bp difference at the
3'-end (58). To determine if this sequence might be involved in
insulin-stimulated splicing, an antisense oligonucleotide was designed.
The modification used to synthesize the oligonucleotide ensured that it
was RNase H-resistant and would not result in destabilizing the
pre-mRNA. A 2'-O-methoxyethyl oligonucleotide was
targeted to bind to the potential SRp40-binding site in the intron
spanning
II-
I exons as shown in Fig.
6A. This antisense sequence
blocked insulin-stimulated exon inclusion in a
dose-dependent manner (Fig. 6, B and
C). A control oligonucleotide containing a 4-bp mismatch
failed to block exon inclusion in the presence of insulin and confirmed
the specificity of the antisense for the target sequence. Thus, by
blocking SRp40 protein interaction with the element with the
2'-O-methoxyethyl oligonucleotide, insulin-induced splicing
was directed away from exon inclusion to the alternative product, PKC
I mRNA.
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Fig. 6.
Antisense oligonucleotide directed toward
SRp40-binding motif block insulin-induced exon inclusion in
PKC II mRNA. A, schematic
diagram of relevant portions of the PKC
II gene
and location of the primers used to amplify both PKC
I and
II
mRNA. B, analysis of PKC
I and
II mRNA in cells
transfected with antisense (AS 34, 100 nM) corresponding to
an SRp40-binding motif identified in the
II/
I intron.
-Actin
levels in corresponding samples are shown in the lower
panel.
-Actin levels in corresponding samples are shown.
C, dose dependence of AS34 to block insulin effects on exon
inclusion at 50, 100, and 150 nM concentrations of
oligonucleotides transfected into cells. The experiment was repeated
independently on three occasions with similar results.
II-mediated metabolic changes induced by insulin action (51),
PKC
II-mediated response on glucose uptake by L6 cells was examined.
II mRNA and protein levels were demonstrated to be low (Fig. 5C). Under these
conditions, glucose uptake reflects a requirement for new protein
synthesis if the rapid effect of insulin on splicing of PKC
II is
relevant to a physiological event. To demonstrate this requirement, L6 myotubes were serum-starved for 18 h prior to 2-deoxyglucose
uptake (56). As shown in Table I, cycloheximide pretreatment
blocked insulin effects on 2-[3H]deoxyglucose uptake. The
transient overexpression of SRp40 cDNA in L6 myotubes also mimicked
insulin effects by increasing basal glucose uptake, and there was no
further stimulation in the presence of insulin. Basal glucose uptake
was also highly elevated in cells stably transfected with SRp40, and
insulin had no further stimulatory effect (data not shown.) To
demonstrate further the importance of the switch in PKC
isozymes,
cells were transiently transfected with the antisense oligonucleotide
(AS 34) shown to block splicing of the pre-mRNA (Fig. 6). At a
concentration shown to block exon inclusion and the switch to PKC
II,
insulin effects on glucose transport were totally blocked. The control
antisense oligonucleotide with the 4-bp mismatch (AS35) had no effect
on glucose transport. Hence, under conditions where insulin stimulation
of alternative splicing of PKC
II mRNA was controlled, glucose
uptake was linked to the regulation of SRp40 phosphorylation and
PKC
II splicing by insulin.
I (IC50 50 nM) and
PKC
II (IC50 30 nM). At 30 nM,
glucose uptake was blocked 50% of the full insulin effect consistent
with the involvement of PKC
II in insulin-stimulated glucose
transport and previous studies demonstrating the effects of PKC
inhibitors (51, 65).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and PKC
II in skeletal muscle (51,
65-67). Insulin signaling to the nucleus by PI 3-kinase is not as well studied, but it is associated with activation of gene transcription in
addition to its roles in glycogen and protein synthesis and glucose
transport (55). Our study found increased phosphorylation of SR
proteins by insulin-dependent PI 3-kinase pathways. This indicated that factors involved in 5'-splice site selection could be
regulated by insulin signaling mechanisms.
II mRNA, increased PKC
II protein, and to
increased glucose transport. It is premature to suggest which
downstream kinases or phosphatases are activated or inhibited by PI
3-kinase to result in increased SR protein phosphorylation. It is
possible that insulin-activated kinases such as Akt or PKC could
phosphorylate SR proteins (72).
II-specific exon.
The first sequence occurs prior to the 3'-pyrimidine tract (ACDGS). The
second sequence we identified by sequence analysis is longer, occurs
about 350 bp after the first 5'-splice site, and corresponds closely to
one described (58). When the second site was targeted using antisense
oligonucleotides, exon inclusion was inhibited. The use of antisense
oligonucleotides for down-regulating gene expression is well documented
where sequences are targeted to block translation or lead to
destabilization of the message by RNase H or inhibit transcription by
forming triplex structures within the promoter regions of DNA.
Antisense oligonucleotides have also been used to restore splicing of
mutated pre-mRNA in thalassemic
-globin and to redirect splice
site selection for Bcl-xS versus Bcl-xL independent of
down-regulating gene expression (59, 60, 74). Here, blocking one site
for SRp40-RNA interaction provided mutual dependence of a downstream
sequence with insulin-induced changes in SRp40 phosphorylation. This
finding is analogous to studies where antisense toward SF2/ASF-binding
sites blocked splicing of bGH pre-mRNA in vitro
(61).
II on
insulin-stimulated glucose uptake. Cycloheximide blocked the
recruitment of glucose carriers in adipose cells (62, 63). Although
there is a conflicting report in adipocytes (64), differences in the
preparation and pretreatment of cells could be involved. In this study,
cycloheximide treatment blocked insulin effects in serum-depleted myotubes.
II concentrations that occur following SRp40
overexpression. Since increased SRp40 concentrations may be altering
alternative splicing of other pre-mRNA in a nonspecific manner,
this correlation should be interpreted tentatively. For example, the
insulin receptor is also alternatively spliced, and the B form of the
receptor is thought to signal more effectively (76). However, the
ability of antisense oligonucleotides targeting the SRp40-binding site
to block insulin effects on glucose transport suggests that the
alteration in splicing alone is responsible for the increase in
transport. Finally, LY379196, a PKC
inhibitor which blocks glucose
uptake in primary mouse myotubes (65), also inhibited insulin
effects on glucose uptake.
II splicing, inhibition of
its phosphorylation state by LY294002, a compound that blocks insulin
activation of PI 3-kinase, and the ability of
2'-O-methoxyethyl antisense oligonucleotides directed to a
putative SRp40 site to block insulin effects on splicing as well as to
block insulin effects on glucose transport. The demonstration of SRp40
as a factor regulated by a PI 3-kinase signaling cascade provides an
additional mechanism for regulating alternative splicing. SRp40
phosphorylation correlates to alternative splicing of the
II exon in
a manner analogous to insulin treatment and links a signaling pathway
to exon inclusion events in vivo. Unlike systems of
tissue-specific alternative splicing, however, the concentration of
SRp40 did not change with insulin treatment, rather its phosphorylation
state increased. Taken together, the multiple strategies used here to
investigate SRp40 interactions in intact cells indicate a pivotal role
for this trans-factor and PI 3-kinase in insulin-stimulated
alternative splicing of PKC
pre-mRNA and subsequent effects of
insulin on glucose transport.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Rebecca Taub, Department of Genetics, University of Pennsylvania School of Medicine, for generously providing pCMV-SRp40 and polyclonal SRp40 antibody. We thank Dr. James L. Manley, Department of Biological Sciences, Columbia University, for discussions. We also thank Konrad Mebert, Dan Mancu, and David Chappell for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Department of Veterans Affairs Merit Review (to D. R. C.) and the National Science Foundation Grant MCB 9720601 (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: Dept. of
Biochemistry and Molecular Biology, University of South Florida College of Medicine, Research Service 151, James A. Haley Veterans Hospital, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-972-2000 (Ext.
7017); E-mail: dcooper@hsc.usf.edu.
** Present address: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M101260200
2 Acevedo-Duncan, M., Patel, R., and Whelan, S., (2001) Cell Prolif., in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
PKC, protein kinase
C;
PI 3-kinase, phosphatidylinositol-3 kinase;
SR, serine-arginine-rich;
RS, arginine/serine domain;
HRS, hepatic Arg-Ser
protein;
RT-PCR, reverse transcriptase-polymerase chain reaction;
AS, antisense oligonucleotides;
BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis;
-MEM,
-minimum Eagle's
medium;
bp, base pairs;
PCR, polymerase chain reaction.
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