Insulin Regulates Alternative Splicing of Protein Kinase C beta II through a Phosphatidylinositol 3-Kinase-dependent Pathway Involving the Nuclear Serine/Arginine-rich Splicing Factor, SRp40, in Skeletal Muscle Cells*

Niketa A. PatelDagger , Charles E. ChalfantDagger **, James E. Watson§, Jacqueline R. Wyatt, Nicholas M. Dean, Duane C. EichlerDagger , and Denise R. CooperDagger §||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) beta II 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 PKCbeta 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 PKCbeta 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 beta II-beta 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 PKCbeta II exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbeta II mRNA in L6 cells occurs in response to a metabolic change.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin regulates levels of protein kinase C (PKC)1 beta 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 PKCbeta II affecting subcellular localization and substrate specificity of the kinase. The terminal PKCbeta I-specific exon with its 3'-untranslated region is spliced to the PKCbeta II-specific exon via exon inclusion such that a stop codon is introduced at the splice site, and as a result, the PKCbeta I exon becomes part of an extended 3'-untranslated region of PKCbeta II mRNA (1, 2). Therefore, PKCbeta II and PKCbeta I differ only by their C-terminal 52-50 amino acids, respectively. In contrast to PKCbeta I, increased expression of PKCbeta II results in activation/inactivation of the mitogen-activated kinase cascade (3), glycogen kinase synthase 3beta (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).

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 PKCbeta 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).

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 PKCbeta 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 PKCbeta II expression was examined.

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 PKCbeta 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 PKCbeta II mRNA, there must be another mechanism that influences SRp40 activity, other than changes in concentration.

In the present study, we provide evidence to support SRp40 involvement in the regulation of PKCbeta II exon inclusion by insulin via its increased phosphorylation by a PI 3-kinase-dependent pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Rat L6 skeletal myoblasts (obtained from Dr. Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown on alpha -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 alpha -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 alpha -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.

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-PKCbeta II antibody. Following incubation with anti-rabbit IgG or anti-mouse IgM-horseradish peroxidase, detection was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

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 beta -galactosidase (50).

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 beta 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 alpha -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).

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 PKCbeta II-specific exon was detected using an upstream sense primer corresponding to the C4 kinase domain, common to both PKCbeta I and -beta II (5'-GTTGTGGGCCTGAAGGGGAACG-3'), and an antisense primer to the -beta IV5 exon common to both transcripts, (5'-TGCCTGGTGAACTCTTTGTCG-3'). The PCR products would be 159 bp for PKCbeta I, 374 bp for PKCbeta II (where the first splice site was activated, SSI), and 510 bp for PKCbeta 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 PKCbeta II versus PKCbeta 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 PKCbeta II mRNA in some experiments. A sense primer corresponding to the coding region of the beta II exon (5'-CACCCGCCATCCACCAGTCCT-3') was used with antisense corresponding to -beta IV5 as described above. The resulting products would be 227 bp. This assay offers increased sensitivity for detecting the beta II exon since PKCbeta I mRNA was not amplified. For each experiment, beta -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).

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 alpha -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.

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-PKCbeta 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 beta -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

Insulin Activation of PI 3-Kinase Results in Exon Inclusion-- Since insulin regulates the alternative splicing of PKCbeta 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).

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 PKCbeta pre-mRNA splicing and PKCbeta II mRNA production (Fig. 1). Insulin stimulated exon inclusion as evidenced by the presence of the beta 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 PKCbeta pre-mRNA along with the positions of the primers used for detection of PKCbeta II mRNA. C4 represents the last exon (indicated as a box) common to both beta II and beta 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. beta -Actin levels in corresponding samples are shown in the lower panel. beta -Actin levels in corresponding samples are shown in the lower panel. Detection of PKCbeta II mRNA was negligible in cells treated with LY294002. The experiment was repeated on four occasions with the same results.

                              
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Table I
Insulin effects on 2-deoxyglucose uptake in L6 myotubes
L6 myotubes were grown in 24-well plates until confluent and differentiated into myotubes as described. Cells were serum-starved overnight prior to pretreatment with inhibitors for 1-4 h prior to addition of insulin for 30 min.

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 -beta 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.

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.


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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.

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 PKCbeta II-specific exon as determined by RT-PCR analysis, and the effect was analogous to that observed for insulin treatment. Basal levels of PKCbeta II mRNA were negligible in serum-starved L6 myotubes, but upon stimulation with insulin for 30 min, inclusion of the 216-bp PKCbeta II exon was evident. Transient overexpression of SRp40 also mimicked insulin-induced PKCbeta 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 PKCbeta 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 PKCbeta II gene and location of the primers designed to amplify insertion of the 216-bp beta II exon. Predicted splice products and their lengths are indicated. B, RT-PCR analysis of PKCbeta II exon inclusion in L6 myotubes stably expressing either the empty vector or pCMV-SRp40. As a control, beta -actin was amplified. C, RT-PCR analysis of PKCbeta II exon inclusion in L6 myotubes transiently transfected with SRp40 cDNA. The experiments were repeated to ensure reproducible results.

Protein levels of SRp40 and PKCbeta II were also analyzed to determine that the transfected cDNA was expressed and to determine whether the increase in PKCbeta 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 PKCbeta 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-PKCbeta 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-PKCbeta II (detects an 81-kDa protein). The experiment was repeated on at least three occasions with similar results.

Effect of Antisense Oligonucleotide Targeted to Putative SRp40-binding Site in the Intron Spanning beta II-beta 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 PKCbeta 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 beta II-beta 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, PKCbeta I mRNA.


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Fig. 6.   Antisense oligonucleotide directed toward SRp40-binding motif block insulin-induced exon inclusion in PKCbeta II mRNA. A, schematic diagram of relevant portions of the PKCbeta II gene and location of the primers used to amplify both PKCbeta I and beta II mRNA. B, analysis of PKCbeta I and beta II mRNA in cells transfected with antisense (AS 34, 100 nM) corresponding to an SRp40-binding motif identified in the beta II/beta I intron. beta -Actin levels in corresponding samples are shown in the lower panel. beta -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.

Effects of Cycloheximide, SRp40 Overexpression, and Antisense Oligonucleotides on Cellular 2-Deoxyglucose Uptake-- To show that the functional consequences of SRp40 overexpression correlated to PKCbeta II-mediated metabolic changes induced by insulin action (51), PKCbeta II-mediated response on glucose uptake by L6 cells was examined.

By serum-depriving L6 myotubes for longer periods (up to 18 h) before measuring glucose uptake, PKCbeta 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 PKCbeta 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 PKCbeta 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 PKCbeta 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 PKCbeta II mRNA was controlled, glucose uptake was linked to the regulation of SRp40 phosphorylation and PKCbeta II splicing by insulin.

As a control, L6 myotubes were pretreated for 2 h with LY379196, which inhibits PKCbeta I (IC50 50 nM) and PKCbeta II (IC50 30 nM). At 30 nM, glucose uptake was blocked 50% of the full insulin effect consistent with the involvement of PKCbeta II in insulin-stimulated glucose transport and previous studies demonstrating the effects of PKCbeta inhibitors (51, 65).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin is known to activate PI 3-kinase (41), and several downstream kinases are activated in a PI 3-kinase-dependent manner including Akt, PKCzeta , and PKCbeta 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.

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 PKCbeta II mRNA, increased PKCbeta 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).

Two consensus sequences have been proposed for SRp40-binding sites (58, 73). Both are present in the introns flanking the beta 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 beta -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).

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 PKCbeta 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.

We demonstrated that overexpression of SRp40 mimicked insulin to increase basal glucose uptake. This is consistent with the effects of increased PKCbeta 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 PKCbeta inhibitor which blocks glucose uptake in primary mouse myotubes (65), also inhibited insulin effects on glucose uptake.

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 PKCbeta 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 beta 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 PKCbeta pre-mRNA and subsequent effects of insulin on glucose transport.

    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.

    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.

    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; alpha -MEM, alpha -minimum Eagle's medium; bp, base pairs; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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