Insulin Regulates Protein Kinase Cbeta II Expression through Enhanced Exon Inclusion in L6 Skeletal Muscle Cells
A NOVEL MECHANISM OF INSULIN- AND INSULIN-LIKE GROWTH FACTOR-I-INDUCED 5' SPLICE SITE SELECTION*

Charles E. ChalfantDagger , James E. Watson§, Linda D. Bisnauth§, Jordan Brown Kang§, Niketa PatelDagger , Lina M. Obeid, Duane C. EichlerDagger , and Denise R. CooperDagger §par **

From the Dagger  Departments of Biochemistry and Molecular Biology and par  Internal Medicine, University of South Florida College of Medicine and § James A. Haley Veterans Hospital, Tampa, Florida 33612 and the  Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710-0001

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The protein kinase Cbeta (PKCbeta ) gene encodes two isoforms, PKCbeta I and PKCbeta II, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCbeta pre-mRNA was examined in L6 myotubes. Mature PKCbeta II mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCbeta I mRNA and protein levels remained unchanged. Mature PKCbeta II mRNA resulted from inclusion of the PKCbeta II-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCbeta II expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCbeta II mRNA, and suggest that PKCbeta 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 PKCbeta 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 PKCbeta II mRNA species is presently unknown, inclusion of either PKCbeta II-specific exon results in the same PKCbeta II protein.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein kinase Cbeta (PKCbeta )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 PKCbeta gene encodes two mRNAs that originate from alternative splicing of exons encoding the carboxyl terminus (see Fig. 1A) (4). The resulting polypeptides, PKCbeta I and PKCbeta II, diverge in the sequence of their COOH-terminal 50 (PKCbeta I) or 52 (PKCbeta II) amino acids, respectively (4, 5). PKCbeta 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 PKCbeta I and -beta II have different and distinct functions in response to insulin in stable transfectants of NIH-3T3 fibroblasts (12). PKCbeta II overexpression enhanced insulin-stimulated 2-deoxyglucose uptake significantly above control cells or stable transfectants overexpressing PKCbeta I (12). The contribution of PKCbeta 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 PKCbeta II-specific dominant negative blocked insulin-stimulated 2-deoxyglucose uptake (8). CG53353, a PKCbeta II-specific inhibitor at 1 µM, also inhibited insulin-stimulated 2-deoxyglucose uptake (8). Thus, one alternative splice variant of the PKCbeta gene is more effective than the other as a positive transducer for glucose transport responses.

The regulation of PKCbeta 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 PKCbeta mRNA, thereby switching expression from PKCbeta I to PKCbeta II mRNA (12). The switch in mRNA was reflected by increased protein levels of PKCbeta II (12). However, the mechanism of how insulin regulated the post-transcriptional processing of PKCbeta 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 PKCbeta II-specific exon into the mature message since the PKCbeta II-specific exon includes a translation stop codon. Increased transcription or changes in PKCbeta 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 PKCbeta II. Insulin also activated two 5' splice sites, a previously known site and a second newly identified 5' splice site, to include the PKCbeta II-specific exon, which contains a stop codon, thereby encoding only the PKCbeta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture-- L6 rat skeletal myoblasts (obtained from Dr. Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown in alpha -minimum essential medium (alpha -MEM) supplemented with 10% fetal bovine serum (Sigma) to confluence. Myoblasts were fused into myotubes by changing media to alpha -MEM supplemented with 2% fetal bovine serum for 2-4 days after confluence. Cells were incubated in alpha -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.

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 10-2 amol of mimic DNA using primers specific for PKCbeta I, PKCbeta II, or beta -actin and Taq DNA polymerase from Perkin-Elmer. The PKCbeta II-specific primers correspond to Primer A, a sense primer to the V3 region of PKCbeta (5'-ATGAAACTGACCGATTTTAACTTCCTG-3'), and Primer B, an antisense primer corresponding to the V5 region of PKCbeta II (5'-CGGAGGTCTACAGATCTACTTAGCTCT-3') (Fig. 1B). The PKCbeta 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 PKCbeta I (5'-TGCCTGGTGAACTCTTTGTCG-3') (Fig. 1B). Sense and antisense primers for beta -actin (CLONTECH) were used to normalize for total RNA. Mimics (competitors) for PKCbeta I and PKCbeta II were constructed using the CLONTECH MIMIC construction kit (CLONTECH). The mimic is a neutral piece of DNA containing primer sequences for either PKCbeta I or PKCbeta II on its 5' and 3' ends. The mimic will specifically compete for primer binding sites with the target PKCbeta I or PKCbeta 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 (beta -actin and PKCbeta II: 94 °C, 1 min; 58 °C, 1 min; and 72 °C, 3 min; PKCbeta 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.

For evaluating PKCbeta II-specific exon inclusion, Primer E, an upstream sense primer corresponding to the C4 kinase domain common to both PKCbeta I and -beta II (5'-GTTGTGGGCCTGAAGGGGAACG-3'), and Primer D (described above) were used. This RT-PCR assay allows for relative comparison of PKCbeta II versus PKCbeta I mRNA levels.

For evaluating PKCbeta II 5' splice site selection, Primer F, a sense primer corresponding to the PKCbeta II exon (5'-CACCCGCCATCCACCAGTCCT-3') and Primer D (described above) were used. This method allows for comparing different PKCbeta II alternative splice products.

To examine potential expression of the polyadenylated form of PKCbeta II, Primer E (described above) and Primer G, an antisense primer corresponding to intronic sequences upstream from the PKCbeta II polyadenylation signal and downstream of the PKCbeta II exon (5'-CGGGAAGGTGGAAGAATGTTGC-3'), were used.

Following 35 cycles of a two-step PCR amplification program (95 °C, 30 s; and 68 °C, 2 min) using Taq-Gold DNA polymerase from Perkin-Elmer, 15% of the PCR reactions were resolved by electrophoresis on 1.5% agarose gels. The gel was then denatured 30 min in a 0.5 M NaOH, 1 M NaCl solution, neutralized 30 min in a 1 M Tris-HCl, M NaCl solution, and blotted via capillary transfer to nylon membranes. Membranes were hybridized with a 32P-labeled PKCbeta cDNA probe as described below.

For insulin receptor isoform analysis, primers specific for the exons flanking the alternatively spliced exon 11 of the insulin receptor were used to amplify reverse transcribed total RNA. The sequences are (5'-GTCCCCACCTTTTGAGTCTGA-3') for the sense primer to exon 10 and (5'-AAATGGTCTGTGCTCTTCGTG-3') for the antisense primer to exon 12. Following 35 cycles of a three-step PCR amplification program (95 °C, 30 s; 64 °C, 30 s; and 72 °C, 2 min), PCR reactions were resolved and quantitated as described above.

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 PKCbeta II or PKCbeta 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-- PKCbeta II cDNA (25 ng) was labeled with [alpha -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 PKCbeta II 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-- PKCbeta 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 PKCbeta promotor/luciferase construct and 1 µg of SV40 beta -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 beta -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Insulin Increased PKCbeta II Mature mRNA and Had No Effect on PKCbeta I Mature mRNA Levels-- Although insulin regulation of alternative splicing of PKCbeta 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 PKCbeta 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 PKCbeta II mRNA was observed (Fig. 2). The up-regulation of PKCbeta 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.


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Fig. 1.   A, PKCbeta 3' gene structure. Diagram depicts the 3' end of the PKCbeta gene with approximate sizes of exons and introns. Polyadenylation sites are designated AATAAA for PKCbeta II and ATTAAA for PKCbeta I. B, the domain structure of PKCbeta I and -beta II and regions amplified by RT-PCR. A schematic representation of PKCbeta as deduced from cDNA sequence analysis is shown. PKCbeta I is a 671-amino acid product, and PKCbeta II is a 673-amino acid product, respectively. C1-C4 represent the conserved regions common to calcium, diacylglycerol, and phospholipid-dependent PKCs. V1-V5 are variable regions specific for each isoform. Arrows indicate the regions that correspond to oligonucleotide primer sequences (A-D) in the PKCbeta cDNA used to amplify specific products of differing lengths as indicated (see "Materials and Methods" for primer sequences).


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Fig. 2.   Competitive RT-PCR analysis of PKCbeta I and -beta II mRNA levels in L6 myotubes after insulin treatment. L6 myotubes were removed from serum for 6 h prior to treatment with either 200 nM insulin or PBS (sham control) for various times. Total RNA was extracted, reverse transcribed, and subjected to competitive PCR using synthetic competitors for PKCbeta I or PKCbeta II primer binding sites (see Fig. 1B). Target designates the reverse transcribed mature mRNA, and mimic designates synthetic competitor DNA. The PKCbeta II target length was 1030 bp, and the mimic length was 748 bp using primers A and B. The PKCbeta I target length was 684 bp, and the mimic length was 398 bp using primers C and D. PCR products were resolved by fractionation on 1.2% agarose gels and visualized via ethidium bromide staining. PCR products were verified by sequencing. Shown is a representative analysis that was repeated on five separate occasions with different L6 myotube preparations.

To quantify potential changes in PKCbeta I mRNA levels via competitive RT-PCR, cycling conditions were optimized to efficiently amplify the 684-bp PKCbeta I PCR product and not other species. Unlike the BC3H-1 myocytes, no change occurred in PKCbeta I mRNA with acute insulin treatment (Fig. 2). This may reflect the difference between an undifferentiated cell line such as the the BC3H-1 myocytes and a differentiated cell type like the L6 myotubes. This may also suggest that there is a functional role for PKCbeta I in insulin signal transduction in the L6 myotubes. PKCbeta I and -beta II mRNA levels were normalized to beta -actin mRNA levels using competitive RT-PCR (data not shown).

The PKCbeta II mRNA Increase Is Reflected at the Protein Level-- To examine whether insulin effects on PKCbeta I and -beta II mRNA were reflected at the protein level, polyclonal antibodies specific for the COOH terminus of either PKCbeta I or PKCbeta II proteins were used for detection on Western blots. Insulin treatment increased PKCbeta II immunoreactive protein levels greater than 3.5-fold (350%), while PKCbeta 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 PKCbeta 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 PKCbeta II immunoreactive protein between experiments are attributed to variables in cell culture and sensitivity of chemiluminescence detection. The overall increase in PKCbeta II immunoreactive protein following insulin treatment was consistent.


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Fig. 3.   Effect of insulin on PKCbeta I and -beta II protein expression. Whole cell lysates (20 µg) from L6 myotubes treated for various times as indicated with insulin (200 nM) or PBS (sham control) were prepared, subjected to SDS-PAGE fractionation, and Western blot analysis. Blots were developed as described under "Material and Methods." Each panel represents a separate experiment with its own control. Data are composites from four different experiments reproduced on separate occasions.

The Rapid Up-regulation in PKCbeta II mRNA Was Not Reflected by Increased Transcriptional Activity-- If the alternative splicing that leads to mature PKCbeta II mRNA is under negative regulation by a limited amount of RNA transactivating factors, a rapid increase in PKCbeta pre-mRNA via transcriptional activation may account for increased PKCbeta II expression. To examine whether the up-regulation of PKCbeta II mRNA was related to an increase in transcriptional activity, several PKCbeta promotor constructs of varying lengths including a (-2200 to +43) construct were examined for insulin responsiveness. The PKCbeta 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 PKCbeta 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 PKCbeta I mRNA is not increased in response to insulin, the early increase in PKCbeta II mRNA was not likely due to an increase in transcriptional activity of the PKCbeta 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-beta -galactosidase activity.

The Early Up-regulation in PKCbeta II mRNA Was Not Likely Due to Increased Message Stability-- The possibility of post-transcriptional regulation of PKCbeta II expression at the level of message stability was also evaluated. To demonstrate whether PKCbeta II mRNA was up-regulated via increases in message stability, insulin effects on PKCbeta I and PKCbeta 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 PKCbeta II message were not likely due to rapid increases in mRNA stabilization. Also of note is the observation that insulin did not destabilize PKCbeta I message as in the BC3H-1 myocytes, demonstrating either the lack of a hormone-induced destabilization system or a requirement for PKCbeta 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 PKCbeta II expression was regulated primarily at the level of alternative splicing was likely. Early studies by Nishizuka and co-workers (4) suggested PKCbeta II mRNA is encoded via inclusion of the PKCbeta 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 PKCbeta II splicing. Based on the information available about the arrangement of terminal exons in the PKCbeta gene, it was possible that the alternatively spliced PKCbeta II mRNA resulted from one of these two mechanisms, inclusion of the PKCbeta 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 PKCbeta II exon, thereby producing mature PKCbeta II mRNA. In the case of an exon inclusion mechanism, the PKCbeta I polyadenylation site would be used, but a stop codon in the PKCbeta II exon terminates translation and results in a protein with the PKCbeta II COOH terminus. If the PKCbeta II exon were skipped, then PKCbeta I would be encoded. To determine if the increase in PKCbeta II mRNA was due to regulation of PKCbeta 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 PKCbeta II exon and near the PKCbeta II polyadenylation site, only a polyadenylated form of PKCbeta II mRNA could be amplified. Neither control L6 myotubes nor insulin-treated L6 myotubes expressed a polyadenylated form of PKCbeta II even after 55 PCR cycles (Fig. 4). Therefore, the increase in PKCbeta II mRNA in response to insulin was not likely due to regulation of PKCbeta II polyadenylation site selection. As a positive control, 10-3 amol of a PKCbeta II cDNA containing the 3'-untranslated region of the polyadenylated form of PKCbeta II was amplified (obtained from Dr. S. Ohno, Yokohama City University School of Medicine, Yokohama, Japan).


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Fig. 4.   The PKCbeta II mature mRNA encoded using the PKCbeta II polyadenylation site is not expressed in L6 myotubes. RT-PCR analysis of L6 myotubes treated with PBS (sham control) or insulin (200 nM) for 15 min using primer E, a primer to the C4 region common to both PKCbeta I and -beta II, and primer G, a primer specific for the downstream PKCbeta II intronic sequence between the polyadenylation site and the nearest 5' splice site (see "Materials and Methods" for primer sequences). Prior to assay, the cells were removed from serum for 6 h. C indicates control, I indicates insulin, and + reflects 10-3 amol of polyadenylated PKCbeta II positive control (PKCbeta II cDNA). Arrow designates the expected product size for polyadenylated mature PKCbeta II mRNA. Data are representative of three separate determinations from an experiment reproduced on two occasions.

Insulin Enhanced the Inclusion of the PKCbeta II Exon into the Mature mRNA Transcript-- Since alternative polyadenylation site selection did not likely account for the effect of insulin on PKCbeta II expression, the possibility that insulin affected inclusion of the PKCbeta II-specific exon in L6 myotubes was a likely alternative. An RT-PCR assay that allowed for the simultaneous detection of PKCbeta II and PKCbeta I mRNA utilizing primer E, a sense primer to the upstream PKCbeta common exon (C4 kinase domain), and primer D, an antisense downstream primer specific for PKCbeta I (V5 domain) was designed (Fig. 5, A and B). In response to acute insulin treatment, PKCbeta II message (374-bp PCR product) increased to levels that exceeded PKCbeta I message (153-bp PCR product) (Fig. 6). The amount of mature PKCbeta 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 PKCbeta II-specific exon.


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Fig. 5.   Structure of PKCbeta I and -beta II splice variants and regions amplified by RT-PCR for PKCbeta II exon inclusion/exclusion and 5' splice site activation assays. A, a diagram depicting the 3' end of the PKCbeta gene showing the mRNA processing schemes for inclusion of the PKCbeta II exon in skeletal muscle. Arrows indicate the position of oligonucleotide sequences for primers (E, F, and D) used in RT-PCR analyzing PKCbeta II exon inclusion into mature mRNA transcripts and PKCbeta II exon 5' splice site activation (see "Materials and Methods" for primers sequences). B, the three possible PCR products for the PKCbeta gene in L6 myotubes using primers E and D are depicted with predicted molecular weights. C, the two possible PCR products using primers F and D reflecting the activation of two 5' splice sites are depicted with predicted molecular weights.


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Fig. 6.   The effects of insulin on PKCbeta II exon inclusion into mature mRNA. L6 myotubes treated with PBS (sham control) or insulin (200 nM) for 15 min were analyzed by RT-PCR using primer E, a sense primer to the C4 region common to both PKCbeta I and -beta II, and primer D, an antisense primer specific for the PKCbeta I V5 exon (depicted in Fig. 5, A and B) (see "Materials and Methods" for primer sequences). Prior to assay, the cells were removed from serum for 6 h. C indicates control, I indicates insulin, and + indicates PKCbeta I positive control (PKCbeta I cDNA). Arrows indicate PKCbeta I or PKCbeta II mature mRNA. Data are representative of three separate determinations from an experiment reproduced on three occasions.

Identification of a Previously Unidentified 5' Splice Site for the PKCbeta II Exon Encoding an Additional Splice Variant of PKCbeta II-- An additional, longer PCR product was also observed following exon inclusion analysis, which did not correspond to the anticipated PKCbeta II amplification product (Figs. 6) (4). The new fragment hybridized with a labeled PKCbeta cDNA probe upon Southern blot analysis suggesting that the amplified fragment was not artifactual. Sequence analysis revealed it was a new splice variant of PKCbeta II, which included approximately 136 nucleotides of additional extended 3'-untranslated sequence (Fig. 5C). After comparing this sequence to the published sequence of the PKCbeta 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).


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Fig. 7.   A, revised PKCbeta 3' gene structure. Schematic diagram depicts the 3' end of the PKCbeta gene with approximate sizes of exons and introns from sequence analysis (4). Polyadenylation sites are designated AATAAA for PKCbeta II and ATTAAA for PKCbeta I. Enlarged schematic of the PKCbeta II exon depicts 5' splice site sequences along with RNA cis-elements. Black dots represent short (10-14 bp) purine-rich exon splicing enhancers, ESE designates a large 38-bp purine-rich exon splicing enhancer, Stem loop structure reflects a 44-bp AUUUA stem loop structure, PYR designates a 30-bp pyrimidine-rich tract, and SRp40 depicts a binding site for phosphorylated SRp40. B, structure of PKCbeta I and -beta II splice variants. Schematic represents the 3' end of PKCbeta I and PKCbeta II mature mRNA. I) reflects PKCbeta I mature mRNA; II) reflects PKCbeta II mature mRNA derived from exon inclusion and deduced from rat brain; III) reflects a splice variant for PKCbeta II with extended 5'-untranslated region derived from L6 myotubes; IV) reflects PKCbeta II mRNA derived from alternative polyadenylation selection. beta C, the nearest exon shared by both PKCbeta I and -beta II.

Insulin Sequentially Activates a Second 5' Splice Site in the PKCbeta 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 PKCbeta II exon and the PKCbeta I terminal exon (Fig. 5, A and C). This RT-PCR-based assay examines specifically exon-included PKCbeta II mRNA and therefore increases our detection sensitivity for PKCbeta 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 PKCbeta 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.


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Fig. 8.   The effects of insulin and IGF-I on 5' splice site selection of the PKCbeta II exon. RT-PCR analysis of L6 myotubes treated with PBS (sham control), insulin 200 nM, or IGF-I (Long R3 IGF-I, GroPep) for various times using primer F, a sense primer to the PKCbeta II exon, and primer D, an antisense primer specific for the PKCbeta I V5 exon (depicted in Fig. 5, A and C) (see "Materials and Methods" for primer sequences). Prior to assay, cells were maintained in serum-free medium for 6 h. Arrows designate PKCbeta II 5' splice site variants. B indicates the product corresponding to activation of the brain PKCbeta II 5' splice site, and A indicates the product corresponding to activation of the second PKCbeta II 5' splice site with 136 bp of extended untranslated region. Data are representative of three separate determinations from an experiment reproduced on three occasions.

Insulin Effects on mRNA Splicing Are Specific for PKCbeta II 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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Fig. 9.   Effect of insulin on insulin receptor exon 11 inclusion. RT-PCR analysis of L6 myotube mRNA from cells treated with insulin (200 nM) or PBS (sham control) for 30 min using primers corresponding to exons 10 and 12 of the insulin receptor (see "Materials and Methods" for primer sequences). Cells were cultured as described under "Materials and Methods." Arrows designate either type A (exon 11 excluded) or type B (exon 11 included) mRNA. C indicates control, I indicates insulin-treated, and P indicates primer control. Data are representative of three separate determinations from an experiment repeated on two occasions.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our earlier studies in the BC3H-1 myocytes demonstrated that PKCbeta II expression was regulated by insulin (12). L6 myotubes were used to determine the mechanism responsible for insulin effects on PKCbeta 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 PKCbeta I and PKCbeta 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 PKCbeta II mRNA and protein increased more than 3.5-fold following acute insulin treatment. Levels of PKCbeta I mature mRNA and immunoreactive protein remained unchanged following insulin treatment. Prior studies in the BC3H-1 myocytes showed that PKCbeta I mRNA and protein levels decreased in response to insulin. This was due to destabilization of PKCbeta 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 PKCbeta I in insulin action in the L6 myotubes. Other findings from our laboratory suggest a role for PKCbeta I in insulin action. We found that overexpression of PKCbeta I in L6 myotubes overcomes the inhibition of insulin-stimulated 2-deoxyglucose uptake by wortmannin, a phosphatidylinositol 3-kinase inhibitor (19). Overexpression of PKCbeta I in L6 myotubes also increases insulin-stimulated glycogen synthase activation.2 These data suggest that PKCbeta I as well as PKCbeta II have distinct roles in the metabolic effects of insulin. However, the mechanism by which insulin affected PKCbeta pre-mRNA splicing had not been elucidated, and whether PKCbeta pre-mRNA preferentially processed to PKCbeta I or PKCbeta II mature mRNA was not known. It also remained to be elucidated whether PKCbeta 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 PKCbeta II mRNA expression could be through increased transcriptional activation in response to insulin. In this case, the RNA splicing of PKCbeta pre-mRNA to mature PKCbeta 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 PKCbeta II-specific exon or recognition of the PKCbeta II polyadenylation site, thereby leading to the production of mature PKCbeta I mRNA. A rapid increase in the transcription of the PKCbeta gene in response to insulin may increase the levels of PKCbeta pre-mRNA. If the inhibitory RNA transactivating factors are limiting, the newly transcribed PKCbeta pre-mRNA would be processed to mature PKCbeta II mRNA. The PKCbeta 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 PKCbeta promotor/luciferase constructs spanning -2200 to +43 bp (15). Only a -511/+43 PKCbeta 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 PKCbeta promotor construct occurred, however, at later times (16 h), and induction was minimal. Hence, the increase in PKCbeta 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 PKCbeta II mRNA stability could also increase PKCbeta II mRNA levels in response to insulin. To determine if increases in PKCbeta II mRNA were due to increased mRNA stability, relative decay rates of PKCbeta I and PKCbeta II mRNA were measured following actinomycin D treatment. Relative decay rates did not change within 1 h of insulin treatment, and the increase in PKCbeta II mRNA levels were not likely justified by increased PKCbeta II mRNA stability.

Mature PKCbeta II 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 PKCbeta II mRNA was produced via inclusion of an exon that encodes a stop codon defining the V5 region of PKCbeta II in response to insulin treatment. This mechanism produces a message with the PKCbeta I exon including a polyadenylation site spliced onto the 3' end of the PKCbeta II exon (Fig. 7B). Insulin regulation of PKCbeta II exon inclusion changed the ratio of PKCbeta I to PKCbeta II mRNA following insulin treatment. The levels of newly processed PKCbeta II mRNA exceeded the synthesis of PKCbeta I mRNA, demonstrating that insulin enhanced PKCbeta II exon inclusion into the mature mRNA transcript.

The molecular mechanism through which insulin affects PKCbeta II exon inclusion has not been addressed. Our findings suggest that alterations in splicing factors associated with the PKCbeta 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 PKCbeta 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 PKCbeta pre-mRNA splicing was the observation that insulin activated a second 5' splice site downstream of the previously reported 5' splice site for the PKCbeta 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 PKCbeta II extended the 3'-untranslated region by approximately 136 nucleotides. The observation that insulin activated two 5' splice sites for PKCbeta 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 PKCbeta 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 PKCbeta pre-mRNA splicing compared with insulin receptor mRNA splicing.

PKCbeta is activated and translocated by insulin in L6 myotubes, and we have previously reported that PKCbeta II is associated with enhanced insulin-stimulated 2-deoxyglucose uptake, thus suggesting a physiological significance for increasing PKCbeta II expression (8, 12, 24). A possible link of PKCbeta II to insulin-stimulated 2-deoxyglucose uptake could be hypothesized by the fact that PKCbeta II specifically encodes an F-actin binding site in its V5 region that PKCbeta I does not encode. PKCbeta 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, PKCbeta 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, PKCbeta II 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 PKCbeta 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.

    ACKNOWLEDGEMENTS

We thank Dr. Harald Mischak for providing the design of the PKCbeta II primers used in the competitive RT-PCR assay and Dr. Yoshiko Akita for the gracious gift of the PKCbeta I- and PKCbeta II-specific antibodies.

    FOOTNOTES

* 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); alpha -MEM, alpha -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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